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Nadia Zaffaroni

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DOI: 10.1158/0008-5472.can-05-0626
2005
Cited 1,656 times
Isolation and <i>In vitro</i> Propagation of Tumorigenic Breast Cancer Cells with Stem/Progenitor Cell Properties
Breast cancer-initiating cells have been recently identified in breast carcinoma as CD44+/CD24(-/low) cells, which exclusively retain tumorigenic activity and display stem cell-like properties. However, at present, direct evidence that breast cancer-initiating cells can be propagated in vitro is still lacking. We report here the isolation and in vitro propagation of breast cancer-initiating cells from three breast cancer lesions and from an established breast carcinoma cell line. Our breast carcinoma-derived cultures encompassed undifferentiated cells capable of self-renewal, extensive proliferation as clonal nonadherent spherical clusters, and differentiation along different mammary epithelial lineages (ductal and myoepithelial). Interestingly, cultured cells were CD44+/CD24- and Cx43-, overexpressed neoangiogenic and cytoprotective factors, expressed the putative stem cell marker Oct-4, and gave rise to new tumors when as few as 10(3) cells were injected into the mammary fat pad of SCID mice. Long-term cultures of breast tumorigenic cells with stem/progenitor cell properties represent a suitable in vitro model to study breast cancer-initiating cells and to develop therapeutic strategies aimed at eradicating the tumorigenic subpopulation within breast cancer.
DOI: 10.1158/0008-5472.can-06-4690
2007
Cited 657 times
Human Bone Marrow–Derived Mesenchymal Stem Cells Do Not Undergo Transformation after Long-term<i>In vitro</i>Culture and Do Not Exhibit Telomere Maintenance Mechanisms
Significant improvement in the understanding of mesenchymal stem cell (MSC) biology has opened the way to their clinical use. However, concerns regarding the possibility that MSCs undergo malignant transformation have been raised. We investigated the susceptibility to transformation of human bone marrow (BM)-derived MSCs at different in vitro culture time points. MSCs were isolated from BM of 10 healthy donors and propagated in vitro until reaching either senescence or passage (P) 25. MSCs in the senescence phase were closely monitored for 8 to 12 weeks before interrupting the cultures. The genetic characterization of MSCs was investigated through array-comparative genomic hybridization (array-CGH), conventional karyotyping, and subtelomeric fluorescent in situ hybridization analysis both before and after prolonged culture. MSCs were tested for the expression of telomerase activity, human telomerase reverse transcriptase (hTERT) transcripts, and alternative lengthening of telomere (ALT) mechanism at different passages. A huge variability in terms of proliferative capacity and MSCs life span was noted between donors. In eight of 10 donors, MSCs displayed a progressive decrease in proliferative capacity until reaching senescence. In the remaining two MSC samples, the cultures were interrupted at P25 to pursue data analysis. Array-CGH and cytogenetic analyses showed that MSCs expanded in vitro did not show chromosomal abnormalities. Telomerase activity and hTERT transcripts were not expressed in any of the examined cultures and telomeres shortened during the culture period. ALT was not evidenced in the MSCs tested. BM-derived MSCs can be safely expanded in vitro and are not susceptible to malignant transformation, thus rendering these cells suitable for cell therapy approaches.
DOI: 10.1158/0008-5472.can-08-2894
2009
Cited 337 times
miR-205 Exerts Tumor-Suppressive Functions in Human Prostate through Down-regulation of Protein Kinase Cε
Abstract Limited information is available concerning the expression and role of microRNAs in prostate cancer. In this study, we investigated the involvement of miR-205 in prostate carcinogenesis. Significantly lower miR-205 expression levels were found in cancer than in normal prostate cell lines as well as in tumor compared with matched normal prostate tissues, with a particularly pronounced reduction in carcinomas from patients with local-regionally disseminated disease. Restoring the expression of miR-205 in prostate cancer cells resulted in cell rearrangements consistent with a mesenchymal-to-epithelial transition, such as up-regulation of E-cadherin and reduction of cell locomotion and invasion, and in the down-regulation of several oncogenes known to be involved in disease progression (i.e., interleukin 6, caveolin-1, EZH2). Our evidence suggests that these events are driven by the concurrent repression of specific predicted miR-205 targets, namely N-chimaerin, ErbB3, E2F1, E2F5, ZEB2, and protein kinase Cε. Strikingly, the latter seemed to play a direct role in regulating epithelial-to-mesenchymal transition. In fact, its down-regulation led to a cell phenotype largely reminiscent of that of cells ectopically expressing miR-205. Overall, we showed for the first time that miR-205 exerts a tumor-suppressive effect in human prostate by counteracting epithelial-to-mesenchymal transition and reducing cell migration/invasion, at least in part through the down-regulation of protein kinase Cε. [Cancer Res 2009;69(6):2287–95]
DOI: 10.1016/j.ccr.2005.03.035
2005
Cited 307 times
Rational design of shepherdin, a novel anticancer agent
Anticancer agents that selectively kill tumor cells and spare normal tissues are urgently needed. Here, we engineered a cell-permeable peptidomimetic, shepherdin, modeled on the binding interface between the molecular chaperone Hsp90 and the antiapoptotic and mitotic regulator, survivin. Shepherdin makes extensive contacts with the ATP pocket of Hsp90, destabilizes its client proteins, and induces massive death of tumor cells by apoptotic and nonapoptotic mechanisms. Conversely, shepherdin does not reduce the viability of normal cells, and does not affect colony formation of purified hematopoietic progenitors. Systemic administration of shepherdin in vivo is well tolerated, and inhibits human tumor growth in mice without toxicity. Shepherdin could provide a potent and selective anticancer agent in humans.
DOI: 10.1158/1078-0432.ccr-15-2170
2016
Cited 157 times
TNF-Related Apoptosis-Inducing Ligand (TRAIL)–Armed Exosomes Deliver Proapoptotic Signals to Tumor Site
Exosomes deliver signals to target cells and could thus be exploited as an innovative therapeutic tool. We investigated the ability of membrane TRAIL-armed exosomes to deliver proapoptotic signals to cancer cells and mediate growth inhibition in different tumor models.K562 cells, transduced with lentiviral human membrane TRAIL, were used for the production of TRAIL(+) exosomes, which were studied by nanoparticle tracking analysis, cytofluorimetry, immunoelectronmicroscopy, Western blot, and ELISA. In vitro, TRAIL(+) exosomes induced more pronounced apoptosis (detected by Annexin V/propidium iodide and activated caspase-3) in TRAIL-death receptor (DR)5(+) cells (SUDHL4 lymphoma and INT12 melanoma), with respect to the DR5(-)DR4(+)KMS11 multiple myeloma. Intratumor injection of TRAIL(+) exosomes, but not mock exosomes, induced growth inhibition of SUDHL4 (68%) and INT12 (51%), and necrosis in KMS11 tumors. After rapid blood clearance, systemically administered TRAIL(+) exosomes accumulated in the liver, lungs, and spleen and homed to the tumor site, leading to a significant reduction of tumor growth (58%) in SUDHL4-bearing mice. The treatment of INT12-bearing animals promoted tumor necrosis and a not statistically significant tumor volume reduction. In KMS11-bearing mice, despite massive perivascular necrosis, no significant tumor growth inhibition was detected.TRAIL-armed exosomes can induce apoptosis in cancer cells and control tumor progression in vivo Therapeutic efficacy was particularly evident in intratumor setting, while depended on tumor model upon systemic administration. Thanks to their ability to deliver multiple signals, exosomes thus represent a promising therapeutic tool in cancer. Clin Cancer Res; 22(14); 3499-512. ©2016 AACR.
DOI: 10.1007/s00018-002-8518-3
2002
Cited 256 times
Expression of the anti-apoptotic gene survivin correlates with taxol resistance in human ovarian cancer
DOI: 10.1186/1476-4598-9-12
2010
Cited 192 times
miR-21: an oncomir on strike in prostate cancer
Aberrant expression of microRNAs, small non-coding RNA molecules that post-transcriptionally repress gene expression, seems to be causatively linked to the pathogenesis of cancer. In this context, miR-21 was found to be overexpressed in different human cancers (e.g. glioblastoma, breast cancer). In addition, it is thought to be endowed with oncogenic properties due to its ability to negatively modulate the expression of tumor-suppressor genes (e.g. PTEN) and to cause the reversion of malignant phenotype when knocked- down in several tumor models. On the basis of these findings, miR-21 has been proposed as a widely exploitable cancer-related target. However, scanty information is available concerning the relevance of miR-21 for prostate cancer. In the present study, we investigated the role of miR-21 and its potential as a therapeutic target in two prostate cancer cell lines, characterized by different miR-21 expression levels and PTEN gene status.We provide evidence that miR-21 knockdown in prostate cancer cells is not sufficient per se i) to affect the proliferative and invasive potential or the chemo- and radiosensitivity profiles or ii) to modulate the expression of the tumor-suppressors PTEN and Pdcd4, which in other tumor types were found to be regulated by miR-21. We also show that miR-21 is not differently expressed in carcinomas and matched normal tissues obtained from 36 untreated prostate cancer patients subjected to radical prostatectomy.Overall, our data suggest that miR-21 is not a central player in the onset of prostate cancer and that its single hitting is not a valuable therapeutic strategy in the disease. This supports the notion that the oncogenic properties of miR-21 could be cell and tissue dependent and that the potential role of a given miRNA as a therapeutic target should be contextualized with respect to the disease.
DOI: 10.1093/annonc/mdq644
2011
Cited 190 times
Sunitinib in advanced alveolar soft part sarcoma: evidence of a direct antitumor effect
The purpose of this study was to confirm sunitinib activity in alveolar soft part sarcoma (ASPS) and to report on new insights into the molecular bases thereof.From July 2007, nine patients with progressive metastatic ASPS received sunitinib 37.5 mg/day, within a named use program. Cryopreserved material was available for five naive patients, among whom three received sunitinib. Immunofluorescence (IF)/confocal microscopy, biochemical, and molecular/cytogenetic analyses were carried out, complemented by antiproliferative and activation assays in a short-term culture derived from one case.All patients were eligible for response. Best RECIST response was partial response in five cases, stable disease in three, and progression in one. The median progression-free survival was 17 months. Positron emission tomography results were consistent. Two cases of interval progressions were recorded. Antiproliferative assays and biochemistry on short-term culture showed that sunitinib is able to markedly impair ASPS cells growth and switch-off PDGFRB. IF/confocal microscopy demonstrated coexpression and physical association between PDGFRB/vascular endothelial growth factor receptor 2 (VEGFR2) and RET/VEGFR2 in ASPS cells, which was validated by biochemistry. PDGFRB, RET, and MET ligand-dependent activation was confirmed.We confirm the clinical efficacy of sunitinib in ASPS, mediated by PDGFRB, VEGFR2, and RET, which are all expressed in tumor cells. A direct antitumor effect was shown in a short-term cell culture.
DOI: 10.1016/s1368-7646(02)00049-3
2002
Cited 189 times
Survivin expression and resistance to anticancer treatments: perspectives for new therapeutic interventions
Survivin is a structurally unique member of the inhibitors of apoptosis protein (IAP) family that is involved in both control of cell division and inhibition of apoptosis. Its anti-apoptotic function seems to be related to its ability to directly or indirectly inhibit caspases, although the precise role of survivin in the modulation of the caspase cascade has not been fully elucidated. Survivin has been described to be selectively expressed in the most common human neoplasms and to be associated with clinical tumour progression. Moreover, the protein appears to be involved in tumour cell resistance to some anticancer agents and ionizing radiation. On the basis of these findings survivin has been proposed as a promising target for new anticancer interventions. In in vitro and in vivo studies targeting survivin with antisense oligonucleotides, dominant negative mutants or ribozymes have shown to induce apoptosis, reduce tumour-growth potential and sensitise tumour cells to chemotherapeutic drugs such as Taxol, cisplatin and etoposide, gamma-irradiation, and immunotherapy.
DOI: 10.1517/14728222.12.4.463
2008
Cited 163 times
Targeting survivin in cancer therapy
Background: Survivin is a structurally unique member of the inhibitor of apoptosis protein (IAP) family that acts as a suppressor of apoptosis and plays a central role in cell division. Owing to its massive upregulation in human tumors and its involvement in cancer progression and treatment resistance, survivin is currently undergoing extensive investigation as a novel therapeutic target. Objective: The purpose of this review is to define the potential of survivin as a therapeutic target for new anticancer interventions. Methods: The literature dealing with the therapeutic targeting of survivin has been carefully reviewed. Results/conclusion: Several preclinical studies have demonstrated that downregulation of survivin expression or function, accomplished by means of various strategies, reduced tumor growth potential, increased the apoptotic rate and sensitized tumor cells to chemotherapeutic drugs and radiation in different human tumor models. Moreover, the first survivin inhibitors are being currently evaluated in clinical settings.
DOI: 10.3390/molecules181012368
2013
Cited 128 times
G-Quadruplex Structures in the Human Genome as Novel Therapeutic Targets
G-quadruplexes are secondary structures that may form within guanine-rich nucleic acid sequences. Telomeres have received much attention in this regard since they can fold into several distinct intramolecular G-quadruplexes, leading to the rational design and development of G-quadruplex‑stabilizing molecules. These ligands were shown to selectively exert an antiproliferative and chemosensitizing activity in in vitro and in vivo tumor models, without appreciably affecting normal cells. Such findings point to them as possible drug candidates for clinical applications. Other than in telomeres, G-quadruplexes may form at additional locations in the human genome, including gene promoters and untranslated regions. For instance, stabilization of G-quadruplex structures within the promoter of MYC, KIT, or KRAS resulted in the down-regulation of the corresponding oncogene either in gene reporter assays or in selected experimental models. In addition, the alternative splicing of a number of genes may be affected for a therapeutic benefit through the stabilization of G-quadruplexes located within pre-mRNAs. It is now emerging that G-quadruplex structures may act as key regulators of several biological processes. Consequently, they are considered as attractive targets for broad-spectrum anticancer therapies, and much effort is being made to develop a variety of ligands with improved G-quadruplex recognition properties. Quarfloxin, a fluoroquinolone derivative designed to target a G-quadruplex within ribosomal DNA and disrupt protein-DNA interactions, has entered clinical trials for different malignancies. This review will provide some hints on the role of G-quadruplex structures in biological processes and will evaluate their implications as novel therapeutic targets.
DOI: 10.1016/s1470-2045(12)70294-2
2012
Cited 126 times
Pazopanib in advanced and platinum-resistant urothelial cancer: an open-label, single group, phase 2 trial
Background The development of new drugs for patients with refractory urothelial cancer is still an unmet medical need. Preclinical evidence lends support to a rationale for targeting of the VEGF or platelet-derived growth-factor axis. We therefore investigated the activity and safety of pazopanib, a multitarget drug with antiangiogenic activity, in patients with urothelial cancer. Methods In an open-label, single-group, phase 2 study, patients (aged ≥18 years) with relapsed or refractory urothelial cancer were given pazopanib 800 mg per day, orally. They were treated until disease progression or prohibitive toxicity occurred. The primary endpoint was the proportion of patients who achieved a confirmed objective response, defined as complete or partial response, after independent review, and was analysed by intention to treat. The trial is registered with ClinicalTrials.gov, number NCT01031875. Findings The trial has been completed. 21 (51%) of 41 patients enrolled were given pazopanib as third-line or further-line treatment. 26 (63%) patients had an Eastern Cooperative Oncology Group performance status of 1 or 2. Seven patients had a confirmed objective response (17·1%, 95% CI 7·2–32·1), all of which were partial responses. The most frequent treatment-related grade 3 adverse events were hypertension (three [7%]), fatigue (two [5%]), and gastrointestinal and vaginal fistulisations (two each [5%]). One patient died as a result of duodenal fistulisation that was related to tissue response of bulky tumour masses. Interpretation Pazopanib has single-agent activity in patients with heavily pretreated metastatic urothelial cancer, and warrants further study in this setting. Particular attention should be paid to patients with bulky tumour masses adjacent to viscera because fistulisation is probably related to the response to pazopanib and is the most frequent serious adverse event. Funding Fondazione IRCCS Istituto Nazionale dei Tumori provided the grant. GlaxoSmithKline provided the study drug and provided funding for the independent radiological review.
DOI: 10.1016/j.molonc.2014.07.009
2014
Cited 107 times
Senescent stroma promotes prostate cancer progression: The role of miR‐210
We focused our interest on senescent human-derived fibroblasts in the progression of prostate cancer. Hypoxic senescent fibroblasts promote prostate cancer aggressiveness by inducing epithelial to mesenchymal transition (EMT) and by secreting energy-rich compounds to support cancer cell growth. Hypoxic senescent fibroblasts additionally increase: i) the recruitment of monocytes and their M2-macrophage polarization, ii) the recruitment of bone marrow-derived endothelial precursor cells, facilitating their vasculogenic ability and iii) capillary morphogenesis, proliferation and invasion of human mature endothelial cells. In addition, we highlight that overexpression of the hypoxia-induced miR-210 in young fibroblasts increases their senescence-associated features and converts them into cancer associated fibroblast (CAF)-like cells, able to promote cancer cells EMT, to support angiogenesis and to recruit endothelial precursor cells and monocytes/macrophages.
DOI: 10.1021/jm400842x
2013
Cited 93 times
Novel 1<i>H</i>-Pyrrolo[2,3-<i>b</i>]pyridine Derivative Nortopsentin Analogues: Synthesis and Antitumor Activity in Peritoneal Mesothelioma Experimental Models
In this study, we describe the synthesis of new nortopsentin analogues, 1H-pyrrolo[2,3-b]pyridine derivatives and their biological effects in experimental models of diffuse malignant peritoneal mesothelioma (DMPM), a rare and rapidly fatal disease, poorly responsive to conventional therapies. The three most active compounds, 1f (3-[2-(5-fluoro-1-methyl-1H-indol-3-yl)-1,3-thiazol-4-yl]-1H-pyrrolo[2,3-b]pyridine), 3f (3-[2-(1H-indol-3-yl)-1,3-thiazol-4-yl]-1-methyl-1H-pyrrolo[2,3-b]pyridine), and 1l (3-[2-(5-fluoro-1-methyl-1H-indol-3-yl)-1,3-thiazol-4-yl]-1-methyl-1H-pyrrolo[2,3-b] pyridine), which were shown to act as cyclin-dependent kinase 1 inhibitors, consistently reduced DMPM cell proliferation and induced a caspase-dependent apoptotic response, with a concomitant reduction of the expression of the active Thr34-phosphorylated form of the antiapoptotic protein survivin. Moreover, the combined treatment of DMPM cells with 3f derivative and paclitaxel produced a synergistic cytotoxic effect, which was paralleled by an enhanced apoptotic response. In the mouse model, i.p. administration of 1f, 3f, and 1l derivatives was effective, resulting in a significant tumor volume inhibition of DMPM xenografts (range, 58–75%) at well-tolerated doses, and two complete responses were observed in each treatment group.
DOI: 10.1016/j.bcp.2013.12.009
2014
Cited 83 times
miR-205 impairs the autophagic flux and enhances cisplatin cytotoxicity in castration-resistant prostate cancer cells
Compelling evidence suggests that epithelial-to-mesenchymal transition is involved in the resistance of human cancer cells to chemotherapy. We previously reported that the expression of miR-205, a miRNA down-regulated in prostate cancer, is further repressed in prostate cancer cells undergoing epithelial-to-mesenchymal transition, suggesting a possible involvement of the miRNA in the acquisition of the chemoresistant phenotype. In the present study, we show that miR-205 replacement in castration-resistant mesenchymal prostate cancer cells caused an enhancement of cisplatin cytotoxic activity in vitro and in vivo, as a consequence of autophagy impairment. Specifically, the constraints on the autophagic flux were associated to the miRNA-dependent down-regulation of the lysosome-associated proteins RAB27A and LAMP3. These findings suggest that miR-205-mediated impairment of the autophagic pathway may interfere with the detoxifying capabilities of prostate cancer cells in their attempt to cope with cisplatin-induced detrimental effects. Overall, our data indicate that (i) loss of miR-205 may indeed contribute to acquire mesenchymal tracts and concomitantly establish a permissive autophagic milieu that confers a chemotherapy resistant phenotype to prostate cancer cells, and (ii) strategies aimed at restoring miR-205 expression levels may represent a successful approach to overcome resistance of prostate cancer to platinum compounds.
DOI: 10.1016/j.canlet.2017.02.033
2017
Cited 81 times
miR-875-5p counteracts epithelial-to-mesenchymal transition and enhances radiation response in prostate cancer through repression of the EGFR-ZEB1 axis
Radiotherapy is one of the main treatment choices for non-metastatic prostate cancer (PCa), although development of radioresistance limits its effectiveness. Mounting evidence supports the ability of microRNAs to interfere with different radioresistance-associated pathways, suggesting their potential as radiosensitizers. Here, we demonstrate that reconstitution of miR-875-5p, whose expression is down-regulated in PCa clinical samples and directly correlates with that of E-cadherin, was able to enhance radiation response in PCa cell lines and xenografts through EGFR direct targeting. Consistent with the established role of EGFR in sustaining epithelial-to-mesenchymal transition (EMT) and promoting DNA repair following radiation-induced nuclear translocation, we found that miR-875-5p reconstitution in PCa cells counteracted EMT and impaired DNA lesion clearance. Down-regulation of the EMT-inducing transcription factor ZEB1, which also plays a role in homologous recombination-mediated repair of DNA lesions by regulating CHK1 expression, was found to be a major determinant of miR-875-5p-induced radiosensitization, as confirmed by phenocopy experiments showing that siRNA-mediated ZEB1 knock-down was able to reproduce the microRNA radiosensitizing effect. Overall, our data support the clinical interest in developing a novel therapeutic approach based on miR-875-5p reconstitution to increase PCa response to radiotherapy.
DOI: 10.3390/cancers11010090
2019
Cited 61 times
Role of FoxO Proteins in Cellular Response to Antitumor Agents
FoxO proteins (FoxOs) are transcription factors with a common DNA binding domain that confers selectivity for DNA interaction. In human cells, four proteins (FoxO1, FoxO3, FoxO4 and FoxO6), with redundant activity, exhibit mainly a positive effect on genes involved in cell cycle, apoptosis regulation and drug resistance. Thus, FoxOs can affect cell response to antitumor agent treatment. Their transcriptional activity depends on post-translational modifications, including phosphorylation, acetylation, and mono/poly-ubiquitination. Additionally, alterations in microRNA network impact on FoxO transcripts and in turn on FoxO levels. Reduced expression of FoxO1 has been associated with resistance to conventional agents (e.g., cisplatin) and with reduced efficacy of drug combinations in ovarian carcinoma cells. FoxO3 has been shown as a mediator of cisplatin toxicity in colorectal cancer. A requirement for FoxO3-induced apoptosis has been reported in cells exposed to targeted agents (e.g., gefitinib). Recently, the possibility to interfere with FoxO1 localization has been proposed as a valuable approach to improve cell sensitivity to cisplatin, because nuclear retention of FoxO1 may favor the induction of pro-apoptotic genes. This review focuses on the role of FoxOs in drug treatment response in tumor cells and discusses the impact of the expression of these transcription factors on drug resistance/sensitivity.
DOI: 10.1186/s13046-019-1060-z
2019
Cited 60 times
miR-205 enhances radiation sensitivity of prostate cancer cells by impairing DNA damage repair through PKCε and ZEB1 inhibition
Radiotherapy is one of the main treatment options for non-metastatic prostate cancer (PCa). Although treatment technical optimization has greatly improved local tumor control, a considerable fraction of patients still experience relapse due to the development of resistance. Radioresistance is a complex and still poorly understood phenomenon involving the deregulation of a variety of signaling pathways as a consequence of several genetic and epigenetic abnormalities. In this context, cumulative evidence supports a functional role of microRNAs in affecting radioresistance, suggesting the modulation of their expression as a novel radiosensitizing approach. Here, we investigated for the first time the ability of miR-205 to enhance the radiation response of PCa models.miR-205 reconstitution by a miRNA mimic in PCa cell lines (DU145 and PC-3) was used to elucidate miR-205 biological role. Radiation response in miRNA-reconstituted and control cells was assessed by clonogenic assay, immunofluorescence-based detection of nuclear γ-H2AX foci and comet assay. RNAi was used to silence the miRNA targets PKCε or ZEB1. In addition, target-protection experiments were carried out using a custom oligonucleotide designed to physically disrupt the pairing between the miR-205 and PKCε. For in vivo experiments, xenografts generated in SCID mice by implanting DU145 cells stably expressing miR-205 were exposed to 5-Gy single dose irradiation using an image-guided animal micro-irradiator.miR-205 reconstitution was able to significantly enhance the radiation response of prostate cancer cell lines and xenografts through the impairment of radiation-induced DNA damage repair, as a consequence of PKCε and ZEB1 inhibition. Indeed, phenocopy experiments based on knock-down of either PKCε or ZEB1 reproduced miR-205 radiosensitizing effect, hence confirming a functional role of both targets in the process. At the molecular level, miR-205-induced suppression of PKCε counteracted radioresistance through the impairment of EGFR nuclear translocation and the consequent DNA-PK activation. Consistently, disruption of miR-205-PKCε 3'UTR pairing almost completely abrogated the radiosensitizing effect.Our results uncovered the molecular and cellular mechanisms underlying the radiosensitizing effect of miR-205. These findings support the clinical interest in developing a novel therapeutic approach based on miR-205 reconstitution to increase PCa response to radiotherapy.
DOI: 10.3390/ijms22115947
2021
Cited 47 times
On the Road to Fight Cancer: The Potential of G-Quadruplex Ligands as Novel Therapeutic Agents
Nucleic acid sequences able to adopt a G-quadruplex conformation are overrepresented within the human genome. This evidence strongly suggests that these genomic regions have been evolutionary selected to play a pivotal role in several aspects of cell biology. In the present review article, we provide an overview on the biological impact of targeting G-quadruplexes in cancer. A variety of small molecules showing good G-quadruplex stabilizing properties has been reported to exert an antitumor activity in several preclinical models of human cancers. Moreover, promiscuous binders and multiple targeting G-quadruplex ligands, cancer cell defense responses and synthetic lethal interactions of G-quadruplex targeting have been also highlighted. Overall, evidence gathered thus far indicates that targeting G-quadruplex may represent an innovative and fascinating therapeutic approach for cancer. The continued methodological improvements, the development of specific tools and a careful consideration of the experimental settings in living systems will be useful to deepen our knowledge of G-quadruplex biology in cancer, to better define their role as therapeutic targets and to help design and develop novel and reliable G-quadruplex-based anticancer strategies.
DOI: 10.1158/2159-8290.cd-23-0110
2024
Cited 3 times
Overcoming clinical resistance to EZH2 inhibition using rational epigenetic combination therapy
Abstract Epigenetic dependencies have become evident in many cancers. Based on antagonism between BAF/SWI/SNF and PRC2 in SMARCB1-deficient sarcomas, we recently completed the clinical trial of the EZH2 inhibitor tazemetostat. However, the principles of tumor response to epigenetic therapy in general, and tazemetostat in particular, remain unknown. Using functional genomics and diverse experimental models, we define molecular mechanisms of tazemetostat resistance in SMARCB1-deficient tumors. We found distinct acquired mutations that converge on the RB1/E2F axis and decouple EZH2-dependent differentiation and cell cycle control. This allows tumor cells to escape tazemetostat-induced G1 arrest, suggests a general mechanism for effective therapy, and provides prospective biomarkers for therapy stratification, including PRICKLE1. Based on this, we develop a combination strategy to circumvent tazemetostat resistance using bypass targeting of AURKB. This offers a paradigm for rational epigenetic combination therapy suitable for translation to clinical trials for epithelioid sarcomas, rhabdoid tumors, and other epigenetically dysregulated cancers.
DOI: 10.1016/j.ejca.2006.01.031
2006
Cited 128 times
Breast cancer stem cells: An overview
The theory that cancer may be originated and sustained by a small proportion of stem-like, self-renewing cells (termed ‘cancer stem cells’) has gained support in recent years. Breast cancer stem cells have been identified as CD44+CD24− breast tumour cells and have recently been isolated and propagated in vitro. It has been demonstrated that these cells exclusively retain the ability to form new tumours in mouse models and that they display stem/progenitor cell properties. The ability to identify breast cancer stem cells in vivo and to propagate them in vitro provides the means to compare them with normal cells, in order to investigate from which cell they originate, which molecular alterations critically affect them, and how they interact with the microenvironment. Elucidation of these critical points is essential to develop new therapeutic strategies and to improve diagnosis and prognosis for breast cancer patients.
DOI: 10.1158/0008-5472.can-06-0273
2006
Cited 123 times
Telomere Maintenance Mechanisms in Liposarcomas: Association with Histologic Subtypes and Disease Progression
Abstract Human cancer cells maintain telomeres by telomerase activity (TA) or by alternative lengthening of telomeres (ALT). We proposed to define the prevalence of the two telomere maintenance mechanisms (TMM), to assess their association with histology, and to compare their prognostic relevance in a series of 93 patients with liposarcoma. ALT was detected by assaying ALT-associated promyelocytic leukemia nuclear bodies and TA was assayed using the telomeric repeat amplification protocol. ALT or TA was found in 25.9% or 26.6% of 139 tested liposarcoma lesions, respectively. Three lesions were ALT+/TA+ whereas ∼50% of lesions did not show any known TMM. TMM phenotype was consistent during disease progression. ALT was prevalent in dedifferentiated and in grade 3 liposarcomas whereas TA prevailed in most round-cell myxoid and in grade 2 liposarcomas. ALT and TA incidence was similar in primary and recurrent lesions whereas metastases were more frequently TA+ than ALT+ (59% versus 18%; P = 0.04). TMM presence negatively affected patient prognosis (P = 0.001): increased mortality was associated with positivity for TA (P = 0.038) or ALT (P &amp;lt; 0.0001) compared with TMM absence. ALT proved to be a stronger prognostic discriminant of increased mortality than TA even when adjusted for tumor location, grade, and histology (hazard ratio for cause-specific death, 3.58 versus 1.15). Our results indicate that ALT can support fully malignant liposarcomas and is associated with unfavorable disease outcome. (Cancer Res 2006; 66(17): 8918-24)
DOI: 10.2174/138920107783018354
2007
Cited 120 times
Photochemical Internalization: A New Tool for Drug Delivery
The utilisation of macromolecules in the therapy of cancer and other diseases is becoming increasingly important. Recent advances in molecular biology and biotechnology have made it possible to improve targeting and design of cytotoxic agents, DNA complexes and other macromolecules for clinical applications. In many cases the targets of macromolecular therapeutics are intracellular. However, degradation of macromolecules in endocytic vesicles after uptake by endocytosis is a major intracellular barrier for the therapeutic application of macromolecules having intracellular targets of action. Photochemical internalisation (PCI) is a novel technology for the release of endocytosed macromolecules into the cytosol. The technology is based on the activation by light of photosensitizers located in endocytic vesicles to induce the release of macromolecules from the endocytic vesicles. Thereby, endocytosed molecules can be released to reach their target of action before being degraded in lysosomes. PCI has been shown to stimulate intracellular delivery of a large variety of macromolecules and other molecules that do not readily penetrate the plasma membrane, including type I ribosome-inactivating proteins (RIPs), DNA delivered as gene-encoding plasmids or by means of adenovirus or adeno-associated virus, peptide nucleic acids (PNAs) and chemotherapeutic agents such as bleomycin and in some cases doxorubicin. PCI of PNA may be of particular importance due to the low therapeutic efficacy of PNA in the absence of an efficient delivery technology and the 10-100-fold increased efficacy in combination with PCI. The efficacy and specificity of PCI of macromolecular therapeutics has been improved by combining the macromolecules with targeting moieties, such as the epidermal growth factor. In general, PCI can induce efficient light-directed delivery of macromolecules into the cytosol, indicating that it may have a variety of useful applications for site-specific drug delivery as for example in gene therapy, vaccination and cancer treatment. Keywords: Photodynamic, photochemical internalisation, photosensitizer, macromolecule, peptide nucleic acid, gene therapy, drug delivery, protein toxin
DOI: 10.1016/j.mee.2006.01.113
2006
Cited 115 times
Porous silicon as drug carrier for controlled delivery of doxorubicin anticancer agent
One of the topics of the biomedical research is to design and develop new drug delivery systems able to perform time-controlled release of medicals with target specificity. Porous silicon, characterized by macropores of 2 μm average diameter, with a 200 nm thick nanoporous coverage, was produced and its potential applications as drug-carrier were investigated. Doxorubicin loaded porous silicon bits exhibited a time-depending drug delivery profile that is in trend with the cytotoxicity of adenocarcinoma cancer cells exposed to bits.
DOI: 10.3324/haematol.2009.006171
2009
Cited 109 times
Generation of mesenchymal stromal cells in the presence of platelet lysate: a phenotypic and functional comparison of umbilical cord blood- and bone marrow-derived progenitors
Mesenchymal stromal cells are employed in various different clinical settings in order to modulate immune response. However, relatively little is known about the mechanisms responsible for their immunomodulatory effects, which could be influenced by both the cell source and culture conditions.We tested the ability of a 5% platelet lysate-supplemented medium to support isolation and ex vivo expansion of mesenchymal stromal cells from full-term umbilical-cord blood. We also investigated the biological/functional properties of umbilical cord blood mesenchymal stromal cells, in comparison with platelet lysate-expanded bone marrow mesenchymal stromal cells.The success rate of isolation of mesenchymal stromal cells from umbilical cord blood was in the order of 20%. These cells exhibited typical morphology, immunophenotype and differentiation capacity. Although they have a low clonogenic efficiency, umbilical cord blood mesenchymal stromal cells may possess high proliferative potential. The genetic stability of these cells from umbilical cord blood was demonstrated by a normal molecular karyotype; in addition, these cells do not express hTERT and telomerase activity, do express p16(ink4a) protein and do not show anchorage-independent cell growth. Concerning alloantigen-specific immune responses, umbilical cord blood mesenchymal stromal cells were able to: (i) suppress T- and NK-lymphocyte proliferation, (ii) decrease cytotoxic activity and (iii) only slightly increase interleukin-10, while decreasing interferon-gamma secretion, in mixed lymphocyte culture supernatants. While an indoleamine 2,3-dioxygenase-specific inhibitor did not reverse mesenchymal stromal cell-induced suppressive effects, a prostaglandin E(2)-specific inhibitor hampered the suppressive effect of both umbilical cord blood- and bone marrow-mesenchymal stromal cells on alloantigen-induced cytotoxic activity. Mesenchymal stromal cells from both sources expressed HLA-G.Umbilical cord blood- and bone marrow-mesenchymal stromal cells may differ in terms of clonogenic efficiency, proliferative capacity and immunomodulatory properties; these differences may be relevant for clinical applications.
DOI: 10.1021/nn103044z
2011
Cited 94 times
Redox-Active Polymer Microcapsules for the Delivery of a Survivin-Specific siRNA in Prostate Cancer Cells
In this report, we describe the delivery of small interfering RNA (siRNA) using LbL-assembled microcapsules. The microcapsules are based on negatively charged poly(methacrylic acid) nanometer thin films containing cross-linking disulfide bonds. One system is polycation-free and another contains polylysine for siRNA complexation in the microcapsule void. When microcapsules containing a siRNA targeting survivin were delivered to PC-3 prostate cancer cells, a significant inhibition of the expression of the antiapoptotic protein was observed. However, down-regulation of survivin was also observed in PC-3 cells exposed to microcapsules embedded with a scrambled siRNA as well as in cells treated with empty microcapsules. These findings indicate a capsule-dependent off-target effect, which is supported by a reduction in the expression of other survivin-unrelated proteins. The microcapsules and their polymeric constituents do not affect cell proliferation, as determined by a metabolic assay, even after 4 days of exposure. In addition, in PC-3 cells exposed to microcapsules, we observed a marked accumulation of LC3b, a marker related to autophagy (i.e., self-digestion), a degradation pathway involved in the maintenance of cell homeostasis in response to different stresses. This evidence suggests that empty microcapsules can induce a perturbation of the intracellular environment, which causes the activation of a cell safeguard mechanism that may limit the therapeutic effect of the microcapsules in tumor cells.
DOI: 10.4161/cc.10.18.17183
2011
Cited 91 times
Characterization of novel antisense HIF-1α transcripts in human cancers
Whole transcriptome analyses have revealed new classes of long ncRNA (lncRNA), the functions of which are however largely unknown. Recently, we showed that the antitumor DNA topoisomerase I (Top1) inhibitor camptothecin (CPT) increases the cellular levels of two antisense lncRNAs at the 5' (5'aHIF-1α) and 3' (3'aHIF-1α) ends of the human HIF-1α gene. To gain insights into their functions, we have here determined structural and functional aspects of the two antisense RNAs in human cancer cell lines and kidney tumor specimen. We found that the antisense transcripts are activated in response to partially different kinds of stress, and that the 5'aHIF-1α has a 5'Cap and a poly(A+) tail, while the 3'aHIF-1α is known to lack both modifications. Cell fractionation experiments showed that 5' and 3' antisense RNAs are nuclear transcripts. Further analyses by RNA-FISH showed that the 5'aHIF-1α accumulates at the perinuclear cellular compartment and co-localizes with the nuclear pore complex Nup62 protein, suggesting a role in nuclear membrane trafficking. Finally, we provide evidence that the studied antisense lncRNAs are expressed in human kidney cancer tissues, highlighting their possible roles in cancer development. Altogether, our findings may suggest a novel function of 5'aHIF-1α in membrane transport that may regulate the cancer-relevant HIF-1α pathway.
DOI: 10.1039/c2ob06816h
2012
Cited 91 times
Hybrid ligand–alkylating agents targeting telomeric G-quadruplex structures
The synthesis, physico-chemical properties and biological effects of a new class of naphthalene diimides (NDIs) capable of reversibly binding telomeric DNA and alkylate it through an electrophilic quinone methide moiety (QM), are reported. FRET and circular dichroism assays showed a marked stabilization and selectivity towards telomeric G4 DNA folded in a hybrid topology. NDI–QMs' alkylating properties revealed a good reactivity on single nucleosides and selectivity towards telomeric G4. A selected NDI was able to significantly impair the growth of melanoma cells by causing telomere dysfunction and down-regulation of telomerase expression. These findings points to our hybrid ligand–alkylating NDIs as possible tools for the development of novel targeted anticancer therapies.
DOI: 10.1517/14728222.2011.550878
2011
Cited 90 times
MicroRNAs as new therapeutic targets and tools in cancer
MicroRNAs (miRNAs) are a class of endogenous, non-coding small RNAs that negatively regulate gene expression at the post-transcriptional level. Several studies have provided evidence that abnormal expression of selected miRNAs is associated with the pathogenesis of cancer. As they can act as either oncogenes or tumor suppressors, miRNAs have been proposed as potential new therapeutic targets or tools for cancer therapy.This paper reviews a significant body of the experimental data collected to date which indicate that specific miRNA inhibition or replacement can successfully modify the proliferative and invasive properties of tumor cells. It discusses recent evidence that has also revealed a direct involvement of miRNAs in drug resistance, underlying an entirely new mechanism by which tumor cells may be refractory to the treatment with cytotoxic agents. Based on these findings, in the therapeutic setting, interference with cancer-specific miRNAs could be exploited not only to produce a direct anticancer effect but also to improve the response of tumor cells to conventional treatments.Overall, manipulation of miRNA functions, either by mimicking or inhibiting them, is emerging as a highly promising therapeutic strategy. However, before miRNA-based therapeutics enters the clinical armamentarium, important issues concerning specific delivery to cells/tissues of interest, safety as well as pharmacokinetic profiles needs to be addressed.
DOI: 10.1021/bm800225v
2008
Cited 88 times
Novel PVA-Based Hydrogel Microparticles for Doxorubicin Delivery
Micro- and nanoparticles are considered suitable drug delivery systems for their unique features, such as a large surface to volume ratio, and for the possibility to tune their size and hydrophobicity. A polymer/polymer/water emulsion method was used for producing a chemically cross-linked hydrogel made of poly(vinyl alcohol) and of poly(methacrylate) moieties. Mesoscopic investigation of the microparticles was accomplished by laser scanning confocal microscopy. Dynamics of confined water within the gel meshes was studied by quasi-elastic incoherent neutron scattering. Succinoylation of these particles allowed an efficient loading with a maximum doxorubicin payload of about 50% (w/w) of dry microparticles. To evaluate the potentials of such a microdevice for drug delivery, LoVo colon cancer cells have been exposed to doxorubicin loaded microparticles to study the in vitro efficiency of the payload release and the consequent cytotoxic effect.
DOI: 10.2174/09298673113209990106
2013
Cited 86 times
Targeting the Akt Kinase to Modulate Survival, Invasiveness and Drug Resistance of Cancer Cells
The deregulation of oncogenic signaling pathways which provide survival advantages to tumor cells is mediated by multiple cellular networks. Among them, the PI3K-Akt-mTOR axis, in particular the serine/threonine kinase Akt, is recognized as a key player. The kinase is hyperactivated due to a variety of mechanisms including loss of PTEN, mutations in the PI3K catalytic subunit, receptor tyrosine kinase and Ras activation. Indeed, inappropriate activation of the Akt kinase is a common event in human tumors and Akt appears to be a critical player in cell survival that may also account for the therapeutic resistance and the invasive phenotype of tumors. Inhibition of Akt signalling results in apoptosis and growth inhibition of tumour cells with elevated Akt activity. A functional role in drug resistance is supported by evidence that tumor cells with acquired resistance to antitumor agents may display increased Akt activation and that treatment with molecularly targeted agents can activate feed-back loops involving Akt. This serine/threonine kinase may therefore represent an amenable target for modulation of sensitivity to compounds with different molecular features due to its pleiotropic role in cell survival. Different types of Akt inhibitors [i.e., ATP mimetics and pleckstrin-homology (PH) domain binders] have been generated and some of them have reached the clinical setting. The present review focuses on the i) mechanisms implicating Akt in increased survival and invasive potential of tumor cells of different tumor types and ii) on the development of Akt inhibitors as modulators of drug resistance.
DOI: 10.1002/chem.201203097
2012
Cited 80 times
Targeting Loop Adenines in G‐Quadruplex by a Selective Oxirane
Caught in the oxirane: Naphthalene diimides conjugated to a quinone methide and an oxirane have been synthesized and investigated as selective DNA G-quadruplex alkylating agents. The oxirane derivative generates a stable adduct with a G-quadruplex and shows selective alkylation of the loop adenines, as illustrated. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
DOI: 10.1038/cdd.2012.56
2012
Cited 78 times
miR-205 regulates basement membrane deposition in human prostate: implications for cancer development
The basement membrane (BM) is a layer of specialized extracellular matrix that surrounds normal prostate glands and preserves tissue integrity. Lack or discontinuity of the BM is a prerequisite for tumor cell invasion into interstitial spaces, thus favoring metastasis. Therefore, BM maintenance represents a barrier against cancer development and progression. In the study, we show that miR-205 participates in a network involving ΔNp63α, which is essential for maintenance of the BM in prostate epithelium. At the molecular level, ΔNp63α is able to enhance miR-205 transcription by binding to its promoter, whereas the microRNA can post-transcriptionally limit the amount of ΔNp63α protein, mostly by affecting ΔNp63α proteasomal degradation rather than through a canonical miRNA/target interaction. Functionally, miR-205 is able to control the deposition of laminin-332 and its receptor integrin-β4. Hence, pathological loss of miR-205, as widely observed in prostate cancer, may favor tumorigenesis by creating discontinuities in the BM. Here we demonstrate that therapeutic replacement of miR-205 in prostate cancer (PCa) cells can restore BM deposition and 3D organization into normal-like acinar structures, thus hampering cancer progression.
DOI: 10.2174/0929867311320120006
2013
Cited 76 times
Camptothecin Resistance in Cancer: Insights into the Molecular Mechanisms of a DNA-Damaging Drug
Poisoning of DNA topoisomerase I is the mechanism by which camptothecins interfere with tumor growth. Although the clinical use of camptothecins has had a significant impact on cancer therapy, de novo or acquired clinical resistance to these drugs is common. Clinical resistance to camptothecins is still a poorly understood phenomenon, likely involving pharmacological and tumor-related factors. Experimental models including yeast and mammalian cell cultures suggest three general mechanisms of camptothecin resistance: i) reduced cellular accumulation of drugs, ii) alteration in the structure/expression of topoisomerase I, and iii) alterations in the cellular response to camptothecin-DNA-ternary complex formation. Some lines of evidence have also suggested links between cellular camptothecin resistance, the existence of a subset of tumor-initiating cells and miRNA deregulation. In this regard, a better definition of the molecular events clarifying the regulation of tumorigenesis and gene expression might contribute to gain insight into the molecular mechanisms on the basis of camptothecin resistance of tumors and to identify new molecular tools for targeting cancer cells. The relevance of these mechanisms to clinical drug resistance has not yet been completely defined, but their evaluation in clinical specimens should help to define personalized treatments including camptothecins as single agents or in combination with other cytotoxic and target-specific anticancer agents. The present review focuses on the cellular/ molecular aspects involved in resistance of tumor cells to camptothecins, including the potential role of cancer stem cells and deregulated miRNAs, and on the approaches proposed for overcoming resistance. Keywords: ABC transporters, antitumor therapy, camptothecins, cellular pharmacology, drug combination studies, drug resistance, miRNA, non-camptothecin poisons, topoisomerase I, tumor cells
DOI: 10.1016/j.bcp.2013.02.023
2013
Cited 75 times
Antitumor efficacy of the heparanase inhibitor SST0001 alone and in combination with antiangiogenic agents in the treatment of human pediatric sarcoma models
The activity of heparanase is responsible for heparan sulfate cleavage, thus resulting in the release of heparan sulfate-bound growth factors. Since heparanase activity is upregulated in several tumor types and is implicated in the malignant behavior, the enzyme is regarded as a promising target for antitumor therapy. Based on previous evidence that the heparanase inhibitor SST0001, a non-anticoagulant N-acetylated glycol split heparin, is effective against an Ewing's sarcoma model, the present study was performed to extend the preclinical evaluation of SST0001 to a panel of pediatric sarcoma models, representative of various tumor histotypes (soft tissue and bone sarcomas) and to further elucidate its mode of action. SST0001 treatment downregulated several angiogenic factors in the conditioned media of sarcoma cells, inhibited the pro-invasive effect of heparin-binding factors (VEGF, bFGF, HGF, PDGF), and abrogated PDGF receptor tyrosine phosphorylation. Subcutaneous administration of SST0001 was very effective, resulting in a significant growth inhibition (range, 64–95%) of all tested tumor xenografts. The efficacy of SST0001 was enhanced in combination with antiangiogenic agents (bevacizumab, sunitinib) as documented by the high rate of complete response. The synergistic effect of SST0001 in combination with antiangiogenic agents is consistent with the heparanase mode of action and with the relevant role of heparin-binding proangiogenic/growth factors in the malignant behavior of sarcoma cells.
DOI: 10.1021/jm301058f
2012
Cited 67 times
Synthesis and Biological Evaluation (in Vitro and in Vivo) of Cyclic Arginine–Glycine–Aspartate (RGD) Peptidomimetic–Paclitaxel Conjugates Targeting Integrin α<sub>V</sub>β<sub>3</sub>
A small library of integrin ligand-paclitaxel conjugates 10-13 was synthesized with the aim of using the tumor-homing cyclo[DKP-RGD] peptidomimetics for site-directed delivery of the cytotoxic drug. All the paclitaxel-RGD constructs 10-13 inhibited biotinylated vitronectin binding to the purified αVβ3 integrin receptor at low nanomolar concentration and showed in vitro cytotoxic activity against a panel of human tumor cell lines similar to that of paclitaxel. Among the cell lines, the cisplatin-resistant IGROV-1/Pt1 cells expressed high levels of integrin αVβ3, making them attractive to be tested in in vivo models. cyclo[DKP-f3-RGD]-PTX 11 displayed sufficient stability in physiological solution and in both human and murine plasma to be a good candidate for in vivo testing. In tumor-targeting experiments against the IGROV-1/Pt1 human ovarian carcinoma xenotransplanted in nude mice, compound 11 exhibited a superior activity compared with paclitaxel, despite the lower (about half) molar dosage used.
DOI: 10.1002/chem.201502211
2015
Cited 65 times
Activation of Hsp90 Enzymatic Activity and Conformational Dynamics through Rationally Designed Allosteric Ligands
Abstract Hsp90 is a molecular chaperone of pivotal importance for multiple cell pathways. ATP‐regulated internal dynamics are critical for its function and current pharmacological approaches block the chaperone with ATP‐competitive inhibitors. Herein, a general approach to perturb Hsp90 through design of new allosteric ligands aimed at modulating its functional dynamics is proposed. Based on the characterization of a first set of 2‐phenylbenzofurans showing stimulatory effects on Hsp90 ATPase and conformational dynamics, new ligands were developed that activate Hsp90 by targeting an allosteric site, located 65 Å from the active site. Specifically, analysis of protein responses to first‐generation activators was exploited to guide the design of novel derivatives with improved ability to stimulate ATP hydrolysis. The molecules’ effects on Hsp90 enzymatic, conformational, co‐chaperone and client‐binding properties were characterized through biochemical, biophysical and cellular approaches. These designed probes act as allosteric activators of the chaperone and affect the viability of cancer cell lines for which proper functioning of Hsp90 is necessary.
DOI: 10.2174/1381612822666160831101031
2017
Cited 63 times
Emerging Role of G-quadruplex DNA as Target in Anticancer Therapy
DNA has represented the most exploited target for the development of anticancer agents. It is now established that DNA may assume a variety of non-B conformations. This evidence has generated a total novel wave of interest in DNA as a cancer-associated target, since its distinct non-B structures may be regarded as sites for selective therapeutic intervention. G-quadruplexes are peculiar non-B DNA conformations that may form within guaninerich nucleic acid sequences. They are generated by a core of two or more vertically stacked G-quartets (i.e., the square planar arrangement of four guanine residues) held together by intervening loops of variable length. The evidence that G-quadruplexes are highly polymorphic and overrepresented within human genome points out at such non-B DNA conformations as druggable sites amenable of targeting by small molecules. In the present paper we will provide a concise overview on the emerging role of G-quadruplex structures forming within telomeres, gene promoters and mitochondrial DNA as a promising therapeutic target in cancer. In this context, a variety of small molecules has been documented to have excellent G-quadruplex binding/stabilizing properties and to exert good antiproliferative and antitumor activity in several in vitro and in vivo models of human cancers. Pieces of evidence indicate that targeting G-quadruplexes may represent an innovative and fascinating approach for the therapeutic management of the neoplastic disease. However, several issues still need to be addressed both at chemical and biological level before G-quadruplex-interacting molecules will turn out into effective therapeutic agents. Nevertheless, this has been an exciting, though sometime subdued, field of research over the last century. The continued improvements in methodologies and the development of specific tools will contribute not only to achieve the design and development of potentially novel anticancer approaches but also to deepen our knowledge of G-quadruplex biology and, consequently, of cancer at molecular level. Keywords: Gene promoter, G-quadruplex, mitochondrial DNA, non-B DNA, telomeres.
DOI: 10.1186/1756-8722-7-46
2014
Cited 60 times
XPO1/CRM1-Selective Inhibitors of Nuclear Export (SINE) reduce tumor spreading and improve overall survival in preclinical models of prostate cancer (PCa)
Exportin 1 (XPO1), also called chromosome region maintenance 1 (CRM1), is the sole exportin mediating transport of many multiple tumor suppressor proteins out of the nucleus. To verify the hypothesis that XPO1 inhibition affects prostate cancer (PCa) metastatic potential, orally available, potent and selective, SINE compounds, Selinexor (KPT- 330) and KPT-251, were tested in preclinical models known to generate bone lesions and systemic tumor spread. In vitro, Selinexor reduced both secretion of proteases and ability to migrate and invade of PCa cells. SINEs impaired secretion of pro-angiogenic and pro-osteolytic cytokines and reduced osteoclastogenesis in RAW264.7 cells. In the intra-prostatic growth model, Selinexor reduced DU145 tumor growth by 41% and 61% at the doses of 4 mg/Kg qd/5 days and 10 mg/Kg q2dx3 weeks, respectively, as well as the incidence of macroscopic visceral metastases. In a systemic metastasis model, following intracardiac injection of PCb2 cells, 80% (8/10) of controls, 10% (1/10) Selinexor- and 20% (2/10) KPT-251-treated animals developed radiographic evidence of lytic bone lesions. Similarly, after intra-tibial injection, the lytic areas were higher in controls than in Selinexor and KPT-251 groups. Analogously, the serum levels of osteoclast markers (mTRAP and type I collagen fragment, CTX), were significantly higher in controls than in Selinexor- and KPT-251-treated animals. Importantly, overall survival and disease-free survival were significantly higher in Selinexor- and KPT-251-treated animals when compared to controls. Selective blockade of XPO1-dependent nuclear export represents a completely novel approach for the treatment of advanced and metastatic PCa.
DOI: 10.1089/ars.2013.5292
2014
Cited 59 times
<i>miR-205</i> Hinders the Malignant Interplay Between Prostate Cancer Cells and Associated Fibroblasts
Aims: Tumor microenvironment is a strong determinant for the acquisition of metastatic potential of cancer cells. We have recently demonstrated that cancer-associated fibroblasts (CAFs) elicit a redox-dependent epithelial-mesenchymal transition (EMT) in prostate cancer (PCa) cells, driven by cycloxygenase-2/hypoxia-inducible factor-1 (HIF-1)/nuclear factor-κB pathway and enhancing tumor aggressiveness. Here, we investigated the involvement of microRNAs (miRNAs) in tumor-stroma interplay to identify possible tools to counteract oxidative stress and metastasis dissemination. Results: We found that miR-205 is the most downmodulated miRNA in PCa cells upon CAF stimulation, due to direct transcriptional repression by HIF-1, a known redox-sensitive transcription factor. Rescue experiments demonstrated that ectopic miR-205 overexpression in PCa cells counteracts CAF-induced EMT, thus impairing enhancement of cell invasion, acquisition of stem cell traits, tumorigenicity, and metastatic dissemination. In addition, miR-205 blocks tumor-driven activation of surrounding fibroblasts by reducing pro-inflammatory cytokine secretion. Innovation: Overall, such findings suggest miR-205 as a brake against PCa metastasis by blocking both the afferent and efferent arms of the circuit between tumor cells and associated fibroblasts, thus interrupting the pro-oxidant and pro-inflammatory circuitries engaged by reactive stroma. Conclusion: The evidence that miR-205 replacement in PCa cells is able not only to prevent but also to revert the oxidative/pro-inflammatory axis leading to EMT induced by CAFs sets the rationale for developing miRNA-based approaches to prevent and treat metastatic disease. Antioxid. Redox Signal. 20, 1045–1059.
DOI: 10.1038/s41598-017-17364-4
2018
Cited 55 times
A Computational Assay of Estrogen Receptor α Antagonists Reveals the Key Common Structural Traits of Drugs Effectively Fighting Refractory Breast Cancers
Abstract Somatic mutations of the Estrogen Receptor α (ERα) occur with an up to 40% incidence in ER sensitive breast cancer (BC) patients undergoing prolonged endocrine treatments. These polymorphisms are implicated in acquired resistance, disease relapse, and increased mortality rates, hence representing a current major clinical challenge. Here, multi-microseconds (12.5 µs) molecular dynamics simulations revealed that recurrent ERα polymorphisms ( i . e . L536Q, Y537S, Y537N, D538G) (mERα) are constitutively active in their apo form and that they prompt the selection of an agonist (active)-like conformation even upon antagonists binding. Interestingly, our simulations rationalize, for the first time , the efficacy profile of (pre)clinically used Selective Estrogen Receptor Modulators/Downregulators (SERMs/SERDs) against these variants, enlightening, at atomistic level of detail, the key common structural traits needed by drugs able to effectively fight refractory BC types. This knowledge represents a key advancement for mechanism-based therapeutics targeting resistant ERα isoforms, potentially allowing the community to move a step closer to ‘precision medicine’ calibrated on patients’ genetic profiles and disease progression.
DOI: 10.18632/oncotarget.5056
2015
Cited 54 times
Integrated gene and miRNA expression analysis of prostate cancer associated fibroblasts supports a prominent role for interleukin-6 in fibroblast activation
Tumor microenvironment coevolves with and simultaneously sustains cancer progression. In prostate carcinoma (PCa), cancer associated fibroblasts (CAF) have been shown to fuel tumor development and metastasis by mutually interacting with tumor cells. Molecular mechanisms leading to activation of CAFs from tissue-resident fibroblasts, circulating bone marrow-derived fibroblast progenitors or mesenchymal stem cells are largely unknown. Through integrated gene and microRNA expression profiling, we showed that PCa-derived CAF transcriptome strictly resembles that of normal fibroblasts stimulated in vitro with interleukin-6 (IL6), thus proving evidence, for the first time, that the cytokine is able per se to induce most of the transcriptional changes characteristic of patient-derived CAFs. Comparison with publicly available datasets, however, suggested that prostate CAFs may be alternatively characterized by IL6 and TGFβ-related signatures, indicating that either signal, depending on the context, may concur to fibroblast activation. Our analyses also highlighted novel pathways potentially relevant for induction of a reactive stroma. In addition, we revealed a role for muscle-specific miR-133b as a soluble factor secreted by activated fibroblasts to support paracrine activation of non-activated fibroblasts or promote tumor progression.Overall, we provided insights into the molecular mechanisms driving fibroblast activation in PCa, thus contributing to identify novel hits for the development of therapeutic strategies targeting the crucial interplay between tumor cells and their microenvironment.
DOI: 10.1038/s41467-018-08153-2
2019
Cited 44 times
LEADeR role of miR-205 host gene as long noncoding RNA in prostate basal cell differentiation
Though miR-205 function has been largely characterized, the nature of its host gene, MIR205HG, is still completely unknown. Here, we show that only lowly expressed alternatively spliced MIR205HG transcripts act as de facto pri-miRNAs, through a process that involves Drosha to prevent unfavorable splicing and directly mediate miR-205 excision. Notably, MIR205HG-specific processed transcripts revealed to be functional per se as nuclear long noncoding RNA capable of regulating differentiation of human prostate basal cells through control of the interferon pathway. At molecular level, MIR205HG directly binds the promoters of its target genes, which have an Alu element in proximity of the Interferon-Regulatory Factor (IRF) binding site, and represses their transcription likely buffering IRF1 activity, with the ultimate effect of preventing luminal differentiation. As MIR205HG functions autonomously from (albeit complementing) miR-205 in preserving the basal identity of prostate epithelial cells, it warrants reannotation as LEADeR (Long Epithelial Alu-interacting Differentiation-related RNA).
DOI: 10.3390/cells11071221
2022
Cited 18 times
Necroptosis and Prostate Cancer: Molecular Mechanisms and Therapeutic Potential
Necroptosis is a programmed form of necrosis characterized by mitochondrial alterations and plasma membrane permeabilization resulting in the release of cytoplasmic content into extracellular space, and leading to inflammatory reactions. Besides its critical role in viral defense mechanisms and inflammatory diseases, necroptosis plays pivotal functions in the drug response of tumors, including prostate cancer. Necroptosis is mainly governed by kinase enzymes, including RIP1, RIP3, and MLKL, and conversely to apoptosis, is a caspase-independent mechanism of cell death. Numerous compounds induce necroptosis in prostate cancer models, including (i) compounds of natural origin, (ii) synthetic and semisynthetic small molecules, and (iii) selenium and selenium-based nanoparticles. Here, we overview the molecular mechanisms underlying necroptosis and discuss the possible implications of drugs inducing necroptosis for prostate cancer therapy.
DOI: 10.1038/sj.onc.1207071
2004
Cited 86 times
Ribozyme-mediated inhibition of survivin expression increases spontaneous and drug-induced apoptosis and decreases the tumorigenic potential of human prostate cancer cells
DOI: 10.1016/j.ejca.2004.12.002
2005
Cited 85 times
Antisense oligonucleotide-mediated inhibition of hTERT, but not hTERC, induces rapid cell growth decline and apoptosis in the absence of telomere shortening in human prostate cancer cells
Recent evidence points to a novel function of human telomerase reverse transcriptase (hTERT) in promoting tumour cell survival, which might be independent of the telomere-elongating activity of the enzyme. To test this hypothesis, we evaluated comparatively the effects of telomerase inhibition, accomplished through antisense oligonucleotide-mediated interference with hTERT or human telomerase RNA component (hTERC), on the proliferative potential of DU145 human prostate cancer cells. Exposure of cells to a 2'-O-methyl-RNA phosphorothioate oligonucleotide targeting a splicing site within hTERT pre-mRNA induced almost complete inhibition of telomerase activity as a consequence of a marked reduction of the hTERT mRNA expression level, an early decline of DU145 cell growth and apoptotic cell death without any appreciable telomere shortening. Conversely, exposure of DU145 cells to a 2'-O-methyl-RNA phosphorothioate oligonucleotide targeting the template region of hTERC failed to interfere with cell proliferation in spite of the almost complete abrogation of telomerase activity. These results extend and corroborate earlier evidence in favour of an enzymatic activity-independent mechanism by which hTERT maintains tumour cell survival and proliferation.
DOI: 10.1021/jm060836y
2006
Cited 83 times
Small-Molecule Targeting of Heat Shock Protein 90 Chaperone Function: Rational Identification of a New Anticancer Lead
Heat shock protein 90 (Hsp90) is a significant target in the development of rational cancer therapy due to its role at the crossroads of multiple signaling pathways associated with cell proliferation and cell viability. Here we present a combined structure- and dynamics-based computational design strategy, taking the flexibility of the receptor and of a lead peptidic antagonist into account explicitely, to identify the nonpeptidic small molecule 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) as a structurally novel inhibitor of Hsp90. The compound is selected to bind the Hsp90 N-terminal domain, mimicking the chemical and conformational properties of the recently described peptidic antagonist of the survivin−Hsp90 complex, shepherdin [Plescia et al. Cancer Cell 2005, 7, 457−468]. Experimental tests show that AICAR binds the Hsp90 N-domain, destabilizes multiple Hsp90 client proteins in vivo, including survivin, and exhibits antiproliferative and proapoptotic activity in multiple tumor cell lines, while not affecting proliferation of normal human fibroblasts. We propose that AICAR represents a viable lead for further development of anticancer drugs with wide therapeutic opportunities.
DOI: 10.1021/bm050723g
2006
Cited 75 times
Tethering Functional Ligands onto Shell of Ultrasound Active Polymeric Microbubbles
Hollow (air-filled) microparticles, i.e., microbubbles, provide a promising novel vehicle for both local delivery of therapeutic agents and simultaneous diagnostic ultrasound echo investigations. In this paper, we describe the synthetic routes for decorating the polymeric shell of a poly(vinyl alcohol)-based microbubble with low and high molecular weight ligands with pharmacological relevance. Investigations on physical properties of microbubbles and surface chemical coupling with different cargo molecules such as l-cysteine, l-lysine, poly(l-lysine), chitosan, and β-cyclodextrin were carried out by CD and NMR spectroscopies, confocal laser scanning microscopy, and microcalorimetry. The in vitro cytotoxicity and biocompatibility of the polymer microbubbles have been also determined toward different cell lines. The results are discussed in terms of the features shown by this device, i.e., injectability, long shelf life, ease of preparation, biocompatibility, loading and cargo capacities, and functional properties.
DOI: 10.1038/onc.2009.238
2009
Cited 65 times
A gene expression signature classifying telomerase and ALT immortalization reveals an hTERT regulatory network and suggests a mesenchymal stem cell origin for ALT
Telomere length is maintained by two known mechanisms, the activation of telomerase or alternative lengthening of telomeres (ALT). The molecular mechanisms regulating the ALT phenotype are poorly understood and it is unknown how the decision of which pathway to activate is made at the cellular level. We have shown earlier that active repression of telomerase gene expression by chromatin remodelling of the promoters is one mechanism of regulation; however, other genes and signalling networks are likely to be required to regulate telomerase and maintain the ALT phenotype. Using gene expression profiling, we have uncovered a signature of 1305 genes to distinguish telomerase-positive and ALT cell lines. By combining this with the gene expression profiles of liposarcoma tissue samples, we refined this signature to 297 genes. A network analysis of known interactions between genes within this signature revealed a regulatory signalling network consistent with a model of human telomerase reverse transcriptase (hTERT) repression in ALT cell lines and liposarcomas. This network expands on our existing knowledge of hTERT regulation and provides a platform to understand differential regulation of hTERT in different tumour types and normal tissues. We also show evidence to suggest a novel mesenchymal stem cell origin for ALT immortalization in cell lines and mesenchymal tissues.
DOI: 10.1002/cmdc.201100078
2011
Cited 54 times
Synthesis and Antitumor Activity of 3‐(2‐Phenyl‐1,3‐thiazol‐4‐yl)‐1<i>H</i>‐indoles and 3‐(2‐Phenyl‐1,3‐thiazol‐4‐yl)‐1<i>H</i>‐7‐azaindoles
Abstract Given the potent antimicrobial, antiviral, and antitumor activities of many natural products, there is an increasing interest in the synthesis of new molecules based on natural compound scaffolds. Based on a 2,4‐bis(3′‐indolyl)imidazole skeleton, two new series of phenylthiazolylindoles and phenylthiazolyl‐7‐azaindoles were obtained by Hantzsch reaction between substituted phenylthioamides and the α‐bromoacetyl derivatives. Some azaindole derivatives, tested at the National Cancer Institute against a panel of ∼60 tumor cell lines derived from nine human cancer cell types, showed inhibitory effects against all cell lines investigated at micromolar to nanomolar concentrations. Two of them exhibited a high affinity for CDK1, with IC 50 values of 0.41 and 0.85 μ M . These promising results will set the foundation for future investigations into the development of anticancer therapies.
DOI: 10.1371/journal.pone.0029085
2011
Cited 54 times
Synergistic Antitumor Effects of Novel HDAC Inhibitors and Paclitaxel In Vitro and In Vivo
Preclinical studies support the therapeutic potential of histone deacetylases inhibitors (HDACi) in combination with taxanes. The efficacy of combination has been mainly ascribed to a cooperative effect on microtubule stabilization following tubulin acetylation. In the present study we investigated the effect of paclitaxel in combination with two novel HDACi, ST2782 or ST3595, able to induce p53 and tubulin hyperacetylation. A synergistic effect of the paclitaxel/ST2782 (or ST3595) combination was found in wild-type p53 ovarian carcinoma cells, but not in a p53 mutant subline, in spite of a marked tubulin acetylation. Such a synergistic interaction was confirmed in additional human solid tumor cell lines harboring wild-type p53 but not in those expressing mutant or null p53. In addition, a synergistic cytotoxic effect was found when ST2782 was combined with the depolymerising agent vinorelbine. In contrast to SAHA, which was substantially less effective in sensitizing cells to paclitaxel-induced apoptosis, ST2782 prevented up-regulation of p21(WAF1/Cip1) by paclitaxel, which has a protective role in response to taxanes, and caused p53 down-regulation, acetylation and mitochondrial localization of acetylated p53. The synergistic antitumor effects of the paclitaxel/ST3595 combination were confirmed in two tumor xenograft models. Our results support the relevance of p53 modulation as a major determinant of the synergistic interaction observed between paclitaxel and novel HDACi and emphasize the therapeutic interest of this combination.
DOI: 10.1021/jm5005122
2014
Cited 54 times
A New Avenue toward Androgen Receptor Pan-antagonists: C2 Sterically Hindered Substitution of Hydroxy-propanamides
The androgen receptor (AR) represents the primary target for prostate cancer (PC) treatment even when the disease progresses toward androgen-independent (AIPC) or castration-resistant (CRPC) forms. Because small chemical changes in the structure of nonsteroidal AR ligands determine the pharmacological responses of AR, we developed a novel stereoselective synthetic strategy that allows sterically hindered C2-substituted bicalutamide analogues to be obtained. Biological and theoretical evaluations demonstrate that C2-substitution with benzyl and phenyl moieties is a new, valuable option toward improving pan-antagonist behavior. Among the synthesized compounds, (R)-16m, when compared to casodex, (R)-bicalutamide, and enzalutamide, displayed very promising in vitro activity toward five different prostate cancer cell lines, all representative of CPRC and AIPC typical mutations. Despite being less active than (R)-bicalutamide, (R)-16m also displayed marked in vivo antitumor activity on VCaP xenografts and thus it may serve as starting point for developing novel AR pan-antagonists.
DOI: 10.1016/j.ejca.2014.09.004
2014
Cited 53 times
Preclinical and clinical evidence of activity of pazopanib in solitary fibrous tumour
Background To explore the activity of pazopanib in solitary fibrous tumour (SFT). Patients and methods In a preclinical study, we compared the activity of pazopanib, sorafenib, sunitinib, regorafenib, axitinib and bevacizumab in a dedifferentiated-SFT (DSFT) xenotransplanted into Severe Combined Immunodeficiency (SCID) mice. Antiangiogenics were administered at their reported optimal doses when mean tumour volume (TV) was 80 mm3. Drug activity was assessed as TV inhibition percentage (TVI%). From May 2012, six consecutive patients with advanced SFT received pazopanib, on a national name-based programme. In one case sunitinib was administered after pazopanib failure. Results In the xenograft model, pazopanib showed the lowest antitumour activity (21%TVI), while regorafenib was the most active (95%TVI). Sorafenib, bevacizumab, sunitinib were markedly active (78/70/65%TVI). Axitinib was marginally active (51%TVI). In the retrospective case-series, three patients carried malignant-SFT (MSFT), three DSFT. Best Response Evaluation Criteria in Solid Tumour (RECIST) responses were: three stable disease (SD), all MSFT, three progressive disease (PD), all DSFT, corresponding to one partial response (PR), two SD, three PD by Choi criteria. Median-progression-free survival was 3 months (range 1–15). In one patient, sunitinib was started after pazopanib failure, with a response. Conclusions In dedifferentiated-SFT xenograft pazopanib induced a marginal antitumour activity, while regorafenib appeared the most active and promising agent. When administered in patients, pazopanib showed a modest activity in terms of tumour growth stabilisation, observed only in non-dedifferentiated cases.
DOI: 10.1039/c4ob02054e
2015
Cited 52 times
Naphthalene diimides as red fluorescent pH sensors for functional cell imaging
Water-soluble naphthalene diimides have been designed and synthesized as cell permeable pH “turned-on” fluorescent sensors for cellular applications.
DOI: 10.1016/j.ejmech.2015.03.005
2015
Cited 51 times
Water-soluble isoindolo[2,1-a]quinoxalin-6-imines: In vitro antiproliferative activity and molecular mechanism(s) of action
Water-soluble isoindoloquinoxalin (IIQ) imines and the corresponding acetates were conveniently prepared from the key intermediates 2-(2'-aminophenyl)-2H-isoindole-1-carbonitriles obtained by a Strecker reaction between substituted 1,2-dicarbaldehydes and 1,2-phenylenediamines. Both series were screened by the National Cancer Institute (Bethesda, MD) and showed potent antiproliferative activity against a panel of 60 human tumor cell lines. Several of the novel compounds showed GI50 values at a nanomolar level on the majority of the tested cell lines. Among IIQ derivatives, methoxy substituents at positions 3 and 8 or/and 9 were especially effective in impairing cell cycle progression and inducing apoptosis in cancer cells. These effects were associated to IIQ-mediated impairment of tubulin polymerization at pharmacologically significant concentrations of tested compounds. In addition, impaired DNA topoisomerase I functions and perturbation in telomere architecture were observed in cells exposed to micromolar concentrations of IIQ derivatives. The above results suggest that IIQ derivatives exhibit multi-target cytotoxic activities.
DOI: 10.1016/j.colsurfb.2013.04.022
2013
Cited 51 times
Targeted doxorubicin delivery by chitosan-galactosylated modified polymer microbubbles to hepatocarcinoma cells
Targeted drug delivery is a main issue in cancer treatment. Taking advantage of recently developed polyvinyl alcohol (PVA)-based microbubbles, which are characterized by chemical versatility of the polymeric surface thereby allowing coating with different ligands, we set up a strategy for the targeted delivery of the anticancer agent doxorubicin to hepatocarcinoma cells. Such microbubbles are exceptionally efficient ultrasound scatterers and thus represent also an option as potential ultrasound contrast agents. Moreover, the oscillation of microbubbles induced by ultrasound could contribute to favor the release of drugs allocated on shell. Specifically, PVA-based microbubbles were reacted with a galactosylated chitosan complex and loaded with doxorubicin to enable the localization and drug delivery to HepG2 hepatocarcinoma cells overexpressing asialoglycoprotein receptors. We demonstrated selectivity and greater bioadhesive properties of the functionalized microbubbles for tumor cells than to normal fibroblasts, which were influenced by the degree of galactosylation. The presence of galactosylated chitosan did not modify the rate of doxorubicin release from microbubbles, whichwas almost complete within 48 h. Cellular uptake of doxorubicin loaded on functionalized microbubbles was higher in HepG2 than in normal fibroblasts, which do not over express the asialoglycoprotein receptors. In addition, doxorubicin loaded onto functionalized microbubbles fully retained its cytotoxic activity. Cells were also irradiated with ultrasound, immediately after exposure to microbubbles. An early enhancement of doxorubicin release and cellular drug uptake associated to a concomitant increase in cytotoxicity was observed in HepG2 cells. Overall, results of the study indicate that galactosylated chitosan microbubbles represent promising devices for the targeted delivery of antitumor agents to liver cancer cells.
DOI: 10.1007/s10637-014-0074-9
2014
Cited 50 times
PF-03446962, a fully-human monoclonal antibody against transforming growth-factor β (TGFβ) receptor ALK1, in pre-treated patients with urothelial cancer: an open label, single-group, phase 2 trial
DOI: 10.4161/auto.20519
2012
Cited 50 times
Autophagy acts as a safeguard mechanism against G-quadruplex ligand-mediated DNA damage
G-quadruplex ligands have attracted considerable interest as novel anticancer therapeutics due to their capability to interfere with guanosine-rich DNA/RNA sequences, such as telomeres. Elucidation of the structures of telomeric G-quadruplexes has led, in the past few years, to the rational development of effective G-quadruplex-stabilizing small molecules. In the present study, we showed that short-term exposure of melanoma cells to Ant1,5--an anthracene-based ligand able to stabilize telomeric G-quadruplexes--impaired cell growth without inducing cell senescence or apoptosis. Conversely, drug-treated cells were characterized by the occurrence of typical biochemical and morphological features associated with autophagy, such as an increase in the lipidated form of the autophagic marker LC3B and the accumulation of autophagosomes. Such drug-induced autophagy occurred as a consequence of DNA damage induction, at least in part dependent on drug-mediated telomere uncapping, through a pathway converging on the cyclin-dependent kinase inhibitor 1A (CDKN1A/p21). Indeed, melanoma cells depleted for CDKN1A did not show evidence of autophagic markers upon exposure to Ant1,5. The inhibition of autophagy by a pharmacologic inhibitor or through RNAi-mediated depletion of the ATG5 gene enhanced the cytotoxic activity of Ant1,5, as revealed by the marked increase in drug-induced apoptosis. Our data outline a molecular scenario in which G-quadruplex ligand-induced telomeric dysfunctions and DNA damage are translated into an autophagic response and provide the first evidence of autophagy as a safeguard mechanism activated by melanoma cells to counteract G-quadruplex ligand-mediated cellular stress.
DOI: 10.2174/09298673113206660307
2014
Cited 50 times
Synthesis and Antiproliferative Activity of Substituted 3[2-(1H-indol-3-yl)- 1,3-thiazol-4-yl]-1H-pyrrolo[3,2-b]pyridines, Marine Alkaloid Nortopsentin Analogues
A large number of indolyl-4-azaindolyl thiazoles, nortopsentin analogues, were conveniently synthesized. The antiproliferative activity of the new derivatives was examined against four human tumor cell lines with different histologic origin. Seven derivatives consistently reduced the growth of the experimental models independently of TP53 gene status and exhibited the highest activity against the malignant peritoneal mesothelioma (STO) cell line. The most active compound of this series acts as a CDK1 inhibitor, and was found to cause cell cycle arrest at G2/M phase, to induce apoptosis by preventing the phosphorylation of survivin in Thr34 and to increase the cytotoxic activity of paclitaxel in STO cells. Keywords: Antiproliferative activity, CDK1 inhibitors, diffuse malignant peritoneal mesothelioma, indolyl-4-azaindolyl thiazoles, nortopsentin analogues, survivin.
DOI: 10.1016/j.drup.2015.04.001
2015
Cited 47 times
New mechanisms for old drugs: Insights into DNA-unrelated effects of platinum compounds and drug resistance determinants
Platinum drugs have been widely used for the treatment of several solid tumors. Although DNA has been recognized as the primary cellular target for these agents, there are unresolved issues concerning their effects and the molecular mechanisms underlying the antitumor efficacy. These cytotoxic agents interact with sub-cellular compartments other than the nucleus. Here, we review how such emerging phenomena contribute to the pharmacologic activity as well as to drug resistance phenotypes. DNA-unrelated effects of platinum drugs involve alterations at the plasma membrane and in endo-lysosomal compartments. A direct interaction with the mitochondria also appears to be implicated in drug-induced cell death. Moreover, the pioneering work of a few groups has shown that platinum drugs can act on the tumor microenvironment as well, and potentiate antitumor activity of the immune system. These poorly understood aspects of platinum drug activity sites may be harnessed to enhance their antitumor efficacy. A complete understanding of DNA-unrelated effects of platinum compounds might reveal new aspects of drug resistance allowing the implementation of the antitumor therapeutic efficacy of platinum compound-based regimens and minimization of their toxic side effects.
DOI: 10.1016/j.semcancer.2017.03.005
2017
Cited 43 times
microRNAs as players and signals in the metastatic cascade: Implications for the development of novel anti-metastatic therapies
microRNAs (miRNAs) are small non-coding RNAs that negatively regulate gene expression at the post-transcriptional level. Increasing evidence emerging from human tumor preclinical models clearly indicates that specific miRNAs, collectively termed "metastamirs," play a functional role in different steps of the metastatic cascade, by exerting either pro- or anti-metastatic functions, and behave as signaling mediators to enable tumor cell to colonize a specific organ. miRNAs also actively participate in the proficient interaction of cancer cells with tumor microenvironment, either at the primary or at the metastatic site. Circulating miRNAs, released by multiple cell types, following binding to proteins or encapsulation in extracellular vesicles, play a main role in this cross-talk by acting as transferrable messages. The documented involvement of specific miRNAs in the dissemination process has aroused interest in the development of miRNA-based strategies for the treatment of metastasis. Preclinical research carried out in tumor experimental models, using both miRNA replacement and miRNA inhibitory approaches, is encouraging towards translating miRNA-based strategies into human cancer therapy, based on the observed therapeutic activity in the absence of main toxicity. However, to accelerate their adoption in the clinic, further improvements in terms of efficacy and targeted delivery to the tumor are still necessary.
DOI: 10.1021/acs.jmedchem.6b00777
2016
Cited 42 times
Preclinical Activity of New [1,2]Oxazolo[5,4-<i>e</i>]isoindole Derivatives in Diffuse Malignant Peritoneal Mesothelioma
A series of 22 derivatives of the [1,2]oxazolo[5,4-e]isoindole system were synthesized through an efficient and versatile procedure that involves the annelation of the [1,2]oxazole moiety to the isoindole ring, producing derivatives with a wide substitution pattern. The structure–activity relationship indicates that the N-4-methoxybenzyl group appears crucial for potent activity. In addition, the presence of a 6-phenyl moiety is important and the best activity is reached with a 3,4,5-trimethoxy substituent. The most active compound, bearing both the structural features, was able to inhibit tumor cell proliferation at nanomolar concentrations when tested against the full NCI human tumor cell line panel. Interestingly, this compound was effective in reducing in vitro and in vivo cell growth, impairing cell cycle progression and inducing apoptosis, as a consequence of the inhibition of tubulin polymerization, in experimental models of diffuse malignant peritoneal mesothelioma (DMPM), a rapidly lethal disease, poorly responsive to conventional therapeutic strategies.
DOI: 10.2174/0929867324666170216114248
2017
Cited 42 times
Targeting Heparan Sulfate Proteoglycans and their Modifying Enzymes to Enhance Anticancer Chemotherapy Efficacy and Overcome Drug Resistance
Targeting heparan sulfate proteoglycans (HSPGs) and enzymes involved in heparan sulfate (HS) chain editing is emerging as a new anticancer strategy. The involvement of HSPGs in tumor cell signaling, inflammation, angiogenesis and metastasis indicates that agents able to inhibit aberrant HSPG functions can potentially act as multitarget drugs affecting both tumor cell growth and the supportive boost provided by the microenvironment. Moreover, accumulating evidence supports that an altered expression or function of HSPGs, or of the complex enzyme system regulating their activities, can also depress the tumor response to anticancer treatments in several tumor types. Thereby, targeting HSPGs or HSPG modifying enzymes appears an appealing approach to enhance chemotherapy efficacy. A great deal of effort from academia and industry has led to the development of agents mimicking HS, and/or inhibiting HSPG modifying enzymes. Inhibitors of Sulf-2, an endosulfatase that edits the HS sulfation pattern, and inhibitors of heparanase, the endoglycosidase that produces functional HS fragments, appear particularly promising. In fact, a Sulf-2 inhibitor (OKN-007), and two heparanase inhibitors/HS mimics (roneparstat, PG545) are currently under early clinical investigation. In this review, we summarized preclinical studies in experimental tumor models of the main chemical classes of Sulf-2 and heparanase inhibitors. We described examples of different mechanisms through which heparanase and HSPGs, often in cooperation, may impact tumor sensitivity to various antitumor agents. Finally, we reported a few preclinical studies showing increased antitumor efficacy obtained with the use of candidate clinical HS mimics in combination regimens. Keywords: Heparan sulfate proteoglycan, heparanase, heparan sulfate mimic, heparanase inhibitor, sulfatase inhibitor, anticancer drug resistance, anticancer chemotherapy.
DOI: 10.1186/s13045-016-0387-6
2017
Cited 40 times
Antitumor activity of miR-34a in peritoneal mesothelioma relies on c-MET and AXL inhibition: persistent activation of ERK and AKT signaling as a possible cytoprotective mechanism
The value of microRNAs (miRNAs) as novel targets for cancer therapy is now widely recognized. However, no information is currently available on the expression/functional role of miRNAs in diffuse malignant peritoneal mesothelioma (DMPM), a rapidly lethal disease, poorly responsive to conventional treatments, for which the development of new therapeutic strategies is urgently needed. Here, we evaluated the expression and biological effects of miR-34a—one of the most widely deregulated miRNAs in cancer and for which a lipid-formulated mimic is already clinically available—in a large cohort of DMPM clinical samples and a unique collection of in house-developed preclinical models, with the aim to assess the potential of a miR-34a-based approach for disease treatment. miR-34a expression was determined by qRT-PCR in 45 DMPM and 7 normal peritoneum specimens as well as in 5 DMPM cell lines. Following transfection with miR-34a mimic, the effects on DMPM cell phenotype, in terms of proliferative potential, apoptotic rate, invasion ability, and cell cycle distribution, were assessed. In addition, three subcutaneous and orthotopic DMPM xenograft models were used to examine the effect of miR-34a on tumorigenicity. The expression of miRNA targets and the activation status of relevant pathways were investigated by western blot. miR-34a was found to be down-regulated in DMPM clinical specimens and cell lines compared to normal peritoneal samples. miR-34a reconstitution in DMPM cells significantly inhibited proliferation and tumorigenicity, induced an apoptotic response, and declined invasion ability, mainly through the down-regulation of c-MET and AXL and the interference with the activation of downstream signaling. Interestingly, a persistent activation of ERK1/2 and AKT in miR-34a-reconstituted cells was found to counteract the antiproliferative and proapoptotic effects of miRNA, yet not affecting its anti-invasive activity. Our preclinical data showing impressive inhibitory effects induced by miR-34a on DMPM cell proliferation, invasion, and growth in immunodeficient mice strongly suggest the potential clinical utility of a miR-34a-replacement therapy for the treatment of such a still incurable disease. On the other hand, we provide the first evidence of a potential cytoprotective/resistance mechanism that may arise towards miRNA-based therapies through the persistent activation of RTK downstream signaling.
DOI: 10.1021/acs.jmedchem.7b00105
2017
Cited 40 times
1,4-Substituted Triazoles as Nonsteroidal Anti-Androgens for Prostate Cancer Treatment
Prostate cancer (PC) is the fifth leading cause of cancer death in men, and the androgen receptor (AR) represents the primary target for PC treatment, even though the disease frequently progresses toward androgen-independent forms. Most of the commercially available nonsteroidal antiandrogens show a common scaffold consisting of two aromatic rings connected by a linear or a cyclic spacer. By taking advantage of a facile, one-pot click chemistry reaction, we report herein the preparation of a small library of novel 1,4-substituted triazoles with AR antagonistic activity. Biological and theoretical evaluation demonstrated that the introduction of the triazole core in the scaffold of nonsteroidal antiandrogens allowed the development of small molecules with improved overall AR-antagonist activity. In fact, compound 14d displayed promising in vitro antitumor activity toward three different prostate cancer cell lines and was able to induce 60% tumor growth inhibition of the CW22Rv1 in vivo xenograft model. These results represent a step toward the development of novel and improved AR antagonists.
DOI: 10.1016/j.ebiom.2018.12.025
2019
Cited 40 times
Splicing modulation as novel therapeutic strategy against diffuse malignant peritoneal mesothelioma
IntroductionTherapeutic options for diffuse malignant peritoneal mesothelioma (DMPM) are limited to surgery and locoregional chemotherapy. Despite improvements in survival rates, patients eventually succumb to disease progression. We investigated splicing deregulation both as molecular prognostic factor and potential novel target in DMPM, while we tested modulators of SF3b complex for antitumor activity.MethodsTissue-microarrays of 64 DMPM specimens were subjected to immunohistochemical assessment of SF3B1 expression and correlation to clinical outcome. Two primary cell cultures were used for gene expression profiling and in vitro screening of SF3b modulators. Drug-induced splicing alterations affecting downstream cellular pathways were detected through RNA sequencing. Ultimately, we established bioluminescent orthotopic mouse models to test the efficacy of splicing modulation in vivo.ResultsSpliceosomal genes are differentially upregulated in DMPM cells compared to normal tissues and high expression of SF3B1 correlated with poor clinical outcome in univariate and multivariate analysis. SF3b modulators (Pladienolide-B, E7107, Meayamycin-B) showed potent cytotoxic activity in vitro with IC50 values in the low nanomolar range. Differential splicing analysis of Pladienolide-B-treated cells revealed abundant alterations of transcripts involved in cell cycle, apoptosis and other oncogenic pathways. This was validated by RT-PCR and functional assays. E7107 demonstrated remarkable in vivo antitumor efficacy, with significant improvement of survival rates compared to vehicle-treated controls.ConclusionsSF3B1 emerged as a novel potential prognostic factor in DMPM. Splicing modulators markedly impair cancer cell viability, resulting also in potent antitumor activity in vivo. Our data designate splicing as a promising therapeutic target in DMPM.
DOI: 10.18632/oncotarget.10292
2016
Cited 39 times
Antitumor efficacy of the heparan sulfate mimic roneparstat (SST0001) against sarcoma models involves multi-target inhibition of receptor tyrosine kinases
The heparan sulfate (HS) mimic/heparanase inhibitor roneparstat (SST0001) shows antitumor activity in preclinical sarcoma models. We hypothesized that this 100% N-acetylated and glycol-split heparin could interfere with the functions of several receptor tyrosine kinases (RTK) coexpressed in sarcomas and activated by heparin-binding growth factors. Using a phospho-proteomic approach, we investigated the drug effects on RTK activation in human cell lines representative of different sarcoma subtypes. Inhibition of FGF, IGF, ERBB and PDGF receptors by the drug was biochemically and functionally validated. Roneparstat counteracted the autocrine loop induced by the COL1A1/PDGFB fusion oncogene, expressed in a human dermatofibrosarcoma protuberans primary culture and in NIH3T3COL1A1/PDGFB transfectants, inhibiting cell anchorage-independent growth and invasion. In addition, roneparstat inhibited the activation of cell surface PDGFR and PDGFR-associated FAK, likely contributing to the reversion of NIH3T3COL1A1/PDGFB cell transformed and pro-invasive phenotype. Biochemical and histological/immunohistochemical ex vivo analyses confirmed a reduced activation of ERBB4, EGFR, INSR, IGF1R, associated with apoptosis induction and angiogenesis inhibition in a drug-treated Ewing's sarcoma family tumor xenograft. The combination of roneparstat with irinotecan significantly improved the antitumor effect against A204 rhabdoid xenografts resulting in a high rate of complete responses and cures. These findings reveal that roneparstat exerts a multi-target inhibition of RTKs relevant in the pathobiology of different sarcoma subtypes. These effects, likely cooperating with heparanase inhibition, contribute to the antitumor efficacy of the drug. The study supports heparanase/HS axis targeting as a valuable approach in combination therapies of different sarcoma subtypes providing a preclinical rationale for clinical investigation.
DOI: 10.3390/pharmaceutics13111785
2021
Cited 26 times
Nanoparticles for Ferroptosis Therapy in Cancer
Ferroptosis is a regulated cell death mechanism holding promise for anticancer therapy. Numerous small molecules inducing ferroptosis have been reported thus far. However, these compounds suffer from important drawbacks including poor solubility, systemic toxicity, and scarce tumor targeting ability that have limited their clinical success. The notion that nanoparticles inducing ferroptosis show better preclinical profiles compared to small molecules and overcome resistance to apoptosis has opened a new scenario for cancer treatment. Due to peculiar chemical-physical properties, nanoparticles can be loaded with anticancer drugs or decorated with tumor-selecting molecules. These features allow for drug combination treatment as well as tumor targeting. In the review, we summarize and discuss the available information concerning nanoparticles inducing ferroptosis endowed with different peculiarities and suitable for therapeutic purposes including nanoparticles for (i) antitumor drug delivery, (ii) tumor targeting, (iii) immunomodulation, and (iv) radiofrequency ablation, hyperthermia, and photodynamic therapy.
DOI: 10.1016/s0014-5793(00)01540-4
2000
Cited 90 times
Inhibition of telomerase activity by a cell‐penetrating peptide nucleic acid construct in human melanoma cells
We investigated the effect of two peptide nucleic acids (PNAs), which are complementary to the RNA component of human telomerase, on the catalytic activity of the enzyme. PNAs induced a dose‐dependent reduction of telomerase activity in cell extracts from human melanoma cell lines and surgical specimens. To down‐regulate telomerase in intact cells, we generated a chimeric molecule synthesized by coupling the 13‐mer PNA to the Antennapedia peptide. The PNA construct induced a dose‐ and time‐dependent inhibition of telomerase activity. However, a 20‐day exposure to the PNA construct only caused a slight increase in melanoma cell doubling time and failed to induce any telomere shortening.
DOI: 10.1158/1535-7163.mct-05-0132
2006
Cited 70 times
Silencing of <i>survivin</i> gene by small interfering RNAs produces supra-additive growth suppression in combination with 17-allylamino-17-demethoxygeldanamycin in human prostate cancer cells
Abstract Survivin is an antiapoptotic gene, which is overexpressed in most human tumors and involved in mitotic checkpoint control. Recent evidence points to an essential role for heat shock protein 90 (Hsp90) in survivin function regulation. Although the survivin-Hsp90 association may promote tumor cell proliferation, it may also suggest new opportunities for the design of novel anticancer approaches. We evaluated the effect of small interfering RNA (siRNA)–mediated inhibition of survivin on the proliferative potential of prostate cancer cells and their sensitivity to the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG). Human androgen-independent prostate cancer cell lines (DU145 and PC-3) were transfected with four 21-mer double-stranded siRNAs (100 nmol/L) directed against different portions of survivin mRNA. After transfection, cells were collected and analyzed for survivin mRNA and protein expression, cell proliferation rate, ability to undergo apoptosis, and sensitivity to 17-AAG. Transfection of prostate cancer cells with siRNAs induced a variable extent of inhibition of survivin mRNA expression (39–60% compared with controls), which was paralleled by a 38% to 75% reduction in survivin protein abundance. The three siRNAs able to induce the greatest inhibition of survivin expression also significantly reduced cell proliferation and enhanced the rate of apoptosis, with a concomitant increase in caspase-9 activity. Sequential treatment with siRNA and 17-AAG induced supra-additive antiproliferative effects in all cell lines, with an enhanced caspase-9-dependent apoptotic response. These findings suggest that combined strategies aimed at interfering with the survivin-Hsp90 connection may provide novel approaches for treatment of androgen-independent prostate cancer. [Mol Cancer Ther 2006;5(1):179–86]
DOI: 10.1158/1535-7163.mct-05-0022
2005
Cited 70 times
Potentiation of paclitaxel-induced apoptosis by the novel cyclin-dependent kinase inhibitor NU6140: a possible role for survivin down-regulation
Abstract Cyclin-dependent kinases (CDK) play a crucial role in the control of the cell cycle. Aberrations in the control of cell cycle progression occur in the majority of human malignancies; hence, CDKs are promising targets for anticancer therapy. Here, we define the cellular effects of the novel CDK inhibitor NU6140, alone or in association with paclitaxel, with respect to inhibition of cell proliferation and cell cycle progression and induction of apoptosis in HeLa cervical carcinoma cells and in comparison with purvalanol A. Both CDK inhibitors induced a concentration-dependent cell cycle arrest at the G2-M phase and an increase in the apoptotic rate, with a concomitant down-regulation of the antiapoptotic protein survivin, a member of the inhibitors of apoptosis protein family. Notably, the addition of NU6140 to paclitaxel-treated cells resulted in markedly increased cytotoxic effect and apoptotic response in comparison with the paclitaxel-purvalanol A combination (86 ± 11% and 37 ± 8%, respectively). Similarly, the extent of caspase-9 and caspase-3 activation in paclitaxel-NU6140–treated cells was ∼4-fold higher than after the paclitaxel-purvalanol A combination. Moreover, an almost complete abrogation of the expression of the active, Thr34-phosphorylated form of survivin was observed in cells exposed to the paclitaxel-NU6140 combination. A synergistic effect of the paclitaxel-NU6140 combination, as a consequence of survivin inhibition and increased activation of caspase-9 and caspase-3, was also observed in OAW42/e ovarian cancer line but not in the derived OAW42/Surv subline ectopically expressing survivin. Results from this study indicate that NU6140 significantly potentiates the apoptotic effect of paclitaxel, with inhibition of survivin expression/phosphorylation as the potential mechanism.
DOI: 10.1158/1078-0432.ccr-08-0099
2008
Cited 60 times
Multiple Mechanisms of Telomere Maintenance Exist and Differentially Affect Clinical Outcome in Diffuse Malignant Peritoneal Mesothelioma
Abstract Purpose: This study aims to investigate the prevalence of the two known telomere maintenance mechanisms, telomerase activity (TA) and alternative lengthening of telomeres (ALT), and to assess their prognostic relevance in diffuse malignant peritoneal mesothelioma (DMPM). Experimental Design: In 44 DMPM specimens obtained from 38 patients, TA was determined using the telomeric repeat amplification protocol and ALT was detected by assaying ALT-associated promyelocytic leukemia nuclear bodies. The prognostic significance of telomere maintenance mechanisms was analyzed by Cox regression in the overall series and in a subset of 29 patients who underwent a uniform treatment regimen consisting of cytoreductive surgery and hyperthermic i.p. chemotherapy. Results: Telomere maintenance mechanisms were detectable in 86.4% of DMPM: ALT or TA alone was found in 18.2% or 63.6% of lesions, respectively, whereas two cases (4.6%) were ALT+/TA+. TA and ALT proved to be inversely associated (P = 0.002). In the overall series, TA was prognostic for 4-year relapse (TA+ versus TA−, hazard ratio, 3.30; 95% confidence interval, 1.23-8.86; P = 0.018) and cancer-related death (TA+ versus TA−, hazard ratio, 3.56; 95% confidence interval, 1.03-12.51; P = 0.045), whereas ALT failed to significantly affect clinical outcome. These results held true also in the subset of patients submitted to uniform treatment with cytoreductive surgery and hyperthermic i.p. chemotherapy. Conclusions: Our results indicate that both known telomere maintenance mechanisms, TA and ALT, are present in DMPM and differentially affect patient prognosis.
DOI: 10.1016/j.molmed.2009.07.004
2009
Cited 53 times
Towards the definition of prostate cancer-related microRNAs: where are we now?
Eradication of advanced prostate cancer still represents an unsolved clinical problem, making the development of alternative treatment approaches highly desirable. Understanding the molecular alterations that distinguish non-progressive from progressive disease would provide mechanistic information for the identification of new therapeutic targets. Recent findings indicate that human tumors have deregulated expression of microRNAs, which have thus been proposed as novel oncogenes or tumor suppressors. A few studies have analyzed the expression profiles or the functional role of microRNAs in prostate cancer, generating largely inconsistent data. Here we review the major issues that have hindered the identification of prostate cancer-related microRNAs, outlining an approach for rational validation of candidates that might be clinically relevant in the management of this disease. Eradication of advanced prostate cancer still represents an unsolved clinical problem, making the development of alternative treatment approaches highly desirable. Understanding the molecular alterations that distinguish non-progressive from progressive disease would provide mechanistic information for the identification of new therapeutic targets. Recent findings indicate that human tumors have deregulated expression of microRNAs, which have thus been proposed as novel oncogenes or tumor suppressors. A few studies have analyzed the expression profiles or the functional role of microRNAs in prostate cancer, generating largely inconsistent data. Here we review the major issues that have hindered the identification of prostate cancer-related microRNAs, outlining an approach for rational validation of candidates that might be clinically relevant in the management of this disease. deprivation of testicular androgens, which should contrast the growth of androgen-dependent cells, including prostate cancer cells. Androgen ablation in prostate cancer patients is mainly pursued by: (i) castration (testosterone elimination) through surgical removal of the testicles (orchiectomy) or administration of gonadotropin-releasing hormone agonists (i.e. goserelin) or (ii) administration of androgen-receptor antagonists (flutamide, bicalutamide, nilutamide). cells or tumors that rely on male hormones for their growth. also referred to as androgen-independent or castration-resistant cancer. Prostate cancer that has progressed despite deprivation of androgens, thus becoming resistant to androgen ablation therapy. 2′-O-methyl-antisense oligonucleotide conjugated with a cholesterol moiety at the 3′-end. synthetic small DNA or RNA molecule designed to interact with a specific target mRNA, thus interfering with its translation into protein. It represents a widely used tool to silence gene expression in experimental models in vitro and in vivo. Some antisense oligonucleotides have already entered clinical trials. increased prostate-specific antigen levels in serum of patients previously subjected to eradication of prostate cancer (i.e. radical prostatectomy), which is a sensitive indicator of recurrence of the disease. hyperplasia (overproliferation) of prostatic stromal and epithelial cells, resulting in enlargement of the prostate gland. It is usually associated with aging. numeric system used for prostate cancer grading. It is the sum of two scores, each varying from 1 (well differentiated) to 5 (undifferentiated), representative of the most prevalent and the second most prevalent differentiation grade within the tumor. bicyclic high-affinity RNA analogue in which the furanose ring in the sugar-phosphate backbone is chemically locked in an RNA-mimicking N-type (C3′-endo) conformation by the introduction of a 2′-O,4′-C methylene bridge. endogenous ∼22-nucleotide-long non-coding RNAs that bind to partially complementary sites within the 3′-untranslated regions of target mRNAs, thus inducing their degradation or translational repression. a ∼60–70-nucleotide stem–loop intermediate of miRNA processing, released by the primary transcript after cleavage by Drosha RNAse III endonuclease within the nucleus. The miRNA precursor is then exported into the cytosol, where it is further cleaved by Dicer RNAse III endonuclease to the mature form that is ∼22 nucleotide long. Chemically synthesized miRNA precursors are commonly used to upmodulate miRNA expression in experimental models. also referred to as microRNA star. Passenger strand of mature miRNA generated after Dicer processing of a pre-miRNA duplex. Mature miRNA preferentially enters the multiprotein RNA-induced silencing complex, thus guiding it to complementary sequences in target mRNAs. In some instances, the star form can be active as well, as is the case for miR-126*. serine protease belonging to the family of glandular kallikrein-related peptidases synthesized by prostate tissue and functions to liquefy seminal fluid. Only a small fraction of PSA leaks into the circulatory system under physiological conditions. The onset of prostate cancer, as well as other benign prostate diseases, can increase the release of PSA into blood, thus making its serum levels a commonly used marker for detection and monitoring of prostate tumors. classification of tumor stage, specifically nonpalpable (T1), palpable but confined to the prostate (T2), palpable and extending beyond the prostate (T3) or palpable and invading adjacent structures (T4).
DOI: 10.1111/j.1464-410x.2012.10970.x
2012
Cited 49 times
The 6‐year attendance of a multidisciplinary prostate cancer clinic in Italy: incidence of management changes
Study Type - Therapy (decision analysis) Level of Evidence 2b. What's known on the subject? and What does the study add? The benefits of the multidisciplinary approach in oncology are widely recognised. In particular, managing patients with prostate cancer within a multidisciplinarity and multiprofessional context is of paramount importance, to address the complexity of a disease where patients may be offered multiple therapeutic and observational options handled by different specialists and having severe therapy-induced side-effects. The present study describes the establishing of a multidisciplinary clinic at the Prostate Cancer Programme of Milan Istituto Nazionale dei Tumori, its effects on the quality of care provided, and strategies implemented to meet upcoming needs and improve quality standards. Having analysed the data of the 2260 multidisciplinary clinics held from March 2005 to March 2011, our dynamic and modifiable organisational model was evaluated for ways to optimise the human resources, offer high-quality standards, meet new needs and ultimately reduce costs. The study is focused on the organisational aspects and adds a perspective from one of the major oncological centres of reference in Italy and in Europe.To describe the establishing of a multidisciplinary clinic for men with prostate cancer at the Istituto Nazionale Tumori, Milan. • To evaluate the quality of care provided and to describe the management changes implemented to improve standards and meet new needs.In March 2005, we established a multidisciplinary clinic comprising weekly clinics and case-discussion sessions. • We have altered the organisational model periodically to meet new needs and improve quality.We held 2260 multidisciplinary clinics up to March 2011. • For stage distribution, patients with low-risk prostate cancer increased to a peak of 61% in 2009, probably because of the anticipation of diagnosis and the active surveillance expertise of the Prostate Cancer Programme at Istituto Nazionale Tumori, Milan. The slight decrease in 2010 might be due to the availability of robot-assisted prostatectomy in several hospitals in Milan, and the start of a multicentre active surveillance protocol in December 2009. • In terms of the efficacy of our multidisciplinary strategy, 11% of drug therapies (mostly hormones) prescribed outside our institute were terminated in the multidisciplinary clinic, and 6% of indications formulated in the multidisciplinary clinics were altered during the case-discussion sessions.The multidisciplinary approach needs to be adaptable to meet new needs and improve quality. • Our experience has proved successful for both physicians and patients. The team agrees on strategies; complex cases are managed by a multidisciplinary team; dedicated psychologists contribute their knowledge and perspectives; and patients report the feeling of being cared for.
DOI: 10.1371/journal.pone.0043249
2012
Cited 47 times
Induction of Endoplasmic Reticulum Stress Response by the Indole-3-Carbinol Cyclic Tetrameric Derivative CTet in Human Breast Cancer Cell Lines
Indole-3-carbinol and its metabolic products are considered promising chemopreventive and anticancer agents. Previously we have shown that the indole-3-carbinol cyclic tetrameric derivative CTet induces autophagy and inhibits cell proliferation via inhibition of Akt activity and overexpression of p21/CDKN1A and GADD45A, in both estrogen receptor-positive (MCF-7) and triple negative (MDA-MB-231) breast cancer cell lines. In the present study, we further characterize the autophagic response and investigate the mechanism through which CTet regulates these events.Analysis of gene expression microarray data and subsequent confirmation by quantitative real-time PCR, showed that CTet is able to induce up-regulation of key signaling molecules involved in endoplasmic reticulum (ER) stress response (e.g. DDIT3/CHOP, CHAC1, ATF3, HSPA5/BiP/GRP78, CEBPB, ASNS) and autophagy (e.g. MAP1LC3B), in both MCF-7 and MDA-MB-231 cell lines. Moreover, the monitoring of Xbp-1 splicing confirmed the activation of IRE1/Xbp-1 ER stress response branch after CTet treatment. The role of autophagic processes (known to be induced by ER stress) was investigated further through ATG5 gene silencing and pharmacological inhibition of AVOs formation. CTet was shown to induce an autophagy-related cell death. Moreover, CTet-treated cells stained with Hoechst/PI revealed the presence of necrotic processes without evidence of apoptosis.The ER stress response was identified as the main upstream molecular mechanism through which CTet acts in both hormone-responsive and triple-negative breast cancer cells. Because of its important role in cancer development, ER stress is a potential target in cancer therapy. The abiltiy of CTet to induce ER stress response and subsequently activate a death program in tumor cells confirms this molecule as a promising anticancer agent.
DOI: 10.1517/14728222.2011.556621
2011
Cited 45 times
Telomeres as targets for anticancer therapies
The limitless replicative potential of cancer cells relies on telomere integrity (which is guaranteed by a complex interaction between several specialized proteins and telomeric DNA) and the activation of specific mechanisms for telomere length maintenance. Two mechanisms are currently known in human cancer, namely telomerase activity and the alternative lengthening of telomere pathway.In this review, we summarize the available data concerning the therapeutic strategies proposed thus far and the current challenges posed for the development of innovative telomere-based therapeutic approaches with broad-spectrum anticancer activity and for their translation into the clinical setting.Due to their essential role in tumor cell proliferation, telomere maintenance mechanisms have become extremely attractive targets for the development of new anticancer interventions. Although numerous efforts have been made to identify specific approaches to interfere with telomere maintenance mechanisms in human cancers, the only molecule currently tested in clinical trials is the oligonucleotide GRN163L. However, a growing body of evidence suggests that interfering with telomeres, through the direct targeting of telomeric G-quadruplex structures, may be a valuable antitumor therapeutic strategy, independent of the specific telomere maintenance mechanism operating in the tumor.
DOI: 10.2174/138161213804547187
2013
Cited 43 times
Modulation of Sensitivity to Antitumor Agents by Targeting the MAPK Survival Pathway
DOI: 10.1016/j.ejmech.2013.02.022
2013
Cited 42 times
Design, modeling, synthesis and biological activity evaluation of camptothecin-linked platinum anticancer agents
The design, modeling, synthesis and biological activity evaluation of two hybrid agents formed by 7-oxyiminomethylcamptothecin derivatives and diaminedichloro-platinum (II) complex are reported. The compounds showed growth inhibitory activity against a panel of human tumor cell lines, including sublines resistant to topotecan and platinum compounds. The derivatives were active in all the tested cell lines, and compound 1b, the most active one, was able to overcome cisplatin resistance in the osteosarcoma U2OS/Pt cell line. Platinum-containing camptothecins produced platinum-DNA adducts and topoisomerase I-mediated DNA damage with cleavage pattern and persistence similar to SN38, the active principle of irinotecan. Compound 1b exhibited an appreciable antitumor activity in vivo against human H460 tumor xenograft, comparable to that of irinotecan at lower well-tolerated dose levels and superior to cisplatin. The results support the interpretation that the diaminedichloro-platinum (II) complex conjugated via an oxyiminomethyl linker at the 7-position of the camptothecin resulted in a new class of effective antitumor compounds.
DOI: 10.1016/j.molmed.2014.07.004
2014
Cited 40 times
miRNAs in tumor radiation response: bystanders or participants?
•miRNAs regulate the expression of components of radiation-relevant pathways. •miRNA expression profile changes upon irradiation in normal and cancer cells. •Modulation of specific miRNAs influences tumor cell radiosensitivity. There is increasing interest in defining a functional association between miRNAs and tumor radiation response, with the double aim of rationally designing miRNA-based strategies to increase patient radiosensitivity and identifying novel biomarkers of treatment response. Although it has been demonstrated that several miRNAs directly regulate the expression of components of cell pathways relevant to radiosensitivity, and miRNA expression profiles change upon irradiation, understanding the causal role exerted by individual miRNAs in determining tumor radiation response is still at an early stage. Based on available experimental and clinical evidence, we discuss here the potential of miRNAs as targets and/or tools for modulating radioresponsivity at the clinical level, as well as possible predictive biomarkers, underlining present limits and future perspectives. There is increasing interest in defining a functional association between miRNAs and tumor radiation response, with the double aim of rationally designing miRNA-based strategies to increase patient radiosensitivity and identifying novel biomarkers of treatment response. Although it has been demonstrated that several miRNAs directly regulate the expression of components of cell pathways relevant to radiosensitivity, and miRNA expression profiles change upon irradiation, understanding the causal role exerted by individual miRNAs in determining tumor radiation response is still at an early stage. Based on available experimental and clinical evidence, we discuss here the potential of miRNAs as targets and/or tools for modulating radioresponsivity at the clinical level, as well as possible predictive biomarkers, underlining present limits and future perspectives. generated when the two complementary stands of the DNA double helix are broken simultaneously at sites that are sufficiently close to one another that base pairing and chromatin structure are insufficient to keep the two DNA ends juxtaposed. discontinuities in one strand of the DNA double helix that are usually accompanied by the loss of a single nucleotide, and by damaged 5′- and/or 3′-termini at the site of the break. the intrinsic structure of miRNAs enables the use of Watson–Crick base-pairing interactions to design antisense oligonucleotides for their inhibition. Thus, an anti-miRNA oligonucleotide should be an oligomer that, by strongly binding to a given miRNA, acts as a steric block to prevent RISC loading and mRNA targeting. Several chemical modifications have been introduced onto miRNA inhibitors, either on sugars or on the backbone. As a result of these modifications, miRNA inhibitors are now characterized by a high stability in both intracellular environments and body fluids, and resistance to nucleases. The most used antisense oligonucleotides are antagomirs, which are modified to contain phosphorotioate linkage and a conjugated cholesterol moiety, and locked nucleic acids (LNA), which are bicyclic nucleic acid analogues bearing RNA bases with an extra bridge connecting the 2′ oxygen and 4′ carbon. different types of molecules with the peculiar characteristic of mimicking native miRNAs, used to restore or overexpress given miRNA species. Generally, with the aim to reproduce faithfully a physiological context, double-stranded RNA molecules mimicking natural miRNA precursors are used. Synthetic precursors are usually designed to carry chemical modifications allowing the exclusive production of only the mature miRNA of interest. Moreover, because these molecules have the same sequence as the native miRNAs that need to be re-expressed, they preserve their ability to interact with the natural targets and off-target effects are almost negligible. a locoregional treatment exploiting high-energy ionizing radiation to damage cancer cells and stop them from dividing, while sparing healthy tissue. Radiation can come from a machine (external radiation) or from an implant placed directly into or near the tumor (internal radiation). Current standard radiotherapy is 3D conformal radiotherapy where the beams of radiation used in treatment are shaped to match the tumor. In addition, intensity modulated radiation therapy, an advanced technique that enables the design of steep dose gradients conforming to concave targets, thus allowing delivering of radiation to a tumor with more precision and accuracy, is also used.
DOI: 10.1021/acs.biomac.5b00562
2015
Cited 39 times
Redox-Sensitive PEG–Polypeptide Nanoporous Particles for Survivin Silencing in Prostate Cancer Cells
We report the engineering of intracellular redox-responsive nanoporous poly(ethylene glycol)–poly(l-lysine) particles (NPEG–PLLs). The obtained particles exhibit no toxicity while maintaining the capability to deliver a small interfering RNA sequence (siRNA) targeting the anti-apoptotic factor, survivin, in prostate cancer cells. The redox-mediated cleavage of the disulfide bonds stabilizing the NPEG–PLL–siRNA complex results in the release of bioactive siRNA into the cytosol of prostate cancer PC-3 cells, which, in turn, leads to the effective silencing (∼59 ± 8%) of the target gene. These findings, obtained under optimal conditions, indicate that NPEG–PLLs may protect the therapeutic nucleic acid in the extracellular and intracellular environments, thus preventing the occurrence of competitive interactions with serum and cytosolic proteins as well as degradation by RNase. The intracellular trafficking and final fate of the NPEG–PLLs were investigated by a combination of deconvolution microscopy, fluorescence lifetime imaging microscopy, and super-resolution structured illumination microscopy. A significant impairment of cell survival was observed in cells concomitantly exposed to paclitaxel and siRNA-loaded NPEG–PLLs. Overall, our findings indicate that NPEG–PLLs represent a highly loaded depot for the delivery of therapeutic nucleic acids to cancer cells.
DOI: 10.1182/blood-2014-07-590034
2015
Cited 38 times
HSPH1 inhibition downregulates Bcl-6 and c-Myc and hampers the growth of human aggressive B-cell non-Hodgkin lymphoma
Key Points In human aggressive B-NHLs, HSPH1 favors c-Myc and Bcl-6 expression, and its inhibition provides significant antilymphoma activity. HSPH1 is expressed in function of Bcl-6 and c-Myc and constitutes a valuable alternative lymphoma therapeutic target of aggressive B-NHLs.
DOI: 10.18632/oncotarget.3761
2015
Cited 37 times
Anti-tumor activity of selective inhibitors of XPO1/CRM1-mediated nuclear export in diffuse malignant peritoneal mesothelioma: the role of survivin
Survivin, which is highly expressed and promotes cell survival in diffuse malignant peritoneal mesothelioma (DMPM), exclusively relies on exportin 1 (XPO1/CRM1) to be shuttled into the cytoplasm and perform its anti-apoptotic function. Here, we explored the efficacy of Selective Inhibitors of Nuclear Export (SINE), KPT-251, KPT-276 and the orally available, clinical stage KPT-330 (selinexor), in DMPM preclinical models. Exposure to SINE induced dose-dependent inhibition of cell growth, cell cycle arrest at G1-phase and caspase-dependent apoptosis, which were consequent to a decrease of XPO1/CRM1 protein levels and the concomitant nuclear accumulation of its cargo proteins p53 and CDKN1a. Cell exposure to SINE led to a time-dependent reduction of cytoplasmic survivin levels. In addition, after an initial accumulation, the nuclear protein abundance progressively decreased, as a consequence of an enhanced ubiquitination and proteasome-dependent degradation. SINE and the survivin inhibitor YM155 synergistically cooperated in reducing DMPM cell proliferation. Most importantly, orally administered SINE caused a significant anti-tumor effect in subcutaneous and orthotopic DMPM xenografts without appreciable toxicity. Overall, we have demonstrated a marked efficacy of SINE in DMPM preclinical models that may relay on the interference with survivin intracellular distribution and function. Our study suggests SINE-mediated XPO1/CRM1 inhibition as a novel therapeutic option for DMPM.
DOI: 10.1080/2162402x.2015.1040214
2015
Cited 36 times
Poly(I:C) and CpG-ODN combined aerosolization to treat lung metastases and counter the immunosuppressive microenvironment
The immunostimulatory ability of synthetic oligonucleotides containing CpG motifs (CpG-ODN), agonists of Toll-like receptor 9 (TLR9), can be harnessed to promote antitumor immunity by their application at the tumor site to stimulate local activation of innate immunity; however, particularly in the lung, tumor-associated immunosuppression can subvert such antitumor innate immune responses. To locally maintain continuous activation of innate subpopulations while inhibiting immunosuppressive cells, we evaluated aerosol delivery CpG-ODN combined with Poly(I:C), a TLR3 agonist able to convert tumor-supporting macrophages to tumoricidal effectors, in the treatment of B16 melanoma lung metastases in C57BL/6 mice. Aerosolization of CpG-ODN with Poly(I:C) into the bronchoalveolar space reduced the presence of M2-associated arginase- and IL-10-secreting macrophages in tumor-bearing lungs and increased the antitumor activity of aerosolized CpG-ODN alone against B16 lung metastases without apparent signs of toxicity or injury of the bronchial-bronchiolar structures and alveolar walls. Moreover, CpG-ODN/Poly(I:C) aerosol combined with dacarbazine, a therapeutic agent used in patients with inoperable metastatic melanoma able to exert immunostimulatory effects, led to a significant increase in antitumor activity as compared to treatments with aerosolized CpG-ODN/Poly(I:C) or dacarbazine alone. This effect was related to an enhanced recruitment and cytotoxic activity of tumor-infiltrating NK cells in the lung. Our results point to aerosol delivery as a convenient approach for repeated applications of immunostimulants in patients with lung metastases to maintain a continuous local activation of innate immune cells while suppressing polarization of tumor-infiltrating macrophages to an M2 phenotype.
DOI: 10.1016/j.taap.2016.08.005
2016
Cited 36 times
PKC-alpha modulation by miR-483-3p in platinum-resistant ovarian carcinoma cells
The occurrence of drug resistance limits the efficacy of platinum compounds in the cure of ovarian carcinoma. Since microRNAs (miRNAs) may contribute to this phenomenon by regulating different aspects of tumor cell response, the aim of this study was to exploit the analysis of expression of miRNAs in platinum sensitive/resistant cells in an attempt to identify potential regulators of drug response. MiR-483-3p, which may participate in apoptosis and cell proliferation regulation, was found up-regulated in 4 platinum resistant variants, particularly in the IGROV-1/Pt1 subline, versus parental cells. Transfection of a synthetic precursor of miR-483-3p in IGROV-1 parental cells elicited a marked up-regulation of the miRNA levels. Growth-inhibition and colony-forming assays indicated that miR-483-3p over-expression reduced cell growth and conferred mild levels of cisplatin resistance in IGROV-1 cells, by interference with their proliferative potential. Predicted targets of miR-483-3p included PRKCA (encoding PKC-alpha), previously reported to be associated to platinum-resistance in ovarian carcinoma. We found that miR-483-3p directly targeted PRKCA in IGROV-1 cells. In keeping with this finding, cisplatin sensitivity of IGROV-1 cells decreased upon molecular/pharmacological inhibition of PKC-alpha. Overall, our results suggest that overexpression of miR-483-3p by ovarian carcinoma platinum-resistant cells may interfere with their proliferation, thus protecting them from DNA damage induced by platinum compounds and ultimately representing a drug-resistance mechanism. The impairment of cell growth may account for low levels of drug resistance that could be relevant in the clinical setting.
DOI: 10.1002/chem.201700169
2017
Cited 34 times
Design of Allosteric Stimulators of the Hsp90 ATPase as New Anticancer Leads
Abstract Allosteric compounds that stimulate Hsp90 adenosine triphosphatase (ATPase) activity were rationally designed, showing anticancer potencies in the low micromolar to nanomolar range. In parallel, the mode of action of these compounds was clarified and a quantitative model that links the dynamic ligand–protein cross‐talk to observed cellular and in vitro activities was developed. The results support the potential of using dynamics‐based approaches to develop original mechanism‐based cancer therapeutics.
DOI: 10.1093/annonc/mdx124
2017
Cited 34 times
Pazopanib in advanced germ cell tumors after chemotherapy failure: results of the open-label, single-arm, phase 2 Pazotest trial
Therapeutic options for patients with chemoresistant germ cell tumors (GCTs) are limited. Pazopanib is a selective tyrosine kinase inhibitor with distinct antiangiogenic activity. We aimed to evaluate pazopanib activity in patients with refractory GCT.In the open-label, single-arm, phase 2 Pazotest study (NCT01743482), patient eligibility included failure of ≥2 platinum-based regimens, and allowed prior high-dose chemotherapy administration. Patients were given pazopanib 800 mg/day until disease progression (PD) or onset of unacceptable toxicity. Measurements of serum tumor markers (STM), computed tomography and FDG-PET were carried out at baseline, after 4 weeks of pazopanib treatment, and every 8 weeks thereafter. PD was defined as increasing levels of STM, increasing size of non-teratomatous masses, or appearance of new lesions. The study primary endpoint was progression-free survival (PFS, H0: 3-month PFS ≤ 10%, H1: ≥25%, α = 5%, β = 20%).Forty-three patients were enrolled from May 2013 to July 2016. The number of prior chemotherapy regimens was: 2 (11.6%), 3 (51.2%), >3 (37.2%). Grade 3 adverse events were observed in six patients (13.9%). Overall, 70.3% of patients had reduced levels of STM after 4 weeks. There were 2 partial responses (4.7%), 19 cases of stable disease, and 16 cases of PD (6 not evaluable by RECIST). The median follow-up duration was 29.6 months. The 3-month PFS probability was 12.8% [95% confidence interval (CI): 5.7%-28.9%]. The 24-month OS probability was 14.2% (95% CI: 6.0%-33.7%). In patients with a >50% decline in STM, the 24-month OS probability was 24.1% (95% CI: 8.3%-69.6%). The small sample size was the major limitation.Despite pazopanib showed potent but short-lived activity in refractory GCT, long-term survival was obtained in a proportion of treated patients. According to the kinetics of pazopanib activity, this drug may be investigated in less pre-treated patients as an optimal bridging therapy preceding and/or combined with salvage chemotherapy.
DOI: 10.1016/j.ejca.2018.10.024
2019
Cited 33 times
Activity of axitinib in progressive advanced solitary fibrous tumour: Results from an exploratory, investigator-driven phase 2 clinical study
To explore the activity of axitinib in advanced solitary fibrous tumour (SFT).In this investigator-driven phase II study on axitinib in advanced and progressive SFT, patients received axitinib, 5 mg bis in day (BID), until progression or limiting toxicity. Pathologic diagnosis was centrally reviewed, distinguishing malignant SFT (M-SFT) and high-grade/dedifferentiated SFT (HG/D-SFT) subtypes. The primary end-point was the overall response rate (ORR) by Choi criteria (Choi). Secondary end-points were response by Response Evaluation Criteria in Solid Tumours (RECIST), progression-free survival (PFS) and overall survival (OS).From April 2015 and October 2017, 17 eligible patients entered the study (metastatic: 17; SFT subtype: 13 M-SFT, 4 HG/D-SFT; prior treatment: 9 antiangiogenics, 5 cytotoxics). All patients were evaluable for response. The best Choi response was seven partial response (PR) (ORR, 41.2%), six stable disease (SD) and four progressions. Choi-ORR was 54% (7/13) when only M-SFTs were considered. Four of seven responsive patients were pretreated with pazopanib. No responses were detected in HG/D-SFT. Best RECIST response was one PR (5.9%), 14 SD and two progressions. Toxicity was as expected. Median Choi-PFS was 5.1 (interquartile range [IQR]: 2.5-14.8) months. Median Choi-PFS was 14.8 (IQR: 5.1-18.0) and 2.8 (IQR: 2.0-5.9) months for patients responsive and non-responsive by Choi, respectively (p = 0.0416). At a 14.4-month median follow-up, median OS was 25.3 months.This study showed that axitinib is active in progressive advanced SFT. One-half of patients carrying the malignant variant of the disease responded, with a >12-month median progression arrest. Responses were better detected with Choi and seen even in patients resistant to other antiangiogenics. Tolerability was good.
DOI: 10.1016/j.bcp.2017.11.009
2018
Cited 32 times
FoxO-1 contributes to the efficacy of the combination of the XPO1 inhibitor selinexor and cisplatin in ovarian carcinoma preclinical models
The XPO1/CRM1 inhibitor selinexor (KPT-330), is currently being evaluated in multiple clinical trials as an anticancer agent. XPO1 participates in the nuclear export of FoxO-1, which we previously found to be decreased in platinum-resistant ovarian carcinoma. The aim of this study was to determine whether enriching FoxO-1 nuclear localization using selinexor would increase ovarian cancer cell sensitivity to cisplatin. Selinexor, as a single agent, displayed a striking antiproliferative effect in different ovarian carcinoma cell lines. A schedule-dependent synergistic effect of selinexor in combination with cisplatin was found in cisplatin-sensitive IGROV-1, the combination efficacy being more evident in sensitive than in the resistant cells. In IGROV-1 cells, the combination was more effective when selinexor followed cisplatin exposure. A modulation of proteins involved in apoptosis (p53, Bax) and in cell cycle progression (p21WAF1) was found by Western blotting. Selinexor-treated cells exhibited enriched FoxO-1 nuclear staining. Knock-down experiments with RNA interference indicated that FOXO1-silenced cells displayed a reduced sensitivity to selinexor. FOXO1 silencing also tended to reduce the efficacy of the drug combination at selected cisplatin concentrations. Selinexor significantly inhibited tumor growth, induced FoxO-1 nuclear localization and improved the efficacy of cisplatin in IGROV-1 xenografts. Taken together, our results support FoxO-1 as one of the key factors promoting sensitivity towards selinexor and the synergistic interaction between cisplatin and selinexor in ovarian carcinoma cells with selected molecular backgrounds, highlighting the need for treatment regimens tailored to the molecular tumor features.
DOI: 10.1016/j.ejmech.2019.02.045
2019
Cited 31 times
Rational design of allosteric modulators of the aromatase enzyme: An unprecedented therapeutic strategy to fight breast cancer
Estrogens play a key role in cellular proliferation of estrogen-receptor-positive (ER+) breast cancers (BCs). Suppression of estrogen production by competitive inhibitors of the enzyme aromatase (AIs) is currently one of the most effective therapies against ER + BC. Yet, the development of acquired resistance, after prolonged treatments with AIs, represents a clinical major concern. Serendipitous findings indicate that aromatase may be non-competitively inhibited by clinically employed drugs and/or industrial chemicals. Here, by performing in silico screening on two putative allosteric sites, molecular dynamics and free energy simulations, supported by enzymatic and cell-based assays, we identified five leads inhibiting the enzyme via a non-active site-directed mechanism. This study provides new compelling evidences for the existence of an allosteric regulation of aromatase and for the possibility of exploiting it to modulate estrogens biosynthesis. Such modulation can aptly reduce side effects caused by the complete estrogen deprivation therapy, and, possibly, delay/avoid the onset of resistance.
DOI: 10.3390/cancers12040949
2020
Cited 29 times
The Role of Alternative Lengthening of Telomeres Mechanism in Cancer: Translational and Therapeutic Implications
Telomere maintenance mechanisms (i.e., telomerase activity (TA) and the alternative lengthening of telomere (ALT) mechanism) contribute to tumorigenesis by providing unlimited proliferative capacity to cancer cells. Although the role of either telomere maintenance mechanisms seems to be equivalent in providing a limitless proliferative ability to tumor cells, the contribution of TA and ALT to the clinical outcome of patients may differ prominently. In addition, several strategies have been developed to interfere with TA in cancer, including Imetelstat that has been the first telomerase inhibitor tested in clinical trials. Conversely, the limited information available on the molecular underpinnings of ALT has hindered thus far the development of genuine ALT-targeting agents. Moreover, whether anti-telomerase therapies may be hampered or not by possible adaptive responses is still debatable. Nonetheless, it is plausible hypothesizing that treatment with telomerase inhibitors may exert selective pressure for the emergence of cancer cells that become resistant to treatment by activating the ALT mechanism. This notion, together with the evidence that both telomere maintenance mechanisms may coexist within the same tumor and may distinctly impinge on patients' outcomes, suggests that ALT may exert an unexpected role in tumor biology that still needs to be fully elucidated.
DOI: 10.3390/ijms24021285
2023
Cited 5 times
The Therapeutic Potential of Pyroptosis in Melanoma
Pyroptosis is a programmed cell death characterized by the rupture of the plasma membranes and release of cellular content leading to inflammatory reaction. Four cellular mechanisms inducing pyroptosis have been reported thus far, including the (i) caspase 1-mediated canonical, (ii) caspase 4/5/11-mediated non-canonical, (iii) caspase 3/8-mediated and (iv) caspase-independent pathways. Although discovered as a defense mechanism protecting cells from infections of intracellular pathogens, pyroptosis plays roles in tumor initiation, progression and metastasis of tumors, as well as in treatment response to antitumor drugs and, consequently, patient outcome. Pyroptosis induction following antitumor therapies has been reported in several tumor types, including lung, colorectal and gastric cancer, hepatocellular carcinoma and melanoma. This review provides an overview of the cellular pathways of pyroptosis and discusses the therapeutic potential of pyroptosis induction in cancer, particularly in melanoma.
DOI: 10.1046/j.1523-1747.2000.00870.x
2000
Cited 68 times
Inhibition of Telomerase Activity by a Hammerhead Ribozyme Targeting the RNA Component of Telomerase in Human Melanoma Cells
Reactivation of telomerase, an RNA-dependent DNA polymerase that synthesizes new telomeric repeats at the end of chromosomes, is a very common feature in human cancers. Telomerase is thought to be essential in maintaining the proliferative capacity of tumor cells and, as a consequence, it could represent an attractive target for new anti-cancer therapies. In this study, we generated a hammerhead ribozyme composed of a catalytic domain with flanking sequences complementary to the RNA component of human telomerase and designed to cleave specifically a site located at the end of the telomerase template sequence. In vitro the ribozyme induced cleavage of a synthetic RNA substrate obtained by cloning a portion of the RNA component of human telomerase. The extent of cleavage was dependent on the ribozyme/substrate ratio as well as the Mg2+ concentration. Moreover, when added to cell extracts from two human melanoma cell lines (JR8 and M14), or three melanoma surgical specimens, the ribozyme inhibited telomerase activity in a concentration- and time-dependent manner. When the ribozyme was delivered to growing JR8 melanoma cells by (N-(1-(2,3 dioleoxyloxy)propil)-N,N,N trimethylammonium methylsulfate-mediated transfer, a marked inhibition of telomerase activity was observed. Next, the ribozyme sequence was cloned in an expression vector and JR8 cells were transfected with it. The cell clones obtained showed a reduced telomerase activity and telomerase RNA levels and expressed the ribozyme. Moreover, ribozyme transfectants had significantly longer doubling times than control cells and showed a dendritic appearance in monolayer culture. No telomere shortening, however, was observed in these clones. Overall, our results indicate that the hammerhead ribozyme is a potentially useful tool for the inactivation of telomerase in human tumors. Reactivation of telomerase, an RNA-dependent DNA polymerase that synthesizes new telomeric repeats at the end of chromosomes, is a very common feature in human cancers. Telomerase is thought to be essential in maintaining the proliferative capacity of tumor cells and, as a consequence, it could represent an attractive target for new anti-cancer therapies. In this study, we generated a hammerhead ribozyme composed of a catalytic domain with flanking sequences complementary to the RNA component of human telomerase and designed to cleave specifically a site located at the end of the telomerase template sequence. In vitro the ribozyme induced cleavage of a synthetic RNA substrate obtained by cloning a portion of the RNA component of human telomerase. The extent of cleavage was dependent on the ribozyme/substrate ratio as well as the Mg2+ concentration. Moreover, when added to cell extracts from two human melanoma cell lines (JR8 and M14), or three melanoma surgical specimens, the ribozyme inhibited telomerase activity in a concentration- and time-dependent manner. When the ribozyme was delivered to growing JR8 melanoma cells by (N-(1-(2,3 dioleoxyloxy)propil)-N,N,N trimethylammonium methylsulfate-mediated transfer, a marked inhibition of telomerase activity was observed. Next, the ribozyme sequence was cloned in an expression vector and JR8 cells were transfected with it. The cell clones obtained showed a reduced telomerase activity and telomerase RNA levels and expressed the ribozyme. Moreover, ribozyme transfectants had significantly longer doubling times than control cells and showed a dendritic appearance in monolayer culture. No telomere shortening, however, was observed in these clones. Overall, our results indicate that the hammerhead ribozyme is a potentially useful tool for the inactivation of telomerase in human tumors. (N-(1-(2,3 dioleoxyloxy)propil)-N,N,N trimethylammonium methylsulfate telomeric repeat amplification protocol Human telomeres are specialized structures located at the end of chromosomes, consisting of simple repeated DNA sequences (TTAGGG)n and associated proteins. They are essential to protect chromosomes against exonucleolytic degradation and to prevent aberrant recombination that may cause DNA rearrangements leading to karyotypic changes and genomic instability (Blackburn, 1991Blackburn E.H. Structure and function of telomeres.Nature. 1991; 350: 569-573Crossref PubMed Scopus (2911) Google Scholar). It is known that, due to the ''end replication problem'' (Watson, 1972Watson J. Origin of concatemeric T7 DNA.Nat Biol. 1972; 239: 197-201Crossref Scopus (1298) Google Scholar;Olovnikov, 1973Olovnikov A.M. A theory of marginotomy: the incomplete copying of template margin in enzymic synthesis of polynucleotides and biological significance of the phenomenon.J Theor Biol. 1973; 41: 181-190Crossref PubMed Scopus (1339) Google Scholar), telomeric DNA shortens during the proliferation of human somatic cells. The phenomenon is responsible for cell senescence that occurs by growth arrest as a response to the shortened telomeres (Harley, 1991Harley C.B. Telomere loss: mitotic clock or genetic time bomb?.Mutat Res. 1991; 256: 271-282Crossref PubMed Scopus (1063) Google Scholar;Allsopp et al., 1992Allsopp R.C. Vaziri H. Patterson C. et al.Telomere length predicts replicative capacity of human fibroblasts.Proc Natl Acad Sci USA. 1992; 89: 10114-10118Crossref PubMed Scopus (1885) Google Scholar). To compensate for the telomeric sequences loss, germline, immortalized, and tumor cells express the RNA-dependent DNA polymerase telomerase (Counter et al., 1992Counter C.M. Avilion A.A. LeFeuvre C.E. Stewart N.G. Greider C.W. Harley C.B. Bacchetti S. Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity.EMBO J. 1992; 11: 1921-1929Crossref PubMed Scopus (1881) Google Scholar;Kim et al., 1994Kim N.W. Piatyszek M.A. Prowse K.R. et al.Specific association of human telomerase activity with immortal cells and cancer.Science. 1994; 266: 2011-2015Crossref PubMed Scopus (6308) Google Scholar;Chadeneau et al., 1995Chadeneau C. Hay K. Hirte H.W. Gallinger S. Bacchetti S. Telomerase activity associated with acquisition of malignancy in human colorectal cancer.Cancer Res. 1995; 55: 2533-2536PubMed Google Scholar;Greider, 1998Greider C.W. Telomerase activity, cell proliferation, and cancer.Proc Natl Acad Sci USA. 1998; 95: 90-92Crossref PubMed Scopus (353) Google Scholar). The enzyme is a ribonucleoprotein which contains a short RNA molecule that serves as a template for the synthesis of new telomeric sequences (Collins, 1996Collins K. Structure and function of telomerase.Curr Opin Cell Biol. 1996; 8: 374-380Crossref PubMed Scopus (37) Google Scholar). The enzyme is involved in the attainment of immortality in cancer cells and therefore may contribute to tumorigenesis and neoplastic progression (Autexier and Greider, 1996Autexier C. Greider C.W. Telomerase and cancer: revisiting the telomere hypothesis.Trends Biochem Sci. 1996; 21: 387-391https://doi.org/10.1016/0968-0004(96)10042-6Crossref PubMed Scopus (166) Google Scholar). Thus far, telomerase activity has been detected in about 90% of human tumors screened (Shay and Bacchetti, 1997Shay J.W. Bacchetti S. A survey of telomerase activity in human cancer.Eur J Cancer. 1997; 33: 787-791https://doi.org/10.1016/s0959-8049(97)00062-2Abstract Full Text PDF PubMed Scopus (0) Google Scholar). Owing to its pattern of peculiar expression, telomerase has been proposed as a new diagnostic tool (Sugino et al., 1996Sugino T. Yoshida K. Bolodeoku J. et al.Telomerase activity in human breast cancer and benign breast lesions: diagnostic applications in clinical specimens, including fine needle aspirates.Int J Cancer. 1996; 69: 301-306https://doi.org/10.1002/(sici)1097-0215(19960822)69:4<301::aid-ijc11>3.0.co;2-8Crossref PubMed Scopus (0) Google Scholar;Villa et al., 1998Villa R. Zaffaroni N. Folini M. Martelli G. De Palo G. Daidone M.G. Silvestrini R. Telomerase activity in benign and malignant breast lesions: a pilot prospective study on fine-needle aspirates.J Natl Cancer Inst. 1998; 90: 537-539Crossref PubMed Scopus (27) Google Scholar), as a target for new anti-cancer therapies (Raymond et al., 1996Raymond E. Sun D. Chen S.-F. Von Windle B. Hoff D.D. Agents that target telomerase and telomeres.Curr Opin Biotech. 1996; 7: 583-591Crossref PubMed Scopus (81) Google Scholar) and, possibly, as a putative prognostic factor (Clark et al., 1997Clark G.M. Osborne C.K. Levitt D. Wu F. Kim N.W. Telomerase activity and survival of patients with node-positive breast cancer.J Natl Cancer Inst. 1997; 89: 1874-1881Crossref PubMed Scopus (138) Google Scholar;Hoos et al., 1998Hoos A. Hepp H.H. Kaul S. Ahlert T. Bastert G. Wallwiener D. Telomerase activity correlates with tumor aggressiveness and reflects therapy effect in breast cancer.Int J Cancer. 1998; 79: 8-12https://doi.org/10.1002/(sici)1097-0215(19980220)79:1<8::aid-ijc2>3.0.co;2-5Crossref PubMed Scopus (0) Google Scholar). One possible strategy to identify anti-telomerase compounds is based on the screening of molecules that can interact with the ribonucleoprotein components. Specifically, taking advantage of structural and functional similarities in telomerase and retroviral reverse transcriptase (Lingner et al., 1997Lingner J. Hughes T.R. Shevchenko A. Mann M. Lundblad V. Cech T.R. Reverse transcriptase motifs in the catalytic subunit of telomerase.Science. 1997; 276: 561-567Crossref PubMed Scopus (1003) Google Scholar;Nakamura and Cech, 1998Nakamura T.M. Cech T.R. Reversing time: origin of telomerase.Cell. 1998; 92: 587-590Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar), reverse transcriptase inhibitors, such as nucleoside analogs, have been also used as inhibitors of telomerase activity (Strahl and Blackburn, 1996Strahl C. Blackburn E.H. Effects of reverse transcriptase inhibitors on telomere length and telomerase activity in two immortalized human cell lines.Mol Cell Biol. 1996; 16: 53-65Crossref PubMed Scopus (348) Google Scholar;Yegorov et al., 1996Yegorov Y.E. Chernov D.N. Akimov S.S. Bolsheva N.L. Krayevsky A.A. Zelenin A.V. Reverse transcriptase inhibitors suppress telomerase function and induce senescence-like processes in cultured mouse fibroblasts.FEBS Lett. 1996; 389: 115-118https://doi.org/10.1016/0014-5793(96)00533-9Abstract Full Text PDF PubMed Scopus (79) Google Scholar;Melana et al., 1998Melana S.M. Holland J.F. Pogo B.G.-T. Inhibition of cell growth and telomerase activity of breast cancer cells in vitro by 3′-azido-3′-deoxythymidine.Clin Cancer Res. 1998; 4: 693-696PubMed Google Scholar). An additional targeted strategy deals with the physical blockage of the RNA template. In fact, it has been shown that chemically modified oligonucleotides, such as phosphorothioate oligomers and peptide nucleic acids, efficiently inhibit telomerase activity in vitro (Norton et al., 1996Norton J.C. Piatyszek M.A. Wright W.E. Shay J.W. Corey D.R. Inhibition of human telomerase activity by peptide nucleic acids.Nat Biotechnol. 1996; 14: 615-619Crossref PubMed Scopus (344) Google Scholar). A marked reduction of telomerase catalytic activity was also obtained by using hammerhead ribozymes directed against the RNA component of the enzyme (Kanazawa et al., 1996Kanazawa Y. Ohkawa K. Ueda K. et al.Hammerhead ribozyme-mediated inhibition of telomerase activity in extracts of human hepatocellular carcinoma cells.Biochem Biophys Res Commun. 1996; 225: 570-576https://doi.org/10.1006/bbrc.1996.1213Crossref PubMed Scopus (101) Google Scholar;Yokoyama et al., 1998Yokoyama Y. Takahashi Y. Shinohara A. Wan X. Niwwa K. Tamay A. Attenuation of telomerase activity by a hammerhead ribozyme targeting the template region of the telomerase RNA in endometrial carcinoma cells.Cancer Res. 1998; 59: 5406-5410Google Scholar). Ribozymes are RNA molecules which possess specific endoribonuclease activity and catalyze the hydrolysis of specific phosphodiester bonds, resulting in the cleavage of the RNA target sequences (Irie et al., 1997Irie A. Kijima H. Ohkawa T. et al.Anti-oncogene ribozymes for cancer gene therapy.Adv Pharmacol. 1997; 40: 207-257Crossref PubMed Scopus (18) Google Scholar;James and Gibson, 1998James H.A. Gibson I. The therapeutic potential of ribozymes.Blood. 1998; 91: 371-382Crossref PubMed Google Scholar). Moreover, after the cleavage reaction, the substrate is accessible by ribonucleases, a step that guarantees its permanent inactivation and offers a considerable advantage over the simple physical blockage obtained with complementary oligomers. In this study, we evaluated the ability of a hammerhead ribozyme, directed against the RNA component of human telomerase, to inhibit the catalytic activity of the enzyme. We first verified the ribozyme specificity, in terms of its ability to cleave in vitro a synthetic RNA substrate that corresponds to a portion of telomerase RNA component. Subsequently, the efficiency of the ribozyme in inhibiting telomerase activity was assessed on protein extracts from telomerase-positive cell lines and surgical specimens of human cutaneous melanoma. Lastly, we examined whether cationic liposome as well as expression vector-mediated transfer of the ribozyme inhibited telomerase activity and induced changes in the proliferative and phenotypic features of melanoma cells growing in culture. Two cell lines (JR8 and M14) and three surgical specimens (ML1528, ML1583, ML1584) of human cutaneous melanoma were used in the study. The biologic characteristics of the cell lines have been previously reported (Zupi et al., 1985Zupi G. Mauro F. Balduzzi M.A. Pardini C. Cavaliere R. Greco C. Established melanoma cell lines from different metastatic nodules of a single patient. A useful model for cancer therapy.Proc Am Assoc Cancer Res. 1985; 26: 22Google Scholar). JR8 and M14 cells were maintained in logarithmic growth phase at 37°C in a 5% CO2 humidified atmosphere using RPMI 1640 (Biowhittaker, Verviers, Belgium) supplemented with 10% fetal calf serum, 2 mM L-glutamine and 0.25% gentamycin. Single-stranded synthetic DNA oligonucleotides encoding active ribozyme (telo RZ –) and negative control (telo RZ +) were obtained from M-Medical S.r.l. (Firenze, Italy). The oligonucleotide sequences were the following: telo-RZ – (5′-AGCTTGGCGGCCGCCATTTTTTGTTTCGTCCTCAC-GACTCTTCAGTAACCCTAACGCGGCCGCG-3′) and telo-RZ + (5′- AATTCGCGGCCGCGTTAGGGTTACTGAAGAGTCCGTGA-GGACGAAACAAAAAATGGCGGCCGCCA-3′). Annealing of the complementary oligonucleotides produced a fragment with EcoRI and HindIII protruding ends. The fragment was inserted into the pGEM-3Z vector (Promega, Madison, WI), which had been previously digested with HindIII and EcoRI restriction enzymes. The presence and the correct orientation of the insert was verified by DNA sequencing (AmpliCycle, Perkin Elmer-Roche Molecular System, Branchburg, NJ). The resulting plasmid was named pRZ. To obtain the active ribozyme (RZH) and the negative control oligomer, we linearized the pRZ plasmid with HindIII and EcoRI, respectively. Twenty micrograms of the resulting linear templates were transcribed in vitro in 0.2 ml of a reaction mixture composed of 260 U recombinant RNAsin ribonuclease inhibitor (Promega), 1 mM each of ribonucleotide triphosphates, 10 mM dithiothreitol (DTT), 0.1 mg per ml bovine serum albumin, 1 × transcription optimized buffer (40 mM Tris–HCl, pH 7.9, 10 mM NaCl, 6 mM MgCl2, 2 mM spermidine) and 300 U T7 RNA polymerase for RZH and 300 U SP6 RNA polymerase for negative control oligomer (Figure 1). All reagents were purchased from Promega. The reaction mixtures were incubated for 2 h at 37°C (RZH) or 40°C (negative control oligomer), as suggested bySambrook et al., 1989Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning, a Laboratory Manual.Cold Spring Harbor, NY. Cold Spring Harbor Laboratory Press, 1989Google Scholar, then treated with 20 U RQ1 RNase-free DNase for 15 min at 37°C. The products were extracted with phenol/chloroform saturated with 10 mM Tris-1 mM ethylenediaminetetraacetic acid (EDTA), pH 4.5, precipitated with 100% isopropyl alcohol/5 M ammonium acetate, pH 5.5, washed with 70% pre-chilled ethanol, dried by speed vacuum and resuspended in twice distilled water. The concentration and the purity of the transcription products were assessed by A260 and A260/A280 spectrophotometrical measurement, respectively. The transcription products of expected size were then verified by denaturating polyacrylamide gel electrophoresis. To produce a synthetic RNA substrate, total RNA was isolated from the JR8 melanoma cell line using a Qiagen total RNA kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions. A portion of the human telomerase RNA component containing the telomeric template element (Feng et al., 1995Feng J. Funk W.D. Wang S.-S. et al.The RNA component of human telomerase.Science. 1995; 269: 1236-1241Crossref PubMed Scopus (2018) Google Scholar) was produced by reverse transcriptase–polymerase chain reaction (reverse transcriptase–PCR). Reverse transcription was done using a GeneAmp RNA PCR core kit (Perkin Elmer) according to the manufacturer's instructions. The resulting cDNA was amplified using telomerase RNA sequence specific primers (telo S, 5′-CTGGGAGGGGTGGTGGCCAT-3′ and telo A, 5′-GGAGCAAAAG-CACGGCGCCT-3′ (Kanazawa et al., 1996Kanazawa Y. Ohkawa K. Ueda K. et al.Hammerhead ribozyme-mediated inhibition of telomerase activity in extracts of human hepatocellular carcinoma cells.Biochem Biophys Res Commun. 1996; 225: 570-576https://doi.org/10.1006/bbrc.1996.1213Crossref PubMed Scopus (101) Google Scholar)) and by performing 26 cycles of PCR (95°C for 30 s, 65°C for 45 s, and 72°C for 30 s) followed by a 5 min extension step at 72°C. The product of expected size was gel purified and cloned into pGEM-T-Easy vector (Promega) according to the manufacturer's instructions. The presence and the orientation of the fragment were verified by DNA sequencing. The resulting plasmid was named pRNAtelo. To obtain the internally labelled RNA substrate, pRNAtelo was linearized with NcoI restriction enzyme. The resulting linear template was transcribed in vitro in the presence of [α-32P]cytidine triphosphate (10 μCi per μl, 800 Ci per mmol, Amersham International, Buckinghamshire, U.K.) and SP6 RNA polymerase, using the Riboprobe in vitro transcription system (Promega) according to the manufacturer's instructions. The reaction product was purified and the transcription product of expected size was verified as described above. 32P-substrate RNA (0.1 μM) was mixed with increasing concentrations of ribozyme in 50 mM Tris–HCl, pH 7.5. The mixtures were heated for 2 min at 90°C, cooled at 37°C, and then incubated in the presence of increasing concentrations of MgCl2. The cleavage reactions were performed at 37°C for 2 h and at 23°C for 12 h. Reactions were stopped by adding an equal volume of stop solution (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.02% xylene cyanol FF). Products were resolved on 10% polyacrylamide/8 M urea gel, and results were quantitated by densitometric analysis. DOTAP (N-(1-(2,3 dioleoyloxy)propil)-N,N,N-trimethylammonium methyl sulfate, Boehringer Mannheim, Mannheim, Germany) and the RZH ribozyme, or the negative control oligomer, were mixed at various concentrations in 20 mM HEPES buffer, in a final volume of 1 ml, followed by incubation at room temperature for 15 min. Then, 100 μg per ml of recombinant RNasin ribonuclease inhibitor was added to the complex solution. For the liposome-mediated ribozyme transfection, 8 × 105 cells in 2 ml culture medium were plated in each well of a six-well plate and allowed to attach for 24 h at 37°C in a 5% CO2 humidified atmosphere. Cells were then incubated with DOTAP alone or with the DOTAP-ribozyme complexes. The final concentration of DOTAP alone was 60 μg per 2 ml per well and that of liposomes–ribozyme complexes was 60 μg DOTAP and 10–45 μg ribozyme/2 ml per well. After 48 h of incubation with DOTAP or the DOTAP–ribozyme complexes, cells were trypsinized and counted in a Coulter Counter (Coulter Electronics, Luton, U.K.), and their viability was determined by the Trypan Blue dye exclusion test. Cell extracts were then obtained (see below) for the determination of telomerase activity. The oligonucleotides telo-RZ– and telo-RZ+ were annealed and digested with the NotI restriction enzyme. The resulting fragment, with NotI protruding ends, was inserted into the pRc/CMV (Invitrogen, San Diego, CA) mammalian expression vector, previously digested with NotI. The sequence and the correct orientation of the insert in the vector was verified by DNA sequencing, and the resultant vector was named pRcRz. DOTAP-mediated transfection of JR8 cells was performed according to the manufacturer's instructions. Briefly, cells previously seeded at a density yielding approximately 50% confluency at the time of transfection, were transfected with 5 μg of pRcRz vector (or pRc/CMV control vector) that had been complexed with 30 μg of DOTAP. Six hours after transfection, the culture medium containing the DOTAP/DNA mixture was replaced by a selection medium containing G418 to a final concentration of 2 mg per ml. The transfected cells were exposed to G418 for 1 mo. Total RNA was isolated from the transfectants and parental JR8 cell line, and reverse-transcribed as previously described. To analyze ribozyme expression, the resultant cDNA was amplified using T7 and SP6 primers (M-Medical) and by performing 32 cycles of PCR (95°C for 45 s, 48°C for 30 s, and 72°C for 45 s) followed by a 7 min extension step at 72°C in the presence of 1 μCi/sample of [α-32P]deoxycytidine triphosphate (3000 Ci per mmol, Amersham) in a 50 μl of finale volume. For the analysis of hTR expression, reverse transcriptase–mixtures were diluted 10,000 fold. One microliter of each solution was amplified in the presence of teloS and teloA primers and 1 μCi of [α-32P]deoxycytidine triphosphate, by performing 26 cycles of PCR (95°C for 30 s, 65°C for 45 s, and 72°C for 30 s) followed by a 5 min extension step at 72°C, in a final volume of 20 μl. β-Actin, used as a standard of amplification, was coamplified with hTR RNA using two specific primers (774: 5′-GGGAATTCAAAACTGGAA-CGGTGAAGG-3′ 775: 5′-GGAAGCTTATCAAAGTCCTCGGCCA-CA-3′) (Ulaner et al., 1998Ulaner G.A. Hu J.F. Vu T.H. Giudice L.C. Hoffman A.R. Telomerase activity in human development is regulated by human telomerase reverse transcriptase (hTERT) transcription and by alternate splicing of hTERT transcripts.Cancer Res. 1998; 58: 4168-4172PubMed Google Scholar). The PCR products were analyzed by electrophoresis on a 5% nondenaturating polyacrylamide gel in 1 × Tris-borate EDTA buffer (TBE; 45 mM Tris-borate, pH 8.3, 1 mM EDTA). The gel was dried and autoradiographed. Telomerase activity was measured by the telomeric repeat amplification protocol (TRAP) as described byKim et al., 1994Kim N.W. Piatyszek M.A. Prowse K.R. et al.Specific association of human telomerase activity with immortal cells and cancer.Science. 1994; 266: 2011-2015Crossref PubMed Scopus (6308) Google Scholar. Five to 106 cells (or 50–100 mg of melanoma tissues) were resuspendend in ice-cold lysis buffer (10 mM Tris–HCl, pH 7.5, 1 mM MgCl2, 1 mM ethylene glycol-bis-(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, 0.1 mM phenylmethylsulfonyl fluoride, 5 mMβ-mercaptoethanol, 0.5% 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate) and kept on ice for 30 min. After centrifugation at 25,000 × g at 4°C for 30 min, the supernatants were quick-frozen in liquid nitrogen and stored at -80°C. Protein concentrations of the lysates were determined using standard procedures. Proteins (2 μg) from each extract were assayed in 50 μl of TRAP reaction mixture composed of 20 mM Tris–HCl, pH 8.3, 1.5 mM MgCl2, 63 mM KCl, 0.005% Tween-20, 1 mM ethylene glycol-bis-(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, 50 mM each of deoxynucleoside triphosphates, 0.1 μg telomerase substrate (TS) oligonucleotide, 1 μg of T4g32 protein (Boehringer Mannheim), 0.1 mg bovine serum albumin per ml, 2 U Taq DNA polymerase (AmpliTaq, Perkin Elmer), 0.2 μl of [α-32P]deoxycytidine triphosphate (10 μCi per μl, 3000 Ci per mmol, Amersham International). After 20 min of incubation at 23°C for extension of the TS oligonucleotide by telomerase, the reaction mixture was heated at 94°C for 30 s and then subjected to 30 PCR cycles (94°C for 30 s, 50°C for 30 s, 72°C for 45 s), followed by a 3 min extension step at 72°C. The total reaction mixture was analyzed by electrophoresis on a 10% nondenaturating polyacrylamide gel. For each cell extract, aliquots containing 2 μg of protein were pretreated with 20 μg per ml RNase A for 20 min at 37°C and used as a control to verify the specific telomerase activity. For quantitative analysis, assays were repeated using the Oncor TRAPeze Telomerase Detection Kit (Oncor Appligene, Heidelberg, Germany). Each reaction product was amplified in the presence of a 36 bp internal TRAP assay standard (ITAS), and each sample extract was tested for RNase sensitivity. A TSR8 quantitation standard was included for each set of TRAP assays. The TSR8 control is provided in the kit and serves as a standard for estimating the amount of product extended by telomerase in a given extract. Quantitative analysis was performed with the Image-QuanT software (Molecular Dynamics, Sunnyvale, CA), which allowed densitometric evaluation of the digitized image. Telomerase activity was quantitated by measuring the signal of telomerase ladder bands, and the relative telomerase activity was calculated as the ratio to the internal standard using the following formula relativetelomeraseactivity:[(X-X0)/C]×[(R-R0)/Cr]-1 where X is the untreated sample, X0 is the RNase-treated sample, C is the internal control of untreated samples, Cr is the internal control of TSR8, R is the TSR8 quantitation control, and R0 is the negative control. The effect of ribozyme on telomerase was expressed as the percentage inhibition of enzyme activity in samples exposed to the ribozyme compared with controls. The extent of inhibition as a function of the ribozyme concentration was plotted and these graphs were utilized to derive the IC50 values. Total DNA was isolated using DNAzol (Life Technologies, Gaithersburg, MD). For each sample, 10 μg of DNA was digested with 40 units of HinfI and then electrophoresed on 0.8% agarose gels. Following electrophoresis, gels were denatured, neutralized, transferred to a nylon membrane (Hybond N; Amersham), and then cross-linked with ultraviolet light. The membrane was hybridized with a 5′-end [α-32P]deoxyadenosine triphosphate-labeled telomeric oligonucleotide probe (TTAGGG)4. Hybridization was carried out at 42°C for 1 h in rapid hybridization buffer (Amersham). The membrane was washed in 5 × sodium citrate/chloride buffer, 0.1% sodium dodecyl sulfate at room temperature and 1 × sodium citrate/chloride buffer, 0.1% sodium dodecyl sulfate at 42°C. Filters were autoradiographed (Hyperfilm-MP; Amersham) with an intensifying screen at -80°C for 12–24 h. Autoradiographs were scanned (ScanJet IIcx/T; Hewlett Packard) and mean telomere length was calculated as previously reported (Mehle et al., 1994Mehle C. Ljungberg B. Ross G. Telomere shortening in renal carcinoma.Cancer Res. 1994; 54: 236-241PubMed Google Scholar). As the first step of the study, we verified the catalytic potential of the RZH ribozyme on an internally 32P-labeled synthetic RNA substrate obtained by cloning a portion of the RNA component of human telomerase. The size of the resulting substrate was 165 bp. Preliminary experiments performed by incubating 0.1 μM labeled RNA substrate with increasing concentrations (from 0.01 to 1.0 μM) of the ribozyme for 2 h at 37°C resulted in the expected cleavage products of 102 bp and 63 bp, respectively. The efficiency of cleavage was dependent on the ribozyme/substrate ratio, with cleavage products being detectable at a ratio of 0.5:1, and increased with increasing amounts of the ribozyme relative to the substrate (data not shown). In parallel, we assessed the catalytic activity of the RZH ribozyme as a function of MgCl2 concentration. Cleavage was absolutely dependent on Mg2+, but it was already detectable at MgCl2 concentrations as low as 1 mM (data not shown). Based on these results, we carried out a quantitative analysis of RZH ribozyme catalytic activity under experimental conditions thought to guarantee an efficient cleavage reaction (Figure 2). In addition to being concentration-dependent, ribozyme catalytic activity was more efficient at 37°C than at 23°C. Specifically, whereas 95% of the substrate was cleaved in the presence of 10 μM RZH over a 2 h incubation at 37°C, only 78% of substrate cleavage was observed after 12 h of incubation with the same ribozyme concentration at 23°C. A greater cleavage activity of the ribozyme at the higher temperature was also appreciable at the concentration of 1 μM, whereas the same efficiency of cleavage at 23°C and 37°C was evident at the intermediate ribozyme concentration (3 μM). A weak band, corresponding to a fragment of approximately 102 bp, was observed even in lanes containing only the RNA substrate. It may not have been a specific cleavage product but only an artifact produced during the in vitro transcription for RNA substrate synthesis or, alternatively, a product of substrate degradation. No cleavage was detected when the reaction was performed in the presence of 10 μM of the negative control oligomer at either temperatures. The ability of RZH ribozyme to inhibit the activity of telomerase was assessed on cell extracts from two human melanoma cell lines that displayed a comparable basal level of telomerase activity in the TRAP assay. The JR8 cell extract was mixed with increasing concentrations of RZH and incubated for 3, 6, and 12 h at 23°C. Although the ribozyme is more active at 37°C, the 23°C
DOI: 10.1016/0959-8049(94)90132-5
1994
Cited 66 times
Expression of P-glycoprotein and in vitro or in vivo resistance to doxorubicin and cisplatin in breast and ovarian cancers
The expression of P-glycoprotein (P-gp) was studied by immunocytohistochemistry, using the C219 monoclonal antibody, in 39 locally advanced breast cancers and 20 ovarian cancers from previously untreated patients. P-gp was expressed in 46 and 35% of breast and ovarian tumours, respectively. A significant association was observed in both tumour types between P-gp expression and in vitro resistance to doxorubicin. We also observed a higher clinical response rate to doxorubicin ± vincristine in patients with breast cancers not expressing P-gp. Conversely, no correlation was found between P-gp expression and in vitro resistance to cisplatin or in vivo response to cisplatin ± cyclophosphamide treatment in ovarian cancers. Our results support the relevance of P-gp expression as a specific indicator of resistance to certain drugs, such as doxorubicin and vincrinstine, involved in the phenomenon of multidrug resistance in breast and ovarian cancer cells.
DOI: 10.1093/carcin/bgh107
2004
Cited 62 times
Ribozyme-mediated down-regulation of survivin expression sensitizes human melanoma cells to topotecan in vitro and in vivo
The ability of melanoma cells to evade engagement of apoptosis plays a significant role in their resistance to chemotherapy. In an attempt to lower the apoptotic threshold of melanoma cells as a possible strategy to increase their drug sensitivity, we generated a hammerhead ribozyme to down-regulate the expression of the anti-apoptotic protein survivin. The JR8 human melanoma cell line was stably transfected with the active ribozyme RZsurv (targeting the 3' end of the GUC294 triplet in the exon 3 of the survivin mRNA) or the catalytically inactive ribozyme mutRZsurv (carrying a mutation in the catalytic core of RZsurv). Two polyclonal cell populations expressing the active (JR8/RZsurv) or the mutant (JR8/mutRZsurv) ribozyme were selected for the study. JR8/RZsurv cells were characterized by a markedly lower survivin protein level than JR8 parental cells, whereas a negligible reduction in survivin expression was observed in JR8/mutRZsurv cells. JR8/RZsurv cells showed a significantly increased sensitivity to the topoisomerase-I inhibitor topotecan (as detected by clonogenic cell survival) compared with JR8/mutRZsurv cells. Moreover, the extent of drug-induced apoptosis (in terms of percentage of apoptotic nuclei and level of caspase-9 and caspase-3 catalytic activity) was significantly greater in JR8/RZsurv than in JR8/mutRZsurv cells. Finally, an increased antitumor activity of oral topotecan was observed in JR8/RZsurv cells grown as xenograft tumors in athymic nude mice compared with JR8/mutRZsurv cells. These results demonstrate that attenuation of survivin expression renders human melanoma cells more susceptible to topotecan-induced apoptosis and more responsive to in vivo treatment, and support the concept that survivin is an attractive target for new therapeutic interventions in melanoma.
DOI: 10.1002/ijc.23412
2008
Cited 49 times
Evidence for alternative lengthening of telomeres in liposarcomas in the absence of ALT‐associated PML bodies
Abstract Immortalized and cancer cells maintain their telomeres by activation of a telomere maintenance mechanism (TMM). In ∼85% of cancers telomerase is activated (TA) but in some tumours, in particular sarcomas, an alternative lengthening of telomeres (ALT) pathway is used. Liposarcomas are the most common soft‐tissue sarcoma in adults and they activate ALT or telomerase with equal frequency, however no TMM has been identified in ∼50% of liposarcomas. In our study, we have shown that instability at the minisatellite MS32, usually associated with ALT activation, aids the identification of liposarcomas that have recombination‐like activity at telomeres in absence of ALT associated PML‐bodies (APBs). Furthermore, using single molecule telomere analysis, we have detected complex telomere mutations directly in ALT positive liposarcomas and interestingly in some liposarcomas with an unknown TMM but high MS32 instability. We have shown by sequence analysis that some of these complex telomere mutations must arise by an inter‐molecular recombination‐like process rather than by deletion caused by t ‐loop excision or by unequal telomere‐sister‐chromatid‐exchange (T‐SCE), which is known to be elevated in ALT cell lines. Preliminary evidence also suggests that inter‐molecular recombination events may be processed differently in liposarcomas with APBs compared to those without. In conclusion, we have shown for the first time, that some telomerase negative liposarcomas without APBs have other features associated with ALT, indicating that the incidence of ALT in these tumours has previously been under‐estimated. This has major implications for the use of cancer treatments targeted at TMMs. © 2008 Wiley‐Liss, Inc.
DOI: 10.1038/sj.bjc.6604927
2009
Cited 48 times
Apollon gene silencing induces apoptosis in breast cancer cells through p53 stabilisation and caspase-3 activation
We analysed the effects of small interfering RNA (siRNA)-mediated silencing of Apollon, a member of the inhibitors of apoptosis protein family, on the proliferative potential and ability of human breast cancer cell lines to undergo apoptosis. In wild-type p53 ZR75.1 cells, Apollon knockdown resulted in a marked, time-dependent decline of cell growth and an increased rate of apoptosis, which was associated with p53 stabilisation and activation of the mitochondrial-dependent apoptotic pathway. Pre-incubation of cells with a p53-specific siRNA resulted in a partial rescue of cell growth inhibition, as well as in a marked reduction of the apoptotic response, indicating p53 as a major player in cell growth impairment consequent on Apollon silencing. Apollon knockdown induced consistently less pronounced anti-proliferative and pro-apoptotic effects in mutant p53 MDA-MB-231 cells than in ZR75.1 cells. Furthermore, the activation of caspase-3 seemed to be essential for the induction of apoptosis after Apollon knockdown, as the Apollon-specific siRNA had no effect on the viability of caspase-3-deficient, wild-type p53 MCF-7 cells or the ZR75.1 cells after RNA interference-mediated caspase-3 silencing. Our results indicate that p53 stabilisation and caspase-3 activation concur to determine the apoptotic response mediated by Apollon knockdown in breast cancer cells, and suggest Apollon to be a potential new therapeutic target for this malignancy.
DOI: 10.1016/j.ijrobp.2008.10.021
2009
Cited 43 times
To Bleed or Not to Bleed. A Prediction Based on Individual Gene Profiling Combined With Dose–Volume Histogram Shapes in Prostate Cancer Patients Undergoing Three-Dimensional Conformal Radiation Therapy
Purpose The main purpose of this work was to try to elucidate why, despite excellent rectal dose-volume histograms (DVHs), some patients treated for prostate cancer exhibit late rectal bleeding (LRB) and others with poor DVHs do not. Thirty-five genes involved in DNA repair/radiation response were analyzed in patients accrued in the AIROPROS 0101 trial, which investigated the correlation between LRB and dosimetric parameters. Methods and Materials Thirty patients undergoing conformal radiotherapy with prescription doses higher than 70 Gy (minimum follow-up, 48 months) were selected: 10 patients in the low-risk group (rectal DVH with the percent volume of rectum receiving more than 70 Gy [V70Gy] < 20% and the percent volume of rectum receiving more than 50 Gy [V50Gy] < 55%) with Grade 2 or Grade 3 (G2–G3) LRB, 10 patients in the high-risk group (V70Gy > 25% and V50Gy > 60%) with G2–G3 LRB, and 10 patients in the high-risk group with no toxicity. Quantitative reverse-transcriptase polymerase chain reaction was performed on RNA from lymphoblastoid cell lines obtained from Epstein-Barr virus–immortalized peripheral-blood mononucleated cells and on peripheral blood mononucleated cells. Interexpression levels were compared by using the Kruskal-Wallis test. Results Intergroup comparison showed many constitutive differences: nine genes were significantly down–regulated in the low-risk bleeder group vs. the high-risk bleeder and high-risk nonbleeder groups: AKR1B1 (p = 0.019), BAZ1B (p = 0.042), LSM7 (p = 0.0016), MRPL23 (p = 0.015), NUDT1 (p = 0.0031), PSMB4 (p = 0.079), PSMD1 (p = 0.062), SEC22L1 (p = 0.040), and UBB (p = 0.018). Four genes were significantly upregulated in the high-risk nonbleeder group than in the other groups: DDX17 (p = 0.048), DRAP1 (p = 0.0025), RAD23 (p = 0.015), and SRF (p = 0.024). For most of these genes, it was possible to establish a cut-off value that correctly classified most patients. Conclusions The predictive value of sensitivity and resistance to LRB of the genes identified by the study is promising and should be tested in a larger data set. The main purpose of this work was to try to elucidate why, despite excellent rectal dose-volume histograms (DVHs), some patients treated for prostate cancer exhibit late rectal bleeding (LRB) and others with poor DVHs do not. Thirty-five genes involved in DNA repair/radiation response were analyzed in patients accrued in the AIROPROS 0101 trial, which investigated the correlation between LRB and dosimetric parameters. Thirty patients undergoing conformal radiotherapy with prescription doses higher than 70 Gy (minimum follow-up, 48 months) were selected: 10 patients in the low-risk group (rectal DVH with the percent volume of rectum receiving more than 70 Gy [V70Gy] < 20% and the percent volume of rectum receiving more than 50 Gy [V50Gy] < 55%) with Grade 2 or Grade 3 (G2–G3) LRB, 10 patients in the high-risk group (V70Gy > 25% and V50Gy > 60%) with G2–G3 LRB, and 10 patients in the high-risk group with no toxicity. Quantitative reverse-transcriptase polymerase chain reaction was performed on RNA from lymphoblastoid cell lines obtained from Epstein-Barr virus–immortalized peripheral-blood mononucleated cells and on peripheral blood mononucleated cells. Interexpression levels were compared by using the Kruskal-Wallis test. Intergroup comparison showed many constitutive differences: nine genes were significantly down–regulated in the low-risk bleeder group vs. the high-risk bleeder and high-risk nonbleeder groups: AKR1B1 (p = 0.019), BAZ1B (p = 0.042), LSM7 (p = 0.0016), MRPL23 (p = 0.015), NUDT1 (p = 0.0031), PSMB4 (p = 0.079), PSMD1 (p = 0.062), SEC22L1 (p = 0.040), and UBB (p = 0.018). Four genes were significantly upregulated in the high-risk nonbleeder group than in the other groups: DDX17 (p = 0.048), DRAP1 (p = 0.0025), RAD23 (p = 0.015), and SRF (p = 0.024). For most of these genes, it was possible to establish a cut-off value that correctly classified most patients. The predictive value of sensitivity and resistance to LRB of the genes identified by the study is promising and should be tested in a larger data set.
DOI: 10.1016/j.bcp.2011.09.021
2012
Cited 37 times
Role of tyrosyl-DNA phosphodiesterase 1 and inter-players in regulation of tumor cell sensitivity to topoisomerase I inhibition
Tyrosyl-DNA phosphodiesterase 1 (TDP1) plays a unique function as it catalyzes the repair of topoisomerase I-mediated DNA damage. Thus, ovarian carcinoma cell lines exhibiting increased TDP1 levels and resistance to the topoisomerase I poisons campthotecins were used to clarify the role of this enzyme. The camptothecin gimatecan was employed as a tool to inhibit topoisomerase I because it produces a persistent damage. The resistant sublines displayed an increased capability to repair drug-induced single-strand breaks and a reduced amount of drug-induced double-strand breaks, which was enhanced following TDP1 silencing. In loss of function studies using U2-OS cells, we found that TDP1 knockdown did not produce a change in sensitivity to camptothecin, whereas co-silencing of other pathways cooperating with TDP1 in cell response to topoisomerase I poisons indicated that XRCC1 and BRCA1 were major regulators of sensitivity. No change in cellular sensitivity was observed when TDP1 was silenced concomitantly to RAD17, which participates in the stabilization of collapsed replication forks. The expression of dominant-negative PARP1 in cells with reduced expression of TDP1 due to a constitutively expressed TDP1 targeting microRNA did not modulate cell sensitivity to camptothecin. Mild resistance to gimatecan was observed in cells over-expressing TDP1, a feature associated with decreased levels of drug-induced single-strand breaks. In conclusion, since TDP1 alone can account for mild levels of camptothecin resistance, repair of topoisomerase I-mediated DNA damage likely occurs through redundant pathways mainly implicating BRCA1 and XRCC1, but not RAD17 and PARP1. These findings may be relevant to define novel therapeutic strategies.
DOI: 10.1158/0008-5472.can-11-1285
2011
Cited 36 times
TLR9 Agonists Oppositely Modulate DNA Repair Genes in Tumor versus Immune Cells and Enhance Chemotherapy Effects
Synthetic oligodeoxynucleotides expressing CpG motifs (CpG-ODN) are a Toll-like receptor 9 (TLR9) agonist that can enhance the antitumor activity of DNA-damaging chemotherapy and radiation therapy in preclinical mouse models. We hypothesized that the success of these combinations is related to the ability of CpG-ODN to modulate genes involved in DNA repair. We conducted an in silico analysis of genes implicated in DNA repair in data sets obtained from murine colon carcinoma cells in mice injected intratumorally with CpG-ODN and from splenocytes in mice treated intraperitoneally with CpG-ODN. CpG-ODN treatment caused downregulation of DNA repair genes in tumors. Microarray analyses of human IGROV-1 ovarian carcinoma xenografts in mice treated intraperitoneally with CpG-ODN confirmed in silico findings. When combined with the DNA-damaging drug cisplatin, CpG-ODN significantly increased the life span of mice compared with individual treatments. In contrast, CpG-ODN led to an upregulation of genes involved in DNA repair in immune cells. Cisplatin-treated patients with ovarian carcinoma as well as anthracycline-treated patients with breast cancer who are classified as "CpG-like" for the level of expression of CpG-ODN modulated DNA repair genes have a better outcome than patients classified as "CpG-untreated-like," indicating the relevance of these genes in the tumor cell response to DNA-damaging drugs. Taken together, the findings provide evidence that the tumor microenvironment can sensitize cancer cells to DNA-damaging chemotherapy, thereby expanding the benefits of CpG-ODN therapy beyond induction of a strong immune response.
DOI: 10.1111/j.1582-4934.2012.01652.x
2012
Cited 33 times
Characterization of stress response in human retinal epithelial cells
The pathogenesis of age-related macular degeneration (AMD) involves demise of the retinal pigment epithelium and death of photoreceptors. In this article, we investigated the response of human adult retinal pigmented epithelial (ARPE-19) cells to 5-(N,N-hexamethylene)amiloride (HMA), an inhibitor of Na(+) /H(+) exchangers. We observed that ARPE-19 cells treated with HMA are unable to activate 'classical' apoptosis but they succeed to activate autophagy. In the first 2 hrs of HMA exposure, autophagy is efficient in protecting cells from death. Thereafter, autophagy is impaired, as indicated by p62 accumulation, and this protective mechanism becomes the executioner of cell death. This switch in autophagy property as a function of time for a single stimulus is here shown for the first time. The activation of autophagy was observed, at a lesser extent, with etoposide, suggesting that this event might be a general response of ARPE cells to stress and the most important pathway involved in cell resistance to adverse conditions and toxic stimuli.