A novel BH3 mimetic efficiently induces apoptosis in melanoma cells through direct binding to anti‐apoptotic Bcl‐2 family proteins,including phosphorylated Mcl‐1

The Bcl‐2 family modulates sensitivity to chemotherapy in many cancers, including melanoma, in which the RAS/BRAF/MEK/ERK pathway is constitutively activated. Mcl‐1, a major anti‐apoptotic protein in the Bcl‐2 family, is extensively expressed in melanoma and contributes to melanoma's well‐documented chemoresistance. Here, we provide the first evidence that Mcl‐1 phosphorylation at T163 by ERK1/2 and JNK is associated with the resistance of melanoma cell lines to the existing BH3 mimetics gossypol, S1 and ABT‐737, and a novel anti‐apoptotic mechanism of phosphorylated Mcl‐1 (pMcl‐1) is revealed. pMcl‐1 antagonized the known BH3 mimetics by sequestering pro‐apoptotic proteins that were released from Bcl‐2/Mcl‐1. Furthermore, an anthraquinone BH3 mimetic, compound 6, was identified to be the first small molecule to that induces endogenous apoptosis in melanoma cells by directly binding Bcl‐2, Mcl‐1, and pMcl‐1 and disrupting the heterodimers of these proteins. Although compound 6 induced upregulation of the pro‐apoptotic protein Noxa, its apoptotic induction was independent of Noxa. These data reveal the promising therapeutic potential of targeting pMcl‐1 to treat melanoma. Compound 6 is therefore a potent drug that targets pMcl‐1 in melanoma.     

Mitochondria-independent morphological and biochemical apoptotic alterations promoted by the anti-tumor agent bleomycin in Saccharomyces cerevisiae.

Bleomycin is a highly potent cytotoxic and genotoxic agent used in the chemotherapy of various types of tumors. It is a radiomimetic anticancer drug that produces single- and double-stranded DNA breaks in a catalytic way. Using Saccharomyces cerevisiae as a model system, we show that when a high amount of bleomycin molecules is internalized (100 micromol/L), morphological changes identical to those usually associated with apoptosis, i.e., a sub-G1 region peak, chromatin condensation, and very rapid DNA fragmentation into oligonucleosomal-sized fragments, are observed. The known bleomycin inhibitors cobalt and EDTA were able to prevent bleomycin nucleasic activity and thus apoptotic cell death. However, both oligomycin, a potent inhibitor of the mitochondrial F0F1-ATPase, and antimycin, a drug affecting mitochondria respiration, were unable to prevent the bleomycin-induced apoptotic-like cell death. These results suggest that high bleomycin concentrations induce an apoptosis-like mitochondria-independent cell death in yeast.     

Genistein Enhances the Cisplatin‐Induced Inhibition of Cell Growth and Apoptosis in Human Malignant Melanoma Cells

Genistein, a naturally occurring isoflavone found chiefly in soybeans, has been reported to be a potent antitumor agent. Genistein is presumed to exert multiple effects related to the inhibition of cancer growth. Metastatic melanoma is a chemotherapy‐refractory neoplasm. The present study was designed to explore the possible activity of genistein to inhibit the aberrant proliferation and to induce apoptosis of human malignant melanoma cells in cooperation with cisplatin treatment. Five human melanoma cell lines were utilized for these experiments. Genistein at physiologic concentrations (20 μM) did not induce apoptosis by itself but did enhance cisplatin‐induced apoptosis in all five human melanoma cell lines tested. The enhanced susceptibility among the cell lines was diverse. Changes in the expression of two anti‐apoptotic proteins, bcl‐2 and bcl‐xL, and one pro‐apoptotic protein, apoptotic protease activating factor‐1 (Apaf‐1), were examined. Genistein alone or cisplatin alone generally did not alter bcl‐2 expression or bcl‐xL expression, but slightly increased Apaf‐1 in some cell lines. The combined treatment with genistein and cisplatin significantly reduced bcl‐2 and bcl‐xL protein and increased Apaf‐1 protein expression. These data suggest that genistein therapy may enhance the chemosensitivity of melanoma patients.     

Casein kinase 2 inhibition differentially modulates apoptotic effect of trichostatin A against epithelial ovarian carcinoma cell lines

Histone deacetylase inhibitors and casein kinase 2 inhibitors have been shown to induce apoptosis. However, the combined effect of casein kinase 2 inhibition on the apoptotic effect of histone deacetylase inhibitor is unknown. We assessed the effect of casein kinase 2 inhibition on the apoptotic effect of trichostatin A in human epithelial carcinoma cell lines with respect to cell death signaling pathways. At concentrations that did not induce cell death, the casein kinase 2 inhibitor 4,5,6,7-tetrabromobenzotriazole inhibited activation of apoptotic proteins and changes in mitochondrial membrane permeability induced by the histone deacetylase inhibitor trichostatin A. These results suggest that casein kinase 2 inhibition may reduce trichostatin A-induced apoptosis in ovarian carcinoma cell lines by suppressing activation of apoptotic proteins and changes in mitochondrial membrane permeability, which both lead to caspase-3 activation. Casein kinase 2 inhibition, which does not induce a cytotoxic effect, may prevent histone deacetylase inhibitor-mediated apoptosis.     

α-Pinene isolated from Schinus terebinthifolius Raddi (Anacardiaceae) induces apoptosis and confers antimetastatic protection in a melanoma model

Malignant melanoma is one the most aggressive types of cancer and its incidence has gradually increased in the last years, accounting for about 75% of skin cancer deaths. This poor prognosis results from the tumor resistance to conventional drugs mainly by deregulation of apoptotic pathways. The aim of this work was to investigate the cell death mechanism induced by α-pinene and its therapeutic application. Our results demonstrated that α-pinene was able to induce apoptosis evidenced by early disruption of the mitochondrial potential, production of reactive oxygen species, increase in caspase-3 activity, heterochromatin aggregation, DNA fragmentation and exposure of phosphatidyl serine on the cell surface. Most importantly, this molecule was very effective in the treatment of experimental metastatic melanoma reducing the number of lung tumor nodules. This is the first report on the apoptotic and antimetastatic activity of isolated α-pinene.     

Mcl‐1–Bim complexes accommodate surprising point mutations via minor structural changes

Mcl‐1 is an antiapoptotic Bcl‐2‐family protein that protects cells against death. Structures of Mcl‐1, and of other anti‐apoptotic Bcl‐2 proteins, reveal a surface groove into which the α‐helical BH3 regions of certain proapoptotic proteins can bind. Despite high overall structural conservation, differences in this groove afford binding specificity that is important for the mechanism of Bcl‐2 family function. We report the crystal structure of human Mcl‐1 bound to a BH3 peptide derived from human Bim and the structures for three complexes that accommodate large physicochemical changes at conserved Bim sites. The mutations had surprisingly modest effects on complex stability, and the structures show that Mcl‐1 can undergo small changes to accommodate the mutant ligands. For example, a shift in a leucine side chain fills a hole left by an isoleucine‐to‐alanine mutation at the first hydrophobic buried position of Bim BH3. Larger changes are also observed, with shifting of helix α3 accommodating an isoleucine‐to‐tyrosine mutation at this same position. We surveyed the variation in available Mcl‐1 and Bcl‐x_L structures and observed moderate flexibility that is likely critical for facilitating interactions of diverse BH3‐only proteins with Mcl‐1. With the antiapoptotic Bcl‐2 family members attracting significant attention as therapeutic targets, these structures contribute to our growing understanding of how specificity is achieved and can help to guide the design of novel inhibitors that target Mcl‐1.     

Michael adducts of ascorbic acid as inhibitors of protein phosphatase 2A and inducers of apoptosis

Michael adducts of ascorbic acid with alpha,beta-unsaturated carbonyl compounds have been shown to be potent inhibitors of protein phosphatase 1 (PP1) without affecting cell viability at the respective concentrations. Here we were able to show that higher concentrations can partially inhibit PP2A activity and concomitantly induce apoptotic cell death. A nitrostyrene adduct of ascorbic acid proved to be a more potent and effective inhibitor of PP2A as well as a stronger inducer of apoptosis. These adducts only slightly lost their cytotoxic potential in multidrug resistant cells that were 10-fold less sensitive to apoptosis induction by okadaic acid and vinblastine.     

Blockade of the ERK pathway markedly sensitizes tumor cells to HDAC inhibitor-induced cell death

Constitutive activation of the extracellular signal-regulated kinase (ERK) pathway is associated with the neoplastic phenotype of a large number of human tumor cells. Although specific blockade of the ERK pathway by treating such tumor cells with potent mitogen-activated protein kinase/ERK kinase (MEK) inhibitors completely suppresses their proliferation, it by itself shows only a modest effect on the induction of apoptotic cell death. However, these MEK inhibitors markedly enhance the efficacy of histone deacetylase (HDAC) inhibitors to induce apoptotic cell death: such an enhanced cell death is observed only in tumor cells in which the ERK pathway is constitutively activated. Co-administration of MEK inhibitor markedly sensitizes tumor cells to HDAC inhibitor-induced generation of reactive oxygen species, which appears to mediate the enhanced cell death induced by the combination of these agents. These results suggest that the combination of MEK inhibitors and HDAC inhibitors provides an efficient chemotherapeutic strategy for the treatment of tumor cells in which the ERK pathway is constitutively activated.     

PI3K inhibition potentiates Bcl‐2‐dependent apoptosis in renal carcinoma cells

Inhibitors of PI3‐K/Akt are currently being assessed clinically in patients with advanced RCC. Identification of therapeutic strategies that might enhance the efficacy of PI3‐K/Akt inhibitors is therefore of great interest. As PI3‐K inhibition would be expected to have many pro‐apoptotic effects, we hypothesized that there may be unique synergy between PI3‐K inhibitors and BH3‐mimetics. Towards this end, we assessed the combination of the PI3K inhibitor LY 294002 and the Bcl‐2 family inhibitor ABT‐737 in RCC cell lines. We found that the combinatorial treatment with these agents led to a significant increase in PARP cleavage and cell death in all RCC cell lines. The synergized cell death was correlated with decreased levels of Mcl‐1 and XIAP, and increased levels in Bim, and appears critically dependent upon the activation of caspase 3 and 8. The enhanced lethality observed with the combination also appears dependent upon the regulation of XIAP, Mcl‐1 and Bim levels. Our results suggest that the combination of PI3‐K inhibitors with BH3‐mimetics may be a viable therapeutic strategy in RCC.     

TRAIL/TRAIL‐R in hematologic malignancies

Defects in apoptosis are observed in many cancer cell types and contribute in a relevant way to tumorigenesis. Apoptosis is a complex and well‐regulated cell death program that plays a key role in the control of cell homeostasis, particularly at the level of the hematopoietic system. Apoptosis can be initiated through two different mechanisms involving either activation of the death receptors (extrinsic pathway) or activation of a mitochondrial apoptotic process (intrinsic pathway). Among the various death receptors a peculiar role is played by TNF‐related apoptosis‐inducing ligand (TRAIL)‐receptors (TRAIL‐Rs) and their ligand TRAIL. TRAIL recently received considerable interest for its potent anti‐tumor killing activity, sparing normal cells. Here, we will review the expression and the abnormalities of TRAIL/TRAIL‐R system in hematologic malignancies. The large majority of primary hematologic tumors are resistant to TRAIL‐mediated apoptosis, basically due to the activation of anti‐apoptotic signaling pathway (such as NF‐κB), overexpression of anti‐apoptotic proteins (such as FLIP, Bcl‐2, XIAP) or expression of TRAIL decoy receptors or reduced TRAIL‐R1/‐R2 expression. Strategies have been developed to bypass this TRAIL resistance and are based on the combination of TRAIL with chemotherapy or radiotherapy, or with proteasome or histone deacetylase or NF‐κB inhibitors. The agents used in combination with TRAIL either enhance TRAIL‐R1/‐R2 expression or decrease expression of anti‐apoptotic proteins (c‐FLIP, XIAP, Bcl‐2). Many of these combinatorial therapies hold promise for future developments in treatment of hematologic malignancies. J. Cell. Biochem. 110: 21–34, 2010. © 2010 Wiley‐Liss, Inc.     

A possible cross-talk between autophagy and apoptosis in generating an immune response in melanoma

Melanoma is the most aggressive form of skin cancer, responsible for the majority of skin cancer related deaths. Thus, the search for natural molecules which can effectively destroy tumors while promoting immune activation is essential for designing novel therapies against metastatic melanoma. Here, we report for the first time that a natural triterpenoid, Ganoderic acid DM (GA-DM), induces an orchestrated autophagic and apoptotic cell death, as well as enhanced immunological responses via increased HLA class II presentation in melanoma cells. Annexin V staining and flow cytometry showed that GA-DM treatment induced apoptosis of melanoma cells, which was supported by a detection of increased Bax proteins, co-localization and elevation of Apaf-1 and cytochrome c, and a subsequent cleavage of caspases 9 and 3. Furthermore, GA-DM treatment initiated a possible cross-talk between autophagy and apoptosis as evidenced by increased levels of Beclin-1 and LC3 proteins, and their timely interplay with apoptotic and/or anti-apoptotic molecules in melanoma cells. Despite GA-DM's moderate cytotoxicity, viable cells expressed high levels of HLA class II proteins with improved antigen presentation and CD4+ T cell recognition. The antitumor efficacy of GA-DM was also investigated in vivo in murine B16 melanoma model, where GA-DM treatment slowed tumor formation with a significant reduction in tumor volume. Taken together, these findings demonstrate the potential of GA-DM as a natural chemo-immunotherapeutic capable of inducing a possible cross-talk between autophagy and apoptosis, as well as improved immune recognition for sustained melanoma tumor clearance.     

Utility of Clostridium difficile Toxin B for Inducing Anti-Tumor Immunity

Clostridium difficile toxin B (TcdB) is a key virulence factor of bacterium and induces intestinal inflammatory disease. Because of its potent cytotoxic and proinflammatory activities, we investigated the utility of TcdB in developing anti-tumor immunity. TcdB induced cell death in mouse colorectal cancer CT26 cells, and the intoxicated cells stimulated the activation of mouse bone marrow-derived dendritic cells and subsequent T cell activation in vitro. Immunization of BALB/c mice with toxin-treated CT26 cells elicited potent anti-tumor immunity that protected mice from a lethal challenge of the same tumor cells and rejected pre-injected tumors. The anti-tumor immunity generated was cell-mediated, long-term, and tumor-specific. Further experiments demonstrated that the intact cell bodies were important for the immunogenicity since lysing the toxin-treated tumor cells reduced their ability to induce antitumor immunity. Finally, we showed that TcdB is able to induce potent anti-tumor immunity in B16-F10 melanoma model. Taken together, these data demonstrate the utility of C. difficile toxin B for developing anti-tumor immunity.     

Organopalladium compound 7b targets mitochondrial thiols and induces caspase-dependent apoptosis in human myeloid leukemia cells

The advances in the treatment of chronic myeloid leukemia (CML) during the last years were also accompanied by the development of evading strategies by tumor cells, resulting in chemotherapy resistance in some patients. Patented organopalladium compounds derived from the reaction of N,N-dimethyl-1-phenethylamine (dmpa) with [1,2-ethanebis(diphenylphosphine)] (dppe) exhibited a potent antitumor activity in vivo and in vitro in melanoma cells. We showed here that the cyclopalladated derivative [Pd₂(R₍₊₎)C², N-dmpa)₂(μ-dppe)Cl₂], named compound 7b, was highly effective to promote cell death in the K562 human leukemia cells and its mechanisms of action were investigated. It was shown that compound 7b was able to promote exclusively apoptotic cell death in K562 cells associated to cytochrome c release and caspase 3 activation. This cytotoxic effect was not observed in normal peripheral mononuclear blood cells. The compound 7b-induced intrinsic apoptotic pathway was triggered by the protein thiol oxidation that resulted in the dissipation of the mitochondrial transmembrane potential. The preventive effect of the dithiothreitol on the compound 7b-induced cell death and all downstream events associated to apoptosis confirmed that death signal was elicited by the thiol oxidation. These findings contribute to the elucidation of the palladacycle 7b-induced cell death mechanism and present this compound as a promising drug in the CML antitumor chemotherapy.     

Targeting FcαRI on polymorphonuclear cells induces tumor cell killing through autophagy

Neutrophils are the most abundant circulating FcR-expressing WBCs with potent cytotoxic ability. Currently, they are recognized as promising effector cells for Ab-mediated immunotherapy of cancer, because their capacity to kill tumor cells is greatly enhanced by tumor Ag-specific mAbs. The FcαRI represents the most potent FcR on neutrophils for induction of Ab-mediated tumor cell killing. However, the mechanisms of cell death that are induced are poorly understood. Because these mechanisms can be used for modulation of anticancer treatment, we investigated the tumor cell death induced by neutrophil-mediated Ab-dependent killing via FcαRI. Human mammary carcinoma cells were efficiently killed when incubated with human neutrophils and tumor-specific FcαRI bispecific or IgA Abs. Interestingly, we observed characteristics of autophagy such as autophagic structures by electron microscopy and LC3B(+) autophagosomes in different human epithelial carcinoma cells, which resulted in tumor cell death. To a lesser extent, necrotic features, such as cellular membrane breakdown and spillage of intracellular content, were found. By contrast, apoptotic features including fragmented nuclei, Annexin V-positivity, and presence of cleaved caspase-3 were not observed. These findings indicate that neutrophils mainly facilitate autophagy to induce tumor cell death rather than the more commonly recognized apoptotic cell death mechanisms induced by NK cells or cytotoxic T cells. This knowledge not only reveals the type of tumor cell death induced in neutrophil-mediated, Ab-dependent cellular cytotoxicity, but importantly opens up additional perspectives for modulation of anticancer therapy in, for example, apoptosis-resistant tumor cells.     

Hypertonicity primes malignant melanoma cells for apoptosis

The tumor environment critically influences responsiveness of cancer cells to chemotherapies, most of which activate the mitochondria-regulated (intrinsic) apoptotic cascade to kill malignant cells. Especially skin tumors encounter an environment with remarkable biophysical properties. Cutaneous accumulation of Na⁺ locally establishes osmotic pressure gradients in vivo (hypertonicity or hyperosmotic stress), but whether cutaneous hypertonicity is a factor that modulates the responsiveness of skin cancers to therapeutic apoptosis-induction has thus far not been investigated. Here, we show that hyperosmotic stress lowers the threshold for apoptosis induction in malignant melanoma, the deadliest form of skin cancer. Hypertonic conditions enforce addiction to BCL-2-like proteins to prevent initiation of the mitochondria-regulated (intrinsic) apoptotic pathway. Essentially, hyperosmotic stress primes mitochondria for death. Our work identifies osmotic pressure in the tumor microenvironment as a cell extrinsic factor that modulates responsiveness of malignant melanoma cells to therapy.     

BI‐69A11‐mediated inhibition of AKT leads to effective regression of xenograft melanoma

The AKT/PKB pathway plays a central role in tumor development and progression and is often up‐regulated in different tumor types, including melanomas. We have recently reported on the in silico approach to identify putative inhibitors for AKT/PKB. Of the reported hits, we selected BI‐69A11, a compound which was shown to inhibit AKT activity in in vitro kinase assays. Analysis of BI‐69A11 was performed in melanoma cells, a tumor type that commonly exhibits up‐regulation of AKT. Treatment of the UACC903 human melanoma cells, harboring the PTEN mutation, with BI‐69A11 caused efficient inhibition of AKT S473 phosphorylation with concomitant inhibition of AKT phosphorylation of PRAS40. Treatment of melanoma cells with BI‐69A11 also reduced AKT protein expression, which coincided with inhibition of AKT association with HSP‐90. BI‐69A11 treatment not only caused cell death of melanoma, but also prostate tumor cell lines. Notably, the effect of BI‐69A11 on cell death was more pronounced in cells that express an active form of AKT. Significantly, intra‐peritoneal injection of BI‐69A11 caused effective regression of melanoma tumor xenografts, which coincided with elevated levels of cell death. These findings identify BI‐69A11 as a potent inhibitor of AKT that is capable of eliciting effective regression of xenograft melanoma tumors.     

Chemical blockage of the proteasome inhibitory function of bortezomib: impact on tumor cell death

The proteasome inhibitor bortezomib is emerging as a potent anti-cancer agent. Still, recent clinical trials have revealed a significant secondary toxicity of bortezomib. Consequently, there is much interest in dissecting the mechanism of action of this compound to rationally improve its therapeutic index. The cytotoxic effect of bortezomib is frequently characterized by interfering with downstream events derived from the accumulation of proteasomal targets. Here we identify the first chemical agent able to act upstream of the proteasome to prevent cell killing by bortezomib. Specifically, we show that the polyhydroxyl compound Tiron can function as a competitive inhibitor of bortezomib. This effect of Tiron was surprising, since it is a classical radical spin trap and was expected to scavenge reactive oxygen species produced as a consequence of bortezomib action. The inhibitory effect of Tiron against bortezomib was selective, since it was not shared by other antioxidants, such as vitamin E, MnTBAP, L-N-acetyl-cysteine, and FK-506. Comparative analyses with nonboronated proteasome inhibitors (i.e. MG132) revealed a specificity of Tiron for bortezomib. We exploited this novel feature of Tiron to define the "point of no return" of proteasome inhibition in melanoma cells and to block cell death in a three-dimensional model of human skin. Cells from T-cell lymphoma, breast carcinoma, and non-small cell lung cancer were also responsive to Tiron, suggesting a broad impact of this agent as a bortezomib blocker. These results may have important implications for the analysis of bortezomib in vivo and for the design of drug mixtures containing proteasome inhibitors.     

Selective induction of cell death in melanoma cell lines through targeting of Mcl-1 and A1

Melanoma is an often fatal form of skin cancer which is remarkably resistant against radio- and chemotherapy. Even new strategies that target RAS/RAF signaling and display unprecedented efficacy are characterized by resistance mechanisms. The targeting of survival pathways would be an attractive alternative strategy, if tumor-specific cell death can be achieved. Bcl-2 proteins play a central role in regulating survival of tumor cells. In this study, we systematically investigated the relevance of antiapoptotic Bcl-2 proteins, i.e., Bcl-2, Bcl-xL, Bcl-w, Mcl-1, and A1, in melanoma cell lines and non-malignant cells using RNAi. We found that melanoma cells required the presence of specific antiapoptotic Bcl-2 proteins: Inhibition of Mcl-1 and A1 strongly induced cell death in some melanoma cell lines, whereas non-malignant cells, i.e., primary human fibroblasts or keratinocytes were not affected. This specific sensitivity of melanoma cells was further enhanced by the combined inhibition of Mcl-1 and A1 and resulted in 60% to 80% cell death in all melanoma cell lines tested. This treatment was successfully combined with chemotherapy, which killed a substantial proportion of cells that survived Mcl-1 and A1 inhibition. Together, these results identify antiapoptotic proteins on which specifically melanoma cells rely on and, thus, provide a basis for the development of new Bcl-2 protein-targeting therapies.