Modulation of pro- and anti-apoptotic factors in human melanoma cells exposed to histone deacetylase inhibitors

Valproic acid (VPA, 2-propylpentanoic acid) is an established drug in the long-term therapy of epilepsy. Recently, VPA was demonstrated to inhibit histone deacetylases (HDACs) class I enzyme at therapeutically relevant concentrations, thereby, mimicking the prototypical histone deacetylase inhibitors, tricostatin A (TSA) or suberoylanilide hydroxamic acid (SAHA). In the present study, we investigated the cellular effects of VPA, TSA and SAHA on four human melanoma cell lines (WM115, WM266, A375, SK-Mel28) with particular reference to the modulation of regulators of apoptosis, including Bcl-2, BclXL, Mcl-1, Apaf-1, BclXs, NOXA, TRAIL-R1, TRAIL-R2, caspase 8, and survivin). Firstly, we found that VPA induced apoptosis in two of the four human melanoma cell lines, while both TSA and SAHA exhibited an antiproliferative and apoptotic effects in all four cell lines, a different expression of Bcl-2 and BclX(L/S) occurred. On the other hand, SAHA and VPA modulated differently pro- and anti-apoptotic factors. In particular, the treatment with VPA enhanced the level of expression of survivin only in VPA-resistant cell lines, whereas down-regulation of survivin was induced by VPA and SAHA in VPA-sensitive cells. In the latter, since activation of caspase 8 was documented, a receptor-mediated apoptosis was suggested. Taken together, our results suggest that HDAC inhibitors may represent a promising therapeutic strategy to treat melanoma.     

c-Myc overexpression sensitizes Bim-mediated Bax activation for apoptosis induced by histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) through regulating Bcl-2/Bcl-xL expression

Overexpression of the oncogene c-Myc sensitizes many apoptotic signals through the activation of mitochondrial apoptosis pathway. However, the underling mechanism has not been clearly defined. Here, we investigated the effect of c-Myc expression on histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA)-induced apoptosis in rat fibroblast cells possessing various c-Myc levels. In Rat 1a cells overexpressing c-Myc, SAHA-induced enhanced the cell death response relative to the parental cells; whereas Rat 1a cells lacking c-Myc were refractory to SAHA treatment. We demonstrated that SAHA selectively induced the expression of pro-apoptotic BH3-only protein Bim, leading to Bax activation in c-Myc-expressing cells. Where c-Myc was absent, Bim, despite its induction by SAHA, failed to activate Bax and was unable to induce apoptosis. These results indicate that c-Myc is dispensable for Bim induction by SAHA, but is required for subsequent Bax activation. We further show that the expression levels of anti-apoptotic Bcl-2/Bcl2-xL were much elevated in Myc-null cells compared with the c-Myc-expressing cells; furthermore, depletion of Bcl-2/Bcl-xL in these cells restored the ability of SAHA to induce apoptosis by enhancing Bax activation. These data indicate that SAHA induces apoptosis through Bim-triggered Bax activation and that c-Myc regulates this process by modulating Bcl-2/Bcl-xL. Our results provide novel insight into the mechanism whereby Myc sensitizes the apoptotic signals; furthermore, our data suggest that cancer cells with deregulated Myc might be more sensitive to SAHA treatment.     

Chk1 suppresses a caspase-2 apoptotic response to DNA damage that bypasses p53, Bcl-2, and caspase-3

Evasion of DNA damage-induced cell death, via mutation of the p53 tumor suppressor or overexpression of prosurvival Bcl-2 family proteins, is a key step toward malignant transformation and therapeutic resistance. We report that depletion or acute inhibition of checkpoint kinase 1 (Chk1) is sufficient to restore gamma-radiation-induced apoptosis in p53 mutant zebrafish embryos. Surprisingly, caspase-3 is not activated prior to DNA fragmentation, in contrast to classical intrinsic or extrinsic apoptosis. Rather, an alternative apoptotic program is engaged that cell autonomously requires atm (ataxia telangiectasia mutated), atr (ATM and Rad3-related) and caspase-2, and is not affected by p53 loss or overexpression of bcl-2/xl. Similarly, Chk1 inhibitor-treated human tumor cells hyperactivate ATM, ATR, and caspase-2 after gamma-radiation and trigger a caspase-2-dependent apoptotic program that bypasses p53 deficiency and excess Bcl-2. The evolutionarily conserved "Chk1-suppressed" pathway defines a novel apoptotic process, whose responsiveness to Chk1 inhibitors and insensitivity to p53 and BCL2 alterations have important implications for cancer therapy.     

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.     

Occurrence of pre-MBT synthesis of caspase-8 mRNA and activation of caspase-8 prior to execution of SAMDC (S-adenosylmethionine decarboxylase)-induced, but not p53-induced, apoptosis in Xenopus late blastulae

Overexpression of S-adenosylmethionine decarboxylase (SAMDC) in Xenopus fertilized eggs activates caspase-9 and executes maternal program of apoptosis shortly after midblastula transition (MBT). We find that overexpression of caspase-8 and p53, like that of SAMDC, induces apoptosis in Xenopus late blastulae. The apoptosis induced by p53 was abolished by injection of mRNA for xdm-2, a negative regulator of p53, and by injection of a peptide inhibitor or a dominant-negative type mutant of caspase-9, but not caspase-8. The apoptosis induced by SAMDC was not abolished by injection of xdm-2 mRNA, but was abolished by injection of a peptide inhibitor or a dominant-negative type mutant mRNA of both caspase-9 and caspase-8. Unlike caspase-9 mRNA, caspase-8 mRNA did not occur as a maternal mRNA rather induced to be expressed during cleavage stage (pre-MBT stage) by overexpression of SAMDC but not p53. Furthermore, while activities to process procaspase-8 and procaspase-9 appeared in SAMDC-overexpressed apoptotic embryos, the activity to process procaspase-8 did not appear in p53-overexpressed apoptotic embryos. We conclude there are at least two pathways in the execution of the maternal program of apoptosis in Xenopus embryos; one being through do novo expression of caspase-8 gene during cleavage stage, and the other without involvement of caspase-8.     

Role of mitochondria in aspirin-induced apoptosis in human gastric epithelial cells

This study was undertaken to determine whether the Bcl-2 family proteins and Smac are regulators of aspirin-mediated apoptosis in a gastric mucosal cell line known as AGS cells. Cells were incubated with varying concentrations of acetylsalicylic acid (ASA; 2-40 mM), with or without preincubation of caspase inhibitors. Apoptosis was characterized by Hoechst staining and DNA-histone-associated complex formation. Antiapoptotic Bcl-2, proapoptotic Bax and Bid, Smac, and cytochrome-c oxidase (COX IV) were analyzed by Western blot analyses from cytosol and mitochondrial fractions. ASA downregulated Bcl-2 protein expression and induced Bax translocation into the mitochondria and cleavage of Bid. In contrast, expression of Smac was significantly decreased in mitochondrial fractions of ASA-treated cells. Bax and Bid involvement in apoptosis regulation was dependent on caspase activation, because caspase-8 inhibition suppressed Bax translocation and Bid processing. Caspase-9 inhibition prevented Smac release from mitochondria. Additionally, increased expression of the oxidative phosphorylation enzyme COX IV was observed in mitochondrial fractions exposed to ASA at concentrations >5 mM. Although caspase-8 inhibition had no effect on aspirin-induced apoptosis and DNA-histone complex formation, caspase-9 inhibition significantly decreased both of these events. We conclude that Bcl-2 protein family members and Smac regulate the apoptotic pathway in a caspase-dependent manner. Our results indicate also that mitochondrial integration and oxidative phosphorylation play a critical role in the pathogenesis of apoptosis in human gastric epithelial cells.     

Histone Deacetylase Inhibitors Inhibit the Proliferation of Gallbladder Carcinoma Cells by Suppressing AKT/mTOR Signaling

Gallbladder carcinoma is an aggressive malignancy with high mortality mainly due to the limited potential for curative resection and its resistance to chemotherapeutic agents. Here, we show that the histone deacetylase inhibitors (HDACIs) trichostatin-A (TSA) and suberoylanilide hydroxamic acid (SAHA) reduce the proliferation and induce apoptosis of gallbladder carcinoma cells by suppressing the AKT/mammalian target of rapamycin (mTOR) signaling. Gallbladder carcinoma SGC-996 cells were treated with different concentrations of TSA and SAHA for different lengths of time. Cell proliferation and morphology were assessed with MTT assay and microscopy, respectively. Cell cycle distribution and cell apoptosis were analyzed with flow cytometry. Western blotting was used to detect the proteins related to apoptosis, cell cycle, and the AKT/mTOR signaling pathway. Our data showed that TSA and SAHA reduced SGC-996 cell viability and arrested cell cycle at the G1 phase in a dose- and time-dependent manner. TSA and SAHA promoted apoptosis of SGC-996 cells, down-regulated the expression of cyclin D1, c-Myc and Bmi1, and decreased the phosphorylation of AKT, mTOR p70S6K1, S6 and 4E-BP1. Additionally, the mTOR inhibitor rapamycin further reduced the cell viability of TSA- and SAHA-treated SGC-996 cells and the phosphorylation of mTOR, whereas the mTOR activator 1,2-dioctanoyl-sn-glycero-3-phosphate (C8-PA) exerted the opposite influence. Our results demonstrate that histone deacetylase inhibitors (HDACIs) suppress the proliferation of gallbladder carcinoma cell via inhibition of AKT/mTOR signaling. These findings offer a mechanistic rationale for the application of HDACIs in gallbladder carcinoma treatment.     

Analysis of apoptosis regulatory genes altered by histone deacetylase inhibitors in chronic lymphocytic leukemia cells

Histone deacetylases (HDACs) play a key role in the regulation of acetylation status not only of histones but also of many other non-histone proteins involved in cell cycle regulation, differentiation or apoptosis. Therefore, histone deacetylase inhibitors (HDACi) have emerged as promising anticancer agents. Herein, we report the characterization of apoptosis in B-cell chronic lymphocytic leukemia (CLL) induced by two HDACi, Kendine 92 and SAHA. Both inhibitors induce dose-, time- and caspase-dependent apoptosis through the mitochondrial pathway. Interestingly, Kendine 92 and SAHA show a selective cytotoxicity for B lymphocytes and induce apoptosis in CLL cells with mutated or deleted TP53 as effectively as in tumor cells harboring wild-type TP53. The pattern of apoptosis-related gene and protein expression profile has been characterized. It has shown to be irrespective of TP53 status and highly similar between SAHA and Kendine 92 exposure. The balance between the increased BAD, BNIP3L, BNIP3, BIM, PUMA and AIF mRNA expression levels, and decreased expression of BCL-W, BCL-2, BFL-1, XIAP and FLIP indicates global changes in the apoptosis mRNA expression profile consistent with the apoptotic outcome. Protein expression analysis shows increased levels of NOXA, BIM and PUMA proteins upon Kendine 92 and SAHA treatment. Our results highlight the capability of these molecules to induce apoptosis not only in a selective manner but also in those cells frequently resistant to standard treatments. Thus, Kendine 92 is a novel HDACi with anticancer efficacy for non-proliferating CLL cells.     

Polyamine depletion prevents camptothecin-induced apoptosis by inhibiting the release of cytochrome c

We have shown previously that depletion of polyamines delays apoptosis induced by camptothecin in rat intestinal epithelial cells (IEC-6). Mitochondria play an important role in the regulation of apoptosis in mammalian cells because apoptotic signals induce mitochondria to release cytochrome c. The latter interacts with Apaf-1 to activate caspase-9, which in turn activates downstream caspase-3. Bcl-2 family proteins are involved in the regulation of cytochrome c release from mitochondria. In this study, we examined the effects of polyamine depletion on the activation of the caspase cascade, release of cytochrome c from mitochondria, and expression and translocation of Bcl-2 family proteins. We inhibited ornithine decarboxylase, the first rate-limiting enzyme in polyamine synthesis, with alpha-difluoromethylornithine (DFMO) to deplete cells of polyamines. Depletion of polyamines prevented camptothecin-induced release of cytochrome c from mitochondria and decreased the activity of caspase-9 and caspase-3. The mitochondrial membrane potential was not disrupted when cytochrome c was released. Depletion of polyamines decreased translocation of Bax to mitochondria during apoptosis. The expression of antiapoptotic proteins Bcl-x(L) and Bcl-2 was increased in DFMO-treated cells. Caspase-8 activity and cleavage of Bid were decreased in cells depleted of polyamines. These results suggest that polyamine depletion prevents IEC-6 cells from apoptosis by preventing the translocation of Bax to mitochondria, thus preventing the release of cytochrome c.     

Caspase-3 activation is an early event and initiates apoptotic damage in a human leukemia cell line

Many apoptotic pathways culminate in the activation of caspase cascades usually triggered by the apical caspases-8 or -9. We describe a paradigm where apoptosis is initiated by the effector caspase-3. Diethylmaleate (DEM)-induced apoptotic damage in Jurkat cells was blocked by the anti-apoptotic protein Bcl-2, whereas, a peptide inhibitor of caspase-3 but not caspase-9 blocked DEM-induced mitochondrial damage. Isogenic Jurkat cell lines deficient for caspase-8 or the adaptor FADD (Fas associated death domain) were not protected from DEM-induced apoptosis. Caspase-3 activation preceded that of caspase-9 and initial processing of caspase-3 was regulated independent of caspase-9 and Bcl-2. However, inhibitors of caspase-9 or caspase-6 regulated caspase-3 later in the pathway. We explored the mechanism by which caspase-3 processing is regulated in this system. DEM triggered a loss of Erk-1/2 phosphorylation and XIAP (X-linked inhibitor of apoptosis protein) expression. The phorbol ester PMA activated a MEK-dependent pathway to block caspase-3 processing and cell death. Constitutively active MEK-1 (CA-MEK) upregulated XIAP expression and exogenous XIAP inhibited DEM-induced apoptotic damage. Thus, we describe a pathway where caspase-3 functions to initiate apoptotic damage and caspase-9 and caspase-6 amplify the apoptotic cascade. Further, we show that MEK may regulate caspase-3 activation via the regulation of XIAP expression in these cells.     

Analysis of apoptosis regulatory genes altered by histone deacetylase inhibitors in chronic lymphocytic leukemia cells

Histone deacetylases (HDACs) play a key role in the regulation of acetylation status not only of histones but also of many other non-histone proteins involved in cell cycle regulation, differentiation or apoptosis. Therefore, histone deacetylase inhibitors (HDACi) have emerged as promising anticancer agents. Herein, we report the characterization of apoptosis in B-cell chronic lymphocytic leukemia (CLL) induced by two HDACi, Kendine 92 and SAHA. Both inhibitors induce dose-, time- and caspase-dependent apoptosis through the mitochondrial pathway. Interestingly, Kendine 92 and SAHA show a selective cytotoxicity for B lymphocytes and induce apoptosis in CLL cells with mutated or deleted TP53 as effectively as in tumor cells harboring wild-type TP53. The pattern of apoptosis-related gene and protein expression profile has been characterized. It has shown to be irrespective of TP53 status and highly similar between SAHA and Kendine 92 exposure. The balance between the increased BAD, BNIP3L, BNIP3, BIM, PUMA and AIF mRNA expression levels, and decreased expression of BCL-W, BCL-2, BFL-1, XIAP and FLIP indicates global changes in the apoptosis mRNA expression profile consistent with the apoptotic outcome. Protein expression analysis shows increased levels of NOXA, BIM and PUMA proteins upon Kendine 92 and SAHA treatment. Our results highlight the capability of these molecules to induce apoptosis not only in a selective manner but also in those cells frequently resistant to standard treatments. Thus, Kendine 92 is a novel HDACi with anticancer efficacy for non-proliferating CLL cells.     

Benzo[a]pyrene-7,8-diol-9,10-epoxide causes caspase-mediated apoptosis in H460 human lung cancer cell line

We have shown previously that wild-type p53 renders H460 human lung cancer cells more sensitive to apoptosis induction by environmental carcinogen benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), but the mechanism of cell death is not fully understood. The present study provides insights into the mechanism by which BPDE causes apoptosis in H460 cells. Exposure of H460 cells to BPDE resulted in a concentration-dependent apoptotic cell death characterized by cleavage of poly(ADP-ribose)polymerase, DNA condensation, and apoptotic histone-associated DNA fragments released into the cytosol. The BPDE-mediated release of apoptotic histone-associated DNA fragments into the cytosol was also observed in a normal bronchial epithelial cell line BEAS-2B. The BPDE-induced apoptosis in H460 cells correlated with up-regulation of pro-apoptotic protein Bak, down-regulation of anti-apoptotic Bcl-2 family members Bcl-2 and Bcl-xL, release of cytochrome c from mitochondria to the cytosol without a change in mitochondrial membrane potential or mitochondrial morphology (electron microscopy), and cleavage of caspase-8, -9, and -3. Ectopic expression of Bcl-2 failed to confer significant protection against BPDE-induced apoptosis in H460 cells. The SV40 immortalized mouse embryonic fibroblasts (MEFs) derived from Bak and Bax double knockout mice, but not Bid knockout mice, were significantly more resistant to BPDE-induced apoptosis compared with the MEFs derived from wild-type mice. The BPDE-induced apoptosis was partially but statistically significantly attenuated in the presence of specific inhibitors of caspase-9 (z-LEHDfmk) and caspase-8 (z-IETDfmk). In conclusion, the present study reveals that BPDE-induced apoptosis in H460 cells is associated with Bak induction and caspase activation but independent of Bcl-2.     

Resveratrol induces p53-independent, X-linked inhibitor of apoptosis protein (XIAP)-mediated Bax protein oligomerization on mitochondria to initiate cytochrome c release and caspase activation

Resveratrol, a naturally occurring phytoalexin, is known to induce apoptosis in multiple cancer cell types, but the underlying molecular mechanisms remain unclear. Here, we show that resveratrol induced p53-independent, X-linked inhibitor of apoptosis protein (XIAP)-mediated translocation of Bax to mitochondria where it underwent oligomerization to initiate apoptosis. Resveratrol treatment promoted interaction between Bax and XIAP in the cytosol and on mitochondria, suggesting that XIAP plays a critical role in the activation and translocation of Bax to mitochondria. This process did not involve p53 but required accumulation of Bim and t-Bid on mitochondria. Bax primarily underwent homo-oligomerization on mitochondria and played a major role in release of cytochrome c to the cytosol. Bak, another key protein that regulates the mitochondrial membrane permeabilization, did not interact with p53 but continued to associate with Bcl-xL. Thus, the proapoptotic function of Bak remained suppressed during resveratrol-induced apoptosis. Caspase-9 silencing inhibited resveratrol-induced caspase activation, whereas caspase-8 knockdown did not affect caspase activity, suggesting that resveratrol induces caspase-9-dependent apoptosis. Together, our findings characterize the molecular mechanisms of resveratrol-induced caspase activation and subsequent apoptosis in cancer cells.