Claudia Cerella

It is well admitted that the link between chronic inflammation and cancer involves cytokines and mediators of inflammatory pathways, which act during the different steps of tumorigenesis. The cyclooxygenases (COXs) are a family of enzymes, which catalyze the rate-limiting step of prostaglandin biosynthesis. This family contains three members: ubiquitously expressed COX-1, which is involved in homeostasis; the inducible COX-2 isoform, which is upregulated during both inflammation and cancer; and COX-3, expressed in brain and spinal cord, whose functions remain to be elucidated. COX-2 was described to modulate cell proliferation and apoptosis mainly in solid tumors, that is, colorectal, breast, and prostate cancers, and, more recently, in hematological malignancies. These findings prompt us to analyze here the effects of a combination of COX-2 inhibitors together with different clinically used therapeutic strategies in order to further improve the efficiency of future anticancer treatments. COX-2 modulation is a promising field investigated by many research groups.

Ca(2+) is an important second messenger participating in many cellular activities; when physicochemical insults deregulate its delicate homeostasis, it acts as an intrinsic stressor, producing/increasing cell damage. Damage elicits both repair and death responses; intriguingly, in those responses Ca(2+) also participates as second messenger. This delineates a dual role for Ca(2+) in cell stress, making difficult to separate the different and multiple mechanisms required for Ca(2+)-mediated control of cell survival and apoptosis. Here we attempt to disentangle the two scenarios, examining on the one side, the events implicated in deregulated Ca(2+) toxicity and the mechanisms through which this elicits reparative or death pathways; on the other, reviewing the role of Ca(2+) as a messenger in the transduction of these same signaling events.

Cardiac glycosides (CGs) are natural compounds widely used in the treatment of several cardiac conditions and more recently have been recognized as potential antitumor compounds. They are known to be ligands for Na/K-ATPase, which is a promising drug target in cancer. More recently, in addition to their antitumor effects, it has been suggested that CGs activate tumor-specific immune responses. This review summarizes the anticancer aspects of CGs as new strategies for immunotherapy and drug repositioning (new horizons for old players), and the possible new targets for CGs in cancer cells.

Cardiac glycosides (CGs) are approved for the treatment of cardiovascular alterations and their known cellular target is the alpha subunit of the sodium (Na+)/potassium (K+)-ATPase (NKA). Pharmacologically, they represent a well-known generation of drugs for treating cardiovascular problems, thus allowing the investigation of potential dose-dependent side effects. Interestingly, since the end of the 1960s, epidemiological studies have indicated that anti-cancer effects were associated with the regular use of these compounds. Since then, a large body of evidence has been accumulated on the in vitro and in vivo effects of CGs in various experimental models, thus confirming their selective action on cancer cell proliferation and viability. CGs have the potential for targeted therapeutic applications. Many of the anti-cancer activities of these compounds have been linked to the inhibition of their primary target, the NKA. A number of studies have shown a correlation between the overexpression of specific alpha subunits in cancerous versus non-cancerous cells and cancer cell responsiveness. Other findings have provided evidence of the on-target nature of the ascribed anti-cancer effects. More recently, studies have indicated additional intracellular targets for these agents, whose modulation might be, at least in some instances, unrelated to NKA targeting. These include endosomal trafficking of both NKA and Src kinase, downregulation of pro-survival Mcl-1 and Bcl-xL pro-survival proteins, and immunogenic cell death induction, among others. This research update summarizes the current knowledge about CGs as new, targeted anti-cancer agents, alone or in combination with other chemotherapeutic compounds.

Bax is a cytosolic protein, which in response to stressing apoptotic stimuli, is activated and translocates to mitochondria, thus initiating the intrinsic apoptotic pathway. In spite of many studies and the importance of the issue, the molecular mechanisms that trigger Bax translocation are still obscure. We show by computer simulation that the two cysteine residues of Bax may form disulfide bridges, producing conformational changes that favor Bax translocation. Oxidative, nonapoptogenic treatments produce an up-shift of Bax migration compatible with homodimerization, which is reverted by reducing agents; this is accompanied by translocation to mitochondria. Dimers also appear in pure cytosolic fractions of cell lysates treated with H2O2, showing that Bax dimerization may take place in the cytosol. Bax dimer-enriched lysates support Bax translocation to isolated mitochondria much more efficiently than untreated lysates, indicating that dimerization may promote Bax translocation. The absence of apoptosis in our system allows the demonstration that Bax moves because of oxidations, even in the absence of apoptosis. This provides the first evidence that Bax dimerization and translocation respond to oxidative stimuli, suggesting a novel role for Bax as a sensor of redox imbalance.

We have recently shown that melatonin antagonizes damage-induced apoptosis by interaction with the MT-1/MT-2 plasma membrane receptors. Here, we show that melatonin interferes with the intrinsic pathway of apoptosis at the mitochondrial level. In response to an apoptogenic stimulus, melatonin allows mitochondrial translocation of the pro-apoptotic protein Bax, but it impairs its activation/dimerization The downstream apoptotic events, i.e. cytochrome c release, caspase 9 and 3 activation and nuclear vesiculation are equally impaired, indicating that melatonin interferes with Bax activation within mitochondria. Interestingly, we found that melatonin induces a strong re-localization of Bcl-2, the main Bax antagonist to mitochondria, suggesting that Bax activation may in fact be antagonized by Bcl-2 at the mitochondrial level. Indeed, we inhibit the melatonin anti-apoptotic effect (i) by silencing Bcl-2 with small interfering RNAs, or with small-molecular inhibitors targeted at the BH3 binding pocket in Bcl-2 (i.e. the one interacting with Bax); and (ii) by inhibiting melatonin-induced Bcl-2 mitochondrial re-localization with the MT1/MT2 receptor antagonist luzindole. This evidence provides a mechanism that may explain how melatonin through interaction with the MT1/MT2 receptors, elicits a pathway that interferes with the Bcl-2 family, thus modulating the cell life/death balance.

Selected 7-nitro-2,1,3-benzoxadiazole derivatives have been recently found very efficient inhibitors of glutathione S-transferase (GST) P1-1, an enzyme which displays antiapoptotic activity and is also involved in the cellular resistance to anticancer drugs. These new inhibitors are not tripeptide glutathione-peptidomimetic molecules and display lipophylic properties suitable for crossing the plasma membrane. In the present work, we show the strong cytotoxic activity of these compounds in the following four different cell lines: K562 (human myeloid leukemia), HepG2 (human hepatic carcinoma), CCRF-CEM (human T-lymphoblastic leukemia), and GLC-4 (human small cell lung carcinoma). The LC50 values are in the micromolar/submicromolar range and are close to the IC50 values obtained with GSTP1-1, suggesting that the target of these molecules inside the cell is indeed this enzyme. The cytotoxic mechanism of 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol, the most effective GSTP1-1 inhibitor, has been carefully investigated in leukemic CCRF-CEM and K562 cell lines. Western blot and immunoprecipitation analyzes have shown that 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol promotes in both cell lines the dissociation of the GSTP1-1 in a complex with c-jun NH2-terminal kinase (JNK). This process triggers a reactive oxygen species (ROS)-independent activation of the JNK-mediated pathway that results in a typical process of apoptosis. Besides this main pathway, in K562 cells, a ROS-mediated apoptosis partially occurs (about 30%) which involves the p38MAPK signal transduction pathway. The low concentration of this new compound needed to trigger cytotoxic effects on tumor cells and the low toxicity on mice indicate that the new 7-nitro-2,1,3-benzoxadiazole derivatives are promising anticancer agents.

Cyclooxygenase (COX)-2 is a pro-inflammatory immediate early response protein, chronically up-regulated in many pathological conditions. In autoimmune diseases, it is responsible for degenerative effects whereas in cancer, it correlates with poor prognosis. A constitutive expression of COX-2 is triggered since the earliest steps of carcinogenesis. Consequently, strategies aimed at inhibiting COX-2 enzymatic activity have been clinically applied for the treatment of autoimmune disorders; in addition, the same approaches are currently investigated for anti-cancer purposes. However, COX-2 protein inhibitors (i.e., NSAIDs and COXIBs) are not amenable to prolonged administration since they may cause severe side effects, and efforts are underway to identify alternative approaches for chemoprevention/therapy. COX-2 expression is a multi-step process, highly regulated at transcriptional and post-transcriptional levels. Defects in the modulation of one or both of these steps may be found in pathological conditions. Targeting COX-2 expression may therefore represent a promising strategy, by which the same preventive and therapeutic benefits may be gained while avoiding the severe side effects of COX-2 enzymatic inhibition. Naturally occurring compounds derived from plants/organisms represent a huge source of biologically active molecules, that remains largely unexplored. Derived from plants/organisms used in traditional forms of medicine or as dietary supplements, these compounds have been experimentally investigated for their anti-inflammatory and anti-cancer potential. In this review, we will analyze how natural compounds may modulate the multistep regulation of COX-2 gene expression and discuss their potential as a new generation of COX-2 targeting agents alternative to the synthetic COX-2 inhibitors.

Azadirachta indica (neem tree) is used in traditional Indian medicine for its pharmacological properties including cancer prevention and treatment. Here, we studied a neem extract's anti-inflammatory potential via the nuclear factor-κB (NF-κB) signaling pathway, linked to cancer, inflammation, and apoptosis. Cultured human leukemia cells were treated with a methanolic neem leaf extract with or without tumor necrosis factor (TNF)-α stimulation. Inhibition of NF-κB activity was demonstrated by luciferase assay and electrophoretic mobility shift assay (EMSA). Inhibition of viability by neem extracts was assessed by luminescent assays. Western blot analysis allowed assessing the inhibitory effect of the neem extract on TNF-α-induced degradation of inhibitor of κB (IκB) and nuclear translocation of the NF-κB p50/p65 heterodimer. Inhibition of IκB kinase (IKK) activity was shown as well as the effect of neem extract on the induction of apoptotic cell death mechanisms by nuclear fragmentation analysis and flow cytometry analysis. In conclusion, our data provide evidence for a strong effect of the neem extract on pro-inflammatory cell signaling and apoptotic cell death mechanisms, contributing to a better understanding of the mechanisms triggered by Azadirachta indica.

In this study, we investigated the biological effects of heteronemin, a marine sesterterpene isolated from the sponge Hyrtios sp. on chronic myelogenous leukemia cells. To gain further insight into the molecular mechanisms triggered by this compound, we initially performed DNA microarray profiling and determined which genes respond to heteronemin stimulation in TNFα-treated cells and which genes display an interaction effect between heteronemin and TNFα. Within the differentially regulated genes, we found that heteronemin was affecting cellular processes including cell cycle, apoptosis, mitogen-activated protein kinases (MAPKs) pathway and the nuclear factor κB (NF-κB) signaling cascade. We confirmed in silico experiments regarding NF-κB inhibition by reporter gene analysis, electrophoretic mobility shift analysis and I-κB degradation. In order to assess the underlying molecular mechanisms, we determined that heteronemin inhibits both trypsin and chymotrypsin-like proteasome activity at an IC50 of 0.4 μM. Concomitant to the inhibition of the NF-κB pathway, we also observed a reduction in cellular viability. Heteronemin induces apoptosis as shown by annexin V-FITC/propidium iodide-staining, nuclear morphology analysis, pro-caspase-3, -8 and -9 and poly(ADP-ribose) polymerase (PARP) cleavage as well as truncation of Bid. Altogether, results show that this compound has potential as anti-inflammatory and anti-cancer agent.

Cardiac glycosides (CGs) are compounds used for the treatment of cardiac failure which also display strong anti-cancer activity inducing impairment of cell proliferation or activation of cell death whether by apoptosis or autophagy. Despite the fact that the sodium/potassium (Na+/K+)-ATPase is a well-known target of CGs, its involvement in the anti-cancer effect of these compounds remains to be validated. Importantly, related intracellular sensor(s)/transducer(s) needs further characterization. We will discuss here key mechanisms activated by CGs including their ability to modulate the cell cycle and to regulate the expression of anti-apoptotic Bcl-2 family members, two so far marginally investigated events.

Cardiac steroids are used to treat various diseases including congestive heart failure and cancer. The aim of this study was to investigate the anti-leukemic activity of UNBS1450, a hemi-synthetic cardenolide belonging to the cardiac steroid glycoside family. Here, we report that, at low nanomolar concentrations, UNBS1450 induces apoptotic cell death. Subsequently, we have investigated the molecular mechanisms leading to apoptosis activation. Our results show that UNBS1450 inhibits NF-κB transactivation and triggers apoptosis by cleavage of pro-caspases 8, 9 and 3/7, by decreasing expression of anti-apoptotic Mcl-1 and by recruitment of pro-apoptotic Bak and Bax protein eventually resulting in cell death.

Cancer continues to be a major public health problem despite the efforts that have been made in the search for novel drugs and treatments. The current sources sought for the discovery of new molecules are plants, animals and minerals. During the past decade, the search for anticancer agents of marine origin to fight chemo-resistance has increased greatly. Each year, several novel anticancer molecules are isolated from marine organisms and represent a renewed hope for cancer therapy. The study of structure-function relationships has allowed synthesis of analogues with increased efficacy and less toxicity. In this report, we aim to review 42 compounds of marine origin and their derivatives that were published in 2011 as promising anticancer compounds.

Cell death is an important physiological regulator during development, tissue homeostasis and stress response but it is also a protective tumor suppressive mechanism. Tumor cells almost universally acquire the ability to evade cell death pathways that in normal cells act as a protective mechanism to remove damaged cells. As a result, a population of death-resistant cells with accumulating genetic and epigenetic abnormalities contributes to malignant transformation. Any alteration of the homeostatic balance between survival and death is therefore a critical factor in carcinogenesis. Several forms of cell death exist and cross talk among them is emerging; however, we still miss many molecular details. It becomes essential to revisit the role of each type of cell death to understand interconnections existing between different cell death pathways as well as the network of their mediators to eventually develop new effective strategies to kill cancer cells. More specifically, new therapies based on compounds selectively triggering apoptosis, necrosis or autophagy recently became both appealing and challenging. Despite the rather clear classification of the different cell death modalities according to morphological criteria and the attempt to describe them with distinct signaling pathways, the reality reveals a complex interplay between apoptosis, regulated necrosis and autophagy involving a heterogeneous mix of molecular mediators. Nature, presenting an almost endless plenitude of bioactive scaffolds, can efficiently contribute compounds that allow deciphering the intricate pathways of cell death pathways and thus eventually contribute to selectively target cancer-type specific pathways in an attempt to personalize cancer patient treatment depending on cancer death pathway specificities. The aim of this review is to provide first an overview of molecular cell death specificities and to highlight how compounds of natural origins, with or without hemisynthetic modifications, target unique thanatotic molecular constellations.

Garlic-derived organosulfur compounds including diallyl polysulfides are well known for various health-beneficial properties and recent reports even point to a potential role of diallyl polysulfides as chemopreventive and therapeutic agents in cancer treatment due to their selective antiproliferative effects. In this respect, diallyl tri- and tetrasulfide are reported as strong inducers of an early mitotic arrest and subsequent apoptosis, but the underlying molecular mechanisms and the link between these two events are not yet fully elucidated. Our data revealed that diallyl tetrasulfide acts independently of reactive oxygen species and tubulin represents one of its major cellular targets. Tubulin depolymerization prevents the formation of normal spindle microtubules, thereby leading to G2/M arrest. Here, we provide evidence that c-jun N-terminal kinase, which is activated early in response to diallyl tetrasulfide treatment, mediates multisite phosphorylation and subsequent proteolysis of the anti-apoptotic protein B-cell lymphoma 2. As the latter event occurs concomitantly with the onset of apoptosis and the chemical c-jun N-terminal kinase inhibitor SP600125 not only prevented B-cell lymphoma 2 phosphorylation and proteolysis but also apoptosis following diallyl tetrasulfide treatment, we suggest that these c-jun N-terminal kinase-mediated modulations of B-cell lymphoma 2 represent the missing link connecting early microtubule inactivation to the induction of apoptosis.

Natural compounds are the fundament of pharmacological treatments and more than 50% of all anticancer drugs are of natural origins or at least derived from scaffolds present in Nature. Over the last 25 years, molecular mechanisms triggered by natural anticancer compounds were investigated. Emerging research showed that molecules of natural origins are useful for both preventive and therapeutic purposes by targeting essential hallmarks and enabling characteristics described by Hanahan and Weinberg. Moreover, natural compounds were able to change the differentiation status of selected cell types. One of the earliest response of cells treated by pharmacologically active compounds is the change of its morphology leading to ultra-structural perturbations: changes in membrane composition, cytoskeleton integrity, alterations of the endoplasmic reticulum, mitochondria and of the nucleus lead to formation of morphological alterations that are a characteristic of both compound and cancer type preceding cell death. Apoptosis and autophagy were traditionally considered as the most prominent cell death or cell death-related mechanisms. By now multiple other cell death modalities were described and most likely involved in response to chemotherapeutic treatment. It can be hypothesized that especially necrosis-related phenotypes triggered by various treatments or evolving from apoptotic or autophagic mechanisms, provide a more efficient therapeutic outcome depending on cancer type and genetic phenotype of the patient. In fact, the recent discovery of multiple regulated forms of necrosis and the initial elucidation of the corresponding cell signaling pathways appear nowadays as important tools to clarify the immunogenic potential of non-canonical forms of cell death induction.

A limiting factor in the therapeutic outcome of children with high-risk neuroblastoma is the intrinsic and acquired resistance to common chemotherapeutic treatments. Here we investigated the molecular mechanisms by which the hemisynthetic cardiac glycoside UNBS1450 overcomes this limitation and induces differential cell death modalities in both neuroblastic and stromal neuroblastoma through stimulation of a cell-type-specific autophagic response eventually leading to apoptosis or necroptosis. In neuroblastic SH-SY5Y cells, we observed a time-dependent production of reactive oxygen species that affects lysosomal integrity inducing lysosome-associated membrane protein 2 degradation and cathepsin B and L activation. Subsequent mitochondrial membrane depolarization and accumulation of mitochondria in phagophores occurred after 8h of UNBS1450 treatment. Results were confirmed by mitochondrial mass analysis, electron microscopy and co-localization of mitochondria with GFP-LC3, suggesting the impaired clearance of damaged mitochondria. Thus, a stress-induced defective autophagic flux and the subsequent lack of clearance of damaged mitochondria sensitized SH-SY5Y cells to UNBS1450-induced apoptosis. Inhibition of autophagy with small inhibitory RNAs against ATG5, ATG7 and Beclin-1 protected SH-SY5Y cells against the cytotoxic effect of UNBS1450 by inhibiting apoptosis. In contrast, autophagy progression towards the catabolic state was observed in stromal SK-N-AS cells: here reactive oxygen species (ROS) generation remained undetectable preserving intact lysosomes and engulfing damaged mitochondria after UNBS1450 treatment. Moreover, autophagy inhibition determined sensitization of SK-N-AS to apoptosis. We identified efficient mitophagy as the key mechanism leading to failure of activation of the apoptotic pathway that increased resistance of SK-N-AS to UNBS1450, triggering rather necroptosis at higher doses. Altogether we characterize here the differential modulation of ROS and mitophagy as a main determinant of neuroblastoma resistance with potential relevance for personalized anticancer therapeutic approaches.

Abstract: Among the non‐neurological functions of melatonin, much attention is being directed to the ability of melatonin to modulate the immune system, whose cells possess melatonin‐specific receptors and biosynthetic enzymes. Melatonin controls cell behaviour by eliciting specific signal transduction actions after its interaction with plasma membrane receptors (MT 1 , MT 2 ); additionally, melatonin potently neutralizes free radicals. Melatonin regulates immune cell loss by antagonizing apoptosis. A major unsolved question is whether this is due to receptor involvement, or to radical scavenging considering that apoptosis is often dependent on oxidative alterations. Here, we provide evidence that on U937 monocytic cells, apoptosis is antagonized by melatonin by receptor interaction rather than by radical scavenging. First, melatonin and a set of synthetic analogues prevented apoptosis in a manner that is proportional to their affinity for plasma membrane receptors but not to their antioxidant ability. Secondly, melatonin's antiapoptotic effect required key signal transduction events including G protein, phospholipase C and Ca 2+ influx and, more important, it is sensitive to the specific melatonin receptor antagonist luzindole.

Organic sulfur compounds (OSCs) derived from plants, fungi or bacteria can serve as chemopreventive and/or chemotherapeutic agents and have been attracting medical and research interest as a promising source for novel anti-cancer agents. Garlic, which has long been used as a medicinal plant in different cultures due to its multiple beneficial effects, contains a consistent number of OSCs, the majority of which are currently under investigation for their biological activities. Experimental animal and laboratory studies have shown strong evidence that garlic OSCs may affect cancer cells by promoting early mitotic arrest followed by apoptotic cell death without affecting healthy cells. The ability of OSCs to hinder cancer cell proliferation and viability tightly correlates with the length of the sulfur chain. Current data support a mechanism of mitotic arrest of cancer cells due to the alteration of the microtubule network, possibly as a consequence of the high reactivity of sulfur atoms against the thiol groups of different cellular macromolecules controlling crucial regulatory functions. Taken together, these findings indicate a promising potential for the use of garlic-derived sulfur compounds in chemoprevention and chemotherapy.

Cardiac glycosides (CGs), prescribed to treat cardiovascular alterations, display potent anti-cancer activities. Despite their well-established target, the sodium/potassium (Na(+)/K(+))-ATPase, downstream mechanisms remain poorly elucidated. UNBS1450 is a hemi-synthetic cardenolide derived from 2″-oxovorusharin extracted from the plant Calotropis procera, which is effective against various cancer cell types with an excellent differential toxicity. By comparing adherent and non-adherent cancer cell types, we validated Mcl-1 as a general and early target of UNBS1450. A panel of CGs including cardenolides ouabain, digitoxin and digoxin as well as bufadienolides cinobufagin and proscillaridin A allowed us to generalize our findings. Our results show that Mcl-1, but not Bcl-xL nor Bcl-2, is rapidly downregulated prior to induction of apoptosis. From a mechanistic point of view, we exclude an effect on transcription and demonstrate involvement of a pathway affecting protein stability and requiring the proteasome in the early CG-induced Mcl-1 downregulation, without the involvement of caspases or the BH3-only protein NOXA. Strategies aiming at preventing UNBS1450-induced Mcl-1 downregulation by overexpression of a mutated, non-ubiquitinable form of the protein or the use of the proteasome inhibitor MG132 inhibited the compound's ability to induce apoptosis. Altogether our results point at Mcl-1 as a ubiquitous factor, downregulated by CGs, whose modulation is essential to achieve cell death.

Even though altered metabolism is an "old" physiological mechanism, only recently its targeting became a therapeutically interesting strategy and by now it is considered an emerging hallmark of cancer. Nevertheless, a very poor number of compounds are under investigation as potential modulators of cell metabolism. Candidate agents should display selectivity of action towards cancer cells without side effects. This ideal favorable profile would perfectly overlap the requisites of new anticancer therapies and chemopreventive strategies as well. Nature represents a still largely unexplored source of bioactive molecules with a therapeutic potential. Many of these compounds have already been characterized for their multiple anticancer activities. Many of them are absorbed with the diet and therefore possess a known profile in terms of tolerability and bioavailability compared to newly synthetized chemical compounds. The discovery of important cross-talks between mediators of the most therapeutically targeted aberrancies in cancer (i.e., cell proliferation, survival, and migration) and the metabolic machinery allows to predict the possibility that many anticancer activities ascribed to a number of natural compounds may be due, in part, to their ability of modulating metabolic pathways. In this review, we attempt an overview of what is currently known about the potential of natural compounds as modulators of cancer cell metabolism.

In vitro and in vivo studies reported the anti-cancer potential of organosulfur compounds (OSCs) as they trigger biological effects leading to cell cycle arrest with accumulation of cells in G2/M, alteration of the microtubular network, modulation of Bcl-2 family protein expression patterns and changes of the redox status. Despite these well-described effects, no OSC derivative is yet undergoing clinical trials even though their chemistry is well understood as OSCs act as hydrogen sulfide (H2S) donors. H2S is a biological mediator, synthesized through cysteine degradation and modulates vasodilation, cytoprotection, inflammation and angiogenesis. It is well accepted that H2S plays a biphasic pharmacological role: the inhibition of endogenous synthesis of H2S and paradoxically also the use of H2S donors to increase H2S concentration, induce both anti-cancer effects leading therefore to controversial discussions. Altogether, the role of H2S in the anti-cancer action of OSCs remains poorly understood. In this review, we hypothesize that OSCs act through H2S signaling pathways in cancer cells, and that a clearer understanding of the mechanism of action of H2S in OSC-mediated anti-cancer activity is required for further application of these compounds in translational medicine.

Cancer and aging are two similar processes representing the final outcome of timedependent accumulation of various irreversible dysfunctions, mainly caused by stress-induced DNA and cellular damages. Apoptosis and senescence are two types of cellular response to damages that are altered in both cancer and aging, albeit through different mechanisms. Carcinogenesis is associated with a progressive reduction in the ability of the cells to trigger apoptosis and senescence. In contrast, in aging tissues, there is an increased accumulation of senescent cells, and the nature of apoptosis deregulation varies depending on the tissue. Thus, the prevailing model suggests that apoptosis and cellular senescence function as two essential tumor-suppressor mechanisms, ensuring the health of the individual during early and reproductive stages of life, but become detrimental and promote aging later in life. The recent discovery that various anticancer agents, including canonical inducers of apoptosis, act also as inducers of cellular senescence indicates that pro-senescence strategies may have applications in cancer prevention therapy. Therefore, dissection of the mechanisms mediating the delicate balance between apoptosis and cellular senescence will be beneficial in the therapeutic exploitation of both processes in the development of future anticancer and anti-aging strategies, including minimizing the side effects of such strategies. Here, we provide an overview of the roles of apoptosis and cellular senescence in cancer and aging. Keywords: Aging, apoptosis, cancer, cellular senescence, stress, therapy.

Melatonin is a modified tryptophan with potent biological activity, exerted by stimulation of specific plasma membrane (MT1/MT2) receptors, by lower affinity intracellular enzymatic targets (quinone reductase, calmodulin), or through its strong anti-oxidant ability. Scattered studies also report a perplexing pro-oxidant activity, showing that melatonin is able to stimulate production of intracellular reactive oxygen species (ROS). Here we show that on U937 human monocytes melatonin promotes intracellular ROS in a fast (< 1 min) and transient (up to 5–6 h) way. Melatonin equally elicits its pro-radical effect on a set of normal or tumor leukocytes; intriguingly, ROS production does not lead to oxidative stress, as shown by absence of protein carbonylation, maintenance of free thiols, preservation of viability and regular proliferation rate. ROS production is independent from MT1/MT2 receptor interaction, since a) requires micromolar (as opposed to nanomolar) doses of melatonin; b) is not contrasted by the specific MT1/MT2 antagonist luzindole; c) is not mimicked by a set of MT1/MT2 high affinity melatonin analogues. Instead, chlorpromazine, the calmodulin inhibitor shown to prevent melatonin–calmodulin interaction, also prevents melatonin pro-radical effect, suggesting that the low affinity binding to calmodulin (in the micromolar range) may promote ROS production.

We have shown that melatonin exerts a prooxidant activity in U937 cells, a tumor human promonocytic cell line. (1) Here we show that melatonin induces a strong canonical activation of NF-kappaB, inducing IkappaBalpha degradation and the consequential nuclear translocation of p50/p65 subunits. The timing of NF-kappaB activation overlaps with the timing of reactive oxygen species (ROS) production due to melatonin. Overexpression of dominant-negative IkappaB, which prevents a possible NF-kappaB activation, transformed melatonin in a proapoptotic molecule. These data indicate for the first time that melatonin can trigger NF-kappaB activation and might suggest a possible role for ROS induced by melatonin. Results indicate a possible involvement in the survival pathway of melatonin-generated ROS as secondary messengers.

We recently demonstrated that quercetin, a flavonoid naturally present in food and beverages belonging to the large class of phytochemicals, was able to sensitise leukaemic cells isolated from patients with chronic lymphocytic leukaemia (CLL) when associated with recombinant tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) or anti-CD95. We also showed that quercetin potentiated the effect of fludarabine on resistant B cells from CLL patients. Resistance to therapy in CLL depends on the expression and activity of anti-apoptotic proteins of the Bcl-2 family. Among these, myeloid cell leukaemia-1 (Mcl-1) has been associated with apoptotic resistance in CLL. Therefore, we investigate here whether the sensitising activity of this flavonoid, which leads to increased apoptosis in both cell lines and CLL, could be related to Mcl-1 expression and stability.B cells isolated from CLL patients showed different levels of Mcl-1 protein expression, resulting, in several cases, in increased sensitivity to fludarabine. Quercetin significantly enhanced the downregulation of Mcl-1 in B cells isolated from selected patients expressing detectable levels of Mcl-1. In U-937 cells, quercetin increased Mcl-1 mRNA instability in the presence of actinomycin D. When cells were treated with MG-132, a proteasome inhibitor, Mcl-1 protein level increased. However, quercetin, in the presence of Z-Vad-FMK, continued to lower Mcl-1 protein expression, indicating its independence from caspase-mediated degradation. In contrast, co-treatment of quercetin and MG-132 did not revert the effect of MG-132 mono-treatment, thus suggesting a possible interference of quercetin in regulating the proteasome-dependent degradation of Mcl-1. Gossypol, a small-molecule inhibitor of Bcl-2 family members, mimics the activity of quercetin by lowering Mcl-1 expression and sensitising U-937 cells to apoptosis induced by recombinant TRAIL and the Fas-ligand.This study demonstrates that in U-937 cells, quercetin downregulates Mcl-1 acting directly or indirectly on its mRNA stability and protein degradation, suggesting that the same mechanism may bypass resistance to apoptosis in leukaemic cells isolated from CLL patients and sensitise B cells to apoptosis induced by drugs and death receptor inducers.

Although considerable effort and progress has been made in the search for new anticancer drugs and treatments in the last several decades, cancer remains a major public health problem and one of the major causes of death worldwide.Many sources, including plants, animals, and minerals, are of interest in cancer research because of the possibility of identifying novel molecular therapeutics.Moreover, structure-activity-relationship (SAR) investigations have become a common way to develop naturally derived or semi-synthetic molecular analogues with improved efficacy and decreased toxicity.In 2012, approximately 138 molecules from marine sources, including isolated compounds and their associated analogues, were shown to be promising anticancer drugs.Among these, 62% are novel compounds.In this report, we review the marine compounds identified in 2012 that may serve as novel anticancer drugs.

Coumarins are natural compounds with antioxidant, anti-inflammatory and anti-cancer potential known to modulate inflammatory pathways. Here, non-toxic biscoumarin OT52 strongly inhibited proliferation of non-small cell lung cancer cells with KRAS mutations, inhibited stem-like characteristics by reducing aldehyde dehydrogenase expression and abrogated spheroid formation capacity. This cytostatic effect was characterized by cell cycle arrest and onset of senescence concomitant with endoplasmic reticulum and Golgi stress, leading to metabolic alterations. Mechanistically, this cellular response was associated with the novel capacity of biscoumarin OT52 to inhibit STAT3 transactivation and expression of its target genes linked to proliferation. These results were validated by computational docking of OT52 to the STAT3 DNA-binding domain. Combination treatments of OT52 with subtoxic concentrations of Bcl-xL and Mcl-1-targeting BH3 protein inhibitors triggered synergistic immunogenic cell death validated in colony formation assays as well as in vivo by zebrafish xenografts.

The diversity of natural compounds is essential for their mechanism of action. The source, structures and structure activity relationship of natural compounds contributed to the development of new classes of chemotherapy agents for over 40 years. The availability of combinatorial chemistry and high-throughput screening has fueled the challenge to identify novel compounds that mimic nature's chemistry and to predict their macromolecular targets. Combining conventional and targeted therapies helped to successfully overcome drug resistance and prolong disease-free survival. Here, we aim to provide an overview of preclinical investigated natural compounds alone and in combination to further improve personalization of cancer treatment.

Abstract By comparing imatinib-sensitive and -resistant chronic myeloid leukemia (CML) cell models, we investigated the molecular mechanisms by which tetrahydrobenzimidazole derivative TMQ0153 triggered caspase-dependent apoptosis at low concentrations accompanied by loss of mitochondrial membrane potential (MMP) and increase of cytosolic free Ca 2+ levels. Interestingly, at higher concentrations, TMQ0153 induced necroptotic cell death with accumulation of ROS, both preventable by N -acetyl-L-cysteine (NAC) pretreatment. At necroptosis-inducing concentrations, we observed increased ROS and decreased ATP and GSH levels, concomitant with protective autophagy induction. Inhibitors such as bafilomycin A1 (baf-A1) and siRNA against beclin 1 abrogated autophagy, sensitized CML cells against TMQ0153 and enhanced necroptotic cell death. Importantly, TMQ153-induced necrosis led to cell surface exposure of calreticulin (CRT) and ERp57 as well as the release of extracellular ATP and high mobility group box (HMGB1) demonstrating the capacity of this compound to release immunogenic cell death (ICD) markers. We validated the anti-cancer potential of TMQ0153 by in vivo inhibition of K562 microtumor formation in zebrafish. Taken together, our findings provide evidence that cellular stress and redox modulation by TMQ0153 concentration-dependently leads to different cell death modalities including controlled necrosis in CML cell models.

Epidemiologic data revealed increased brain tumor incidence in workers exposed to magnetic fields (MFs), raising concerns about the possible link between MF exposure and cancer. However, MFs seem to be neither mutagenic nor tumorigenic. The mechanism of their tumorigenic effect has not been elucidated.To evaluate the interference of MFs with physical (heat shock, HS) and chemical (etoposide, VP16) induced apoptoses, respectively, we exposed a human glioblastoma primary culture to 6 mT static MF. We investigated cytosolic Ca(2+) ([Ca(2+)](i)) fluxes and extent of apoptosis as key endpoints. The effect of MFs on HS- and VP16-induced apoptoses in primary glioblastoma cultures from four patients was also tested.Static MFs increased the [Ca(2+)](i) from a basal value of 124 +/- 4 nM to 233 +/- 43 nM (P < 0.05). MF exposure dramatically reduced the extent of HS- and VP16-induced apoptoses in all four glioblastoma primary cultures analyzed by 56% (range, 28-87%) and 44% (range, 38-48%), respectively. However, MF alone did not exert any apoptogenic activity. Differences were observed across the four cultures with regard to apoptotic induction by HS and VP16 and to MF apoptotic reduction, with an individual variability with regard to apoptotic sensitivity.The ability of static MFs to reduce the extent of damage-induced apoptosis in glioblastoma cells might allow the survival of damaged and possibly mutated cells.

Tanzawaic acids M (<bold>1</bold>), N (<bold>2</bold>), O (<bold>3</bold>) and P (<bold>4</bold>) were isolated from an extract of a cultured marine-derived fungus (strain CF07370) identified as a member of the genus <italic>Penicillium</italic>.

Cancer cells depend on an altered energy metabolism characterized by increased rates of both glycolysis and glutaminolysis. Accordingly, corresponding key metabolic enzymes are overexpressed or hyperactivated. As a result, this newly acquired metabolic profile determines most other cancer hallmarks including resistance to cell death. Recent findings highlighted metabolic enzymes as direct modulators of cell death pathways. Conversely, key mediators of cell death mechanisms are emerging as new binding partners of glycolytic actors; moreover, there is evidence that metabolic regulators re-localize to specific subcellular compartments or organelles to modulate various types of cell demise. The final outcome is the resistance against cell death programs. Current findings give a new meaning to metabolic pathways and allow understanding how they affect cancer-specific pathological alterations. Furthermore, they shed light on potentially targetable functions of metabolic actors to restore susceptibility of cancer cells to death. Here, we discuss an emerging interplay between cell metabolism and cell death, focusing on interactions that may offer new options of targeted therapies in cancer treatment involving more specifically hexokinases and glyceraldehyde-3-phosphate dehydrogenase.

We synthetized and investigated the anti-leukemic potential of the novel cytostatic bis(4-hydroxycoumarin) derivative OT-55 which complied with the Lipinski's rule of 5 and induced differential toxicity in various chronic myeloid leukemia (CML) cell models. OT-55 triggered ER stress leading to canonical, caspase-dependent apoptosis and release of danger associated molecular patterns. Consequently, OT-55 promoted phagocytosis of OT-55-treated CML cells by both murine and human monocyte-derived macrophages. Moreover, OT-55 inhibited tumor necrosis factor α-induced activation of nuclear factor-кB and produced synergistic effects when used in combination with imatinib to inhibit colony formation in vitro and Bcr-Abl+ patient blast xenograft growth in zebrafish. Furthermore, OT-55 synergized with omacetaxine in imatinib-resistant KBM-5 R cells to inhibit the expression of Mcl-1, triggering apoptosis. In imatinib-resistant K562 R cells, OT-55 triggered necrosis and blocked tumor formation in zebrafish in combination with omacetaxine.

Cardiac glycosides (CGs) are natural compounds used traditionally to treat congestive heart diseases. Recent investigations repositioned CGs as potential anticancer agents. To discover novel cytotoxic CG scaffolds, we selected the cardenolide glucoevatromonoside (GEV) out of 46 CGs for its low nanomolar anti-lung cancer activity. GEV presented reduced toxicity towards non-cancerous cell types (lung MRC-5 and PBMC) and high-affinity binding to the Na+/K+-ATPase alpha subunit, assessed by computational docking. GEV-induced cell death was caspase-independent, as investigated by a multiparametric approach, and culminates in severe morphological alterations in A549 cells, monitored by transmission electron microscopy, live cell imaging and flow cytometry. This non-canonical cell death was not preceded or accompanied by exacerbation of autophagy. In the presence of GEV, markers of autophagic flux (e.g. LC3I-II conversion) were impacted, even in presence of bafilomycin A1. Cell death induction remained unaffected by calpain, cathepsin, parthanatos or necroptosis inhibitors. Interestingly, GEV triggered caspase-dependent apoptosis in U937 acute myeloid leukemia cells, witnessing cancer-type specific cell death induction. Differential cell cycle modulation by this CG led to a G2/M arrest, cyclin B1 and p53 downregulation in A549, but not in U937 cells. We further extended the anti-cancer potential of GEV to 3D cell culture using clonogenic and spheroid formation assays and validated our findings in vivo by zebrafish xenografts. Altogether, GEV shows an interesting anticancer profile with the ability to exert cytotoxic effects via induction of different cell death modalities.

Glutathione depletion by inhibition of its synthesis with buthionine sulfoximine (BSO) is a focus of the current research in antitumor therapy, BSO being used as chemosensitizer. We had previously shown that two human tumor cell lines (U937 and HepG2) survive to treatment with BSO: BSO can elicit an apoptotic response, but the apoptotic process is aborted after cytochrome c release and before caspase activation, suggesting the development of an adaptive response (FASEB J., 1999, 13, 2031-2036). Here, we investigate the mechanisms of such an adaptation. We found that following BSO, U937 up-regulate Bcl-2 mRNA and protein levels, by a mechanism possibly involving NF-kappaB transcription factor; the increase in protein level is limited by a rapid decay of Bcl-2 in BSO-treated cells, suggesting that redox imbalance speeds up Bcl-2 turnover. BSO-dependent Bcl-2 up-regulation is associated with the ability to survive to BSO. Indeed, 1) its abrogation by CAPE or protein synthesis inhibition sensitizes U937 to BSO; 2) in a panel of four tumor lines, BSO-resistant (U937, HepG2, and HGB1) but not BSO-sensitive (BL41) cells can up-regulate Bcl-2 following GSH depletion; remarkably, only the latter are chemosensitized by BSO.

Magnetic fields (MFs) are receiving much attention in basic research due to their emerging ability to alter intracellular signaling. We show here that static MFs with intensity of 6 mT significantly alter the intracellular redox balance of U937 cells. A strong increase of reactive oxygen species (ROS) and a decrease of glutathione (GSH) intracellular levels were found after 2 h of MF exposure and maintained thereafter. We found that also other types of MFs, such as extremely-low-frequency (ELF) MFs affect intracellular GSH starting from a threshold at 0.09 mT. We previously reported that static MFs in the intensity range of 0.3-60 mT reduce apoptosis induced by damaging agents (Fanelli et al., 1998). Here, we show that ELF-MFs are also able to protect U937 from apoptosis. Interestingly, this ability is limited to the ELF intensities able to alter redox equilibrium, indicating a link between MF's antiapoptotic effect and the MF alteration of intracellular redox balance. This suggests that MF-produced redox alterations may be part of the signaling pathway leading to apoptosis antagonism. Thus, we tested whether MFs may still exert an antiapoptotic action in cells where the redox state was artificially altered in both directions, that is, by creating an oxidative (via GSH depletion with BSO) or a reducing (with DTT) cellular environment. In both instances, MFs fail to affect apoptosis. Thus, a correct intracellular redox state is required in order for MFs to exert their antiapoptotic effect.

Carbon nanotubes (CNTs) are a focus of intense research for their potential applications in multiple diverse applications, including innovative biomedical applications. Due to their very recent discovery, little information is available about the biocompatibility and toxicity of this new class of nanoparticle, and a systematic study on biological interference is lacking. Thus, we decided to explore the toxicity of three different types of carbon nanotube, differing in preparation (arc discharge versus catalysed chemical vapour deposition); size (10–50 versus 100–150 nm wide × 1–10 µm long); contaminants (amorphous C, graphite, fullerenes or iron) and morphological type (multi-walled, MW, or single-walled, SW) on human leukemic U937 cells. We found that these carbon nanotubes exert a strong effect on the proliferation of the reporter U937 monocytic cell. However, these CNTs did not significantly affect the cell viability. These results show that CNTs, though not directly exerting a direct cytotoxic effect, are nonetheless able to deeply alter cell behaviour, and thus we recommend thorough analyses to limit health risk due to uncontrolled exposure.

Cell division cycle (CDC) 25 proteins are key phosphatases regulating cell cycle transition and proliferation by regulating CDK/cyclin complexes. Overexpression of these enzymes is frequently observed in cancer and is related to aggressiveness, high-grade tumors and poor prognosis. Thus, targeting CDC25 by compounds, able to inhibit their activity, appears a good therapeutic approach. Here, we describe the synthesis of a new inhibitor (SV37) whose structure is based on both coumarin and quinone moieties. An analytical in vitro approach shows that this compound efficiently inhibits all three purified human CDC25 isoforms (IC50 1-9 µM) in a mixed-type mode. Moreover, SV37 inhibits growth of breast cancer cell lines. In MDA-MB-231 cells, reactive oxygen species generation is followed by pCDK accumulation, a mark of CDC25 dysfunction. Eventually, SV37 treatment leads to activation of apoptosis and DNA cleavage, underlining the potential of this new type of coumarin-quinone structure.

(R)-(+)-Goniothalamin (GTN), a styryl-lactone isolated from the medicinal plant Goniothalamus macrophyllus, exhibits pharmacological activities including cytotoxic and anti-inflammatory effects. In this study, GTN modulated TNF-α induced NF-κB activation. GTN concentrations up to 20 μM showed low cytotoxic effects in K562 chronic myelogenous leukemia and in Jurkat T cells. Importantly, at these concentrations, no cytotoxicity was observed in healthy peripheral blood mononuclear cells. Our results confirmed that GTN inhibited tumor necrosis factor-α (TNF-α)-induced NF-κB activation in Jurkat and K562 leukemia cells at concentrations as low as 5 μM as shown by reporter gene assays and western blots. Moreover, GTN down-regulated translocation of the p50/p65 heterodimer to the nucleus, prevented binding of NF-κB to its DNA response element and reduced TNF-α-activated interleukin-8 (IL-8) expression. In conclusion, GTN inhibits TNF-α-induced NF-κB activation at non-apoptogenic concentrations in different leukemia cell models without presenting toxicity towards healthy blood cells underlining the anti-leukemic potential of this natural compound.

Natural polysulfanes including diallyltrisulfide (DATS) and diallyltetrasulfide (DATTS) from garlic possess antimicrobial, chemopreventive and anticancer properties. However these compounds exhibit chemical instability and reduced solubility, which prevents their potential clinical applicability. We synthesized six DATS and DATTS derivatives, based on the polysulfane motif, expected to exhibit improved physical and chemical properties and verified their biological activity on human leukemia cells. We identified four novel cytotoxic compounds (IC50 values: compound 1, 24.96 ± 12.37 μM; compound 2, 22.82 ± 4.20 μM; compound 3, 3.86 ± 1.64 μM and compound 5, 40.62 ± 10.07 μM, compared to DATTS: IC50: 9.33 ± 3.86 μM). These polysulfanes possess excellent differential toxicity, as they did not affect proliferating mononuclear blood cells from healthy donors. We further demonstrated ability of active compounds to induce apoptosis in leukemia cells by analysis of nuclear fragmentation and of cleavage of effector and executioner caspases. Apoptosis was preceded by accumulation of cells in G2/M phase with a pro-metaphase-like nuclear pattern as well as microtubular alterations. Prolonged and persistent arrest of cancer cells in early mitosis by the benzyl derivative identifies this compound as the most stable and effective one for further mechanistic and in vivo studies.

Acute myeloid leukemia (AML) is efficiently targeted by combinatorial regimens including B cell lymphoma 2 (BCL2) protein antagonists.BCL2 homology domain 3 (BH3) mimetics selectively target leukemic stem cells.Strategies that inhibit BCL2 proteins impair metabolic routes that are preferentially exploited in AML.BCL2 protein antagonists prevent BCL2 family member-dependent paracrine signaling to the AML tumor microenvironment.Inhibition of MCL1 expression/activity modulates the antitumor immune response. B cell lymphoma 2 (BCL2) homology domain 3 (BH3) mimetics are targeted therapeutic agents that allow response prediction and patient stratification. BH3 mimetics are prototypical activators of the mitochondrial death program in cancer. They emerged as important modulators of cellular mechanisms contributing to poor therapeutic responses, including cancer cell stemness, cancer-specific metabolic routes, paracrine signaling to the tumor microenvironment, and immune modulation. We present an overview of the antagonism between BH3 mimetics and antiapoptotic BCL2 proteins. We focus on acute myeloid leukemia (AML), a cancer with reduced therapeutic options that have recently been improved by BH3 mimetics. B cell lymphoma 2 (BCL2) homology domain 3 (BH3) mimetics are targeted therapeutic agents that allow response prediction and patient stratification. BH3 mimetics are prototypical activators of the mitochondrial death program in cancer. They emerged as important modulators of cellular mechanisms contributing to poor therapeutic responses, including cancer cell stemness, cancer-specific metabolic routes, paracrine signaling to the tumor microenvironment, and immune modulation. We present an overview of the antagonism between BH3 mimetics and antiapoptotic BCL2 proteins. We focus on acute myeloid leukemia (AML), a cancer with reduced therapeutic options that have recently been improved by BH3 mimetics. BCL2 family proteins are crucial cell-survival modulators that are aberrantly expressed in several forms of cancer and contribute to disease progression and therapeutic resistance. The development of synthetic small-molecule inhibitors, specifically BH3 mimetics, provides a successful strategy to reactivate cell death signaling in cancer cells. Their mechanism of action is conceptually simple. BH3 mimetics imitate short sequences derived from the BH3 domain of the canonical binding partners of prosurvival BCL2 proteins (the proapoptotic BH3-only proteins), thus preventing BH3-only/prosurvival BCL2 protein family interaction, and eventually inhibiting cell death modulation (Box 1).Box 1The BCL2 Protein Family in CancerCancer cells cope with permanent cellular stress and can withstand a higher stress threshold before dying. Their refractoriness to cell death commitment is a crucial hallmark. The mitochondria are the stage for this prosurvival strategy, and BCL2 family proteins are the major players. In this context, the antiapoptotic members of the BCL2 family, including BCL2L1 (also known as BCLXL), BCL2L2, MCL1, and BCL2-related protein A1 (BCL2A1), antagonize the activation of the proapoptotic members BCL2-associated X apoptosis regulator (BAX) and BCL2 antagonist/killer 1 (BAK1) that are essential for triggering the mitochondrial apoptotic pathway (Figure I). BH3-only proteins act as liaisons between pro- and antiapoptotic family members. Arising in stress conditions, they are functionally different, where the activators (including BCL2-like 11/BCL2L11, also known as BIM), truncated BH3 interacting-domain death agonist (tBID), and BCL2-binding component 3 (BBC3, also known as PUMA) directly bind to BAK1 and BAX to promote their activation, and the sensitizers (including BCL2-associated agonist of cell death, BAD; BCL2-interacting killer, BIK; phorbol-12-myristate-13-acetate-induced protein 1, PMAIP1, also known as NOXA; BCL2-modifying factor, BMF; and harakiri or BCL2-interacting protein, HRK), interact with antiapoptotic BCL2 members to displace them and enable the activating BH3-only proteins and BAK1/BAX to trigger mitochondrial outer membrane permeabilization (MOMP). BH3 mimetics mimic the sensitizers (Figure I). They interact with antiapoptotic BCL2 proteins in a competitive manner, eventually reactivating the death signal. The ability of BH3 mimetics to induce MOMP in the absence of other stimuli is proof of concept that most cancer cells are primed to die and depend on antiapoptotic BCL2 proteins for survival. This vulnerability can be exploited for therapeutic purposes. There are preferential patterns of interaction between anti- and proapoptotic BCL2 family members. Cancer cells derived from different patients (intertumoral heterogeneity), or belonging to distinct subpopulations within the same patient (intratumoral heterogeneity), may rely on different BCL2 proteins for survival. Furthermore, the patterns of BCL2 protein dependency can vary, and therapeutic treatments can change these patterns through the clonal expansion of specific malignant subpopulations. Approaches to monitor the BCL2 dependency of cancer cells could be used to predict responders and to personalize therapies, and these strategies could also be applied after therapy to monitor the potential onset of unresponsiveness (Box 2). Cancer cells cope with permanent cellular stress and can withstand a higher stress threshold before dying. Their refractoriness to cell death commitment is a crucial hallmark. The mitochondria are the stage for this prosurvival strategy, and BCL2 family proteins are the major players. In this context, the antiapoptotic members of the BCL2 family, including BCL2L1 (also known as BCLXL), BCL2L2, MCL1, and BCL2-related protein A1 (BCL2A1), antagonize the activation of the proapoptotic members BCL2-associated X apoptosis regulator (BAX) and BCL2 antagonist/killer 1 (BAK1) that are essential for triggering the mitochondrial apoptotic pathway (Figure I). BH3-only proteins act as liaisons between pro- and antiapoptotic family members. Arising in stress conditions, they are functionally different, where the activators (including BCL2-like 11/BCL2L11, also known as BIM), truncated BH3 interacting-domain death agonist (tBID), and BCL2-binding component 3 (BBC3, also known as PUMA) directly bind to BAK1 and BAX to promote their activation, and the sensitizers (including BCL2-associated agonist of cell death, BAD; BCL2-interacting killer, BIK; phorbol-12-myristate-13-acetate-induced protein 1, PMAIP1, also known as NOXA; BCL2-modifying factor, BMF; and harakiri or BCL2-interacting protein, HRK), interact with antiapoptotic BCL2 members to displace them and enable the activating BH3-only proteins and BAK1/BAX to trigger mitochondrial outer membrane permeabilization (MOMP). BH3 mimetics mimic the sensitizers (Figure I). They interact with antiapoptotic BCL2 proteins in a competitive manner, eventually reactivating the death signal. The ability of BH3 mimetics to induce MOMP in the absence of other stimuli is proof of concept that most cancer cells are primed to die and depend on antiapoptotic BCL2 proteins for survival. This vulnerability can be exploited for therapeutic purposes. There are preferential patterns of interaction between anti- and proapoptotic BCL2 family members. Cancer cells derived from different patients (intertumoral heterogeneity), or belonging to distinct subpopulations within the same patient (intratumoral heterogeneity), may rely on different BCL2 proteins for survival. Furthermore, the patterns of BCL2 protein dependency can vary, and therapeutic treatments can change these patterns through the clonal expansion of specific malignant subpopulations. Approaches to monitor the BCL2 dependency of cancer cells could be used to predict responders and to personalize therapies, and these strategies could also be applied after therapy to monitor the potential onset of unresponsiveness (Box 2). Approval of the first selective BCL2 inhibitor, venetoclax (ABT-199, Venclexta®), by the FDA in 2016 provided an important impulse to the personalized therapeutic use of BH3 mimetics. From a clinical perspective, BCL2 protein dependency became a marker of therapeutic response, thus allowing patient stratification (see Glossary; Box 2). Mechanistically, its emergence fostered the discovery of additional natural and synthetic BH3 mimetics that are selective for other BCL2 family members. Despite the generally positive response to these drugs, primary or secondary forms of resistance have been described. Indeed, pre-existing clones could be dependent on alternative or multiple BCL2 members (BCL2 codependency), and therapy-induced selection of such clones cannot be excluded. Identifying and administrating appropriate BH3 mimetics alone or in combination is currently under preclinical and clinical investigation as a treatment strategy to overcome these forms of resistance.Box 2Diagnostic Tools To Monitor BCL2 Protein DependencyBH3 profiling uses peptides derived from the BH3 domains of BH3-only proteins that recapitulate all possible interaction patterns between BH3-only proteins and antiapoptotic BCL2 members (Box 1). In the original protocol, mitochondria are isolated and probed using the entire panel of peptides. The release of cytochrome c, detected by enzyme-linked immunosorbent assay (ELISA), indicates MOMP and identifies effective peptides derived from sensitizing BH3-only proteins. The concomitant use of peptides derived from BH3-only protein activators confirms the presence of functional BAK1 and BAX in the isolated mitochondria [74.Certo M. et al.Mitochondria primed by death signals determine cellular addiction to antiapoptotic BCL-2 family members.Cancer Cell. 2006; 9: 351-365Abstract Full Text Full Text PDF PubMed Scopus (873) Google Scholar,75.Deng J. et al.BH3 profiling identifies three distinct classes of apoptotic blocks to predict response to ABT-737 and conventional chemotherapeutic agents.Cancer Cell. 2007; 12: 171-185Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar]. More recently, this technique has been adapted to primary cells to serve as a therapeutic biomarker for BCL2 dependency. Patient cells undergo partial permeabilization to preserve mitochondrial functionality while allowing the intracellular diffusion of BH3 peptides. MOMP is directly monitored through the use of fluorescent probes (JC-1,5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbo-cyanine iodide or tetramethylrhodamine, TMRE) [76.Ryan J. Letai A. BH3 profiling in whole cells by fluorimeter or FACS.Methods. 2013; 61: 156-164Crossref PubMed Scopus (87) Google Scholar]. BH3 profiling contributed to the elucidation of the mechanisms underlying BCL2 protein dependency. In contrast to lymphoid malignancies, AML cells do not rely exclusively on unique antiapoptotic BCL2 members to survive, and instead frequently develop codependencies [75.Deng J. et al.BH3 profiling identifies three distinct classes of apoptotic blocks to predict response to ABT-737 and conventional chemotherapeutic agents.Cancer Cell. 2007; 12: 171-185Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar,77.Butterworth M. et al.BH3 profiling and a toolkit of BH3-mimetic drugs predict anti-apoptotic dependence of cancer cells.Br. J. Cancer. 2016; 114: 638-641Crossref PubMed Scopus (7) Google Scholar] or turnover of BCL2 proteins [78.Moujalled D.M. et al.Combining BH3-mimetics to target both BCL-2 and MCL1 has potent activity in pre-clinical models of acute myeloid leukemia.Leukemia. 2019; 33: 905-917Crossref PubMed Scopus (0) Google Scholar]. BH3 profiling also allows monitoring of resistance that arises from the selection and expansion of pre-existing minor clones bearing different patterns of BCL2 dependency [79.Ramsey H.E. et al.A novel MCL1 inhibitor combined with venetoclax rescues venetoclax-resistant acute myelogenous leukemia.Cancer Discov. 2018; 8: 1566-1581Crossref PubMed Scopus (67) Google Scholar]. This outcome suggests that a combination of agents targeting diverse dependencies (not limited to BCL2 family proteins) is an essential strategy to improve CR rates and OS in AML patients.The concept of BCL2 dependency has inspired several approaches aimed at engineering BCL2 addiction to test the efficacy and selectivity of new BCL2 inhibitors. Cells are engineered to coexpress specific pairs of pro- and antiapoptotic BCL2 proteins and become 'mito-primed' [80.Lopez J. et al.Mito-priming as a method to engineer Bcl-2 addiction.Nat. Commun. 2016; 710538Crossref PubMed Google Scholar, 81.Koss B. et al.Defining specificity and on-target activity of BH3-mimetics using engineered B-ALL cell lines.Oncotarget. 2016; 7: 11500-11511Crossref PubMed Scopus (15) Google Scholar, 82.Lopez J. Tait S.W.G. Application of mito-priming to generate BCL-2 addicted cells.Methods Mol. Biol. 2019; 1877: 45-60Crossref PubMed Scopus (0) Google Scholar]. The proapoptotic member is tagged with GFP, allowing the monitoring of its localization to mitochondria and any perturbation of this condition by immunofluorescence. In addition to assessing the selectivity and potency of new inhibitors, mito-priming profiles any preferential interactions between BH3-only proteins and their effectors BAX and BAK1. Importantly, this technique can be combined with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based genome editing to corroborate mechanistic aspects. CRISPR/Cas9 screening can also be used in combination with drug screening approaches. For example, combining this methodology with a BCL2 inhibitor screen on a broad panel of cell models helped to identify the epithelial–mesenchymal transition (EMT) as a novel mechanism influencing BCL2 dependency in solid tumors [83.Soderquist R.S. et al.Systematic mapping of BCL-2 gene dependencies in cancer reveals molecular determinants of BH3 mimetic sensitivity.Nat. Commun. 2018; 9: 3513Crossref PubMed Scopus (28) Google Scholar]. BH3 profiling uses peptides derived from the BH3 domains of BH3-only proteins that recapitulate all possible interaction patterns between BH3-only proteins and antiapoptotic BCL2 members (Box 1). In the original protocol, mitochondria are isolated and probed using the entire panel of peptides. The release of cytochrome c, detected by enzyme-linked immunosorbent assay (ELISA), indicates MOMP and identifies effective peptides derived from sensitizing BH3-only proteins. The concomitant use of peptides derived from BH3-only protein activators confirms the presence of functional BAK1 and BAX in the isolated mitochondria [74.Certo M. et al.Mitochondria primed by death signals determine cellular addiction to antiapoptotic BCL-2 family members.Cancer Cell. 2006; 9: 351-365Abstract Full Text Full Text PDF PubMed Scopus (873) Google Scholar,75.Deng J. et al.BH3 profiling identifies three distinct classes of apoptotic blocks to predict response to ABT-737 and conventional chemotherapeutic agents.Cancer Cell. 2007; 12: 171-185Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar]. More recently, this technique has been adapted to primary cells to serve as a therapeutic biomarker for BCL2 dependency. Patient cells undergo partial permeabilization to preserve mitochondrial functionality while allowing the intracellular diffusion of BH3 peptides. MOMP is directly monitored through the use of fluorescent probes (JC-1,5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbo-cyanine iodide or tetramethylrhodamine, TMRE) [76.Ryan J. Letai A. BH3 profiling in whole cells by fluorimeter or FACS.Methods. 2013; 61: 156-164Crossref PubMed Scopus (87) Google Scholar]. BH3 profiling contributed to the elucidation of the mechanisms underlying BCL2 protein dependency. In contrast to lymphoid malignancies, AML cells do not rely exclusively on unique antiapoptotic BCL2 members to survive, and instead frequently develop codependencies [75.Deng J. et al.BH3 profiling identifies three distinct classes of apoptotic blocks to predict response to ABT-737 and conventional chemotherapeutic agents.Cancer Cell. 2007; 12: 171-185Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar,77.Butterworth M. et al.BH3 profiling and a toolkit of BH3-mimetic drugs predict anti-apoptotic dependence of cancer cells.Br. J. Cancer. 2016; 114: 638-641Crossref PubMed Scopus (7) Google Scholar] or turnover of BCL2 proteins [78.Moujalled D.M. et al.Combining BH3-mimetics to target both BCL-2 and MCL1 has potent activity in pre-clinical models of acute myeloid leukemia.Leukemia. 2019; 33: 905-917Crossref PubMed Scopus (0) Google Scholar]. BH3 profiling also allows monitoring of resistance that arises from the selection and expansion of pre-existing minor clones bearing different patterns of BCL2 dependency [79.Ramsey H.E. et al.A novel MCL1 inhibitor combined with venetoclax rescues venetoclax-resistant acute myelogenous leukemia.Cancer Discov. 2018; 8: 1566-1581Crossref PubMed Scopus (67) Google Scholar]. This outcome suggests that a combination of agents targeting diverse dependencies (not limited to BCL2 family proteins) is an essential strategy to improve CR rates and OS in AML patients. The concept of BCL2 dependency has inspired several approaches aimed at engineering BCL2 addiction to test the efficacy and selectivity of new BCL2 inhibitors. Cells are engineered to coexpress specific pairs of pro- and antiapoptotic BCL2 proteins and become 'mito-primed' [80.Lopez J. et al.Mito-priming as a method to engineer Bcl-2 addiction.Nat. Commun. 2016; 710538Crossref PubMed Google Scholar, 81.Koss B. et al.Defining specificity and on-target activity of BH3-mimetics using engineered B-ALL cell lines.Oncotarget. 2016; 7: 11500-11511Crossref PubMed Scopus (15) Google Scholar, 82.Lopez J. Tait S.W.G. Application of mito-priming to generate BCL-2 addicted cells.Methods Mol. Biol. 2019; 1877: 45-60Crossref PubMed Scopus (0) Google Scholar]. The proapoptotic member is tagged with GFP, allowing the monitoring of its localization to mitochondria and any perturbation of this condition by immunofluorescence. In addition to assessing the selectivity and potency of new inhibitors, mito-priming profiles any preferential interactions between BH3-only proteins and their effectors BAX and BAK1. Importantly, this technique can be combined with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based genome editing to corroborate mechanistic aspects. CRISPR/Cas9 screening can also be used in combination with drug screening approaches. For example, combining this methodology with a BCL2 inhibitor screen on a broad panel of cell models helped to identify the epithelial–mesenchymal transition (EMT) as a novel mechanism influencing BCL2 dependency in solid tumors [83.Soderquist R.S. et al.Systematic mapping of BCL-2 gene dependencies in cancer reveals molecular determinants of BH3 mimetic sensitivity.Nat. Commun. 2018; 9: 3513Crossref PubMed Scopus (28) Google Scholar]. BH3 mimetics became a suitable tool to explore the regulatory functions of BCL2 family proteins beyond their role in BCL2 resistance; in addition, they helped to extend the network of BCL2 protein binding partners. These research efforts allowed the discovery of modulatory mechanisms that are potentially relevant for innovating therapeutic approaches by targeting processes such as cancer cell stemness, metabolic functions, signaling to the microenvironment, and immune interactions. Elucidation of these mechanisms, whether interconnected or not with the canonical function of BCL2 family proteins in the modulation of mitochondrial outer-membrane permeabilization (MOMP), will provide new rationales for the therapeutic use of BH3 mimetics. In this context, AML, a disease exhibiting dismal survival rates, has become an important model for investigation. AML is a hematological type of cancer characterized by inhibition of myeloid differentiation, with consequent clonal expansion and accumulation of immature myeloid blasts within the bone marrow (BM) and blood [1.Short N.J. et al.Acute myeloid leukaemia.Lancet. 2018; 392: 593-606Abstract Full Text Full Text PDF PubMed Google Scholar]. This disease typically affects adults (80% of cases) and is particularly prevalent among elderly individuals. AML is one of the most aggressive forms of blood cancer, and patients exhibit poor prognosis and limited 5 year overall survival (OS; 27.8%). The ineligibility of a subset of patients to undergo intensive cycles of chemotherapy, a high rate of relapse even in patients with apparent complete remission (CR) after therapy, and limited access to allogeneic hematopoietic stem cell transplantation (HSCT) are the major reasons for these unfavorable outcomes. AML was treated for many years using a standard 'one-size-fits-all' therapeutic regimen. Since 2017, new drugs, including targeted agents, have been approved for the treatment of AML. These new drugs have substantially replaced standard therapies during the induction therapy and/or consolidation therapy phases, ultimately allowing patient stratification before the implementation of novel personalized therapeutic approaches [1.Short N.J. et al.Acute myeloid leukaemia.Lancet. 2018; 392: 593-606Abstract Full Text Full Text PDF PubMed Google Scholar]. Venetoclax is one such newly approved therapeutic agent. The public availability of data regarding patients who were enrolled into clinical trials has progressively shed light on the mechanisms responsible for the high AML relapse rate and allowed researchers and clinicians to highlight new targetable mechanisms, including BCL2 protein inhibition. Venetoclax (ABT-199; GDC-0199) was approved in 2018 in combination with the hypomethylating agent azacytidine or low-dose cytarabine (LDAC) for the treatment of newly diagnosed AML patients who are unfit for intensive chemotherapy (Table 1 and Box 3). It is also under evaluation for the treatment of myelodysplastic syndrome [2.Jilg S. et al.Venetoclax with azacitidine targets refractory MDS but spares healthy hematopoiesis at tailored dose.Exp. Hematol. Oncol. 2019; 8: 9Crossref PubMed Scopus (8) Google Scholar]. Initially developed to overcome the on-target side effects of thrombocytopenia caused by the broader BCL2/BCL2-like 1 (BCL2L1)/BCL2-like 2 (BCL2L2) inhibitor navitoclax (ABT-263; Table 1) [3.Souers A.J. et al.ABT-199, a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing platelets.Nat. Med. 2013; 19: 202-208Crossref PubMed Scopus (1476) Google Scholar], its safety and pharmacokinetics have been improved through the use of suitable dosing protocols and prophylaxis to avoid its most dangerous side effects [4.Konopleva M. et al.Efficacy and biological correlates of response in a phase II study of venetoclax monotherapy in patients with acute myelogenous leukemia.Cancer Discov. 2016; 6: 1106-1117Crossref PubMed Scopus (337) Google Scholar, 5.DiNardo C.D. et al.Safety and preliminary efficacy of venetoclax with decitabine or azacitidine in elderly patients with previously untreated acute myeloid leukaemia: a non-randomised, open-label, phase 1b study.Lancet Oncol. 2018; 19: 216-228Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 6.Pollyea D.A. et al.Venetoclax with azacitidine disrupts energy metabolism and targets leukemia stem cells in patients with acute myeloid leukemia.Nat. Med. 2018; 24: 1859-1866Crossref PubMed Scopus (110) Google Scholar]. Recent clinical developments in regard to venetoclax allow this drug to target BCL2-dependent AML subgroups, including those possessing isocitrate dehydrogenase 1/2 (IDH1/2) mutations [7.Chan S.M. et al.Isocitrate dehydrogenase 1 and 2 mutations induce BCL-2 dependence in acute myeloid leukemia.Nat. Med. 2015; 21: 178-184Crossref PubMed Scopus (235) Google Scholar], or patients with a dependency in response to other therapeutic regimens (synthetic lethality [8.Pan R. et al.Synthetic lethality of combined Bcl-2 inhibition and p53 activation in AML: mechanisms and superior antileukemic efficacy.Cancer Cell. 2017; 32: 748-760Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar]). Although this drug achieves excellent CR levels and fast responses, a limitation of venetoclax is the durability of its clinical response [9.Leverson J.D. et al.Found in translation: how preclinical research is guiding the clinical development of the BCL2–selective inhibitor venetoclax.Cancer Discov. 2017; 7: 1376-1393Crossref PubMed Scopus (0) Google Scholar,10.Muller-Tidow C. Schlenk R.F. A new option for remission induction in acute myeloid leukaemia.Lancet Oncol. 2018; 19: 156-157Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. The outgrowth of AML clones expressing other BCL2 family members that replace BCL2 as prosurvival factors is a major reason for this limitation. In particular, MCL1 (apoptosis regulator, BCL2 family member) is the Achilles heel of venetoclax-based therapy, and this has driven research efforts to develop selective MCL1-targeting approaches.Table 1Chemical Structures of BCL2 Antagonists (Direct Inhibitors and Downregulators)aAbbreviations: ALL, acute lymphoblastic leukemia; DNMT1, DNA methyltransferase 1; eIF4A, eukaryotic initiation factor 4A; MAPK, mitogen-activated protein kinase; MDM2, MDM2 proto-oncogene; MM, multiple myeloma; NKA, sodium/potassium-ATPase; R/R, relapsed/refractory.Compound nameStructureTargetsCommentClinical statusRefsBH3 mimetics targeting BCL2Venetoclax(ABT-199,GDC-0199)BCL2Ki <0.01 nMPotent and selective BCL2 inhibitor; BAD-like BH3 mimeticIt is associated with a fast response in elderly AML patients that are ineligible for other therapies. Good tolerabilitySuitable for AML with IDH1/2 mutations or developing BCL2 dependency in response to other therapeutic regimensLimitations: fast but limited durability of response owing to outgrowth of resistant AML clones; tumor lysis syndrome now controlled with dose escalation administration and suitable prophylaxisApproved since 2017 in combination with the hypomethylating agent azacytidine or LDAC for the treatment of patients with AML who are unfit for chemotherapyUnder evaluation for the treatment of MDSIn clinical trials involving combinational regimens[2.Jilg S. et al.Venetoclax with azacitidine targets refractory MDS but spares healthy hematopoiesis at tailored dose.Exp. Hematol. Oncol. 2019; 8: 9Crossref PubMed Scopus (8) Google Scholar,4.Konopleva M. et al.Efficacy and biological correlates of response in a phase II study of venetoclax monotherapy in patients with acute myelogenous leukemia.Cancer Discov. 2016; 6: 1106-1117Crossref PubMed Scopus (337) Google Scholar,5.DiNardo C.D. et al.Safety and preliminary efficacy of venetoclax with decitabine or azacitidine in elderly patients with previously untreated acute myeloid leukaemia: a non-randomised, open-label, phase 1b study.Lancet Oncol. 2018; 19: 216-228Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar,10.Muller-Tidow C. Schlenk R.F. A new option for remission induction in acute myeloid leukaemia.Lancet Oncol. 2018; 19: 156-157Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar,67.DiNardo C.D. et al.Molecular patterns of response and treatment failure after frontline venetoclax combinations in older patients with AML.Blood. 2020; 135: 791-803Crossref PubMed Google Scholar]Navitoclax(ABT-263)BCL2, BCL2L1, and BCL2L2Ki ≤0.5–3.3 nMOral selective inhibitor, BAD-like BH3 mimetic, orally available derivative of ABT-737Limitations: on-target effects on platelets of thrombocytopenia, currently manageable with suitable prophylaxis; more recently found to impact on healthy blood progenitors depending on BCL2L1In clinical trials involving solid tumors or hematological malignancies other than AML (e.g., ALL)(NCT03592576)(NCT03181126)[96.Kivioja J.L. et al.Dasatinib and navitoclax act synergistically to target NUP98-NSD1+/FLT3-ITD+ acute myeloid leukemia.Leukemia. 2019; 33: 1360-1372Crossref PubMed Scopus (5) Google Scholar,97.Anstee N.S. et al.Impact of elevated anti-apoptotic MCL-1 and BCL-2 on the development and treatment of MLL-AF9 AML in mice.Cell Death Differ. 2019; 26: 1316-1331Crossref PubMed Scopus (10) Google Scholar]ABT-737BCL2, BCL2L1, and BCL2L2Ki <0.1 nMPrototypical BH3 mimeticInduces the disruption of BCL2/BAK1 complexes by mimicking BADBinds with much lower affinity to BCL2A1 and MCL1 (Ki 0.46 μM)Not orally available; intraperitoneal administration in mouse modelsExperimental[97.Anstee N.S. et al.Impact of elevated anti-apoptotic MCL-1 and BCL-2 on the development and treatment of MLL-AF9 AML in mice.Cell Death Differ. 2019; 26: 1316-1331Crossref PubMed Scopus (10) Google Scholar,98.Pan R. et al.Inhibition of Mcl-1 with the pan-Bcl-2 family inhibitor (−)BI97D6 overcomes ABT-737 resistance in acute myeloid leukemia.Blood. 2015; 126: 363-372Crossref PubMed Scopus (0) Google Scholar]BH3 mimetics targeting MCL1A-1210477MCL1Ki 0.45 nMFirst direct MCL1 binder and inhibitor with relevant selective potencyDerived from indole-2-carboxylic acids, disrupts both MCL1–BCL2L11 and MCL1–PMAIP1 complexes. Synergy with venetoclax and navitoclaxLimitations: bioavailability and potency compared to more recent MCL1 inhibitors; lacks in vivo studiesResearch toolPrecluded from further clinical consideration[99.Leverson J.D. et al.Exploiting selective BCL-2 family inhibitors to dissect cell survival dependencies and define improved strategies for cancer therapy.Sci. Transl. Med. 2015; 7279ra240Crossref Scopus (249) Google Scholar]AZD5991MCL1Ki 0.13 nMMacrocyclic molecule;particularly active against hematological malignancies including MM and AMLCytotoxic activity against sensitive cell lines, synergy in combination with venetoclax in AML (and bortezomib in MM); tumor regression in vivo (in murine and rat models), with evidence of good tolerability even at high dosesIn clin

The mechanisms of cell killing by oxidative stress, in particular by hydrogen peroxide, are not yet well clarified. Here, we show that during recovery after H(2)O(2) treatment, apoptosis occurs in two different waves, peaking at 8 h (early) and 18 h (late) of recovery from oxidative stress. The two peaks are differentially modulated by a set of inhibitors of metabolic processes, which suggests that the first peak depends on DNA break formation, whereas the second may be correlated with H(2)O(2)-induced mitochondrial alterations.

Plumbagin is a plant naphtoquinone exerting anti-cancer properties including apoptotic cell death induction and generation of reactive oxygen species (ROS). The aim of this study was to elucidate parameters explaining the differential leukemia cell sensitivity towards this compound. Among several leukemia cell lines, U937 monocytic leukemia cells appeared more sensitive to plumbagin treatment in terms of cytotoxicity and level of apoptotic cell death compared to more resistant Raji Burkitt lymphoma cells. Moreover, U937 cells exhibited a ten-fold higher ROS production compared to Raji. Neither differential incorporation, nor efflux of plumbagin was detected. Pre-treatment with thiol-containing antioxidants prevented ROS production and subsequent induction of cell death by apoptosis whereas non-thiol-containing antioxidants remained ineffective in both cellular models. We conclude that the anticancer potential of plumbagin is driven by pro-oxidant activities related to the cellular thiolstat.

Abstract A new alkaloid, oximoaspergillimide ( 1 ), and the known neohydroxyaspergillic ( 2 ) and neoaspergillic ( 3 ) acids, have been isolated from the extract of a cultured marine‐derived fungus (strain CF07002) identified as a member of the genus Aspergillus . The structure of 1 was determined by interpretation of NMR spectroscopic data, and confirmed by synthesis. The antimicrobial and cytotoxic activities of compounds 1 – 3 were evaluated.

Cyclooxygenase-2 (COX-2) is an essential regulator of cancer promotion and progression. Extensive efforts to target this enzyme have been developed to reduce growth of cancer cells for chemopreventive and therapeutic reasons. In this context, cyclooxygenase-2 inhibitors present interesting antitumor effects. However, inhibition of COX-2 by anti-COX-2 compounds such as celecoxib was recently associated with detrimental cardiovascular side effects limiting their clinical use. As many anticancer effects of celecoxib are COX-2 independent, analogs such as 2,5-dimethyl-celecoxib (DMC), which lacks COX-2-inhibitory activity, represent a promising alternative strategy. In this study, we investigated the effect of this molecule on growth of hematologic cancer cell lines (U937, Jurkat, Hel, Raji, and K562). We found that this molecule is able to reduce the growth and induces apoptosis more efficiently than celecoxib in all the leukemic cell lines tested. Cell death was associated with downregulation of Mcl-1 protein expression. We also found that DMC induces endoplasmic reticulum stress, which is associated with a decreased of GRP78 protein expression and an alteration of cell cycle progression at the G1/S transition in U937 cells. Accordingly, typical downregulation of c-Myc and cyclin D1 and an upregulation of p27 were observed. Interestingly, for shorter time points, an alteration of mitotic progression, associated with the downregulation of survivin protein expression was observed. Altogether, our data provide new evidence about the mode of action of this compound on hematologic malignancies.

Cells reprogram their metabolism very early during carcinogenesis; this event is critical for the establishment of other cancer hallmarks. Many oncogenes and tumor suppressor genes control metabolism by interplaying with the existing nutrient-sensing intracellular pathways. Mammalian target of rapamycin, mTOR, is emerging as a collector and sorter of a metabolic network controlling upstream and downstream modulation of these same genes. Natural compounds represent a source of anti-cancer molecules with chemopreventive and therapeutic properties. This review describes selected pathways and genes orchestrating the metabolic reprogramming and discusses the potential of natural compounds to target oncogenic metabolic aberrations.

Bcl-2 protein family expression pattern determines synergistic pro-apoptotic effects of BH3 mimetics with hemisynthetic cardiac glycoside UNBS1450 in acute myeloid leukemia

Stemphol (STP) is a novel druggable phytotoxin triggering mixed apoptotic and non-apoptotic necrotic-like cell death in human acute myeloid leukemia (AML). Use of several chemical inhibitors highlighted that STP-induced non-canonical programmed cell death was Ca2+-dependent but independent of caspases, poly (ADP-ribose) polymerase-1, cathepsin, or calpains. Similar to thapsigargin, STP led to increased cytosolic Ca2+ levels and computational docking confirmed binding of STP within the thapsigargin binding pocket of the sarco/endoplasmic reticulum (ER) Ca2+-ATPase (SERCA). Moreover, the inositol 1,4,5-trisphosphate receptor is implicated in STP-modulated cytosolic Ca2+ accumulation leading to ER stress and mitochondrial swelling associated with collapsed cristae as observed by electron microscopy. Confocal fluorescent microscopy allowed identifying mitochondrial Ca2+ overload as initiator of STP-induced cell death insensitive to necrostatin-1 or cycloheximide. Finally, we observed that STP-induced necrosis is dependent of mitochondrial permeability transition pore (mPTP) opening. Importantly, the translational immunogenic potential of STP was validated by HMGB1 release of STP-treated AML patient cells. STP reduced colony and in vivo tumor forming potential and impaired the development of AML patient-derived xenografts in zebrafish.

Abstract: It was long believed that melatonin might counteract intracellular oxidative stress because it was shown to potentiate antioxidant endogenous defences, and to increase the activity of many antioxidant enzymes. However, it is now becoming evident that when radicals are measured within cells, melatonin increases, rather than decreasing, radical production. Herein we demonstrate a pro‐oxidant effect of melatonin in U937 cells by showing an increase of intracellular oxidative species and a depletion of glutathione (GSH). The activity of glutathione peroxidase is not modified by melatonin treatment as it does occur in other experimental models.

It is well described that cyclooxygenase-2 (COX-2) inhibitors counteract cancer cell proliferation by preventing the G1/S transition. This effect has been associated with the inhibition of COX-2 enzymatic activity but also as an off-target effect essentially in adherent cancer cell models. In this study, we investigated the effect of three COX-2 inhibitors (nimesulide, NS-398 and celecoxib) on cell proliferation of leukemic and lymphoblastic cells expressing COX-2 at high (U937, Jurkat, Hel and Raji) and very low (K562) protein levels. We found that the inhibitors reduce cell proliferation in all COX-2-expressing cells leading to an accumulation in the G0/G1 phase of the cell cycle. We provide evidence that this modulation corresponds to an accumulation of cells in G0 paralleled by the expression of cell differentiation markers in U937 (CD15) and Hel (CD41a and CD61) cells but not in the insensitive K562. These events are associated with a rapid down-regulation (within one hour) of c-Myc expression, accompanied by the up-regulation of p27 and the down-regulation of PCNA and cyclin D1. Our study suggests c-Myc as a crucial early target of COX-2 inhibitors.

Garlic-derived organo sulphur compounds such as diallylsulfides provide a significant protection against carcinogenesis. Chemically synthesized, and highly pure diallylsulfides with a chain of 1-4 sulphur atoms, as well as a range of control compounds, were employed to investigate the influence of these agents on cell viability, cell cycle arrest and induction of apoptosis in HCT116 human colon cancer cells. Diallyltrisulfide, and even more efficiently diallyltetrasulfide treatment of HCT116 cells led to a reduced cell viability, cell cycle arrest and apoptosis. A similar activity was found for the propyl-analogues, while mono- and disulfides were considerably less active. Initial calculations point toward the ability of tri- and tetrasulfides to form reactive oxygen species (ROS). Here, we found that the induction of apoptosis was indeed dependent on the redox-state of the cell, with anti-oxidants being able to prevent sulfide-induced apoptosis. Furthermore, using HCT116 cells which were either positive or negative for p53 revealed that p53 is clearly dispensable for induction of apoptosis. Growth arrest and induction of apoptosis is associated with a considerable reduction of the level of cdc25C. These results support the therapeutic potential of polysulfides and allow insight into the mechanisms based on the polysulfide biochemistry.

Enzymatic inhibitors of pro-inflammatory cyclooxygenase-2 (COX-2) possess multiple anti-cancer effects, including chemosensitization. These effects are not always linked to the inhibition of the COX-2 enzyme. Here we analyze the effects of three COX-2 enzyme inhibitors (nimesulide, NS-398 and celecoxib) on apoptosis in different hematopoietic cancer models. Surprisingly, COX-2 inhibitors strongly prevent apoptosis induced by a panel of chemotherapeutic agents. We selected U937 cells as a model of sensitive cells for further studies. Here, we provide evidence that the protective effect is COX-independent. No suppression of the low basal prostaglandin (PG)E(2) production may be observed upon treatment by COX-2 inhibitors. Besides, the non-active celecoxib analog 2,5-dimethyl-celecoxib is able to protect from apoptosis as well. We demonstrate early prevention of the stress-induced apoptotic signaling, prior to Bax/Bak activation. This preventive effect fits with an impairment of the ability of chemotherapeutic agents to trigger apoptogenic stress. Accordingly, etoposide-induced DNA damage is strongly attenuated in the presence of COX-2 inhibitors. In contrast, COX-2 inhibitors do not exert any anti-apoptotic activity when cells are challenged with physiological stimuli (anti-Fas, TNFα or Trail) or with hydrogen peroxide, which do not require internalization and/or are not targeted by chemoresistance proteins. Altogether, our findings show a differential off-target anti-apoptotic effect of COX-2 inhibitors on intrinsic vs. extrinsic apoptosis at the very early steps of intracellular signaling, prior to commitment. The results imply that an exacerbation of the chemoresistance phenomena may be implicated.

Our aim was to better understand peroxisome proliferator-activated receptor gamma (PPARγ)-independent pathways involved in anti-cancer effects of thiazolidinediones (TZDs). We focused on Δ2-troglitazone (Δ2-TGZ), a PPARγ inactive TZD that affects breast cancer cell viability. Appearance of TUNEL positive cells, changes in mitochondrial membrane potential, cleavage of poly(ADP-ribose) polymerase (PARP)-1 and caspase-7 revealed that apoptosis occurred in both hormone-dependent MCF7 and hormone-independent MDA-MB-231 breast cancer cells after 24 and 48 h of treatment. A microarray study identified endoplasmic reticulum (ER) stress as an essential cellular function since many genes involved in ER stress were upregulated in MCF7 cells following Δ2-TGZ treatment. Δ2-TGZ-induced ER stress was further confirmed in MCF7 cells by phosphorylation of pancreatic endoplasmic reticulum kinase-like endoplasmic reticulum kinase (PERK) and its target eIF2α after 1.5 h, rapid increase in activating transcription factor (ATF) 3 mRNA levels, splicing of X-box binding protein 1 (XBP1) after 3 h, accumulation of binding immunogloblulin protein (BiP) and CCAAT-enhancer-binding protein homologous protein (CHOP) after 6 h. Immunofluorescence microscopy indicated that CHOP was relocalized to the nucleus of treated cells. Similarly, in MDA-MB-231 cells, overexpression of ATF3, splicing of XBP1, and accumulation of BiP and CHOP were observed following Δ2-TGZ treatment. In MCF7 cells, knock-down of CHOP or the inhibition of c-Jun N-terminal kinase (JNK) did not impair cleavage of PARP-1 and caspase-7. Altogether, our results show that ER stress is an early response of major types of breast cancer cells to Δ2-TGZ, prior to, but not causative of apoptosis.

The use of energy restriction mimetic agents (ERMAs) to selectively target cancer cells addicted to glycolysis could be a promising therapeutic approach. Thiazolidinediones (TZDs) are synthetic agonists of the nuclear receptor peroxisome proliferator-activated receptor (PPAR)γ that were developed to treat type II diabetes. These compounds also display anticancer effects which appear mainly to be independent of their PPARγ agonist activity but the molecular mechanisms involved in the anticancer action are not yet well understood. Results obtained on ciglitazone derivatives, mainly in prostate cancer cell models, suggest that these compounds could act as ERMAs. In the present paper, we introduce how compounds like 2-deoxyglucose target the Warburg effect and then we discuss the possibility that the PPARγ-independent effects of various TZD could result from their action as ERMAs.

Polysulfanes show chemopreventive effects against gastrointestinal tumors. We identified diallyl tetrasulfide and its derivative, dibenzyl tetrasulfide (DBTTS), to be mitotic inhibitors and apoptosis inducers. Here, we translate their application in colorectal cancer (CRC). MALDI-TOF-MS analysis identified both compounds as reversible tubulin binders, validated by in cellulo α-tubulin degradation. BRAF(V600E)-mutated HT-29 cells were resistant to DBTTS, as evidenced by mitotic arrest for 48 h prior to apoptosis induction compared to KRAS(G12V)-mutated SW480/620 cells, which committed to death earlier. The prolonged mitotic block correlated with autophagy impairment and p62 protein accumulation in HT-29 but not in SW480/620 cells, whereas siRNA-mediated p62 inhibition sensitized HT-29 cells to death. In silico analysis with 484 colorectal cancer patients associated higher p62 expression and reduced autophagic flux with greater overall survival. Accordingly, we hypothesized that DBTTS targets CRC survival/death through autophagy interference in cell types with differential autophagic capacities. We confirmed the therapeutic potential of DBTTS by the inhibition of spheroid and colony formation capacities in CRC cells, as well as in HT-29 zebrafish xenografts in vivo.

Immune checkpoint inhibitors (ICIs) are an expanding class of immunotherapeutic agents with the potential to cure cancer. Despite the outstanding clinical response in patient subsets, most individuals become refractory or develop resistance. Patient stratification and personalized immunotherapies are limited by the absence of predictive response markers. Recent findings show that dominant patterns of immune cell composition, T-cell status and heterogeneity, and spatiotemporal distribution of immune cells within the tumor microenvironment (TME) are becoming essential determinants of prognosis and therapeutic response. In this context, ICIs also function as investigational tools and proof of concept, allowing the validation of the identified mechanisms. After reviewing the current state of ICIs, this article will explore new comprehensive predictive markers for ICIs based on recent discoveries. We will discuss the recent establishment of a classification of TMEs into immune archetypes as a tool for personalized immune profiling, allowing patient stratification before ICI treatment. We will discuss the developing comprehension of T-cell diversity and its role in shaping the immune profile of patients. We describe the potential of strategies that score the mutual spatiotemporal modulation between T-cells and other cellular components of the TME. Additionally, we will provide an overview of a range of synthetic and naturally occurring or derived small molecules. We will compare compounds that were recently identified by in silico prediction to wet lab-validated drug candidates with the potential to function as ICIs and/or modulators of the cellular components of the TME.

Abstract: The pineal hormone melatonin (Mel), in addition to having a well‐established role as a regulator of circadian rhythms, modulates nonneural compartments by acting on specific plasma membrane receptors (MT1/MT2) present in many different cell types. Mel plays immunomodulatory roles and is an oncostatic and antiproliferative agent; this led to the widespread belief that Mel may induce or potentiate apoptosis on tumor cells, even though no clear indications have been presented so far. Here we report that Mel is not apoptogenic on U937 human monocytic cells, which are known to possess MT1 receptors at the times (up to 48 h) and doses (up to 1 mM) tested. Mel does not even potentiate apoptosis, but instead, significantly reduces apoptosis induced by both cell‐damaging agents (intrinsic pathway) and physiological means (extrinsic pathway). The doses required for the antiapoptotic effect (≥100 μM) are apparently not compatible with receptor stimulation (receptor affinity &lt;1 nM). However, receptor involvement cannot be ruled out, because we discovered that the actual Mel concentration active on cells was lower than the nominal one because of sequestration by fetal calf serum (FCS). Accordingly, in FCS‐free conditions, Mel doses required for a significant antiapoptotic effect are much lower.

Reactive oxygen species (ROS) are involved in many forms of apoptosis and mediate apoptosis in a number of cell types. In this paper, we use a variant of U937 monocytic cells (U937 HX) that show different biochemical features with respect to standard U937. Apoptotic standard U937 extrude reduced glutathione (GSH) and generate free radicals concomitantly with loss of mitochondria transmembrane potential (mt Deltapsi). These events are correlated with the extrusion of intracellular GSH. Conversely, apoptotic U937 HX cells retain GSH, and the loss of mt Deltapsi is not accompanied by generation of free radicals. The perfect inverse correlation between (a) ROS generation and (b) the presence of intracellular GSH during apoptosis suggests novel mechanisms to finely tune ROS generation in apoptosis.

Chemical/physical agents able to prevent apoptosis are receiving much attention for their potential health hazard as tumor promoters. Magnetic fields (MFs), which have been shown to increase the occurrence of some tumors, reduce damage-induced apoptosis by a mechanism involving Ca2+ entry into cells. In order to discover the mechanism of such effect of MFs, we investigated the interference of MFs on cell metabolism and analyzed cell parameters that are involved in apoptotic signaling and regulation of Ca2+ fluxes. Here we show that different types (static and extremely low-frequency, ELF pulsating) of MFs of different intensities alter plasma membrane potential. Interestingly, MFs induce plasma membrane hyperpolarization in cells sensitive to the antiapoptotic effect of MFs, whereas cells that are insensitive showed a plasma membrane depolarization. These opposite effects suggest that protection against apoptosis and membrane potential modulation are correlated, plasma membrane hyperpolarization possibly being part of the signal transduction chain determining MFs' antiapoptotic effect.

Aplysinopsins are a class of marine indole alkaloids that exhibit a wide range of biological activities. Although both the indole and N-benzyl moieties of aplysinopsins are known to possess antiproliferative activity against cancer cells, their mechanism of action remains unclear. Through in vitro and in vivo proliferation and viability screening of newly synthesized aplysinopsin analogs on myelogenous leukemia cell lines and zebrafish toxicity tests, as well as analysis of differential toxicity in noncancerous RPMI 1788 cells and PBMCs, we identified EE-84 as a promising novel drug candidate against chronic myeloid leukemia. This indole derivative demonstrated drug-likeness in agreement with Lipinski's rule of five. Furthermore, EE-84 induced a senescent-like phenotype in K562 cells in line with its cytostatic effect. EE-84-treated K562 cells underwent morphological changes in line with mitochondrial dysfunction concomitant with autophagy and ER stress induction. Finally, we demonstrated the synergistic cytotoxic effect of EE-84 with a BH3 mimetic, the Mcl-1 inhibitor A-1210477, against imatinib-sensitive and resistant K562 cells, highlighting the inhibition of antiapoptotic Bcl-2 proteins as a promising novel senolytic approach against chronic myeloid leukemia.

Abstract Background Despite advancements in chronic myeloid leukemia (CML) therapy with tyrosine kinase inhibitors (TKIs), resistance and intolerance remain significant challenges. Leukemia stem cells (LSCs) and TKI-resistant cells rely on altered mitochondrial metabolism and oxidative phosphorylation. Targeting rewired energy metabolism and inducing non-apoptotic cell death, along with the release of damage-associated molecular patterns (DAMPs), can enhance therapeutic strategies and immunogenic therapies against CML and prevent the emergence of TKI-resistant cells and LSC persistence. Methods Transcriptomic analysis was conducted using datasets of CML patients' stem cells and healthy cells. DNA damage was evaluated by fluorescent microscopy and flow cytometry. Cell death was assessed by trypan blue exclusion test, fluorescent microscopy, flow cytometry, colony formation assay, and in vivo Zebrafish xenografts. Energy metabolism was determined by measuring NAD + and NADH levels, ATP production rate by Seahorse analyzer, and intracellular ATP content. Mitochondrial fitness was estimated by measurements of mitochondrial membrane potential, ROS, and calcium accumulation by flow cytometry, and morphology was visualized by TEM. Bioinformatic analysis, real-time qPCR, western blotting, chemical reaction prediction, and molecular docking were utilized to identify the drug target. The immunogenic potential was assessed by high mobility group box (HMGB)1 ELISA assay, luciferase-based extracellular ATP assay, ectopic calreticulin expression by flow cytometry, and validated by phagocytosis assay, and in vivo vaccination assay using syngeneic C57BL/6 mice. Results Transcriptomic analysis identified metabolic alterations and DNA repair deficiency signatures in CML patients. CML patients exhibited enrichment in immune system, DNA repair, and metabolic pathways. The gene signature associated with BRCA mutated tumors was enriched in CML datasets, suggesting a deficiency in double-strand break repair pathways. Additionally, poly(ADP-ribose) polymerase (PARP)1 was significantly upregulated in CML patients’ stem cells compared to healthy counterparts. Consistent with the CML patient DNA repair signature, treatment with the methylated indolequinone MAC681 induced DNA damage, mitochondrial dysfunction, calcium homeostasis disruption, metabolic catastrophe, and necroptotic-like cell death. In parallel, MAC681 led to PARP1 degradation that was prevented by 3-aminobenzamide. MAC681-treated myeloid leukemia cells released DAMPs and demonstrated the potential to generate an immunogenic vaccine in C57BL/6 mice. MAC681 and asciminib exhibited synergistic effects in killing both imatinib-sensitive and -resistant CML, opening new therapeutic opportunities. Conclusions Overall, increasing the tumor mutational burden by PARP1 degradation and mitochondrial deregulation makes CML suitable for immunotherapy.

During apoptosis, an increase in cytosolic Ca(2+) concentration ([Ca(2+)](c)) accompanies the depletion of endoplasmic reticulum (ER). The actual roles of each of the two events in apoptosis are difficult to understand. In this work, we have modulated the basal [Ca(2+)](c) and the thapsigargin (THG)-dependent reticular flux (i.e., by chelating extracellular Ca(2+) or by modulating intracellular Ca(2+) by 3-aminobenzamide [3-ABA]). We have found that these treatments alter these Ca(2+) parameters in a differential way and, accordingly, affect apoptosis differentially. We have found that the increase in [Ca(2+)](c) is related to the extent of apoptosis, whereas the ER depletion affects the apoptotic nuclear morphology by shifting it towards the cleavage mode.

U937 monocytic cells show two main apoptotic nuclear morphologies, budding and cleavage, that are the result of two independent morphological routes, since they never interconvert one into the other, and are differently modulated by stressing or physiological apoptogenic agents [Exp Cell Res 1996; 223:340-347]. With the aim of understanding which biochemical alterations are at the basis of these alternative apoptotic morphologies, we performed an in situ analysis that showed that in U937 cells intracellular glutathione (GSH) is lost in cells undergoing apoptosis by cleavage, whereas it is maintained in apoptotic budding cells. Lymphoma cells BL41 lose GSH in apoptosis, and show the cleavage nuclear morphology; the same cells latently infected with Epstein Barr Virus (E2r line) undergo apoptosis without GSH depletion and show the budding nuclear morphology. GSH depletion is not only concomitant to, but is the determinant of the cleavage route, since the inhibition of apoptotic GSH efflux with cystathionine or methionine shifts the apoptotic morphology from cleavage to budding. Accordingly, cystathionine or methionine antagonizes apoptosis in the all-cleavage BL41, without affecting the all-budding E2r.

Lantana ukambensis (Vatke) Verdc. is an African food and medicinal plant. Its red fruits are eaten and highly appreciated by the rural population. This plant was extensively used in African folk medicinal traditions to treat chronic wounds but also as anti-leishmanial or cytotoxic remedies, especially in Burkina Faso, Tanzania, Kenya, or Ethiopia. This study investigates the in vitro bioactivity of polymethoxyflavones extracted from a L. ukambensis as anti-proliferative and pro-apoptotic agents. We isolated two known polymethoxyflavones, 5,6,7,3',4',5'-hexamethoxyflavone (1) and 5-hydroxy-6,7,3',4',5'-pentamethoxyflavone (2) from the whole plant of L. ukambensis. Their chemical structures were determined by spectroscopic analysis and comparison with published data. These molecules were tested for the anti-proliferative, cytotoxic and pro-apoptotic effects on human cancer cells. Among them, 5-hydroxy-6,7,3',4',5'-pentamethoxyflavone (2) was selectively cytotoxic against monocytic lymphoma (U937), acute T cell leukemia (Jurkat), and chronic myelogenous leukemia (K562) cell lines, but not against peripheral blood mononuclear cells (PBMCs) from healthy donors, at all tested concentrations. Moreover, this compound exhibited significant anti-proliferative and pro-apoptotic effects against U937 acute myelogenous leukemia cells. This study highlights the anti-proliferative and pro-apoptotic effects of 5-hydroxy-6,7,3',4',5'-pentamethoxyflavone (2) and provides a scientific basis of traditional use of L. ukambensis.

Treatment of acute myeloid leukemia (AML) remains inefficient due to drug resistance and relapse, particularly in patients with FMS-like tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD). Marine-derived natural products have recently been used for drug development against AML. We show in this study that petromurin C, which was isolated from the culture extract of the marine-derived fungus Aspergillus candidus KUFA0062, isolated from the marine sponge Epipolasis sp., induces early autophagy followed by apoptotic cell death via activation of the intrinsic cell death pathway concomitant with mitochondrial stress and downregulation of Mcl-1 in FLT3-ITD mutated MV4-11 cells. Moreover, petromurin C synergized with the clinically-used FLT3 inhibitor gilteritinib at sub-toxic concentrations. Altogether, our results provide preliminary indications that petromurin C provides anti-leukemic effects alone or in combination with gilteritinib.

We have previously shown that oxidative stress induced by an apoptogenic dose of H 2 O 2 leads to a temporary block of glycolytic flux via inactivation of the glycolytic key enzyme glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) in U937 cells. This corresponds to the activation of a cell defense pathway that is triggered to repair stress‐induced damage and to rescue cells from death. Here, we show that subapoptogenic doses of H 2 O 2 affect GAPDH activity in an opposite way, leading to strong hyperactivation. This phenomenon is related to milder oxidative stress because induction of a moderate oxidative stress with an alternative approach (i.e., by decreasing glutathione content in the cells with buthionine sulphoximine) gives similar results. U937 cells hyperactivate GAPDH with the same timing observed for GAPDH alterations from apoptogenic doses of H 2 O 2 . Additionally, the prevention of the glycolytic flux sensitizes stressed cells to apoptosis. This suggests that GAPDH hyperactivity might also be an active cell response to stress, thus depicting multiple roles for glycolytic flux in different prosurvival pathways where activation depends on the strength of the oxidative stress.

Allium plants, especially garlic (Allium sativum), have been cultivated since thousands of years all over the world not only as spicy food but also as medicinal plant. According to Block, the garlic plant was for the first time referred to in an Egyptian medical papyrus 1550 BC. In this Codex Ebers, 22 formulas of garlic were specified for the treatment of various disorders including heart problems, headache, bites, parasites and even tumors (Block, 1985). Today, the therapeutic value of garlic and other Allium vegetables is confirmed by multiple epidemiological and experimental studies. Especially prevention of cardiovascular diseases has been attributed to regular garlic consumption (Galeone et al., 2009; KrisEtherton et al., 2002; Rahman & Lowe, 2006). Moreover, cholesterol lowering, hypoglycemic, immune-stimulatory, anti-microbial and even anti-cancer properties have been reported for garlic compounds (Agarwal, 1996; Amagase et al., 2001; Balkwill & Mantovani, 2001; Borrelli et al., 2007; Goncagul & Ayaz, 2010; Kalra et al., 2006). Epidemiological studies clearly show the correlation between moderate garlic intake and a low cancer incidence (Galeone et al., 2006; Kim, J.Y. & Kwon, 2009; Salem et al., 2011). A case-control study conducted in the 1980s in Italy revealed for example that people living in high-risk areas for gastric cancer consumed less garlic compared to people in low-risk regions where stomach and colon cancers were three times less frequent (Buiatti et al., 1989). The health beneficial effects of garlic and other Allium species make this plant family an extremely interesting research topic.

Molecules targeting pro‐inflammatory pathways have demonstrated beneficial effects in cancer treatment. More recently, combination of natural and synthetic anti‐inflammatory drugs was suggested as an appealing strategy to inhibit tumor growth. Herein, we show that curcumin, a polyphenol from Curcuma longa and celecoxib induce apoptosis in hematopoietic cancer cell lines (Hel, Jurkat, K562, Raji, and U937). Further investigations on the most sensitive cell line, U937, indicated that these effects were tightly associated with an accumulation of the cells in S and G2/M for curcumin and in G0/G1 phase of cell cycle for celecoxib, respectively. The effect of celecoxib on cell cycle is associated with an induction of p27 and the down‐regulation of cyclin D1. However, in the case of combination experiments, the pretreatment of U937 cells with celecoxib at non‐apoptogenic concentrations counteracted curcumin‐induced apoptosis. We found that this effect correlated with the prevention of the accumulation in S and G2/M phase of cell cycle induced by curcumin. Similar results have been obtained when celecoxib and curcumin were co‐administrated at the same time. Overall our data suggest that this natural and synthetic drug combination is detrimental for cell death induction. © 2014 Wiley Periodicals, Inc.

Cancer development is mostly due to a deregulation of cell death as cancer cells become resistant to apoptosis by increasing expression of anti-apoptotic proteins belonging to the Bcl-2 family. Mcl-1 is one anti-apoptotic protein, which is mainly responsible for cancer cell resistance as it is overexpressed by most cancer cell types. Many research projects aim to restore cancer cell death by using natural pharmacological scaffolds targeting anti-apoptotic proteins to inhibit their effect in cancer development. This review introduces natural compound inhibitors of the Bcl-2 protein family with a focus on Mcl-1.

The ability of magnetic fields (MFs) to promote/increase Ca(2+) influx into cells is widely recognized, but the underlying mechanisms remain obscure. Here we analyze how static MFs of 6 mT modulates thapsigargin-induced Ca(2+) movements in non-excitable U937 monocytes, and how this relates to the anti-apoptotic effect of MFs. Magnetic fields do not affect thapsigargin-induced Ca(2+) mobilization from endoplasmic reticulum, but significantly increase the resulting Ca(2+) influx; this increase requires intracellular signal transduction actors including G protein, phospholipase C, diacylglycerol lipase and nitric oxide synthase, and behaves as a non-capacitative Ca(2+) entry (NCCE), a type of influx with an inherent signaling function, rather than a capacitative Ca(2+) entry (CCE). All treatments abrogating the extra Ca(2+) influx also abrogate the anti-apoptotic effect of MFs, demonstrating that MF-induced NCCE elicits an anti-apoptotic survival pathway.

Palytoxin is considered one of the most potent biotoxins. As palytoxin-induced cancer cell death mechanisms remain to be elucidated, we investigated this effect on various leukemia and solid tumor cell lines at low picomolar concentrations. As palytoxin did not affect the viability of peripheral blood mononuclear cells (PBMC) from healthy donors and did not create systemic toxicity in zebrafish, we confirmed excellent differential toxicity. Cell death was characterized by a multi-parametric approach involving the detection of nuclear condensation and caspase activation assays. zVAD-sensitive apoptotic cell death was concomitant with a dose-dependent downregulation of antiapoptotic Bcl-2 family proteins Mcl-1 and Bcl-xL. Proteasome inhibitor MG-132 prevented the proteolysis of Mcl-1, whereas the three major proteasomal enzymatic activities were upregulated by palytoxin. Palytoxin-induced dephosphorylation of Bcl-2 further exacerbated the proapoptotic effect of Mcl-1 and Bcl-xL degradation in a range of leukemia cell lines. As okadaic acid rescued cell death triggered by palytoxin, protein phosphatase (PP)2A was involved in Bcl-2 dephosphorylation and induction of apoptosis by palytoxin. At a translational level, palytoxin abrogated the colony formation capacity of leukemia cell types. Moreover, palytoxin abrogated tumor formation in a zebrafish xenograft assay at concentrations between 10 and 30 pM. Altogether, we provide evidence of the role of palytoxin as a very potent and promising anti-leukemic agent, acting at low picomolar concentrations in cellulo and in vivo.

Antioxidants-that is, scavengers of free radicals and anaerobic conditions (5%CO(2), 95% N(2))-protect monocytic U937 cells from damage-induced apoptosis. Antioxidants rescue the cells acting on the apoptotic pathway at a step downstream from gluthatione extrusion. Reducing agents, such as DTT, also reduce stress-induced apoptosis. Thus, apoptotic GSH extrusion triggers the downstream events of apoptosis by leaving cells unprotected against thiol\s oxidation and radical production.

Acacia macrostachya is used in Burkina Faso folk medicine for the treatment of inflammation and cancer. The purpose of this study was to evaluate the antioxidant and cytotoxic effects of this plant. The cytotoxic effects of root (dichloromethane B1 and methanol B2) and stem (dichloromethane B3 and methanol B4) bark extracts of A. macrostachya were assessed on chronic K562 and acute U937 myeloid leukemia cancer cells using trypan blue, Hoechst, and MitoTracker Red staining methods. The antioxidant content of extracts was evaluated using DPPH (2,2-diphenyl-1-picryl-hydrazyl) and FRAP (ferric reducing antioxidant power) methods. The root bark extracts B1 and B2 of A. macrostachya demonstrated higher cytotoxicity with IC50 values in a low µg/mL range on both U937 and K562 cells, while the stem bark B4 extract selectively affected U937 cells. Overall, healthy proliferating peripheral blood mononuclear cells (pPBMCs) were not or barely impacted in the range of concentrations cytotoxic to cancer cells. In addition, A. macrostachya exhibited significant antioxidant content with 646.06 and 428.08 µg ET/mg of extract for the B4 and B2 extracts, respectively. Phytochemical screening showed the presence of flavonoids, tannins, alkaloids, and terpenoids/steroids. The results of this study highlight the interest of A. macrostachya extracts for the isolation of anticancer molecules.

Abstract: Tumor cells often develop molecular strategies for survival to anoxia/reoxygenation stress as part of tumor progression. Here we describe that the B lymphoma Epstein–Barr‐positive cells E2r survive reoxygenation in spite of a very high and long‐lasting increase in cytosolic Ca 2+ and the loss of about half of their mitochondria due to specific extrusion of the organelles from the cells. The extrusion typically occurs 3 days after reoxygenation, and a regular mitochondrial asset is regained after further 24 h.

Altered metabolism represents one of the oldest hallmarks associated with cancer. The aberrant metabolic profile deals primarily with the well-known switch of transformed cells towards aerobic glycolysis from mitochondrial oxidative phosphorylation. In addition, it deals with the exacerbation of several biosynthetic pathways interconnected with the increased glycolytic flux, eventually conferring selective metabolic advantages. The distinctive and ubiquitous nature of this altered metabolic profile expressed by cancer cells from various origins or tissues turns the modulation/reversion of these aberrations as an amenable strategy for new anticancer therapies. Despite Otto Warburg's pioneering observations, targeting cancer cell metabolism for therapeutic purposes still remains theoretical. Several factors have contributed to this situation. Primarily, for many years, cancer has been treated as a homogeneous and too simplistic cellular system, where the impairment of proliferation/viability of the most abundant differentiated cancer cells has been extensively pursued. This approach, however, has not been successful in preventing tumour relapse. Cancer is nowadays considered a complex network, including cancer cells at different stages of differentiation, as well as noncancer cells from the tumour microenvironment. These cells exert specific functions further sustaining cancer progression, by maintaining a proinflammatory environment, by inducing activation of angiogenesis and by evading immune responses. The cellular heterogeneity implies more subtle forms of mutual interaction and communication among different cells and with the microenvironment, which may be the niche for future innovative anticancer therapeutic interventions. In this renewed view of cancer etiology, unexpected crosstalk is emerging between metabolic mediators/processes and pathological alterations. This special issue focuses on “Metabolism and cancer: old and new players” and intends to stimulate the discussion about new emerging targetable mediators of cancer metabolism and to evaluate possible noncanonical roles of old players in tumorigenesis as well as in cancer metabolism. The impairment of cancer cell viability by induction of cell death remains the final outcome of many anticancer strategies. Besides apoptosis, other modes of cell death have been defined and are attracting interest as alternative therapeutically exploitable approaches to impact apoptosis-resistant forms of cancer. The review by S. Fulda, “Alternative cell death pathways and cell metabolism,” draws attention to the programmed form of necrosis, namely, necroptosis, and discusses the relevance of metabolic pathways involved in its modulation. The author suggests a possible interconnection between alterations of redox signaling pathways mediating necroptosis and mitochondrial impairment, which implies the latter as a crucial regulator of redox imbalance. An ideal anticancer therapy should be able to efficiently target cancer stem cells. This requires the discovery of markers selectively identifying malignant stem cells. The review by C. Pecqueur et al., “Targeting metabolism to induce cell death in cancer cells and cancer stem cells,” addresses the relationship between cancer cell metabolism and evasion from apoptosis by comparing the main features of cancer cells and cancer stem cells. The authors correlate the peculiar metabolic characteristics of these two types of cancer cells with their ability to evade apoptosis. Over the last 10 years, noncanonical roles for many factors implicated in apoptosis were published. The family of B-cell lymphoma-2 (Bcl-2) proteins represents one of the most interesting examples, with several members exhibiting essentially pro- and apoptotic functions. The paper by J. Michels et al., “Functions of Bcl-xL at the interface between cell death and metabolism,” reviews new and intriguing properties of the Bcl-2 homolog Bcl-xL (B-cell lymphoma extra-large) linked to regulation of bioenergetic metabolism, which in turn controls important processes including mitochondrial ATP synthesis, Ca2+ flux, autophagy, mitosis, and protein acetylation. Cancer cells rely on glycolysis rather than on oxidative phosphorylation to fulfill their energy needs. In conditions of glucose deprivation, however, cancer cells may reactivate mitochondrial bioenergetics as part of a prosurvival strategy. The study by R. Palorini et al., “Mitochondrial complex I inhibitors and forced oxidative phosphorylation synergize in inducing cancer cell death,” shows that mitochondrial complex I inhibitors sensitize cancer cells to cell death under glucose depletion. Interestingly, combined treatments affecting glycolysis and leading to mitochondria impairment are ineffective on immortalized cells or cancer cells cultivated under high glucose conditions. Their results suggest that the forced switch from glycolysis to oxidative phosphorylation combined with the use of mitochondrial inhibitors may be used as alternative therapeutic approach to increase the sensitivity of cancer cells to death. Identification of specific metabolic intermediates as targets for future anticancer treatments may be at the basis for attempting a reversal of tumor metabolism to normal conditions, with the rationale of impairing cancer growth. Pyruvate kinase M2 (PKM2) catalyzing the final rate-limiting reaction of glycolysis currently attracts much interest because of its multiple emerging intracellular functions. The review by N. Wong et al., “PKM2, a central point of regulation in cancer metabolism,” gives an overview of our current knowledge about this important enzyme, discussing several of its potential contributions to tumorigenesis. A limiting factor in exploiting cancer metabolism for therapeutic purposes is the lack of availability of agents acting as specific modulators of cancer metabolism. The review by C. Cerella et al., “Natural compounds as regulators of the cancer cell metabolism,” covers this aspect, focusing on natural compounds as untapped potential regulators of cancer cell metabolism. The authors discuss some of the most important pathways and factors implicated in altered cancer metabolism and give an overview of compounds extracted from natural sources potentially targeting these different aberrantly regulated metabolic key intermediates. The link between obesity, insulin resistance, and cancer is explored from different sides in three papers of this special issue, interestingly dealing all with hormonal forms of cancer. The paper by C. Brosseau et al., “Role of insulin-like growth factor binding protein-3 in 1, 25-dihydroxyvitamin-D3-induced breast cancer cell apoptosis,” aims at identifying the mechanisms involved in 1, 25-dihydroxyvitamin D3-induced apoptosis in breast cancer cells. Their strategy suggests a role for insulin-like growth factor binding protein-3 (IGFBP-3) in vitamin D-induced apoptotic signaling and the involvement of impaired secretion of IGFBP-3 in acquired chemoresistance. The review by J. S. Byun and K. Gardner, “C-terminal binding protein: a molecular link between metabolic imbalance and epigenetic regulation in breast cancer,” introduces the reader to the concept of metabolic transduction by stressing the link between shifts in carbohydrate metabolism and alterations in epigenetic regulatory mechanisms. In particular, these authors investigate the family of transcriptional repressors called C-terminal binding proteins (CtBPs), whose function is controlled by NAD+ levels, as an example of factors interacting and modulating the activity of different histone deacetylases (HDACs) depending on the metabolic status. Finally, the review by J. H. Gunter et al., “New players for advanced prostate cancer and the rationalisation of insulin-sensitising medication,” points to the roles played by metabolic disorders (especially related to insulin resistance) in favoring anticancer treatment failure and consequent higher cancer-specific mortality. The authors discuss the promising use of insulin-sensitizing drugs as potential anticancer agents to be used in combinational therapies. Although alterations in cellular metabolism are an old acquaintance in cancer etiology, we still need to do more before we can translate our knowledge into new and specific anticancer therapies. This final outcome will mostly depend on an in-depth identification of the mutual modulation between metabolism processes and factors that regulate proliferation and death/survival of cancer cells. With this special issue, we hope to offer new and relevant insights towards a critical reevaluation of this rapidly moving field. Claudia Cerella Carine Michiels Roderick H. Dashwood Young-Joon Surh Marc Diederich

Abstract: In U937 monocytic cells induced to apoptosis, plasma membrane blebbing of different intensities appears, before the development of nuclear alterations; this latter phenomenon can occur through two major pathways, namely the cleavage and the budding mode (Dini et al ., 1996). Strongly blebbing cells develop deep nuclear constrictions leading to nuclear fragmentation according to the cleavage mode, while cells with milder forms of blebbing, or no blebbing at all, undergo nuclear fragmentation along the budding mode. Compounds interfering with different cytoskeletal components affect blebbing, which is completely inhibited by the actin polymerization inhibitors, cytochalasins, while disturbance of tubulin network with taxol limits blebbing to milder forms. At the same time, the cytoskeletal poisons affect the type of nuclear fragmentation, abolishing the cleavage mode, shifting all events into the budding pathway. Adherent cells, which possess a more structured cytoskeleton, do not develop strong blebs and undergo nuclear fragmentation via budding. These observations suggest that the deep cytoskeletal movements that cause the strongest forms of plasma membrane blebbing strangle the nucleus, leading to the constrictions that later evolve into nuclear fragmentation by cleavage. The trigger for the cytoskeletal movements, known to be redox‐sensitive, is probably the apoptotic GSH extrusion.

Abstract: Many studies suggest that endoplasmic reticulum (ER) Ca 2+ pool rather than cytosolic Ca 2+ may play a crucial role in triggering apoptosis. In this study, we performed an image analysis of cells loaded with the fluorescent dye chlortetracycline (CTC) to in situ analyze Ca 2+ changes within the ER in apoptosing promonocytic U937 cells. The results, validated through the use of thapsigargin (THG) as ER Ca 2+ depletor, confirm the findings that apoptotic cells have a Ca 2+ ‐depleted ER, in contrast with treated but still viable cells.

15-Deoxy-∆12,14-prostaglandin J2 (15d‑PGJ2) is a natural agonist of peroxisome proliferator-activated receptor γ (PPARγ) that displays anticancer activity. Various studies have indicated that the effects of 15d‑PGJ2 are due to both PPARγ-dependent and -independent mechanisms. In the present study, we examined the effects of a biotinylated form of 15d‑PGJ2 (b‑15d‑PGJ2) on hormone-dependent MCF‑7 and triple‑negative MDA‑MB‑231 breast cancer cell lines. b‑15d‑PGJ2 inhibited cell proliferation more efficiently than 15d‑PGJ2 or the synthetic PPARγ agonist, efatutazone. b‑15d‑PGJ2 was also more potent than its non-biotinylated counterpart in inducing apoptosis. We then analyzed the mechanisms underlying this improved efficiency. It was found not to be the result of biotin receptor-mediated increased incorporation, since free biotin in the culture medium did not decrease the anti-proliferative activity of b‑15d‑PGJ2 in competition assays. Of note, b‑15d‑PGJ2 displayed an improved PPARγ agonist activity, as measured by transactivation experiments. Molecular docking analyses revealed a similar insertion of b‑15d‑PGJ2 and 15d‑PGJ2 into the ligand binding domain of PPARγ via a covalent bond with Cys285. Finally, PPARγ silencing markedly decreased the cleavage of the apoptotic markers, poly(ADP-ribose) polymerase 1 (PARP‑1) and caspase‑7, that usually occurs following b‑15d‑PGJ2 treatment. Taken together, our data indicate that biotinylation enhances the anti-proliferative and pro-apoptotic activity of 15d‑PGJ2, and that this effect is partly mediated via a PPARγ-dependent pathway. These results may aid in the development of novel therapeutic strategies for breast cancer treatment.

Abstract: In many cell systems, pharmacological glutathione (GSH) depletion with the GSH neosynthesis inhibitor buthionine sulfoximine (BSO) leads to cell death and highly sensitizes tumor cells to apoptosis induced by standard chemotherapeutic agents. However, some tumor cells upregulate Bcl‐2 in response to BSO, thus surviving the treatment and failing to be chemosensitized. Cell lines of monocytic and lymphocytic origins respond to BSO treatment in an opposite way, lymphocytes being chemosensitized and unable to transactivate Bcl‐2. In this article we investigate the response to BSO of lymphocytes freshly isolated from peripheral blood of healthy donors. After ensuring that standard separation procedures do not alter per se lymphocytes redox equilibrium nor Bcl‐2 levels in the first 24 h of culture, we show that BSO treatment promotes the upregulation of Bcl‐2, with a mechanism involving the increased radical production consequent to GSH depletion. Thus, BSO treatment may increase the differential cytocidal effect of cytotoxic drugs in tumor versus normal lymphocytes.

Abstract: Thapsigargin (THG), a selective inhibitor of endoplasmic reticulum (ER) Ca 2+ ‐ATPases, causes the rapid emptying of ER Ca 2+ ; in some cell types, this is accompanied by apoptosis, whereas other cells maintain viability. In order to understand the molecular determinants of such a different behavior, we explored the role of oxygen versus nitrogen radicals, by analyzing the apoptogenic ability of THG in the presence of inhibitors of glutathione or nitric oxide (NO) synthesis, respectively. We observed that oxygen radicals play a sensitizing role whereas nitrogen radicals prevent THG‐dependent apoptosis, showing that the apoptogenic effect of THG is redox sensitive.

Abstract: ADP‐ribosylations are reversible posttranslational modifications that regulate the activity of target proteins, catalyzed by two different classes of enzymes, namely poly(ADP‐ribosyl)polymerases (PARPs) and mono(ADP‐ribosyl)transferases (ADPRTs). It is now emerging that ADP‐ribosylation reactions control signal transduction pathways, mostly as a response to cell damage, aimed at both cell repair and apoptosis. Inhibition of ADPRTs, but not PARPs, increases the extent of apoptosis induced by cytocidal treatments, at the same time delaying secondary necrosis, the process leading to plasma membrane collapse in apoptotic cells, and responsible for apoptosis‐related inflammation in vivo . Thus, ADPRT inhibitors may be ideal as adjuvants to cytocidal therapies; to this purpose, we investigated the molecular determinant(s) for such effects by probing a set of molecules with similar structures. We found that the apoptosis‐modulating effects were mimicked by those compounds possessing an amidic group in the same position as two of the most popular ADPRT inhibitors, namely, 3‐aminobenzamide and nicotinamide. This study may provide useful suggestions in designing molecules with therapeutic potential to be used as adjuvant in cytocidal therapies.

Myelomonocytic and monocytic acute myeloid leukemia (AML) subtypes are intrinsically resistant to venetoclax-based regimens. Identifying targetable vulnerabilities would limit resistance and relapse. We previously documented the synergism of venetoclax and cardiac glycoside (CG) combination in AML. Despite preclinical evidence, the repurposing of cardiac glycosides (CGs) in cancer therapy remained unsuccessful due to a lack of predictive biomarkers. We report that the ex vivo response of AML patient blasts and the in vitro sensitivity of established cell lines to the hemi-synthetic CG UNBS1450 correlates with the ATPase Na+/K+ transporting subunit alpha 1 (ATP1A1)/BCL2 like 1 (BCL2L1) expression ratio. Publicly available AML datasets identify myelomonocytic/monocytic differentiation as the most robust prognostic feature, along with core-binding factor subunit beta (CBFB), lysine methyltransferase 2A (KMT2A) rearrangements, and missense Fms-related receptor tyrosine kinase 3 (FLT3) mutations. Mechanistically, BCL2L1 protects from cell death commitment induced by the CG-mediated stepwise triggering of ionic perturbation, protein synthesis inhibition, and MCL1 downregulation. In vivo, CGs showed an overall tolerable profile while impacting tumor growth with an effect ranging from tumor growth inhibition to regression. These findings suggest a predictive marker for CG repurposing in specific AML subtypes.

Despite the efforts of scientific research, cancer remains one of the dreaded diseases in the world. Resistance to chemotherapy has forced researchers to investigate medicinal plants in the search for new anticancer drugs. Our project is part of this dynamic by studying Lantana ukambensis, plant used in Burkina' traditional medicine for the treatment of old wounds. The objective of our study was to isolate anticancer molecules from this plant using bioguided fractionation method and to study their pharmacological properties of interest for cancer treatment. Maceration of shred leaves of L. ukambensis was done and the extract was fractionated by HPLC. The isolated compounds were then tested on K562 cancer cells and healthy cells PBMC using trypan blue test to evaluate the selectivity of their toxicity. Finally inhibition of NF-κB pathway was tested to find a probable anti-inflammatory effect of these molecules. L. ukambensis exhibits a strong antiproliferative effect against K562 cells. Two HPLC fractions (F10 and F12) were found to be cytotoxic against K562 at 5 µg/mL. Four sub-fractions were isolated from F10 and two others from F12. The cytotoxic effect of isolated sub-fractions against healthy cells (PBMC) is found to be low, compared to that on cancer cells (K562), meaning that there is specificity in the toxicity of these compounds. The NF-κB inhibition assay showed that the isolated compounds exhibit high inhibition effect on NF-κB pathway except compound F10C that showed a moderate activity. Phytochemical analyses are ongoing in order to determine the chemical structure of each isolated compounds.

Nanotubes have a great therapeutic potential due to their astounding physico-chemical features, the possibility to be funtionalised for ad hoc uses, and the specific interaction of nanotubes as such with life molecules (DNA and proteins). These features recommend a thorough toxicological study before widespread pharmaceutic use. We provide evidence that culture cells with phagocytic potential internalise multi wall nanotubes (10-50 nm average size). This is not accompanied by cytotoxicity in terms of induction of &apoptosis or necrosis at the doses used (up to 125 microg/mI).

Small-molecule drugs that induce apoptosis in tumor cells by activation of the BCL-2-regulated mitochondrial outer membrane permeabilization (MOMP) pathway hold promise for rational anticancer therapies. Accumulating evidence indicates that the natural product gossypol and its derivatives can kill tumor cells by targeting antiapoptotic BCL-2 family members in such a manner as to trigger MOMP. However, due to the inherent complexity of the cellular apoptotic network, the precise mechanisms by which interactions between gossypol and individual BCL-2 family members lead to MOMP remain poorly understood. Here, we used simplified systems bearing physiological relevance to examine the impact of gossypol on the function of MCL-1, a key determinant for survival of various human malignancies that has become a highly attractive target for anticancer drug design. First, using a reconstituted liposomal system that recapitulates basic aspects of the BCL-2-regulated MOMP pathway, we demonstrate that MCL-1 inhibits BAX permeabilizing function via a “dual-interaction” mechanism, while submicromolar concentrations of gossypol reverse MCL-1-mediated inhibition of functional BAX activation. Solution-based studies showed that gossypol competes with BAX/BID BH3 ligands for binding to MCL-1 hydrophobic groove, thereby providing with a mechanistic explanation for how gossypol restores BAX permeabilizing function in the presence of MCL-1. By contrast, no evidence was found indicating that gossypol transforms MCL-1 into a BAX-like pore-forming molecule. Altogether, our findings validate MCL-1 as a direct target of gossypol, and highlight that making this antiapoptotic protein unable to inhibit BAX-driven MOMP may represent one important mechanism by which gossypol exerts its cytotoxic effect in selected cancer cells.

Introduction Colon cancer is a major cause of morbidity and mortality worldwide. Epidemiological studies revealed an inverse correlation between colon cancer risk and a garlic-rich diet. Natural organosulfur compounds confer protective effects against a wide range of cancer, including colon. Here, we studied the anti-colon cancer activity of garlic-derived diallyl/dibenzyl tetrasulfur (DATTS/DBTTS) derivatives. Material and methods We validated the ability of DATTS/DBTTS to bind tubulin by MALDI-TOF mass spectrometry and its disruptive effect on the microtubule network by immunofluorescence. We selected cell lines with a defined genetic background including HT-29 (BRAF-mutation), SW480 and metastatic SW620 (both KRAS-mutation). Cell cycle analysis, Hoechst staining and western blots allowed to assess cell death. To further validate the anti-cancer activity of DBTTS, colony and spheroid formation assays as well as zebrafish xenografts were realised. To monitor autophagy, western blots, GFP-LC3 plasmid transfection and transmission electron microscopy (TEM) were conducted. Results and discussions All selected cell models were more sensitive to DBTTS than to DATTS. SW480 and SW620 were more susceptible to DBTTS than HT-29 cells. DBTTS induced mitotic arrest followed by cell death. Its anti-cancer activity was validated in 3D cell culture systems and in vivo. DATTS/DBTTS acted as a direct and reversible tubulin binder inducing microtubule disarrangements in cellulo. As tubulin alterations may affect autophagy progression, we evaluated the effect of DBTTS on autophagy. DBTTS induced LC3-II and p62 protein accumulation concomitantly with mitotic arrest in HT-29 but not in SW480 and SW620 cells. TEM analysis showed accumulation of pre-fusion complexes (phagophores and autophagosomes) indicating inhibition of the autophagic flux. Autophagy inhibitor bafilomycin A1 confirmed the impairment of the autophagy flux by DBTTS in HT-29 cells. Immunofluorescence revealed p62 protein accumulation showing a dotted pattern, similar to the LC3-II puncta formation in HT-29 cells. Furthermore, silencing of p62 protein exacerbated cell death indicating a pro-survival role of p62 overexpression in DBTTS-treated HT-29 cells. Conclusion Altogether, we showed here that DBTTS acts as an anti-cancer agent by targeting tubulin. These results suggest that DBTTS targets colon cancer survival/death through autophagy interference depending on cell types with differential autophagy capacities and genetic signatures.

first_page settings Order Article Reprints Font Type: Arial Georgia Verdana Font Size: Aa Aa Aa Line Spacing:    Column Width:    Background: Open AccessExtended Abstract Targeted Anticancer Strategies with Garlic Derivatives † by Claudia Cerella 1, Esma Yagdi Efe 1, Mario Dicato 1 and Marc Diederich 2,* 1 Laboratoire de Biologie Moléculaire et Cellulaire du Cancer (LBMCC), Hôpital Kirchberg, 9 Rue Edward Steichen, 2540 Luxemburg, Luxemburg 2 College of Pharmacy, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-742, Korea * Author to whom correspondence should be addressed. † Presented at the Natural Products and the Hallmarks of Chronic Diseases—COST Action 16112, Luxemburg, 25–27 March 2019. Proceedings 2019, 11(1), 29; https://doi.org/10.3390/proceedings2019011029 Published: 19 April 2019 (This article belongs to the Proceedings of CA16112 - Luxemburg 2019) Download Download PDF Versions Notes Excerpt Note: In lieu of an abstract, this is an excerpt from the first page. Diallyl polysulfides from edible plants have been widely investigated in cancer research holding the promise of a translational application. [...] Keywords: Diallyl compounds; tubulin; Bcl-2; autophagic flux; apoptosis Diallyl compounds; tubulin; Bcl-2; autophagic flux; apoptosis Share and Cite MDPI and ACS Style Cerella, C.; Efe, E.Y.; Dicato, M.; Diederich, M. Targeted Anticancer Strategies with Garlic Derivatives. Proceedings 2019, 11, 29. https://doi.org/10.3390/proceedings2019011029 AMA Style Cerella C, Efe EY, Dicato M, Diederich M. Targeted Anticancer Strategies with Garlic Derivatives. Proceedings. 2019; 11(1):29. https://doi.org/10.3390/proceedings2019011029 Chicago/Turabian Style Cerella, Claudia, Esma Yagdi Efe, Mario Dicato, and Marc Diederich. 2019. "Targeted Anticancer Strategies with Garlic Derivatives" Proceedings 11, no. 1: 29. https://doi.org/10.3390/proceedings2019011029 Find Other Styles Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here. Article Metrics No No Article Access Statistics Multiple requests from the same IP address are counted as one view.

Les neuroblastomes (NBs) chez l’enfant développent une chimiorésistance largement due à l’autophagie contrôlée qui permet d’éliminer les mitochondries endommagées (mitophagie) et de diminuer le stress oxydatif induit par la chimiothérapie [1]. Nous avons exploré les effets in vitro d’un glycoside hémi-synthétique (UNBS1450) sur le flux auto (mito) phagique et l’induction de mort cellulaire en deux lignes cellulaires issues de NBs. Les effets de l’UNBS1450 sur les lignes cellulaires SH-SY5Y et SK-N-AS ont été étudiés par les colorations DAPI, iodure de propidium (mort cellulaire) ; ‹‹ LysotrackerRed ›› (masse lysosomale) et ‹‹ MitotrackerRed ›› (masse mitochondriale). La colocalisation des mitochondries et des autophagosomes ont été étudiées dans les cellules transfectées avec GFP-LC3 (marqueur des membranes autophagosomales) et colorées par ‹‹ MitotrackerRed ›› et par microscopie électronique à transmission. Ces données morphologiques quantitatives obtenues dans l’intervalle 0–24 heures du traitement ont été corrélées avec la dynamique d’activation des caspases 3 et 7, les cathepsines B et L et les dérivées actifs d’oxygène. Dans la lignée SH-SY5Y, l’UNBS1450 a provoqué la déstabilisation des membranes lysosomales, le retard de la formation des autolysosomes et de l’élimination des mitochondries ; ce retard du flux autophagosomique a eu pour résultat ultime l’apoptose des cellules. Aucun de ces effets sur l’autophagie n’a été constate dans la lignée SK-N-AS ; toutefois, ces cellules mourraient par nécroptose et nécrose après 12–18 heures de traitement. Nous avons montré 2 effets cytotoxiques distincts du glycoside UNBS1450 : inhibition du flux autophagique et induction de nécroptose. Le premier et d’un intérêt particulier car il suggère la possibilité de subvertir un des mécanismes de la chimiorésistance des NBs, la mitophagie contrôlée, mais le 2 sont nouveaux : testé sur plusieurs types de cellules, l’UNBS1450 a invariablement montré un effet proapoptotique médié par l’inactivation de la protéine Myeloid Cell Leukemia-1 (Mcl-1) [2], [3].

Tumor promonocytic U937 cells cultured under a low O(2)/high CO(2) atmosphere display altered characteristics after restoration of normal atmosphere: increased resistance to apoptosis induced by different treatments; apoptotic morphology; lack of glutathione (GSH) extrusion in apoptosis; lack of protection by antioxidants; and lack of Ca(2+) mobilization with thapsigargin. These alterations were stably maintained for many months of culture in normal conditions, originating the stable U937-HX variant. Since the hypoxic treatment did not produce a great selective pressure, the alterations are conceivably the result of stable adaptative response.

Background: The development of de novo and acquired resistance to anticancer therapies and the absence of targeted therapy for some types of tumors are strong motivations for discovering new therapeutic agents. Thiazolidinediones (TZD) are studied in this context although they were initially designed for the treatment of type II diabetes due to their PPAR gamma agonist activity. They display anticancer effects that are independent of PPAR gamma, but that are not well understood. In prostate cancer cells, TZD derivatives were shown to act as energy restriction mimetic agents. Our team studies the effects of delta2 troglitazone (delta2-TGZ), a TZD devoid of PPAR gamma agonist activity, on breast cancer cell lines. The aim of this work was to better characterize delta2-TGZ-induced cell death and to understand the underlying molecular events.Material and Methods: Delta2-TGZ was obtained by chemical synthesis. We used hormone-dependent (MCF-7) and hormone-independent (MDA-MB-231) breast cancer cell lines. Gene expression was studied by RT-PCR, western blotting and immunocytochemistry. RNA interference was used for gene silencing.Results and discussion: Delta2-TGZ-induced endoplasmic reticulum (ER) stress in MCF-7 cells as shown by phosphorylation of Pancreatic endoplasmic reticulum kinase-like Endoplasmic Reticulum Kinase (PERK) detected after 1.5 hours, splicing of XBP1 (X-box binding protein 1) after 3 hours, accumulation of the chaperone BiP (Binding immunogloblulin protein) and the pro-apoptotic protein CHOP (CCAAT-enhancer-binding protein homologous protein) after 6 hours. CHOP was located in the nucleus of treated cells. Similar events were observed in MDA-MB-231 cells exposed to delta2-TGZ. In both cell lines cleavage of PARP-1 and caspase-7 revealed apoptosis. In MCF-7 cells, knock-down of CHOP or inhibition of c-Jun N-terminal kinase (JNK) did not impair apoptosis. Preliminary results also indicated that AMP-dependent kinase (AMPK) is activated early in MCF-7 cells after delta2-TGZ treatment. Conclusions: This work contributes to a better understanding of the PPARgamma-independent effects of TZD in breast cancer cells. ER stress is an early response to delta2-TGZ, occurring prior to, but not causative of apoptosis. We have now to confirm whether AMPK phosphorylation is a marker of the energy restriction mimetic action of delta2-TGZ and if this is responsible for ER stress and apoptosis.

The anticancer effects of thiazolidinediones appear to be independent of their PPAR gamma agonist activity but the molecular mechanisms involved in the anticancer action are not well understood. Results obtained on Ciglitazone derivatives, mainly in prostate cancer cells, suggest that these compounds could act as energy restriction mimetic agents. Our team study the effects of delta 2 troglitazone, a thiazolidinedione devoid of PPAR gamma agonist activity, on breast cancer cell lines. The aim of this work was to better characterize the effects of this compound in terms of apoptosis, ER stress and energy restriction, three events that are observed in response to CGZ derivatives. Our studies performed on two breast cancer cell lines MDA-MB-231 (hormone-independent) and MCF7 (hormone-dependent) show that delta 2-troglitazone triggers ER stress prior to apoptosis but that the two events occur independently. Preliminary results also indicate that the AMP-dependent kinase is activated early. We have now to determine if this is the result of an energy restriction mimetic action.

Abstract isolation and structure elucidation of oximoaspergillimide (I)

ChemInformVolume 46, Issue 44 Natural Products ChemInform Abstract: Tanzawaic Acids Isolated from a Marine-Derived fungus of the Genus Penicillium with Cytotoxic Activities. Faviola Cardoso-Martinez, Faviola Cardoso-Martinez Inst. Prod. Nat. Agrobiol., CSIC, Univ. La Laguna, E-38206 La Laguna, Tenerife, SpainSearch for more papers by this authorJose M. de la Rosa, Jose M. de la Rosa Inst. Prod. Nat. Agrobiol., CSIC, Univ. La Laguna, E-38206 La Laguna, Tenerife, SpainSearch for more papers by this authorAna R. Diaz-Marrero, Ana R. Diaz-Marrero Inst. Prod. Nat. Agrobiol., CSIC, Univ. La Laguna, E-38206 La Laguna, Tenerife, SpainSearch for more papers by this authorJose Darias, Jose Darias Inst. Prod. Nat. Agrobiol., CSIC, Univ. La Laguna, E-38206 La Laguna, Tenerife, SpainSearch for more papers by this authorClaudia Cerella, Claudia Cerella Inst. Prod. Nat. Agrobiol., CSIC, Univ. La Laguna, E-38206 La Laguna, Tenerife, SpainSearch for more papers by this authorMarc Diederich, Marc Diederich Inst. Prod. Nat. Agrobiol., CSIC, Univ. La Laguna, E-38206 La Laguna, Tenerife, SpainSearch for more papers by this authorMercedes Cueto, Mercedes Cueto Inst. Prod. Nat. Agrobiol., CSIC, Univ. La Laguna, E-38206 La Laguna, Tenerife, SpainSearch for more papers by this author Faviola Cardoso-Martinez, Faviola Cardoso-Martinez Inst. Prod. Nat. Agrobiol., CSIC, Univ. La Laguna, E-38206 La Laguna, Tenerife, SpainSearch for more papers by this authorJose M. de la Rosa, Jose M. de la Rosa Inst. Prod. Nat. Agrobiol., CSIC, Univ. La Laguna, E-38206 La Laguna, Tenerife, SpainSearch for more papers by this authorAna R. Diaz-Marrero, Ana R. Diaz-Marrero Inst. Prod. Nat. Agrobiol., CSIC, Univ. La Laguna, E-38206 La Laguna, Tenerife, SpainSearch for more papers by this authorJose Darias, Jose Darias Inst. Prod. Nat. Agrobiol., CSIC, Univ. La Laguna, E-38206 La Laguna, Tenerife, SpainSearch for more papers by this authorClaudia Cerella, Claudia Cerella Inst. Prod. Nat. Agrobiol., CSIC, Univ. La Laguna, E-38206 La Laguna, Tenerife, SpainSearch for more papers by this authorMarc Diederich, Marc Diederich Inst. Prod. Nat. Agrobiol., CSIC, Univ. La Laguna, E-38206 La Laguna, Tenerife, SpainSearch for more papers by this authorMercedes Cueto, Mercedes Cueto Inst. Prod. Nat. Agrobiol., CSIC, Univ. La Laguna, E-38206 La Laguna, Tenerife, SpainSearch for more papers by this author First published: 15 October 2015 https://doi.org/10.1002/chin.201544228Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. References Faviola Cardoso-Martinez, Jose M. de la Rosa, Ana R. Diaz-Marrero, Jose Darias, Claudia Cerella, Marc Diederich, Mercedes Cueto, Tanzawaic Acids Isolated from a Marine-Derived fungus of the Genus Penicillium with Cytotoxic Activities., Org. Biomol. Chem., 2015, 13, 7248–7256. DOI: 10.1039/C5OB00773A; 10.1039/C5OB00773A CASPubMedWeb of Science®Google Scholar Volume46, Issue44November, 2015 ReferencesRelatedInformation