Federica Rizzi

Green tea catechins (GTCs) proved to be effective in inhibiting cancer growth in several experimental models. Recent studies showed that 30% of men with high-grade prostate intraepithelial neoplasia (HG-PIN) would develop prostate cancer (CaP) within 1 year after repeated biopsy. This prompted us to do a proof-of-principle clinical trial to assess the safety and efficacy of GTCs for the chemoprevention of CaP in HG-PIN volunteers. The purity and content of GTCs preparations were assessed by high-performance liquid chromatography [(-)-epigallocathechin, 5.5%; (-)-epicatechin, 12.24%; (-)-epigallocatechin-3-gallate, 51.88%; (-)-epicatechin-3-gallate, 6.12%; total GTCs, 75.7%; caffeine, <1%]. Sixty volunteers with HG-PIN, who were made aware of the study details, agreed to sign an informed consent form and were enrolled in this double-blind, placebo-controlled study. Daily treatment consisted of three GTCs capsules, 200 mg each (total 600 mg/d). After 1 year, only one tumor was diagnosed among the 30 GTCs-treated men (incidence, approximately 3%), whereas nine cancers were found among the 30 placebo-treated men (incidence, 30%). Total prostate-specific antigen did not change significantly between the two arms, but GTCs-treated men showed values constantly lower with respect to placebo-treated ones. International Prostate Symptom Score and quality of life scores of GTCs-treated men with coexistent benign prostate hyperplasia improved, reaching statistical significance in the case of International Prostate Symptom Scores. No significant side effects or adverse effects were documented. To our knowledge, this is the first study showing that GTCs are safe and very effective for treating premalignant lesions before CaP develops. As a secondary observation, administration of GTCs also reduced lower urinary tract symptoms, suggesting that these compounds might also be of help for treating the symptoms of benign prostate hyperplasia.

The efficacy of atmospheric plasma treatment on microbial decontamination of different kinds of food products is already known. Recently, new applications of this innovative technology have been proposed, in order to test the improvement of quality maintenance of minimally processed fruit and vegetables. Nevertheless, the knowledge on the modifications of functional and nutritional properties of minimally processed fruit is still scarce. The objective of this study was to evaluate the effect of atmospheric double barrier discharge (DBD) plasma treatment on the quality maintenance of fresh-cut kiwifruit. Treatments of 10 and 20 min per side were performed and their consequences were evaluated during four days of storage in controlled conditions by monitoring parameters related to visual quality, texture, chlorophyll, carotenoids and polyphenols. The in vitro antioxidant activity was evaluated through a multimodal approach, combining different assays for the analysis of antiradical activity and reducing activity of antioxidants. According to the obtained results, plasma treatments positively influenced the quality maintenance of the product, by improving colour retention and reducing the darkened area formation during storage, not inducing any textural change compared with the control. Plasma treatments caused an immediate slight loss of pigments, but a better retention during storage. No significant changes in antioxidants content and antioxidant activity were observed among treated samples and control ones.

Green tea is a beverage that is widely consumed worldwide and is believed to exert effects on different diseases, including cancer. The major components of green tea are catechins, a family of polyphenols. Among them, epigallocatechin-gallate (EGCG) is the most abundant and biologically active. EGCG is widely studied for its anti-cancer properties. However, the cellular and molecular mechanisms explaining its action have not been completely understood, yet. EGCG is effective in vivo at micromolar concentrations, suggesting that its action is mediated by interaction with specific targets that are involved in the regulation of crucial steps of cell proliferation, survival, and metastatic spread. Recently, several proteins have been identified as EGCG direct interactors. Among them, the trans-membrane receptor 67LR has been identified as a high affinity EGCG receptor. 67LR is a master regulator of many pathways affecting cell proliferation or apoptosis, also regulating cancer stem cells (CSCs) activity. EGCG was also found to be interacting directly with Pin1, TGFR-II, and metalloproteinases (MMPs) (mainly MMP2 and MMP9), which respectively regulate EGCG-dependent inhibition of NF-kB, epithelial-mesenchimal transaction (EMT) and cellular invasion. EGCG interacts with DNA methyltransferases (DNMTs) and histone deacetylases (HDACs), which modulates epigenetic changes. The bulk of this novel knowledge provides information about the mechanisms of action of EGCG and may explain its onco-suppressive function. The identification of crucial signalling pathways that are related to cancer onset and progression whose master regulators interacts with EGCG may disclose intriguing pharmacological targets, and eventually lead to novel combined treatments in which EGCG acts synergistically with known drugs.

Alzheimer’s disease patients may benefit from antiplatelet therapy.

The atmospheric double barrier discharge (DBD) plasma technology is a promising tool in food industry as an alternative to traditional food preservation methods.However, the effect of the reactive species generated during the treatment on the content of bioactive compounds in food is still little studied, as well as there are no data concerning potential deleterious effects of DBD treated foods on human cells.Some functional properties of DBD-treated minimally processed Pink Lady ® apples were evaluated in comparison with untreated samples through different in-vitro and ex-vivo tests.Plasma treatment caused only a slight reduction of antioxidant content and antioxidant capacity (up to 10%), mainly limited to the amphiphilic fraction.Noteworthy, apple treated polyphenols extracts did not reduce cell viability and did not suppress the beneficial physiological cell response to oxidative stress in terms of reactive oxygen species production and phase II enzyme activation in human cultured colonocytes

Cancer is a multifaceted and complex pathology characterized by uncontrolled cell proliferation and decreased apoptosis. Most cancers are recognized by an inflammatory environment rich in a myriad of factors produced by immune infiltrate cells that induce host cells to differentiate and to produce a matrix that is more favorable to tumor cells’ survival and metastasis. As a result, the extracellular matrix (ECM) is changed in terms of macromolecules content, degrading enzymes, and proteins. Altered ECM components, derived from remodeling processes, interact with a variety of surface receptors triggering intracellular signaling that, in turn, cancer cells exploit to their own benefit. This review aims to present the role of different aspects of ECM components in the tumor microenvironment. Particularly, we highlight the effect of pro- and inflammatory factors on ECM degrading enzymes, such as metalloproteases, and in a more detailed manner on hyaluronan metabolism and the signaling pathways triggered by the binding of hyaluronan with its receptors. In addition, we sought to explore the role of extracellular chaperones, especially of clusterin which is one of the most prominent in the extracellular space, in proteostasis and signaling transduction in the tumor microenvironment. Although the described tumor microenvironment components have different biological roles, they may engage common signaling pathways that favor tumor growth and metastasis.

Genomic biomarkers of cancer are based on changes in nucleic acids, which include abnormal expression levels of some miRNAs, point mutations in DNA sequences, and altered levels of DNA methylation. The presence of tumor-related nucleic acids in body fluids (blood, saliva, or urine) makes it possible to achieve a non-invasive early-stage cancer diagnosis. Currently existing techniques for the discovery of nucleic acids require complex, time-consuming, costly assays and have limited multiplexing abilities. Surface-enhanced Raman spectroscopy (SERS) is a vibrational spectroscopy technique that is able to provide molecular specificity combined with trace sensitivity. SERS has gained research attention as a tool for the detection of nucleic acids because of its promising potential: label-free SERS can decrease the complexity of assays currently used with fluorescence-based detection due to the absence of the label, while labeled SERS may outperform the gold standard in terms of the multiplexing ability. The first papers about SERS-based methods for the measurement of genomic biomarkers were written in 2008, and since then, more than 150 papers have been published. The aim of this paper is to review and evaluate the proposed SERS-based methods in terms of their level of development and their potential for liquid biopsy application, as well as to contribute to their further evolution by attracting research attention to the field. This goal will be reached by grouping, on the basis of their experimental protocol, all the published manuscripts on the topic and evaluating each group in terms of its limit of detection and applicability to real body fluids. Thus, the methods are classified according to their working principles into five main groups, including capture-based, displacement-based, sandwich-based, enzyme-assisted, and specialized protocols.

The biological functions of clusterin (CLU, also known as ApoJ, SGP2, TRPM-2, CLI) have been puzzling the researchers since its first discovery in the early 80's. We know that CLU is a single 9-exons gene expressing three protein forms with different sub-cellular localisations and diverse biological functions. Despite the many reports from many research teams on CLU action and its relation to tumourigenesis, contradictions in the data and alternative hypothesis still exist. Understanding the role of CLU in tumourigenesis is complicated not only by the existence of different protein forms but also by the changes of tumours over time and the selection pressures imposed by treatments such as hormone ablation or chemotherapy. This review focuses on recent discoveries concerning the role of CLU in prostate and breast cancer onset and progression. Although CLU acts primarily as a tumour suppressor in the early stages of carcinogenesis, consistent with its role in the involution of the prostate following castration, late stage cancer may overexpress CLU following chemotherapeutic drugs or hormonal ablation therapy. High expression of secreted or cytoplasmic CLU may represent a pro-survival stimulus because it confers increased resistance to killing by anti-cancer drugs or enhances tumour cell survival in specific niches.

The proteasome inhibitors Bortezomib (BZM) and MG132 trigger cancer cell death via induction of endoplasmic reticulum (ER) stress and unfolded protein response. Epigallocatechin gallate (EGCG), the most bioactive green tea polyphenol, is known to display strong anticancer properties as it inhibits proteasome activity and induces ER stress. We investigated whether combined delivery of a proteasome inhibitor with EGCG enhances prostate cancer cell death through increased induction of ER stress. Paradoxically, EGCG antagonized BZM cytotoxicity even when used at low concentrations. Conversely, the MG132 dose-response curve was unaffected by co-administration of EGCG. Moreover, apoptosis, proteasome inhibition and ER stress were inhibited in PC3 cells simultaneously treated with BZM and EGCG but not with a combination of MG132 and EGCG; EGCG enhanced autophagy induction in BZM-treated cells only. Autophagy inhibition restored cytotoxicity concomitantly with CHOP and p-eIF2α up-regulation in cells treated with BZM and EGCG. Overall, these findings demonstrate that EGCG antagonizes BZM toxicity by exacerbating the activation of autophagy, which in turn mitigates ER stress and reduces CHOP up-regulation, finally protecting PC3 cells from cell death.

Among endocrine disruptors, the xenoestrogen bisphenol A (BPA) deserves particular attention due to widespread human exposure. Besides hormonal effects, BPA has been suspected to be involved in breast and prostate carcinogenesis, which share similar estrogen-related mechanisms. We previously demonstrated that administration of BPA to female mice results in the formation of DNA adducts and proteome alterations in the mammary tissue. Here, we evaluated the ability of BPA, given with drinking water, to induce a variety of biomarker alterations in male Sprague-Dawley rats. In addition, we investigated the formation of DNA adducts in human prostate cell lines. In BPA-treated rats, no DNA damage occurred in surrogate cells including peripheral blood lymphocytes and bone marrow erythrocytes, where no increase of single-strand DNA breaks was detectable by comet assay and the frequency of micronucleated cells was unaffected by BPA. Liver cells were positive at transferase dUTP nick end labeling assay, which detects both single-strand and double-strand breaks and early stage apoptosis. BPA upregulated clusterin expression in atrophic prostate epithelial cells and induced lipid peroxidation and DNA fragmentation in spermatozoa. Significant levels of DNA adducts were formed in prostate cell lines treated either with high-dose BPA for 24 h or low-dose BPA for 2 months. The BPA-related increase of DNA adducts was more pronounced in PNT1a nontumorigenic epithelial cells than in PC3 metastatic carcinoma cells. On the whole, these experimental findings support mechanistically the hypothesis that BPA may play a role in prostate carcinogenesis and may, potentially, affect the quality of sperm.

Increasing doses of Polyphenon E®, a standardized green tea extract, were given to PNT1a and PC3 prostate epithelial cells mimicking initial and advanced stages of prostate cancer (PCa), respectively. Cell death occurred in both cell lines, with PNT1a being more sensitive [half-maximal inhibitory concentration (IC50) = 35 μg/ml] than PC3 (IC50 = 145 μg/ml) to Polyphenon E®. Cell cycle arrest occurred at G0/G1 checkpoint for PNT1a, and G2/M for PC3 cells. Endoplasmic reticulum stress (ERS) and unfolded protein response (UPR) occurred in both cell lines, with each exhibiting different timing in response to Polyphenon E®. Autophagy was transiently activated in PNT1a cells within 12 h after treatment as a survival response to overcome ERS; then activation of caspases and cleavage of poly (ADP ribose) polymerase 1 occurred, committing cells to anoikis death. Polyphenon E® induced severe ERS in PC3 cells, causing a dramatic enlargement of the ER; persistent activation of UPR produced strong upregulation of GADD153/CHOP, a key protein of ERS-mediated cell death. Thereafter, GADD153/CHOP activated Puma, a BH3-only protein, committing cells to necroptosis, a programmed caspase-independent mechanism of cell death. Our results provide a foundation for the identification of novel targets and strategies aimed at sensitizing apoptosis-resistant cells to alternative death pathways.

The 15q24.1 locus, including CYP1A2, is associated with blood pressure (BP). The CYP1A2 rs762551 C allele is associated with lower CYP1A2 enzyme activity. CYP1A2 metabolizes caffeine and is induced by smoking. The association of caffeine consumption with hypertension remains controversial. We explored the effects of CYP1A2 variants and CYP1A2 enzyme activity on BP, focusing on caffeine as the potential mediator of CYP1A2 effects. Four observational (n = 16 719) and one quasi-experimental studies (n = 106) including European adults were conducted. Outcome measures were BP, caffeine intake, CYP1A2 activity and polymorphisms rs762551, rs1133323 and rs1378942. CYP1A2 variants were associated with hypertension in non-smokers, but not in smokers (CYP1A2-smoking interaction P = 0.01). Odds ratios (95% CIs) for hypertension for rs762551 CC, CA and AA genotypes were 1 (reference), 0.78 (0.59–1.02) and 0.66 (0.50–0.86), respectively, P = 0.004. Results were similar for the other variants. Higher CYP1A2 activity was linearly associated with lower BP after quitting smoking (P = 0.049 and P = 0.02 for systolic and diastolic BP, respectively), but not while smoking. In non-smokers, the CYP1A2 variants were associated with higher reported caffeine intake, which in turn was associated with lower odds of hypertension and lower BP (P = 0.01). In Mendelian randomization analyses using rs1133323 as instrument, each cup of caffeinated beverage was negatively associated with systolic BP [−9.57 (−16.22, −2.91) mmHg]. The associations of CYP1A2 variants with BP were modified by reported caffeine intake. These observational and quasi-experimental results strongly support a causal role of CYP1A2 in BP control via caffeine intake.

Numerous evidences from prevention studies in humans, support the existence of an association between green tea polyphenols consumption and a reduced cancer risk. Prostate cancer is one of the most frequently diagnosed male neoplasia in the Western countries, which is in agreement with this gland being particularly vulnerable to oxidative stress processes, often associated with tumorigenesis. Tea polyphenols have been extensively studied in cell culture and animal models where they inhibited tumor onset and progression. Prostate cancer appears a suitable target for primary prevention care, since it grows slowly, before symptoms arise, thus offering a relatively long time period for therapeutic interventions. It is, in fact, usually diagnosed in men 50-year-old or older, when even a modest delay in progression of the disease could significantly improve the patients quality of life. Although epidemiological studies have not yet yielded conclusive results on the chemopreventive and anticancer effect of tea polyphenols, there is an increasing trend to employ these substances as conservative management for patients diagnosed with less advanced prostate cancer. Here, we intend to review the most recent observations relating tea polyphenols to human prostate cancer risk, in an attempt to outline better their potential employment for preventing prostate cancer.

Autophagy is a homeostatic mechanism through which intracellular organelles and proteins are degraded and recycled in response to increased metabolic demand or stress. Autophagy dysfunction is often associated with many diseases, including cancer. Because of its role in tumorigenesis, autophagy can represent a new therapeutic target for cancer treatment. Prostate cancer (PCa) is one of the most common cancers in aged men. The evidence on alterations of autophagy related genes and/or protein levels in PCa cells suggests a potential implication of autophagy in PCa onset and progression. The use of natural compounds, characterized by low toxicity to normal tissue associated with specific anticancer effects at physiological levels in vivo, is receiving increasing attention for prevention and/or treatment of PCa. Understanding the mechanism of action of these compounds could be crucial for the development of new therapeutic or chemopreventive options. In this review we focus on the current evidence showing the capacity of natural compounds to exert their action through autophagy modulation in PCa cells.

The progressive neuropathological damage seen in Parkinson's disease (PD) is thought to be related to the spreading of aggregated forms of α-synuclein. Clearance of extracellular α-synuclein released by degenerating neurons may be therefore a key mechanism to control the concentration of α-synuclein in the extracellular space. Several molecular chaperones control misfolded protein accumulation in the extracellular compartment. Among these, clusterin, a glycoprotein associated with Alzheimer's disease, binds α-synuclein aggregated species and is present in Lewy bodies, intraneuronal aggregates mainly composed by fibrillary α-synuclein. In this study, using murine primary astrocytes with clusterin genetic deletion, human-induced pluripotent stem cell (iPSC)-derived astrocytes with clusterin silencing and two animal models relevant for PD we explore how clusterin affects the clearance of α-synuclein aggregates by astrocytes. Our findings showed that astrocytes take up α-synuclein preformed fibrils (pffs) through dynamin-dependent endocytosis and that clusterin levels are modulated in the culture media of cells upon α-synuclein pffs exposure. Specifically, we found that clusterin interacts with α-synuclein pffs in the extracellular compartment and the clusterin/α-synuclein complex can be internalized by astrocytes. Mechanistically, using clusterin knock-out primary astrocytes and clusterin knock-down hiPSC-derived astrocytes we observed that clusterin limits the uptake of α-synuclein pffs by cells. Interestingly, we detected increased levels of clusterin in the adeno-associated virus- and the α-synuclein pffs- injected mouse model, suggesting a crucial role of this chaperone in the pathogenesis of PD. Overall, our observations indicate that clusterin can limit the uptake of extracellular α-synuclein aggregates by astrocytes and, hence, contribute to the spreading of Parkinson pathology.

Androgen deprivation therapy (ADT) is a common treatment for recurrent prostate cancer (PC). However, after a certain period of responsiveness, ADT resistance occurs virtually in all patients and the disease progresses to lethal metastatic castration-resistant prostate cancer (mCRPC). Aberrant expression and function of the epigenetic modifiers EZH2 and BET over activates c-myc, an oncogenic transcription factor critically contributing to mCRPC. In the present work, we tested, for the first time, the combination of an EZH2 inhibitor with a BET inhibitor in metastatic PC cells. The combination outperformed single drugs in inhibiting cell viability, cell proliferation and clonogenic ability, and concomitantly reduced both c-myc and NF-kB expression. Although these promising results will warrant further in vivo validation, they represent the first step to establishing the rationale that the proposed combination might be suitable for mCRPC treatment, by exploiting molecular targets different from androgen receptor.

Green tea catechins (GTCs) are a family of chemically related compounds usually classified as antioxidant molecules. Epidemiological evidences, supported by interventional studies, highlighted a more than promising role for GTCs in human prostate cancer (PCa) chemoprevention. In the last decades, many efforts have been made to gain new insights into the mechanism of action of GTCs. Now it is clear that GTCs' anticancer action can no longer be simplistically limited to their direct antioxidant/pro-oxidant properties. Recent contributions to the advancement of knowledge in this field have shown that GTCs specifically interact with cellular targets, including cell surface receptors, lipid rafts, and endoplasmic reticulum, modulate gene expression through direct effect on transcription factors or indirect epigenetic mechanisms, and interfere with intracellular proteostasis at various levels. Many of the effects observed in vitro are dose and cell context dependent and take place at concentrations that cannot be achieved in vivo. Poor intestinal absorption together with an extensive systemic and enteric metabolism influence GTCs' bioavailability through still poorly understood mechanisms. Recent efforts to develop delivery systems that increase GTCs' overall bioavailability, by means of biopolymeric nanoparticles, represent the main way to translate preclinical results in a real clinical scenario for PCa chemoprevention.

The identification of SERCA (sarco/endoplasmic reticulum calcium ATPase) as a target for modulating gain-of-function NOTCH1 mutations in Notch-dependent cancers has spurred the development of this compound class for cancer therapeutics. Despite the innate toxicity challenge associated with SERCA inhibition, we identified CAD204520, a small molecule with better drug-like properties and reduced off-target Ca2+ toxicity compared with the SERCA inhibitor thapsigargin. In this work, we describe the properties and complex structure of CAD204520 and show that CAD204520 preferentially targets mutated over wild-type NOTCH1 proteins in T cell acute lymphoblastic leukemia (T-ALL) and mantle cell lymphoma (MCL). Uniquely among SERCA inhibitors, CAD204520 suppresses NOTCH1-mutated leukemic cells in a T-ALL xenografted model without causing cardiac toxicity. This study supports the development of SERCA inhibitors for Notch-dependent cancers and extends their application to cases with isolated mutations in the PEST degradation domain of NOTCH1, such as MCL or chronic lymphocytic leukemia (CLL).

Clusterin (CLU) has kept many researchers engaged for a long time since its first discovery and characterization in the attempt to unravel its biological role in mammals. Although there is a general consensus on the fact that CLU is supposed to play important roles in nearly all fundamental biological phenomena and in many human diseases including cancer, after about 10 years of work CLU has been defined as an “enigmatic” protein. This sense of frustration among the researchers is originated by the fact that, despite considerable scientific production concerning CLU, there is still a lack of basic information about the complex regulation of its expression. The CLU gene is a single 9-exon gene expressed at very different levels in almost all major tissues in mammals. The gene produces at least three protein forms with different subcellular localization and diverse biological functions. The molecular mechanism of production of these protein forms remains unclear. The best known is the glycosylated mature form of CLU (sCLU), secreted with very big quantitative differences at different body sites. Hormones and growth factors are the most important regulators of CLU gene expression. Before 2006, it was believed that a unique transcript of about 1.9 kb was originated by transcription of the CLU gene. Now we know that alternative transcriptional initiation, possibly driven by two distinct promoters, may produce at least two distinct CLU mRNA isoforms differing in their unique first exon, named Isoform 1 and Isoform 2. A third transcript, named Isoform 11036, has been recently found as one of the most probable mRNA variants. Approaches like cloning, expression, and functional characterization of the different CLU protein products have generated a critical mass of information teaching us an important lesson about CLU gene expression regulation. Nevertheless, further studies are necessary to better understand the tissue-specific regulation of CLU expression and to identify the specific signals triggering the expression of different/alternative transcript isoforms and protein forms in different cell types at appropriate time.

Abstract The Clusterin (CLU) gene produces different forms of protein products, which vary in their biological properties and distribution within the cell. Both the extra‐ and intracellular CLU forms regulate cell proliferation and apoptosis. Dis‐regulation of CLU expression occurs in many cancer types, including prostate cancer. The role that CLU plays in tumorigenesis is still unclear. We found that CLU over‐expression inhibited cell proliferation and induced apoptosis in prostate cancer cells. Here we show that depletion of CLU affects the growth of PC‐3 prostate cancer cells. Following siRNA targeting all CLU mRNA variants, all protein products quickly disappeared, inducing cell cycle progression and higher expression of specific proliferation markers (i.e., H3 mRNA, PCNA, and cyclins A, B1, and D) as detected by RT‐qPCR and Western blot. Quite surprisingly, we also found that the turnover of CLU protein is very rapid and tightly regulated by ubiquitin–proteasome mediated degradation. Inhibition of protein synthesis by cycloheximide showed that CLU half‐life is less than 2 h. CLU protein products were found poly‐ubiquitinated by co‐immuniprecipitation. Proteasome inhibition by MG132 caused stabilization and accumulation of all CLU protein products, including the nuclear form of CLU (nCLU), and committing cells to caspase‐dependent death. In conclusion, proteasome inhibition may induce prostate cancer cell death through accumulation of nCLU, a potential tumor suppressor factor. J. Cell. Physiol. 219: 314–323, 2009. © 2009 Wiley‐Liss, Inc.

Prostate cancer (CaP) is one of the most relevant causes of cancer death in Western Countries. Although detection of CaP at early curable stage is highly desirable, actual screening methods present limitations and new molecular approaches are needed. Gene expression analysis increases our knowledge about the biology of CaP and may render novel molecular tools, but the identification of accurate biomarkers for reliable molecular diagnosis is a real challenge. We describe here the diagnostic power of a novel 8-genes signature: ornithine decarboxylase (ODC), ornithine decarboxylase antizyme (OAZ), adenosylmethionine decarboxylase (AdoMetDC), spermidine/spermine N(1)-acetyltransferase (SSAT), histone H3 (H3), growth arrest specific gene (GAS1), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and Clusterin (CLU) in tumour detection/classification of human CaP.The 8-gene signature was detected by retrotranscription real-time quantitative PCR (RT-qPCR) in frozen prostate surgical specimens obtained from 41 patients diagnosed with CaP and recommended to undergo radical prostatectomy (RP). No therapy was given to patients at any time before RP. The bio-bank used for the study consisted of 66 specimens: 44 were benign-CaP paired from the same patient. Thirty-five were classified as benign and 31 as CaP after final pathological examination. Only molecular data were used for classification of specimens. The Nearest Neighbour (NN) classifier was used in order to discriminate CaP from benign tissue. Validation of final results was obtained with 10-fold cross-validation procedure. CaP versus benign specimens were discriminated with (80+/-5)% accuracy, (81+/-6)% sensitivity and (78+/-7)% specificity. The method also correctly classified 71% of patients with Gleason score<7 versus > or =7, an important predictor of final outcome.The method showed high sensitivity in a collection of specimens in which a significant portion of the total (13/31, equal to 42%) was considered CaP on the basis of having less than 15% of cancer cells. This result supports the notion of the "cancer field effect", in which transformed cells extend beyond morphologically evident tumour. The molecular diagnosis method here described is objective and less subjected to human error. Although further confirmations are needed, this method poses the potential to enhance conventional diagnosis.

Abstract Melanoma differentiation‐associated gene‐7/interleukin‐24 (mda ‐7/IL‐24), a unique member of the IL‐10 gene family, displays a broad range of antitumor properties including cancer‐specific induction of apoptosis, inhibition of tumor angiogenesis, and modulation of anti‐tumor immune responses. Here, we identify clusterin (CLU) as a MDA‐7/IL‐24 interacting protein in DU‐145 cells and investigate the role of MDA‐7/IL‐24 in regulating CLU expression and mediating the antitumor properties of mda‐ 7/IL‐24 in prostate cancer. Ad. mda‐ 7 decreased expression of soluble CLU (sCLU) and increased expression of nuclear CLU (nCLU). In the initial phase of Ad. mda‐ 7 infection sCLU expression increased and CLU interacted with MDA‐7/IL‐24 producing a cytoprotective effect. Infection of stable clones of DU‐145 prostate cancer cells expressing sCLU with Ad. mda ‐7 resulted in generation of nCLU that correlated with decreased cell viability and increased apoptosis. In the presence of mda ‐7/IL‐24, sCLU‐DU‐145 cells displayed G 2 /M phase arrest followed by apoptosis. Similarly, Ad. mda‐ 7 infection decreased cell migration by altering cytoskeleton in sCLU‐DU‐145 cells. Ad. mda‐ 7‐treated sCLU‐DU‐145 cells displayed a significant reduction in tumor growth in mouse xenograft models and reduced angiogenesis when compared to the vector control group. Tumor tissue lysates demonstrated enhanced nCLU generated from sCLU with increased apoptosis in the presence of MDA‐7/IL‐24. Our findings reveal novel aspects relative to the role of sCLU/nCLU in regulating the anticancer properties of MDA‐7/IL‐24 that may be exploited for developing enhanced therapies for prostate cancer. J. Cell. Physiol. 227: 1805–1813, 2012. © 2011 Wiley Periodicals, Inc.

In the present study, a green tea extract (GTE) was used to enrich minimally processed apples by vacuum impregnation VI in order to obtain a nutritionally fortified product. Apples were impregnated with isotonic sucrose solutions containing 1% GTE and/or 1% ascorbic acid (AA). The impact of GTE enrichment on quality characteristics of the product, stability of the antioxidant compounds and activity during refrigerated storage were assessed. Results showed that a satisfactory impregnation of minimally processed apples was achieved and resulted in a strong increase of the antioxidant compounds content and activity. While other qualitative parameters were only slightly affected, colour of samples was influenced just after the VI treatment with an increase of the yellow/orange colour components but also during storage with a higher degree of browning development. However, the addition of 1% of AA allowed to better preserve colour and antioxidant properties during storage limiting oxidative phenomena. The demand for functional foods characterized by a high nutritional value is constantly increasing. Vacuum impregnation (VI) is a technology that allows the replacement of the gas into the porous food structure with an aqueous solution, promoting compositional changes in short treatment times thus permitting the direct formulation of a porous food matrix, hence representing a promising technology for product innovation for the fruit and vegetable industry. Green tea (GT) is a high source of flavanoids, known to have strong antioxidant properties which has been widely studied both in in-vitro and in-vivo trials. According to the results obtained in this study, the enrichment of apples with GT catechins and AA seemed to be promising in order to obtain a nutritionally fortified fruit product.

Lung cancer is the leading cause of cancer-related mortality. It is categorized into two histological groups that have distinct clinical behaviors, the nonsmall cell lung cancers (NSCLC) and the small cell lung cancer (SCLC). When identified at an early stage, NSCLC is treated by surgical resection. However, patients who undergo surgical resection still have a relative low survival rate, primarily for tumor recurrence. Unfortunately, advances in cytotoxic therapy have reached a plateau and new approaches to treatment are needed together with new and better parameters for more accurate prediction of the outcome and more precise indication of the efficacy of the treatment. Several in vitro studies have examined the role of Clusterin (CLU) in carcinogenesis, lung cancer progression, and response to chemo- and radiotherapy. Studies performed in lung cancer cell lines and animal models showed that CLU is upregulated after exposure to chemo- and radiotherapy. A potential role proposed for the protein is cytoprotective. In vitro, CLU silencing by antisense oligonucleotides (ASO) and small-interfering RNAs (siRNA) directed against CLU mRNA in CLU-rich lung cancer cell lines sensitized cells to chemotherapy and radiotherapy and decreased their metastatic potential. In vivo, a recent work analyzed the prognostic role of CLU in NSCLC, showing that CLU-positive patients with lung cancer had a better overall survival and disease-free survival than those with CLU-negative tumors. These data are contradictory to the promising in vitro results. From the results of these studies we may hypothesize that in early-stage lung cancers CLU represents a positive biomarker correlating with better overall survival. In advanced patients, already treated with chemo- and radiotherapy, the induction of CLU may confer resistance to the treatments. However, many studies are needed to better understand the role of CLU in early-stage and advanced lung cancers with the aim to discriminate patients and specific local conditions that could benefit for a CLU knocking down treatment.

Proteomic studies on anticancer activity of Green Tea Catechins (specifically EGCG) are suggesting a large set of protein targets that may directly interact with EGCG and alter the physiology of diseased cells, including cancer. Of notice, benign cells are usually left untouched. Lipid rafts have been recently recognized as signal processing hubs and suggested to be involved in drug uptake by means of endocytosis. These findings are suggesting new insights on the molecular mechanisms of anticancer drugs action. In the membrane, EGCG is hijacked by the laminin receptor (LamR), a lipid raft protein. Similar to aplidin and edelfosin, EGCG alters membrane domains composition also preventing EGF binding to EGFR, imerization of EGFR and relocation of phosphorylated EGFR to lipid rafts. In vitro studies have recently shown that EGCG also binds both DNA and RNA in GpC-rich regions. This event may importantly affect genes function. Moreover, EGCG was shown to inhibit telomerase, topoisomerase II and DNA methyltransferase 1 (DNMT1), thus ultimately affecting chromatin maintenance and remodeling. But another important alternative pathway besides interaction with specific proteins may play an important role in EGCG action: direct targeting of bioactive membrane platforms, lipid rafts. Structural alteration of the platforms deeply impact (and often inactivates) important pathways involving MAP kinases. The key issue is that, important and specific differences in lipid rafts composition have been found in transformed versus benign cells and apoptotic versus non-apoptotic cells. We suggest here that the anticancer activity of Green Tea Catechins against different kind of cancers may find an explanation in direct targeting of lipid rafts by EGCG.

The role of clusterin (CLU) in prostate tumorigenesis is probably the most highly controversial, with evidence that CLU expression is increased or decreased in different cancer models. For example, some studies showed that CLU expression is increased in advanced stages of prostate cancer and that suppression of CLU expression sensitizes prostate cancer cells to chemotherapeutic drugs killing. In contrast with the hypothesis that CLU is a positive modulator of prostate cancer, we and others have observed that CLU is downregulated during human prostate cancer progression. Accordingly, a meta-analysis of available microarray data shows that CLU mRNA is significantly downregulated in prostate cancer tissue compared to normal prostate in 14 out of 15 independent studies. Recently, it was shown that CLU is silenced by promoter methylation in the murine TRAMP-C2 cell line, as well as in the human prostate cancer cell line LNCaP. Consistently, CLU expression was found to be significantly reduced in untreated and hormone-refractory human prostate carcinomas. This data suggest the importance of epigenetic events in the regulation of CLU in prostate cancer, supporting the idea that prostate cell transformation at early stages requires CLU silencing through chromatin remodeling.

Cold atmospheric plasma (CAP) has shown good potentiality for the decontamination and stabilization of fresh fruit and vegetable products; however, information about its effect on nutritional quality is still scarce. The aim of this research was to evaluate the impact of a form of indirect treatment known as Dielectric Barrier Discharge (DBD) on apple slices—more specifically, the polyphenolic profile and antioxidant activity of fresh-cut Pink Lady apples. Atmospheric plasma was generated using air as feed gas, and directed to apple slices for up to 30 min. The effect of plasma treatment on physico-chemical parameters was mainly observed as a slight acidification of the tissue and reduction of browning after an extended period of exposure. The samples’ phenolic profile was significantly affected after 10 min of treatment, both in quantitative (an approximately 20% increase) and qualitative terms, while with increasing exposure time a progressive decrease of all polyphenol classes was observed. The antioxidant activity, evaluated by different in-vitro methods, followed a similar trend, increasing after 10 min of processing and then decreasing. Results highlighted how plasma exposure promotes a metabolic response of the fresh tissue, and the importance of carefully controlling the exposure time in order to minimize the loss of nutritional properties.

Parkinson’s Disease (PD) is a progressive neurodegenerative disease characterized by the presence of proteinaceous aggregates of αSynuclein (αSyn) in the dopaminergic neurons. Chaperones are key components of the proteostasis network that are able to counteract αSyn’s aggregation, as well as its toxic effects. Clusterin (CLU), a molecular chaperone, was consistently found to interfere with Aβ aggregation in Alzheimer’s Disease (AD). However, its role in PD pathogenesis has yet to be extensively investigated. In this study, we assessed the involvement of CLU in the αSyn aggregation process by using SH-SY5Y cells stably overexpressing αSyn (SH-Syn). First, we showed that αSyn overexpression caused a strong increase in CLU expression without affecting levels of Hsp27, Hsp70, and Hsp90, which are the chaperones widely recognized to counteract αSyn burden. Then, we demonstrated that αSyn aggregation, induced by proteasome inhibition, determines a strong increase of CLU in insoluble aggregates. Remarkably, we revealed that CLU down-regulation results in an increase of αSyn aggregates in SH-Syn without significantly affecting cell viability and the Unfolded Protein Response (UPR). Furthermore, we demonstrated the direct molecular interaction between CLU and αSyn via a co-immunoprecipitation (co-IP) assay. All together, these findings provide incontrovertible evidence that CLU is an important player in the response orchestrated by the cell to cope with αSyn burden.

Biobased composites with peculiar properties offer an attractive route for producing environmentally friendly materials. The reinforcement for poly(butylene adipate-co-terephthalate) (PBAT), based on zein-titanium dioxide (TiO2) complex (ZTC) microparticles, is presented and used to produce composite filaments, successfully 3-dimensionally (3D) printed by fused deposition modeling (FDM). The outcome of ZTC addition, ranging from 5 to 40 wt %, on the thermo-mechanical properties of composite materials was analyzed. Results reveal that storage modulus increased with increasing the ZTC content, leading to a slight increase in the glass transition temperature. The creep compliance varies with the ZTC concentration, denoting a better resistance to deformation under constant stress conditions for composites with higher complex content. Scanning electron microscopy was used to assess the quality of interphase adhesion between PBAT and ZTC, showing good dispersion and distribution of complex microparticles in the polymer matrix. Infrared spectroscopy confirmed the formation of a valid interface due to the formation of hydrogen bonds between filler and polymer matrix. Preliminary tests on the biocompatibility of these materials were also performed, showing no cytotoxic effects on cell viability. Finally, the 3D printability of biobased composites was demonstrated by realizing complex structures with a commercial FDM printer.

The human clusterin (CLU) gene codes for several mRNAs characterized by different sequences at their 5' end. We investigated the expression of two CLU mRNAs, called CLU 1 and CLU 2, in immortalized (PNT1a) and tumorigenic (PC3 and DU145) prostate epithelial cells, as well as in normal fetal fibroblasts (WI38) following the administration of the epigenetic drugs 5-aza-2'-deoxycytidine (AZDC) and trichostatin A (TSA) given either as single or combined treatment (AZDC-TSA). Our experimental evidences show that: a) CLU 1 is the most abundant transcript variant. b) CLU 2 is expressed at a low level in normal fibroblasts and virtually absent in prostate cancer cells. c) CLU 1, and to a greater extent CLU 2 expression, increased by AZDC-TSA treatment in prostate cancer cells. d) Both CLU 1 and CLU 2 encode for secreted CLU. e) P2, a novel promoter that overlaps the CLU 2 Transcription Start Site (TSS), drives CLU 2 expression. f) A CpG island, methylated in prostate cancer cells and not in normal fibroblasts, is responsible for long-term heritable regulation of CLU 1 expression. g) ChIP assay of histone tail modifications at CLU promoters (P1 and P2) shows that treatment of prostate cancer cells with AZDC-TSA causes enrichment of Histone3(Lys9)acetylated (H3K9ac) and reduction of Histone3(Lys27)trimethylated (H3K27me3), inducing active transcription of both CLU variants. In conclusion, we show for the first time that the expression of CLU 2 mRNA is driven by a novel promoter, P2, whose activity responds to epigenetic drugs treatment through changes in histone modifications.

We previously found that human prostate cancer (CaP) progression is accompanied by differential expression of a panel of 8 informative genes, some of which are metabolically related. Gene profiling focused on this 8-gene pack by northern blot analysis in combination with standard clinical information provided reliable prognostic prediction of human CaP. For a better insight into the potential of this 8-gene signature in tumor detection/classification and therapeutic response, we determined, by qPCR, the expression of these informative genes in the TRAMP mice model of CaP progression. The 8-genes signature resulted effective in discriminating, by linear discriminant analysis (LDA), the prostate of wild type mice from transgenic TRAMP mice developing CaP (P < 0.0002). Since it is known that Green Tea Catechin (GTC) administration to TRAMP mice results in a substantial delay of CaP progression in 80% of the animals, while 20% remain unresponsive, we determined the 8-gene signature in the prostates of GTC-sensitive and GTC-resistant mice. LDA discriminated benign tissue from CaP (i.e. wild-type + chemoprevented, GTC-sensitive TRAMP mice, in which CaP progression was delayed, was discriminated from TRAMP mice + GTC-resistant TRAMP mice, in which CaP developed irrespective of GTC administration; P < 0.01). Moreover, GTC-sensitive TRAMP mice bearing CaP were discriminated from GTCs-resistant ones, (P = 0.0001). These results show that qPCR gene profiling, based on the signature of the 8-genes selected by us, could represent an appropriate means for studying the biological behavior of CaP, which may lead to identifying new tools of potential prognostic value, in that a molecular classification for the presence/absence of cancer and for discriminating GTCs-responsive from GTC-resistant CaP is provided.

The possible biological role played by Clusterin (CLU) has been puzzling researchers for a long time since its first discovery and characterization. CLU has been often described as an “enigmatic” gene, a clear indication that too many aspects of this issue have been obscure or difficult to interpret for long. The good news is that this is certainly no longer true. Since the beginning, CLU was believed to play important roles in nearly all most important biological phenomena. The diversity, sometime the contradictions, of its biological action is now likely explained by the existence of different protein products all generated by the same single copy CLU gene. The relatively recent discovery that CLU can be retained inside the cell and targeted to many intracellular sites and organelles, including the nucleus, provided us a very different view from that solely deriving from its possible role in the outer cellular environment. In particular, nuclear localization of CLU (nCLU) was found to trigger cell death in many systems. In this chapter, a critical review of previous work will enable us to reinterpret old data and observations in the attempt to progressively unravelling the CLU “enigma” by considering its localization inside and outside the cell. The final picture would supposedly reconciliate different or alternative hypothesis. Starting with an “historical” approach demonstrating that nCLU was right under our eyes since the beginning, up to the more recent contributions we will describe which stimuli would inhibit secretion and maturation of CLU leading at least one protein product to target the nucleus and kill the cell. A better understanding of this complex issue is not an easy work, considering the thoughtfulness in reviewing the existing literature and the known controversial reports. We hope that the information contained in this article will be useful for the reader to enlighten this field.

In early diabetes, hyperglycemia and the associated metabolic dysregulation promote early changes in the functional properties of cardiomyocytes, progressively leading to the appearance of the diabetic cardiomyopathy phenotype. Recently, the interplay between histone acetyltransferases (HAT) and histone deacetylases (HDAC) has emerged as a crucial factor in the development of cardiac disorders. The present study evaluates whether HDAC inhibition can prevent the development of cardiomyocyte contractile dysfunction induced by a short period of hyperglycemia, with focus on the potential underlying mechanisms. Cell contractility and calcium dynamics were measured in unloaded ventricular myocytes isolated from the heart of control and diabetic rats. Cardiomyocytes were either untreated or exposed to the pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) for 90 min. Then, a fraction of each group of cells was used to evaluate the expression levels of proteins involved in the excitation-contraction coupling, and the cardiomyocyte metabolic activity, ATP content, and reactive oxygen species levels. SAHA treatment was able to counteract the initial functional derangement in cardiomyocytes by reducing cell oxidative damage. These findings suggest that early HDAC inhibition could be a promising adjuvant approach for preventing diabetes-induced cardiomyocyte oxidative damage, which triggers the pro-inflammatory signal cascade, mitochondrial damage, and ventricular dysfunction.

Clusterin expression may change in various human malignancies, including lung cancer. Patients with resectable non-small cell lung cancer (NSCLC), including adenocarcinoma, have a poor prognosis, with a relapse rate of 30-50% within 5 years. Nuclear factor kB (Nf-kB) is an intracellular protein involved in the initiation and progression of several human cancers, including the lung.We investigate the role of clusterin and Nf-kB expression in predicting the prognosis of patients with early-stage surgically resected adenocarcinoma of the lung.The level of clusterin gradually decreased from well-differentiated to poorly differentiated adenocarcinomas. Clusterin expression was significantly higher in patients with low-grade adenocarcinoma, in early-stage disease and in women. Clusterin expression was inversely related to relapse and survival in both univariate and multivariate analyses. Finally, we observed an inverse correlation between Nf-kB and clusterin.Clusterin expression represents an independent prognostic factor in surgically resected lung adenocarcinoma and was proven to be a useful biomarker for fewer relapses and longer survival in patients in the early stage of disease. The inverse correlation between Nf-kB and clusterin expression confirm the previously reported role of clusterin as potent down regulator of Nf-kB.

Genomic studies have identified recurrent somatic alterations in genes involved in DNA methylation and post-translational histone modifications in acute lymphoblastic leukemia (ALL), suggesting new opportunities for therapeutic interventions. In this study, we identified G9a/EHMT2 as a potential target in T-ALL through the intersection of epigenome-centered shRNA and chemical screens. We subsequently validated G9a with low-throughput CRISPR-Cas9-based studies targeting the catalytic G9a SET-domain and the testing of G9a chemical inhibitors in vitro, 3D, and in vivo T-ALL models. Mechanistically we determined that G9a repression promotes lysosomal biogenesis and autophagic degradation associated with the suppression of sestrin2 (SESN2) and inhibition of glycogen synthase kinase-3 (GSK-3), suggesting that in T-ALL glycolytic dependent pathways are at least in part under epigenetic control. Thus, targeting G9a represents a strategy to exhaust the metabolic requirement of T-ALL cells.

Dietary polyphenols from green tea have been shown to possess cardio-protective activities in different experimental models of heart diseases and age-related ventricular dysfunction. The present study was aimed at evaluating whether long term in vivo administration of green tea extracts (GTE), can exert positive effects on the normal heart, with focus on the underlying mechanisms.The study population consisted of 20 male adult Wistar rats. Ten animals were given 40 mL/day tap water solution of GTE (concentration 0.3%) for 4 weeks (GTE group). The same volume of water was administered to the 10 remaining control rats (CTRL). Then, in vivo and ex vivo measurements of cardiac function were performed in the same animal, at the organ (hemodynamics) and cellular (cardiomyocyte mechanical properties and intracellular calcium dynamics) levels. On cardiomyocytes and myocardial tissue samples collected from the same in vivo studied animals, we evaluated: (1) the intracellular content of ATP, (2) the endogenous mitochondrial respiration, (3) the expression levels of the Sarcoplasmic Reticulum Ca2+-dependent ATPase 2a (SERCA2), the Phospholamban (PLB) and the phosphorylated form of PLB, the L-type Ca2+ channel, the Na+-Ca2+ exchanger, and the ryanodine receptor 2.GTE cardiomyocytes exhibited a hyperdynamic contractility compared with CTRL (the rate of shortening and re-lengthening, the fraction of shortening, the amplitude of calcium transient, and the rate of cytosolic calcium removal were significantly increased). A faster isovolumic relaxation was also observed at the organ level. Consistent with functional data, we measured a significant increase in the intracellular ATP content supported by enhanced endogenous mitochondrial respiration in GTE cardiomyocytes, as well as higher values of the ratios phosphorylated-PLB/PLB and SERCA2/PLB.Long-term in vivo administration of GTE improves cell mechanical properties and intracellular calcium dynamics in normal cardiomyocytes, by increasing energy availability and removing the inhibitory effect of PLB on SERCA2.

Prostate cancer (CaP) is a fast-growing health and social problem already representing the second leading cause of cancer-related death among men in Western countries. Lifestyle-related factors and diet are major contributors for CaP promotion. Because of unfavourable prognosis of extra-prostatic CaP, prevention is considered the best approach to fight it at present time. Green Tea Catechins (GTCs) were proven effective at inhibiting cancer growth in several laboratory studies. We recently performed a pilot clinical trial in HG-PIN subjects showing that only 1/30 tumour was diagnosed in subjects treated for 1 year with 600 mg/die GTCs, while 9/30 cancers were found in placebo-treated men. CaP is an elusive disease, whose biological behaviour is difficult to predict. We have recently described and validated a RT-qPCR method based on a 8-genes signature that significantly discriminated benign tissue from CaP in both humans and TRAMP mice spontaneously developing CaP. In the animal model, also GTCs-resistant CaP was significantly discriminated from GTCs-sensitive CaP, i.e. responding to GTCs administration. Preliminary experiments in our laboratory have shown that this method can be successfully applied to a single tissue needle biopsy specimen in humans. The combination of these results may be of particular significance on the field. In fact, GTCs treatment for men at high risk of CaP as first line prevention therapy in combination with the 8-genes signature profiling in tissue needle biopsies for real time monitoring of patient's response might importantly change, in the near future, the clinical managing of this highly diffuse malignancy.

Clusterin (CLU) is a stress-activated glycoprotein, whose expression is altered both in inflammation and cancer. Previously, we showed that abrogation of CLU expression in cancer-prone mice (TRAMP) results in the enhancement of tumor spreading and homing, concomitant with an enhanced expression of NF- κ B. In the present paper, we carried out an extensive experimental work by utilizing microarray gene expression data, as well as in vitro and in vivo models of prostate cancer (PCa). Our results demonstrated that (i) CLU expression is significantly downregulated in human PCa and inversely correlates with the expression of p65 in metastases; (ii) CLU overexpression in PCa cells reduces the Ser 536 phosphorylation of p65, inhibits NF- κ B nuclear translocation, and reduces the transcription of matrix metalloproteinase-9 and metalloproteinase-2 (MMP-9 and MMP-2). Conversely, CLU silencing promotes NF- κ B activation and transcriptional upregulation of MMP-9; and (iii) expression and activity of MMP-2 and MMP-9 are increased in CLU −/− mice (CLUKO) and in TRAMP/CLUKO mice in comparison to their relative Clu +/+ littermates. Taken together, our data support the hypothesis that CLU downregulation, an early and relevant event in PCa onset, may inhibit NF- κ B activation and limit the execution of a transcriptional program that favor the disease progression towards a metastatic stage.

Considering that Aurora kinase inhibitors are currently under clinical investigation in hematologic cancers, the identification of molecular events that limit the response to such agents is essential for enhancing clinical outcomes. Here, we discover a NF-κB-inducing kinase (NIK)-c-Abl-STAT3 signaling-centered feedback loop that restrains the efficacy of Aurora inhibitors in multiple myeloma. Mechanistically, we demonstrate that Aurora inhibition promotes NIK protein stabilization via downregulation of its negative regulator TRAF2. Accumulated NIK converts c-Abl tyrosine kinase from a nuclear proapoptotic into a cytoplasmic antiapoptotic effector by inducing its phosphorylation at Thr735, Tyr245 and Tyr412 residues, and, by entering into a trimeric complex formation with c-Abl and STAT3, increases both the transcriptional activity of STAT3 and expression of the antiapoptotic STAT3 target genes PIM1 and PIM2. This consequently promotes cell survival and limits the response to Aurora inhibition. The functional disruption of any of the components of the trimer NIK-c-Abl-STAT3 or the PIM survival kinases consistently enhances the responsiveness of myeloma cells to Aurora inhibitors. Importantly, concurrent inhibition of NIK or c-Abl disrupts Aurora inhibitor-induced feedback activation of STAT3 and sensitizes myeloma cells to Aurora inhibitors, implicating a combined inhibition of Aurora and NIK or c-Abl kinases as potential therapies for multiple myeloma. Accordingly, pharmacological inhibition of c-Abl together with Aurora resulted in substantial cell death and tumor regression in vivo. The findings reveal an important functional interaction between NIK, Abl and Aurora kinases, and identify the NIK, c-Abl and PIM survival kinases as potential pharmacological targets for improving the efficacy of Aurora inhibitors in myeloma.

We recently showed that the long-term in vivo administration of green tea catechin extract (GTE) resulted in hyperdynamic cardiomyocyte contractility. The present study investigates the mechanisms underlying GTE action in comparison to its major component, epigallocatechin-3-gallate (EGCG), given at the equivalent amount that would be in the entirety of GTE. Twenty-six male Wistar rats were given 40 mL/day of a tap water solution with either standardized GTE or pure EGCG for 4 weeks. Cardiomyocytes were then isolated for the study. Cellular bioenergetics was found to be significantly improved in both GTE- and EGCG-fed rats compared to that in controls as shown by measuring the maximal mitochondrial respiration rate and the cellular ATP level. Notably, the improvement of mitochondrial function was associated with increased levels of oxidative phosphorylation complexes, whereas the cellular mitochondrial mass was unchanged. However, only the GTE supplement improved cardiomyocyte mechanics and intracellular calcium dynamics, by lowering the expression of total phospholamban (PLB), which led to an increase of both the phosphorylated-PLB/PLB and the sarco-endoplasmic reticulum calcium ATPase/PLB ratios. Our findings suggest that GTE might be a valuable adjuvant tool for counteracting the occurrence and/or the progression of cardiomyopathies in which mitochondrial dysfunction and alteration of intracellular calcium dynamics constitute early pathogenic factors.

The biological function of Clusterin has been puzzling researchers for a long time since its first discovery and characterization in the early 80's. CLU plays important roles nearly in all most important biological phenomena including cell proliferation and apoptosis, as well as in many diseases including cancer. Now we know that the CLU gene encodes at least three protein forms with different sub-cellular localization and diverse biological functions. The molecular mechanism of production of these protein isoforms remains unclear. Recent data show that many of the previous hypotheses based on preliminary observations are no longer true. For instance, while alternative splicing of CLU mRNA has never been confirmed, the complex transcriptional regulation of CLU gene is now recognized to produce two distinct transcripts resulting from two independent transcriptional start sites. CLU expression was found deregulated in many type of tumours, including prostate cancer. Considering that prostate cancer is one of the major threats in veterans' life, as well as one of the main causes of cancer related death in western countries, we will also specifically address whether targeting CLU might pave the way for a novel therapeutic intervention against prostate cancer.

Clusterin (CLU) is an ATP-independent small heat shock protein-like chaperone, which functions both intra- and extra-cellularly. Consequently, it has been functionally involved in several physiological (including aging), as well as in pathological conditions and most age-related diseases, e.g., cancer, neurodegeneration, and metabolic syndrome. To address CLU function at an in vivo model we established CLU transgenic (Tg) mice bearing ubiquitous or pancreas-targeted CLU overexpression (OE). Our downstream analyses in established Tg lines showed that ubiquitous or pancreas-targeted CLU OE in mice affected antioxidant, proteostatic and metabolic pathways. Targeted OE of CLU in the pancreas, which also resulted in CLU upregulation in the liver likely via systemic effects, increased basal glucose levels in the circulation and exacerbated diabetic phenotypes. Furthermore, by establishing a syngeneic melanoma mouse tumor model we found that ubiquitous CLU OE suppressed melanoma cells growth, indicating a likely tumor suppressor function in early phases of tumorigenesis. Our observations provide in vivo evidence corroborating the notion that CLU is a potential modulator of metabolic and/or proteostatic pathways playing an important role in diabetes and tumorigenesis.

In [1], the incorrect slides were presented in Figure 6. The corrected figure appears below. This erratum corrects the error.

Green Tea Catechins (GTCs) are a family of chemically related compounds usually classified as antioxidant molecules. Epidemiological evidences, supported by interventional studies, highlighted a more than promising role for GTCs in human Prostate Cancer (PCa) chemoprevention.In the last decades many efforts have been made to gain new insights into the mechanism of action of GTCs. Now it is clear that GTCs anticancer action can no longer be simplistically limited to their direct antioxidant/pro-oxidant properties. Recent contributions to the advancement of knowledge in this field have shown that GTCs specifically interact with cellular targets including, cell surface receptors, lipid rafts and endoplasmic reticulum, modulate gene expression through direct effect on transcription factors or indirect epigenetic mechanisms, interfere with intracellular proteostasis at various levels. Many of the effects observed in vitro are dose and cell context dependent and take place at concentration that cannot be achieved in vivo.Poor intestinal absorption together with an extensive systemic and enteric metabolism influence GTCs bioavailability through still poor understood mechanisms. Recent efforts to develop delivery systems that increase GTCs overall bioavailability, by mean of biopolymeric nanoparticles, represent the main way to translate preclinical results in a real clinical scenario for PCa chemoprevention.

It's known that a magnesium (Mg)-deficient diet is associated with an increased risk of osteoporosis. The aim of this work is to investigate, by a histological approach, the effects of a Mg-deprived diet on the bone of 8-weeks-old C57BL/6J male mice. Treated and control mice were supplied with a Mg-deprived or normal diet for 8 weeks, respectively. Body weight, serum Mg concentration, expression of kidney magnesiotropic genes, and histomorphometry on L5 vertebrae, femurs, and tibiae were evaluated. Body weight gain and serum Mg concentration were significantly reduced, while a trend toward increase was found in gene expression in mice receiving the Mg-deficient diet, suggesting the onset of an adaptive response to Mg depletion. Histomorphometric parameters on the amount of trabecular and cortical bone, number of osteoclasts, and thickness of the growth plate in femoral distal and tibial proximal metaphyses did not differ between groups; these findings partially differ from most data present in the literature showing that animals fed a Mg-deprived diet develop bone loss and may be only in part explained by differences among the experimental protocols. However, the unexpected findings we recorded on bones could be attributed to genetic differences that may have developed after multiple generations of inbreeding.

Androgen deprivation therapy (ADT) is a common treatment for recurrent prostate cancer (PC). However, after a certain period of responsiveness, ADT resistance occurs virtually in all patients and the disease progresses to lethal metastatic castration-resistant prostate cancer (mCRPC). Aberrant expression and function of the epigenetic modifiers EZH2 and BET over activates c-myc, an oncogenic transcription factor critically contributing to mCRPC. In the present work, we tested, for the first time, the combination of an EZH2 inhibitor with a BET inhibitor in metastatic PC cells. The combination outperformed single drugs in inhibiting cell viability, cell proliferation and clonogenic ability, and concomitantly reduced both c-myc and NF-kB expression. Although these promising results will warrant further in vivo validation, they represent the first step to establishing the rationale that the proposed combination might be suitable for mCRPC treatment, by exploiting molecular targets different from androgen receptor.

Morbidity and mortality from preventable, noncommunicable chronic disease (NCD) threatens the health of our populations and our economies. The accumulation of vast amounts of scientific knowledge has done little to change this. New and innovative thinking is essential to foster new creative approaches that leverage and integrate evidence through the support of big data, technology, and design thinking. The purpose of this paper is to summarize the results of a consensus meeting on NCD prevention sponsored by the International Olympic Committee (IOC) in April 2013. Within the context of advocacy for multifaceted systems change, the IOC's focus is to create solutions that gain traction within health care systems. The group of participants attending the meeting achieved consensus on a strategy for the prevention and management of chronic disease that includes the following: 1. Focus on behavioral change as the core component of all clinical programs for the prevention and management of chronic disease. 2. Establish actual centers to design, implement, study, and improve preventive programs for chronic disease. 3. Use human-centered design (HCD) in the creation of prevention programs with an inclination to action, rapid prototyping and multiple iterations. 4. Extend the knowledge and skills of Sports and Exercise Medicine (SEM) professionals to build new programs for the prevention and treatment of chronic disease focused on physical activity, diet, and lifestyle. 5. Mobilize resources and leverage networks to scale and distribute programs of prevention. True innovation lies in the ability to align thinking around these core strategies to ensure successful implementation of NCD prevention and management programs within health care. The IOC and SEM community are in an ideal position to lead this disruptive change. The outcome of the consensus meeting was the creation of the IOC Non-Communicable Diseases ad hoc Working Group charged with the responsibility of moving this agenda forward.

Abstract Abstract 1318 Introduction. Diffuse large B-cell lymphoma (DLBCL) is the most frequently aggressive Non-Hodgkin's lymphoma (NHL). DLBCL first line therapy consists of R-CHOP regimen, which combines classical chemotherapeutical drugs and corticosteroids with Rituximab. Rituximab is a chimeric anti-CD20 monoclonal antibody which targets the CD20 surface antigen expressed on both normal and malignant B cells. Rituximab triggers antibody- and complement-dependent cytotoxicity, growth inhibition and apoptosis. Rituximab inhibits the expression of the anti-apoptotic proteins Bcl-2/Bcl-XL, down-modulating different survival pathways. Clusterin (CLU) is an ubiquitary protein involved in many physiological and pathological processes such as apoptosis and cancer. CLU has different transcriptional isoforms, which share exons from 2 to 9 but have a unique exon 1 and at least two protein forms: The cytoplasmatic precursor of about 60 kDa is secreted outside the cell after post-traslational modifications (sCLU), whereas a truncated protein (50–55 kDa) is produced only in particular conditions and localizes in the nucleus (nCLU). CLU expression is disregulated in almost all cancers and its role has been deeply investigated in solid tumors. Only limited information is currently available about its function in lymphomas. Methods. Toledo, a DLBCL-derived cell line and normal B lymphocytes were chosen as experimental models. B lymphocytes were purified and immuno-magnetically sorted from peripheral blood of 4 healthy volunteers. Toledo and B lymphocytes were treated with 10 or 100 μg/ml Rituximab or doxorubicin (from 0.05 to 1 μM) for 48 hours. Phenotype analysis was performed by flow cytometry; cell viability was assessed by Trypan blue exclusion; apoptosis was evaluated by Annexin V/propidium iodide. CLU expression and localization were evaluated by real time PCR, Western Blot (WB) and immunocytochemistry (ICC). Primers for the real time PCR were design in order to distinguish the unique exon 1 of each CLU transcriptional isoforms or exons common to all variants (CLU3-4). Results. Rituximab treatment doesn't significantly inhibit Toledo proliferation and does not induce apoptosis despite the fact that these cells are CD20+. This suggests that Toledo are resistant to Rituximab. As expected, doxorubicin causes a marked growth inhibition by necrosis and apoptosis. Toledo cells have a very low basal level of the 60 kDa CLU protein form. Both Rituximab and doxorubicin up-regulate the 60 kDa form with 1 μM doxorubicin causing the most dramatic increase. The analysis of transcriptional isoforms by real-time PCR shows that CLU mRNA is almost undetectable in Toledo. CLU transcriptional variant 2 (CLU2) and regions common to all CLU transcripts (CLU3-4) are significantly up-regulated only after 1 μM doxorubicin but not after Rituximab. Fluorescence microscopy clearly shows a cytoplasmic localization of CLU under normal growth conditions. Interestingly, the treatment with both Rituximab and doxorubicin causes the appearance of a nuclear staining. This fact might be consistent with the onset of a process of cell death. 10 and 100 μg/ml Rituximab inhibits B lymphocytes cell survival in a dose-dependent manner (39.7% and 44% respectively after 48 hours). Interestingly, B lymphocytes clearly express high basal levels of the 60 kDa CLU and produce an additional form of about 55 kDa only after Rituximab but not after doxorubicin. Furthermore Rituximab-treated B lymphocytes display a Bcl-2 protein decrease while Toledo cells show high expression of Bcl-2 that doesn't seem to change in response to Rituximab. Conclusions. Toledo are resistant to Rituximab up to 100 μg/ml and express very low levels of CLU under normal growth conditions. 60 kDa CLU is partially up-regulated and Bcl-2 expression is not significantly affected by Rituximab treatment. Doxorubicin strongly up-regulates the 60 kDa CLU isoform. At the transcriptional level CLU2 shows the same expression profile of the 60 kDa protein form. On the contrary, Rituximab changes the CLU profile of responsive B lymphocytes in that leads to the appearance of a 55 kDa form that is not visible after doxorubicin treatment. Rituximab also causes a reduction in the expression of the antiapoptotic protein Bcl-2. The biological role of CLU during the response of peripheral B lymphocytes to Rituximab treatment is yet to be determined and is currently being investigated. Disclosures: No relevant conflicts of interest to declare.

Microtubules are one of the three components of the cell cytoskeleton. They are hollow wires, with a diameter of about 25 nm, formed by 13 laterally associated protofilaments composed by dimers of - and β-tubulin. Microtubules are dynamic structures which constantly undergo constant modifications by shrinking and elongating in a phenomenon called treadmilling. Microtubules intervene in various fundamental aspects of the cell's biology. They contribute to determine the shape of a cell, play a role in the cell movement and in the intracellular transport of organelles during motion and mitotic chromo-some segregation. Despite the relevance of the processes mediated by microtubules most studies on the effects of ionizing radiations (IR) focus their attention on the damages delivered to DNA. In this paper we attempt to assess the effects borne by IRs to the microtubules network as a biological target. In this study we irradiated Hs 578Bst cells (a no-cancer, no-immortalized, human breast epithelial cell line) with a 8Gy single dose of either X-rays or protons. After the irradiated cells fixation the microtubules were imaged by means of Stochastic Optical Reconstruction Microscopy (STORM) to characterize the network disruption. In our results MT fibers integrity appears to not have been significantly affected at the administered dose of protons and X-rays, nonetheless we observed differences in the MT network distribution and fiber curvatures

Prostate cancer (CaP) is now recognized as one of the major cancer killers in aged men living in Western countries. Therapeutic options for advanced CaP are unsatisfactory. Little is known about the molecular mechanisms leading to progression. Targeting pre-neoplastic lesions could be a more rewarding strategy. There is overwhelming evidence that catechins extracted from green tea (GTE) possess anti-cancer activity in vitro . We showed that GTE administration was effective at inhibiting progression of CaP using clinical evidence in patients bearing isolated pre-neoplastic lesions (high grade prostate intraepithelial neoplasia, HGPIN). Improvement was stable two years after suspension of treatment. Molecular data point to a central role played by Clusterin (a pro-apoptotic tumor-suppressor) and MCM7 (a proliferation marker), both specifically regulated by GTE, as seen by validated DNA microarray. Long term administration of GTE has been found to be safe in humans. Therefore, treatment with GTE has to be considered the best option for managing initial CaP.

Clinical progression of Prostate Cancer (CaP) is variable: some tumours are indolent, others&#x0D; rapidly progress. Because, diagnosis is usually in elderly men, CaP is an ideal target for&#x0D; chemoprevention. We showed that Green Tea Catechins (GTC) possess anti-tumour activity,&#x0D; suggesting that it might be beneficial in the early stages of cell transformation. But an experimental&#x0D; confirmation in a real clinical setting was needed. High-Grade Prostatic Intraepithelial Neoplasia&#x0D; (HGPIN) is a pre-invasive stage of CaP for which no treatment options are available until CaP is&#x0D; diagnosed.

Reactive oxygen species (ROS) cause cell damages through protein oxidation leading to pathological processes like cardiovascular disease neurological degeneration and cancer (1). Antioxidants molecules, due to their ability to scavenge and neutralize free radicals, might play a pivotal role in the prevention of these disease. Interestingly, besides neutralizing ROS, it’s now proved that some promising antioxidant molecules, including green tea polyphenols (2), induce the expression of genes involved in the cellular protection against oxidative stress (3). Regulation of these detoxifying genes expression by dietary chemopreventive compounds is mediated by the electrophile-responsive element (EpRE) (4). EpRE is a cis-acting regulatory sequence, involved in the coordinated transcriptional activation of genes associated with phase II biotransformation. For this reason the study of the capacity of anti-oxidants to modulate the EpRE gene battery may increase our knowledge in the regulation of cellular physiology and in mechanism of prevention of cancer. To this extent we constructed a new expression vector, cloning the minimal promoter of NADPH quinone oxydoreductase-1 (NADPH(Q)1), comprising the EpRE element, upstream the Zoanthus sp, a gene codifyng a green fluorescent protein, in a promoterless, commercially available, expression vector (pZSGreen1-1). When transiently transfected in the cells this expression vector acts as a molecular probe, reporting the state of activation of NADP(Q)1, in living cells in response to antioxidant substances. Preliminary results indicates that this new molecular probe could be employed for the rapid screening of novel chemopreventive molecules acting through the activation of phase II detoxifying enzymes.

Background: Several studies support the development of NOTCH1 inhibitors for targeted cancer therapy. This idea is most compelling for T‐cell acute lymphoblastic leukemia/lymphoma (T‐ALL) where activating mutations of NOTCH1 are present in 55–60% of the cases, and cancer dependency has been well established. With the identification of S arco/ E ndoplasmic R eticulum C alcium A TPase as a modulator of gain‐of‐function NOTCH1 mutations, the development of SERCA inhibitors represent a unique opportunity for Notch dependent tumors. Aims: Because SERCA plays a critical role in Ca 2+ in cardiac physiology, inhibition of this pump may increase the risk of heart failure limiting the development of this class of compounds for cancer therapeutics. In this work, we describe CAD204520, a new selective SERCA inhibitor with a tolerable toxicity profile and NOTCH1 on‐target anti leukemia effect both in vitro and in vivo . Methods: From a small molecule screening of 191,000 compounds for inhibition of the fungal S. cerevisiae H + ‐ATPase Pma1, 407 hits were counter screened for their ability to target Na + , K + ‐ATPase and the Ca 2+ ‐ATPase proteins. CAD204520 displayed greater selectivity toward human SERCA and emerged as a lead candidate for the development of novel anti‐ NOTCH1 agents. Because altered Ca 2+ levels may cause heart failure we investigated the effect of CAD204520 on rat cardiomyocyte and performed pharmacokinetics studies in CD1 mice. Finally, we assessed the effects of CAD204520 in a xenografted T‐ALL leukemia model. Results: We previously described that thapsigargin mediated SERCA inhibition affects the proliferation of NOTCH1 mutated T‐ALL compared to wild types. CAD204520 retains this therapeutic index in a panel of lymphoid malignancies carrying activating mutations in the NOTCH1 heterodimerization domain (HD, such as T‐ALL) and/or deletions in the degradation domain (PEST, such as mantle cell lymphoma (MCL)). In addition, CAD204520 induces apoptosis and a G1/G0 arrest both in cell line and primary T‐ALL samples. Similar to thapsigargin, CAD204520 treatment decreased the levels of the activated form of NOTCH1, ICN1, while it increased full‐length NOTCH1. The NOTCH1 targets, MYC , DTX1, were consequently repressed as measured by qRT‐PCR. In contrast, no effect at protein level was observed in T‐ALL wild type NOTCH1. To test for the effect of CAD204520 on cardiac tissue, we performed toxicity studies in isolated rat cardiomyocytes and demonstrated that compared to thapsigargin, CAD204520 minimally alters cell mechanical performance (∼25%) suggesting that the heart will likely tolerate CAD204520 modulation in vivo . Consistently in CD1 mice exposed (30 mg/Kg, BID for 21 days) no adverse clinical symptoms were found. Based on the chemical structure, we hypothesized that CAD204520 locks SERCA in a binding site different to that of thapsigargin. Interestingly, thapsigargin and CAD204520 co‐treatment resulted in synergistic anti‐proliferative effect in T‐ALL. This result suggests that the two molecules inhibit SERCA simultaneously but in different pockets explaining for the variation of Ca 2+ dependent toxicities seen in our studies. Finally, to assess the in vivo efficacy of CAD204520 we established a T‐ALL derived xenografts from SKW‐3/KE‐37 human T‐ALL cell and demonstrated that drug's treatment reduced circulating human T‐ALL cells. Summary/Conclusion: In conclusion, this study supports the development of tolerated SERCA inhibitors for Notch‐dependent cancers and extends its application to cases with PEST mutations such as MCL or chronic lymphocytic leukemia.

Abstract Given its oncogenic role in human cancers, Notch1 signaling has garnered increased attention as a therapeutic target. Several Notch modulators, including γ-secretase inhibitors, have shown therapeutic efficacy in preclinical tumor models. However, despite this promise, few of these candidates have shown clinical benefit in patients, in part due to tissue-dependent on-target toxicities from the repression of both mutant and wild type Notch proteins. The discovery of the P-type ATPase Sarco/Endoplasmic Reticulum Ca2+-ATPase (SERCA) as a modulator of Notch transport and activity suggested a new approach to treat T-cell Acute Lymphoblastic Leukemia (T-ALL) an aggressive cancer dependent on aberrant Notch1 activation. In fact, thapsigargin mediated SERCA inhibitions had a stronger effect on the most common type of activating NOTCH1 mutants compared to wild type. However, the consequence of thapsigargin binding to SERCA is a rapid disruption of Ca2+ homeostasis that might cause cardiac toxicity in human, suggesting the need to identify inhibitors with better drug-like properties and reduced off-target toxicity. From a small molecule screening of 191,000 P-type ATPase modulators for inhibition of the yeast S. cerevisiae H+-ATPase Pma1, 407 hits were counter screened for their ability to target Na+/K+-ATPase and the Ca2+-ATPase proteins. CAD204520 displayed ~25 and ~79 fold greater selectivity toward human SERCA compared to Na+/K+and H+-ATPase and emerged as a candidate for the development of novel anti-NOTCH1 agents. We demonstrated that CAD204520 impairs the proliferation of a panel of lymphoid malignancies carrying activating mutations in the NOTCH1 heterodimerization domain (HD, such as T-ALL) and/or deletions in the degradation domain (PEST, such as mantle cell lymphoma (MCL)). Similar to thapsigargin, CAD204520 causes a defect in NOTCH1 trafficking that results in an accumulation of full-length NOTCH1 and in a depletion of the activated form: ICN1. Consequently, the NOTCH1 targets, MYC, DTX1, were repressed as measured by qRT-PCR. In contrast, no effect at protein level was observed in T-ALL wild type NOTCH1. To assess the in vivo efficacy of CAD204520 we established xenografts from the SKW-3/KE-37 T-ALL cell line and demonstrated that drug’s treatment reduced circulating and tissue infiltrating human leukemia cells without causing heart-related off-target effects. Consistently preclinical toxicity studies in rat cardiomyocytes showed that compared to thapsigargin, CAD204520 minimally alters cell cardiac mechanical performance (~25%) suggesting that the heart will likely tolerate CAD204520 modulation in vivo. In fact, in CD1 mice exposed to CAD204520 (30 mg/Kg, BID for 21 days) no adverse clinical symptoms were found. A tempting hypothesis to explain for the variation of Ca2+ dependent toxicities seen in our studies is that thapsigargin and CAD204520 inhibit SERCA in distinct pockets. Consistently, docking poses showed that CAD204520 binds SERCA in the trans-membrane helices M1, M3 and M4 while thapsigargin in the M3, M5 and M7 groove. Consequently, thapsigargin and CAD204520 co-treatment resulted in synergistic anti-proliferative effect in T-ALL. In conclusion, this study supports the development of tolerated SERCA inhibitors for Notch dependent cancers and extends its application to cases with mutations in the PEST degradation domain such as MCL or chronic lymphocytic leukemia. Citation Format: Marchesini Matteo, Andrea Gherli, Anne-Marie Lund Winter, Samuel Kitara, Anna Montanaro, Chiara Rompietti, Claudia Sorrentino, Donatella Stilli, Federica Rizzi, Luca Pagliaro, Kimberly Stegmaier, William Dalby-Brown, Giovanni Roti. Selective blockade of oncogenic NOTCH1 with the new SERCA inhibitor CAD204520 in T-cell acute lymphoblastic leukemia [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B113. doi:10.1158/1535-7163.TARG-19-B113

Given its oncogenic role in human cancers, Notch1 signaling has garnered increased attention as a therapeutic target. Several Notch modulators, including γ-secretase inhibitors, have shown therapeutic efficacy in preclinical tumor models. However, despite this promise, few of these candidates have shown clinical benefit in patients, in part due to tissue-dependent on-target toxicities from the repression of both mutant and wild type Notch proteins. The discovery of the P-type ATPase Sarco/Endoplasmic Reticulum Ca2+-ATPase (SERCA) as a modulator of oncogenic NOTCH1 suggested a new approach to treat T-cell Acute Lymphoblastic Leukemia (T-ALL). In fact, thapsigargin mediated SERCA inhibition had a stronger effect on the most common type of activating NOTCH1 mutants compared to wild type. However, the consequence of thapsigargin binding to SERCA is a rapid increase in cytosolic Ca2+ and a depletion of Ca2+ stored in the endoplasmic reticulum (ER). This shift of Ca2+ efflux might cause cardiac toxicity in human, suggesting the need to identify inhibitors with better drug-like properties and reduced off-target toxicity. From a small molecule screening of 191,000 P-type ATPase modulators CAD204520 displayed ~25 and ~79-fold greater selectivity toward human SERCA compared to Na+/K+ and H+-ATPase respectively and emerged as a candidate for the development of novel anti-NOTCH1 agents. In a series of in vitro studies, we showed that CAD204520 impairs the proliferation of a panel of lymphoid malignancies carrying activating mutations in the NOTCH1 heterodimerization domain (HD, such as T-ALL) and/or deletions in the degradation domain (PEST, such as mantle cell lymphoma (MCL)). Importantly, T-ALL lymphoblasts from patients carrying NOTCH1 mutations were more sensitive to CAD204520 suppression compared with normal lymphocytes or ALL wild type NOTCH1. Similar to thapsigargin, CAD204520 causes a defect in NOTCH1 trafficking that results in an accumulation of full-length NOTCH1 and in a depletion of the activated form: ICN1. Consequently, the NOTCH1 targets, MYC and DTX1, were repressed as measured by qRT-PCR. In contrast, we observed no effect at protein level in T-ALL wild type NOTCH1 suggesting that clinically relevant NOTCH1 mutations including PEST deletions are more sensitive to CAD204250 treatment than wild type proteins. The next question is whether CAD204520 activity has limitations in vivo due to Ca2+ shifts. Thus we analysed cardiac mechanics in isolated rat cardiomyocytes and showed that compared to thapsigargin, CAD204520 minimally alters myocardial performance (~25%) suggesting that the heart will likely tolerate CAD204520 modulation in vivo. This effect may be caused to differences between Ca2+ mediated ER stress upon thapsigargin or CAD204520 treatment. To test this hypothesis, we treated T-ALL cell lines at equimolar doses of CAD204520 and thapsigargin. We showed that, despite a similar effect on NOTCH1 protein levels, CAD204520 does not induce the unfolded protein response (UPR) pathway as measured by the phosphorylation of eIF2α or the induction of the ER chaperone BIP. This result suggests that CAD204520 retains ATPase activity, responsible for Notch inhibition, decoupled from the Ca2+ transport activity responsible for the off-target effects. A tempting hypothesis to explain these differences is that thapsigargin and CAD204520 inhibit SERCA in distinct pockets. Consistently, docking poses showed that CAD204520 binds SERCA in the trans-membrane helices M1, M3 and M4 while thapsigargin in the M3, M5 and M7 groove. Consequently, thapsigargin and CAD204520 co-treatment resulted in synergistic anti-proliferative effect in T-ALL. To test CAD204520 in vivo we studied its effect in CD1 mice and established a maximum tolerated dose. Mice exposed to CAD204520 for 21 days at 30 mg/Kg BID showed no decrease in body weight and, importantly no effect on cardiac hemodynamic. Finally, to assess the in vivo efficacy of CAD204520 we xenografted SKW-3/KE-37 T-ALL cell line in L-2Rγ null mice and demonstrated that drug's treatment reduced circulating and tissue infiltrating human leukemia T-ALL cells without causing heart-related off target effects. In conclusion, this study supports the development of tolerated SERCA inhibitors for Notch dependent cancers and extends its application to cases with mutations in the PEST degradation domain such as MCL or chronic lymphocytic leukemia. Disclosures Lund Winter: CaDo Biotechnology IvS: Employment. Stegmaier:Novartis: Research Funding; Rigel Pharmaceuticals: Consultancy. Dalby-Brown:CaDo Biotechnology IvS: Employment.

CS15-03 Green Tea Catechins (GTCs) were proven effective at inhibiting cancer growth in several laboratory studies and different in vitro and in vivo experimental models. In addition, pioneer work has already demonstrated feasibility and safety of GTCs administration in healthy human volunteers. Beyond all the possible molecular hypothesis on GTCs action in human cells, and also considering that results obtained by epidemiological studies focused on a lower incidence of prostate cancer (CaP) in the Asian countries (where green tea is consumed regularly) as compared to Western populations are still controversial, a clear cut indication that a beneficial effect could also be obtained in a selected population of patients at high risk of CaP progression was timely needed. Prostate cancer (CaP) has been already ranked as the second leading cause of cancer-related death among men in Western countries, representing a major and fast-growing health and social problem. Lifestyle-related factors, and particularly diet, are considered to be the major contributors for CaP promotion. Because of unfavourable prognosis of high grade organ confined and extra-prostatic CaP, prevention may be the best approach to fight it at present time. The fact that CaP onset and progression takes considerable time to occur can be considered as an important opportunity for prevention studies. In fact, an effective protocol for treating pre-malignant lesions in high risk men, if successful, would result in important benefits for patients and substantial saving of social and health costs. Recent studies have shown that about 30% of men bearing high grade PIN (HG-PIN) lesions would develop CaP within one year as assessed by repeated biopsy. Since at present no treatment is given to these patients until CaP is diagnosed, we decided to fill this therapeutic void, in the hope that working with this clinical model would quickly address whether GTCs administration would be effective or not at preventing CaP progression. Therefore, we performed a pilot clinical trial for assessing the efficacy of GTCs administration for chemoprevention of CaP in HG-PIN volunteers. The cohort of patients included 60 HG-PIN volunteers that were enrolled after making them aware of the study and obtaining their signature on an informed consent form. The study was designed as double-blind and two-arms, placebo-controlled. Oral daily treatment was accomplished by giving three capsules containing 200 mg each of GTCs (total: 600 mg/day) and checking for compliance. After 1 year of treatment, only 1 tumor was diagnosed among the 30 GTCs-treated men (incidence: about 3%), while 9 cancers were found among the 30 placebo-treated men (incidence: 30%). Total PSA in GTCs-treated men was constantly lower with respect to placebo-treated ones, although the difference between the two arms never reached statistical significance. As a secondary observation, GTCs administration also affected IPSS (International Prostate Symptom Score) and QoL (Quality of Life) scores in men with coexistent BPH. Both scores clearly improved, reaching statistical significance in the case of IPSS and suggesting that GTCs administration might also be beneficial for treating BPH symptoms. No significant side or adverse effects were documented. To our knowledge, this is the first clinical study showing that GTCs are safe and very effective for treating pre-malignant lesions before CaP develops. Our finding suggests a new scenery in which the incidence of this disease could be greatly reduced by simply making GTCs available to the elders or high-risk men. This approach may result in a tremendous social and clinical impact, especially in the Western countries, although it appears rather obvious that the chemopreventive effect exerted by GTCs on human CaP development must be quickly confirmed by a larger study. Also because of these clinical results, the quest for a rationale explanation of the molecular alterations linked to GTCs action is important, but still wide open. Molecular or gene signatures are often used to model clinically relevant information (e.g. prognosis, survival time, sensitivity to drugs) as a function of gene expression data obtained at transcriptional level, but instead of individual genes, all signature components are used as predictors. By conventional Northern blot analysis, we previously identified a 8-genes signature that provided reliable prognostic prediction of human CaP. The prediction was further enhanced when molecular data were used in combination with standard clinical information. By this finding, we have shown that molecular characterization of CaP progression is indeed possible. We then proceeded further by determining the 8-genes signature in the well-characterized, pre-clinical TRAMP mouse model of CaP progression by RT-qPCR. The TRAMP model is a well known autochthonous transgenic animal model that was developed as an important tool for understanding the progression of human CaP, in that it displays in situ and invasive carcinoma of the prostate mimicking the whole spectrum of human CaP progression from PIN to androgen independent disease. Recent studies, including ours, have demonstrated that administration of GTCs is effective at inhibiting CaP development in these animals. Thus, we have validated our 8-genes signature in the TRAMP model, and particularly the down-regulation of CLU gene expression (which is a component of the signature) at mRNA and protein level during CaP onset and progression. On the contrary, upon GTCs administration, CLU expression is maintained at high levels in the prostate of TRAMP mice in which CaP development was strongly inhibited. In these mice, induction of pro-apoptotic nuclear CLU was detected at early stage of treatment. Linear discriminant analysis showed that the 8-genes signature was highly effective at discriminating the prostate of wild type from transgenic TRAMP mice spontaneously developing CaP (p

The discovery of the P-type ATPase sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) as a modulator of oncogenic NOTCH1 suggests an innovative approach for treating T-cell acute lymphoblastic leukemia (T-ALL). In fact, SERCA inhibition preferentially affects the maturation and activity of the most common class of oncogenic NOTCH1 mutants. The aim of this study was to identify inhibitors with better drug-like properties and reduced off-target toxicity for SERCA inhibition. We developed a novel oral SERCA inhibitor, CAD204520, through medicinal chemistry optimization and crystal structure-oriented analysis describing its anti-leukemic effects in vitro and in vivo to support a SERCA-based therapeutic modality in T-ALL. From a 191,000 small-molecule screening targeting P-type ATPase, we identified CAD204520 as a selective inhibitor of human SERCA compared to Na+/K+ and H+-ATPase. Crystal structure analysis showed that CAD204520 binds the transmembrane interface of SERCA between helices M1, M2, M3, and M4. CAD204520 minimally alters Ca2+ shift and fails to trigger Ca2+-dependent programs, such as the unfolded protein response. We demonstrated that CAD204520 impairs the proliferation of T-ALL cell lines carrying activating mutations of NOTCH1. Importantly, clinical samples carrying NOTCH1 mutations, including PEST deletions, were more sensitive to CAD204520 compared to normal lymphocytes or wild-type NOTCH1 ALL cells. Mechanistically, CAD204520 treatment reduces the levels of the activated form of NOTCH1 as a consequence of a defect in NOTCH1 trafficking. Next, we demonstrated that somatic hotspot mutations in the SERCA2 ATPase pocket that confer resistance to known SERCA modulators (e.g., thapsigargin) do not interfere with CAD204520 binding, suggesting that the activity of CAD204520 will be unlikely affected by recurrent resistant genetic variants. Finally, we showed that 30 mg/kg BID for 21 days is well-tolerated in vivo in CD1 mice without causing loss of weight and cardiac toxicity. In a xenograft SKW-3/KE-37 T-ALL model, CAD204520 reduces circulating and tissue-infiltrating human leukemia T-ALL cells with no heart-related or gastrointestinal toxicities. In conclusion, we present CAD204520 as a novel orally bioavailable SERCA inhibitor with tolerable off-target toxicity in NOTCH1-dependent tumors. This work provides a foundation for the further development of novel drugs targeting Notch-dependent hematopoietic malignancies. The discovery of the P-type ATPase sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) as a modulator of oncogenic NOTCH1 suggests an innovative approach for treating T-cell acute lymphoblastic leukemia (T-ALL). In fact, SERCA inhibition preferentially affects the maturation and activity of the most common class of oncogenic NOTCH1 mutants. The aim of this study was to identify inhibitors with better drug-like properties and reduced off-target toxicity for SERCA inhibition. We developed a novel oral SERCA inhibitor, CAD204520, through medicinal chemistry optimization and crystal structure-oriented analysis describing its anti-leukemic effects in vitro and in vivo to support a SERCA-based therapeutic modality in T-ALL. From a 191,000 small-molecule screening targeting P-type ATPase, we identified CAD204520 as a selective inhibitor of human SERCA compared to Na+/K+ and H+-ATPase. Crystal structure analysis showed that CAD204520 binds the transmembrane interface of SERCA between helices M1, M2, M3, and M4. CAD204520 minimally alters Ca2+ shift and fails to trigger Ca2+-dependent programs, such as the unfolded protein response. We demonstrated that CAD204520 impairs the proliferation of T-ALL cell lines carrying activating mutations of NOTCH1. Importantly, clinical samples carrying NOTCH1 mutations, including PEST deletions, were more sensitive to CAD204520 compared to normal lymphocytes or wild-type NOTCH1 ALL cells. Mechanistically, CAD204520 treatment reduces the levels of the activated form of NOTCH1 as a consequence of a defect in NOTCH1 trafficking. Next, we demonstrated that somatic hotspot mutations in the SERCA2 ATPase pocket that confer resistance to known SERCA modulators (e.g., thapsigargin) do not interfere with CAD204520 binding, suggesting that the activity of CAD204520 will be unlikely affected by recurrent resistant genetic variants. Finally, we showed that 30 mg/kg BID for 21 days is well-tolerated in vivo in CD1 mice without causing loss of weight and cardiac toxicity. In a xenograft SKW-3/KE-37 T-ALL model, CAD204520 reduces circulating and tissue-infiltrating human leukemia T-ALL cells with no heart-related or gastrointestinal toxicities. In conclusion, we present CAD204520 as a novel orally bioavailable SERCA inhibitor with tolerable off-target toxicity in NOTCH1-dependent tumors. This work provides a foundation for the further development of novel drugs targeting Notch-dependent hematopoietic malignancies.

We report the characterization of rotaxanes based on a carbazole–benzophenone thermally activated delayed fluorescence luminophore. We find that the mechanical bond leads to an improvement in key photophysical properties of the emitter, notably an increase in photoluminescence quantum yield and a decrease in the energy difference between singlet and triplet states, as well as fine tuning of the emission wavelength, a feat that is difficult to achieve when using covalently bound substituents. Computational simulations, supported by X-ray crystallography, suggest that this tuning of properties occurs due to weak interactions between the axle and the macrocycle that are enforced by the mechanical bond. This work highlights the benefits of using the mechanical bond to refine existing luminophores, providing a new avenue for emitter optimization that can ultimately increase the performance of these molecules.