The role of Mcl-1 downregulation in the proapoptotic activity of the multikinase inhibitor BAY 43-9006

BAY 43-9006, a multikinase inhibitor that targets Raf, prevents tumor cell proliferation in vitro and inhibits diverse human tumor xenografts in vivo. The mechanism of action of BAY 43-9006 remains incompletely defined. In the present study, the effects of BAY 43-9006 on the antiapoptotic Bcl-2 family member Mcl-1 were examined. Treatment of A549 lung cancer cells with BAY 43-9006 diminished Mcl-1 levels in a time- and dose-dependent manner without affecting other Bcl-2 family members. Similar BAY 43-9006-induced Mcl-1 downregulation was observed in ACHN (renal cell), HT-29 (colon), MDA-MB-231 (breast), KMCH (cholangiocarcinoma), Jurkat (acute T-cell leukemia), K562 (chronic myelogenous leukemia) and MEC-2 (chronic lymphocytic leukemia) cells. Mcl-1 mRNA levels did not change in BAY 43-9006-treated cells. Instead, BAY 43-9006 enhanced proteasome-mediated Mcl-1 degradation. This Mcl-1 downregulation was followed by mitochondrial cytochrome c release and caspase activation as well as enhanced sensitivity to other proapoptotic agents. The caspase inhibitor Boc-D-fmk inhibited BAY 43-9006-induced caspase activation but not cytochrome c release. In contrast, Mcl-1 overexpression inhibited cytochrome c release and other features of BAY 43-9006-induced apoptosis. Conversely, Mcl-1 downregulation by short hairpin RNA enhanced BAY 43-9006-induced apoptosis. Collectively, these findings demonstrate that drug-induced Mcl-1 downregulation contributes to the proapoptotic effects of BAY 43-9006.     

SMAC/Diablo mediates the proapoptotic function of PUMA by regulating PUMA-induced mitochondrial events

p53-upregulated modulator of apoptosis (PUMA) is a BH3-only Bcl-2 family protein and an essential mediator of DNA damage-induced apoptosis. PUMA is localized in the mitochondria and induces apoptosis through the mitochondrial pathway. However, the mechanisms of PUMA-induced apoptosis remain unclear. In this study, we found that second mitochondria-derived activator of caspase (SMAC)/Diablo, a mitochondrial apoptogenic protein, mediates the proapoptotic function of PUMA by regulating PUMA-induced mitochondrial events. SMAC is consistently released into the cytosol in colon cancer cells undergoing PUMA-induced apoptosis. In SMAC-deficient cells, execution of PUMA-induced apoptosis is abrogated, in company with decreases in caspase activation, cytosolic release of cytochrome c and collapse of mitochondrial membrane potential. Reconstituting SMAC expression restored these events in the SMAC-deficient cells. Furthermore, SMAC and agents that mimic the inhibitor of apoptosis proteins (IAPs) inhibition function of SMAC significantly sensitize cells to PUMA-induced apoptosis. These results demonstrate an important role of SMAC in executing DNA damage-induced and PUMA-mediated apoptosis and suggest that SMAC participates in a feedback amplification loop to promote cytochrome c release and other mitochondrial events in apoptosis.     

Histone, H1.2: another housekeeping protein that kills

Histone H1.2, a housekeeping protein that binds nucleosomal linkers, has recently been identified as an apoptogenic factor released from the nucleus to the cytosol in response to double strand DNA breaks. In the cytosol, it promotes the activation of proapoptotic Bcl-2 family proteins, mitochondrial cytochrome c release, and ultimately, cell death.     

At the gates of death

Apoptosis that proceeds via the mitochondrial pathway involves mitochondrial outer membrane permeabilization (MOMP), responsible for the release of cytochrome c and other proteins of the mitochondrial intermembrane space. This essential step is controlled and mediated by proteins of the Bcl-2 family. The proapoptotic proteins Bax and Bak are required for MOMP, while the antiapoptotic Bcl-2 proteins, including Bcl-2, Bcl-xL, Mcl-1, and others, prevent MOMP. Different proapoptotic BH3-only proteins act to interfere with the function of the antiapoptotic Bcl-2 members and/or activate Bax and Bak. Here, we discuss an emerging view, proposed by Certo et al. in this issue of Cancer Cell, on how these interactions result in MOMP and apoptosis.     

Human colon cancer cells lacking Bax resist curcumin-induced apoptosis and Bax requirement is dispensable with ectopic expression of Smac or downregulation of Bcl-XL

Multiple apoptotic stimuli induce conformational changes in Bax, a proapoptotic protein from the Bcl-2 family and its deficiency is a frequent cause of chemoresistance in colon adenocarcinomas. Curcumin, a dietary compound from turmeric, is known to induce apoptosis in a variety of cancer cells. To understand the role of Bax in curcumin-induced apoptosis we used HCT116 human colon cancer cells with one allele of Bax gene (Bax+/-) and Bax knockout HCT116 (Bax-/-) cells in which Bax gene is inactivated by homologous recombination. Cell viability decreased in a concentration-dependent manner in Bax+/- cells treated with curcumin (0-50 microM) whereas only minimal changes in viability were observed in Bax-/- cells upon curcumin treatment. In Bax-/- cells curcumin-induced activation of caspases 9 and 3 was blocked and that of caspase 8 remained unaltered. Curcumin-induced release of cytochrome c, Second mitochondria derived activator of caspase (Smac) and apoptosis inducing factor (AIF) was also blocked in Bax-/- cells and reintroduction of Bax, downregulation of the antiapoptotic protein Bcl-XL by antisense DNA as well as the overexpression of Smac, highly sensitized the Bax-/- cells toward curcumin-induced apoptosis. There was no considerable difference in the percentage of apoptotic cells in Bak RNAi transfected Bax+/- or Bax-/- cells treated with curcumin when compared with their corresponding vector transfected cells treated with curcumin. The present study demonstrates the role of Bax but not Bak as a critical regulator of curcumin-induced apoptosis and implies the potential of targeting antiapoptotic proteins like Bcl-XL or overexpression of proapoptotic proteins like Smac as interventional approaches to deal with Bax-deficient chemo-resistant cancers for curcumin-based therapy.     

Current status of the molecular mechanisms of anticancer drug-induced apoptosis. The contribution of molecular-level analysis to cancer chemotherapy

Apoptosis is an important phenomenon in cytotoxicity induced by anticancer drugs. Here, we review the current status of the molecular mechanisms of anticancer drug-induced apoptosis in order to assess the contribution of molecular-level analysis to cancer chemotherapy. It is apparent that the molecular mechanisms by which anticancer drugs induce apoptosis are mediated by death receptor-dependent and -independent pathways, which are related to the release of cytochrome c through voltage-dependent anion channels in the mitochondrial inner membrane. The release of cytochrome c is the central gate in turning on/off apoptosis, and is regulated by the interaction of proapoptotic proteins, including Bid, Bax and Bak, and antiapoptotic proteins including Bcl-2 and Bcl-X(L), and a specific class of inhibitors of apoptosis proteins (IAPs) including Akt, survivin, and heat-shock proteins. The caspase cascade is activated by the release of cytochrome c, which is initiated by the formation of apoptosomes consisting of procaspase-9, Apaf-1 and cytochrome c in the presence of dATP, and results in the activation of caspase-9 and caspase-3, thereby leading to apoptosis. Drug sensitivity can be enhanced by the introduction of proapoptotic genes and the inhibition of antiapoptotic proteins. The latter process is mediated by antisense oligonucleotides and is associated with apoptosis. The signal transduction pathways that are triggered by the central gate in mitochondria play a critical role in anticancer drug-induced apoptosis. The modulation of signal transduction pathways targeting the proteins involved in these signal transduction pathways using antisense IAPs, and growth factor antibodies may be a good strategy for enhancing therapeutic efficacy of anticancer drugs in cancer chemotherapy.     

Involvement of proapoptotic Bcl-2 family members in parthenolide-induced mitochondrial dysfunction and apoptosis

Parthenolide is a sesquiterpene lactone responsible for the bioactivities of Feverfew. Besides its potent anti-inflammatory effect, this compound has recently been reported to induce apoptosis in cancer cells, possibly through mitochondrial dysfunction. In the present study, we attempted to examine parthenolide-mediated cell death signaling pathway by focusing on the involvement of Bcl-2 family members. Using a human colorectal cancer cell line COLO205, we first demonstrated that parthenolide acted through the cell death receptor pathway to activate caspase 8. Following caspase 8 activation, Bid, a proapoptotic Bcl-2 member, was cleaved and this cleavage then triggered Bax conformational changes and Bax translocation from cytosol to mitochondrial membrane. Meanwhile, another proapoptotic protein, Bak, was up-regulated and oligomerized on the mitochondrial membrane. All these alterations were found to be prerequisite for the subsequent release of proapopototic mitochondrial proteins, including cytochrome c and Samc, in parthenolide-treated cells. Moreover, selective inhibition of caspase 8 activity by a synthetic caspase inhibitor (IETD-FMK) or overexpression of a viral protein (CrmA) suppressed the cleavage of Bid, conformational changes of Bax, cytochrome c release, and apoptosis. Therefore, the proapoptotic Bcl-2 family members are important mediators relaying the cell death signaling elicited by parthenolide from caspase 8 to downstream effector caspases such as caspase 3, and eventually to cell death.     

Ceramide induces mitochondrial activation and apoptosis via a Bax-dependent pathway in human carcinoma cells

The intracellular pathways leading to mitochondrial activation and subsequent cell death in the ceramide-mediated stress response have been intensively studied in recent years. Experimental evidence has been provided that ceramide-induced apoptosis is inhibited by overexpression of antiapoptotic proteins of the Bcl-2 family. However, the direct effect of proapoptotic gene products, e.g. Bax, on ceramide-induced death signalling has not yet been studied in detail. In the present work, we show by measurement of mitochondrial permeability transition, cytochrome c release, activation of caspase-3 and DNA fragmentation that ceramide-induced apoptosis is marginal in Bax-negative DU 145 cells. Reconstitution of Bax by generation of DU 145 cells stably expressing this proapoptotic factor, clearly enhanced ceramide-induced apoptosis at all levels of the mitochondrial signalling cascade. Using the broad-range caspase inhibitor zVAD-fmk and zDEVD-fmk, an inhibitor of caspase-3-like activities, we demonstrate that the ceramide-induced mitochondrial activation in Bax-transfected DU 145 cells is caspase-independent. On the other hand, apoptotic events located downstream of the mitochondria, e.g. DNA fragmentation, were shown to be caspase-dependent. This influence of Bax on ceramide-induced apoptosis was confirmed in another cellular system: whereas Bax-positive HCT116 wild type cells were very sensitive towards induction of cell death by C(2)-ceramide, sensitivity of Bax knock-out HCT116 cells was significantly reduced. Thus, we conclude that Bax is a key activator of ceramide-mediated death pathways.     

Bax and Bak are required for apoptosis induction by sulforaphane, a cruciferous vegetable-derived cancer chemopreventive agent

Sulforaphane, a constituent of many edible cruciferous vegetables, including broccoli, effectively suppresses proliferation of cancer cells in culture and in vivo by causing apoptosis induction, but the sequence of events leading to cell death is poorly defined. Here, we show that multidomain proapoptotic Bcl-2 family members Bax and Bak play a critical role in apoptosis induction by sulforaphane. This conclusion is based on the following observations: (a) sulforaphane treatment caused a dose- and time-dependent increase in the protein levels of both Bax and Bak and conformational change and mitochondrial translocation of Bax in SV40-transformed mouse embryonic fibroblasts (MEF) derived from wild-type mice to trigger cytosolic release of apoptogenic molecules (cytochrome c and Smac/DIABLO), activation of caspase-9 and caspase-3, and ultimately cell death; (b) MEFs derived from Bax or Bak knockout mice resisted cell death by sulforaphane, and (c) MEFs derived from Bax and Bak double knockout mice exhibited even greater protection against sulforaphane-induced cytochrome c release, caspase activation, and apoptosis compared with wild-type or single knockout cells. Interestingly, sulforaphane treatment also caused a dose- and time-dependent increase in the protein level of Apaf-1 in wild-type, Bax-/-, and Bak-/- MEFs but not in double knockout, suggesting that Bax and Bak might regulate sulforaphane-mediated induction of Apaf-1 protein. A marked decline in the protein level of X-linked inhibitor of apoptosis on treatment with sulforaphane was also observed. Thus, it is reasonable to postulate that sulforaphane-induced apoptosis is amplified by a decrease in X-linked inhibitor of apoptosis level, which functions to block cell death by inhibiting activities of caspases. In conclusion, the results of the present study indicate that Bax and Bak proteins play a critical role in initiation of cell death by sulforaphane.     

Helenalin triggers a CD95 death receptor-independent apoptosis that is not affected by overexpression of Bcl-x(L) or Bcl-2

Apoptosis is required for proper tissue homeostasis. Defects in apoptosis signaling pathways, thus, contribute to carcinogenesis and chemoresistance. A major goal in chemotherapy is, therefore, to find cytotoxic agents that restore the ability of tumor cells to undergo apoptosis. We show here that the sesquiterpene lactone helenalin (10-50 microM) induces apoptosis in leukemia Jurkat T cells even if they lack the CD95 death receptor or overexpress the antiapoptotic proteins Bcl-x(L) or Bcl-2. Activated peripheral blood mononuclear cells, however, are not affected (10-50 microM helenalin). Helenalin led to a time-dependent (0-24 h) cleavage of the specific caspase-3-like substrate Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin as well as to the proteolytic processing of procaspase-3 and -8. Caspase activation was a necessary requirement for apoptosis because the broad-spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk, 50 microM) completely abrogated helenalin-induced DNA fragmentation as well as phosphatidylserin translocation. Although the initiator caspase-8 was activated, the helenalin-induced signaling pathway did not require the CD95 death receptor as shown using cells without or with an antibody (ZB4)-blocked CD95 receptor. Helenalin also did not induce CD95 or CD95-ligand expression. On the other hand, helenalin was found to induce the release of cytochrome c from mitochondria that was not inhibited by the caspase inhibitor zVAD-fmk, which indicated that cytochrome c release precedes caspase activation. Cytochrome c release was accompanied by dissipation of the mitochondrial transmembrane potential (DeltaPsi(m)), which was partly inhibited by zVAD-fmk, which suggests that caspases are involved in loss of DeltaPsi(m). Most importantly, overexpression of the mitochondria protecting proteins Bcl-x(L) or Bcl-2 failed to confer resistance to helenalin-induced apoptosis, although the data presented here suggest that helenalin induces a mitochondria-dependent pathway. Thus, helenalin is a promising experimental cytotoxic agent that possibly points to new strategies to overcome apoptosis resistance attributable to overexpression of antiapoptotic Bcl-2 proteins.     

Tumor necrosis factor alpha induces BID cleavage and bypasses antiapoptotic signals in prostate cancer LNCaP cells

Survival of cancer cells in response to therapy, immune response, or metastasis depends on interactions between pro- and antiapoptotic signals. Two major proapoptotic pathways have been described: (a) a death receptor pathway; and (b) a mitochondrial pathway. We reported previously that Akt and the epidermal growth factor (EGF) receptor send separate, redundant survival signals that act to inhibit the mitochondrial proapoptotic pathway in prostate cancer LNCaP cells. However, it was unclear at what level the pro- and antiapoptotic signals interact in these cells, and it was also unclear whether these signals would inhibit the death receptor pathway. We found that EGF can protect LNCaP cells from apoptosis induced by LY294002 but not from tumor necrosis factor a (TNF-alpha)-induced apoptosis. Furthermore, TNF-alpha induced apoptosis under conditions in which Akt was active. Treatment with TNF-alpha resulted in activation of caspase 8 and cleavage of BID, which in turn induced cytochrome c release and caspase 9-dependent activation of effector caspases. Thus, proapoptotic signals induced by both TNF-alpha and LY294002 converge on mitochondria and trigger cytochrome c release. Because EGF can inhibit cytochrome c release induced by LY294002 but not cytochrome c release induced by TNF-alpha, we suggest that the EGF survival mechanism operates on the mitochondrial pathway at a site upstream of cytochrome c release. The ability of TNF-alpha to bypass survival signals from activated EGF receptor and Akt in prostate cancer cells makes death receptor signaling a promising avenue for therapeutic intervention.     

The Bcl-2 transgene protects T cells from renal cell carcinoma-mediated apoptosis.

PURPOSE: Tumors induce T-cell apoptosis as a mechanism of inhibiting antitumor immunity. Using coculture experiments, it has been shown that tumor lines stimulate T-cell apoptosis by a pathway involving a mitochondrial permeability transition and cytochrome c release. Activated T cells express abundant levels of Bcl-2, an antiapoptotic molecule that would be expected to confer resistance to such tumor-mediated killing. We examined the mechanism by which Bcl-2 is dysregulated in T cells exposed to the renal tumor line SK-RC-45, and we determined whether overexpressing Bcl-2 protects T cells from tumor-mediated apoptosis. EXPERIMENTAL DESIGN: Activated T lymphocytes and Jurkat cells transfected or not transfected with Bcl-2 were exposed to SK-RC-45 for 48-72 h. After coculture, lymphocytes were analyzed for Bcl-2 expression using Western analysis and for tumor-induced apoptosis by terminal deoxynucleotidyl transferase-mediated nick end labeling. The role of SK-RC-45-stimulated caspase activation in degrading T-cell Bcl-2 was assessed using a pan-caspase inhibitor, as well as a specific inhibitor of caspase-9. RESULTS: The renal cell carcinoma cell line SK-RC-45 sensitizes peripheral blood activated T lymphocytes and Jurkat cells to apoptosis by a mechanism that involves degradation of the antiapoptotic protein Bcl-2. The SK-RC-45-induced modulation of lymphocyte Bcl-2 levels was largely caspase independent because pretreatment of T cells with pan-caspase inhibitor III or an inhibitor of caspase-9 had minimal or no effect on stabilizing the protein, although it did provide protection against apoptosis. Overexpression of Bcl-2 protected Jurkat cells from tumor-mediated killing. CONCLUSIONS: Bcl-2 inhibition is a mechanism by which tumors may render lymphocytes sensitive to other tumor-derived, proapoptotic stimuli.     

The hepatitis B virus X protein abrogates Bcl-2-mediated protection against Fas apoptosis in the liver

The role of the hepatitis B virus protein HBx in liver cell proliferation and apoptosis remains controversial. Using a transgenic mouse model, we have recently shown that HBx stimulates the apoptotic turnover of hepatocytes, independently of p53. In this paper, we tested whether the proapoptotic function of HBx can interfere with Bcl-2 during hepatic apoptosis in vivo. HBx transgenic mice were crossed with PK-hBcl-2 mice that are protected against Fas killing by constitutive overexpression of Bcl-2 in hepatocytes. In a lethal challenge with Fas antibodies, HBx expressed at low levels restored sensitivity to Fas-mediated apoptosis and fulminant hepatic failure in mice overexpressing Bcl-2. Furthermore, cytochrome c release from mitochondria and caspase 3 activation were restored to normal levels in HBx/Bcl-2 mice during transduction of the Fas signal. Thus, the proapoptotic activity of HBx overcomes or bypasses the inhibitory effect of Bcl-2 against Fas cytotoxicity. This effect was not apparently mediated through downregulation of the PK-hBcl-2 transgene or via delocalization of the Bcl-2 protein, and a direct interaction of HBx with Bcl-2, Bcl-X(L) or Bax could not be evidenced in yeast two-hybrid assays. We further show that apoptosis induced by ectopic expression of HBx is associated with mitochondrial membrane alterations and caspase 3 activation. Our data indicate that the dominant function of HBx upon Bcl-2-regulated control of apoptosis might play an important role in the pathogenesis of chronic hepatitis B.     

Pro-apoptotic properties of hyperforin in leukemic cells from patients with B-cell chronic lymphocytic leukemia.

The effects of the hyperforin (HF), a natural phloroglucinol purified from Hypericum perforatum, were investigated ex vivo on leukemic cells from patients with B-cell chronic lymphocytic leukemia (B-CLL). HF was found to promote apoptosis of B-CLL cells, as shown by time- and dose-dependent stimulation of phosphatidylserine externalization and DNA fragmentation, by disruption of the mitochondrial transmembrane potential, caspase-3 activation and cleavage of the caspase substrate PARP-1. Moreover, HF-induced downregulation of Bcl-2 and Mcl-1, two antiapoptotic proteins that control mitochondrial permeability. HF also downregulated two proteins which are overexpressed by B-CLL patients' cells, the cell cycle inhibitor p27kip1 through caspase-dependent cleavage into a p23 form, and the nitric oxid (NO) synthase of type 2 (inducible NO synthase). This latter was accompanied by reduction in the production of NO known to be antiapoptotic in B-CLL cells. Preventing effects of the general caspase inhibitor z-VAD-fmk indicated that HF-promoted apoptosis of B-CLL cells was mostly caspase dependent. Furthermore, normal B lymphocytes purified from healthy donors appeared less sensitive to HF-induced apoptosis than B-CLL cells. These results indicate that HF may be of interest in the development of new therapies for B-CLL based on the induction of apoptosis and combination with cell cycle-dependent antitumor drugs.     

The BH3-only protein BimL overrides Bcl-2-mediated apoptosis resistance in melanoma cells

Melanoma cells are characterized by apoptosis deficiency coinciding with reduced expression of the proapoptotic Bcl-2 protein Bim. An adenoviral vector was constructed with the Bim_L cDNA controlled by an inducible promoter. Highly efficient apoptosis induction and abrogated cell proliferation was seen in melanoma cells upon Bim_L overexpression. Loss of mitochondrial membrane potential, release of mitochondrial apoptogenic factors and caspase-9 processing indicated the activation of mitochondrial apoptosis pathways. Bim_L activated both Bax and Bak, as shown by siRNA knockdown and activation-specific antibodies. Of note, Bim_L overrode the apoptosis blockade by Bcl-2 overexpression or by Bax/Bak single knockdown. The high efficacy correlated to Bim_L interaction with all antiapoptotic Bcl-2 family members in melanoma cells, shown by co-immunoprecipitation analyses for Bcl-2, Bcl-x_L, Mcl-1 and Bcl-w. Thus, Bim_L reveals an outstanding proapoptotic potential in melanoma cells, and strategies for its re-expression appear of interest. These have been reported for B-Raf inhibitors, and their efficacy may be partly attributed to Bim_L.     

Rapid induction of mitochondrial events and caspase-independent apoptosis in Survivin-targeted melanoma cells

The inhibitor of apoptosis (IAP) protein Survivin is expressed in most cancers and is a key factor in maintaining apoptosis resistance. Although several IAPs have been shown to act as direct inhibitors of caspases, the precise antiapoptotic function of Survivin remains controversial. To clarify the mechanism by which Survivin protects cells, we investigated the kinetics of apoptosis and apoptotic events following Survivin inhibition utilizing a melanoma cell line harboring a tetracycline-regulated Survivin dominant-negative mutant (Survivin-T34A). Blocking Survivin resulted in both caspase activation and apoptosis; however, the level of apoptosis was only partially reduced by caspase inhibition. Survivin blockade also resulted in mitochondrial events that preceded caspase activation, including depolarization and release of cytochrome c and Smac/DIABLO. Levels of other IAPs were not altered in Survivin-targeted cells, although modest cleavage of XIAP and Livin was observed. The earliest proapoptotic event observed in Survivin-targeted cells was nuclear translocation of mitochondrial apoptosis-inducing factor (AIF), known to trigger both apoptotic mitochondrial events and caspase-independent DNA fragmentation. These findings suggest that a key antiapoptotic function of Survivin relates to inhibition of mitochondrial and AIF-dependent apoptotic pathways, and its expression in melanoma and other cancers likely protects against both caspase-independent and -dependent apoptosis.     

Ectopic expression of Bcl-XL or Ku70 protects human colon cancer cells (SW480) against curcumin-induced apoptosis while their down-regulation potentiates it

Curcumin, the yellow pigment derived from Curcuma longa, is known to induce apoptosis of several cancer cells. However, many cancer cells protect themselves by over-expressing antiapoptotic proteins such as Bcl-XL or Ku70. To study their role in curcumin-induced apoptosis, human colon cancer cells (SW480) were made to over-express or under-express Bcl-XL (by stable transfection) and Ku70 (by transient transfection) using plasmid constructs that express their genes in sense or antisense orientation, respectively. Stable cells that express Bax [Bax-GFP (green fluorescent protein)], a proapoptotic member of the Bcl-2 family, were also established. Curcumin-induced cell death and nuclear condensation was more in AsBcl-XL and AsKu70 cells that under-express Bcl-XL and Ku70, respectively, compared with the vector-transfected cells. Bcl-XL and Ku70 protected the cells by inhibiting the release of cytochrome c, Smac (second mitochondria derived activator of caspase) and apoptosis inducing factor (AIF), and the activation of caspases 9, 8 and 3 triggered by curcumin. AsBcl-XL and AsKu70 cells were more sensitive to curcumin through enhanced activation of caspases 9 and 3 and release of cytochrome c, Smac and AIF. Curcumin-induced activation of caspase 8 was blocked by Ku70 but not by Bcl-XL. However, caspase 8 activation by curcumin was accelerated in both AsBcl-XL and AsKu70 cells suggesting a possible feedback activation of caspase 8 by caspase 3. Bax-GFP cells were highly sensitized when Ku70 was down-regulated supporting the reported role of Ku70 in the retention of Bax within the cytosol. The study reveals the potential of antisense inhibition of antiapoptotic proteins as an effective strategy to tackle chemoresistant cancers with curcumin.     

Apoptotic cytosol facilitates Bax translocation to mitochondria that involves cytosolic factor regulated by Bcl-2.

Proapoptotic members of the Bcl-2 family, including Bax, Bak, and Bid, directly trigger the mitochondrial release of apoptogenic cytochrome c and apoptosis-inducing factor into the cytoplasm. One of the crucial steps before Bax can exert its proapoptotic activity is translocation from the cytoplasm to the mitochondria, but the molecular mechanism of this translocation is not understood. To investigate the mechanism of apoptosis-associated Bax translocation, we used an in vitro system comprising isolated mitochondria and cytosol. We found that both endogenous and exogenous added recombinant Bax translocated to the mitochondria more efficiently in the presence of cytosol from cells with VP16-induced apoptosis than with cytoplasm from normal cells. This apoptosis-dependent promotion of Bax translocation was not seen with cytosol that was prepared from VP16-treated cells expressing Bcl-2. Cytosol from cells with VP16-induced apoptosis, but not that from normal cells or Bcl-2-expressing cells, induced cytochrome c release from isolated mitochondria, which, as assessed by immunodepletion experiments, was mainly mediated by Bax. These results suggest that Bcl-2 exerts its antiapoptotic activity partly by inhibiting the translocation of Bax through the modification of cytosolic factors that are involved in such translocation during apoptosis.     

Bisindolylmaleimide IX is a potent inducer of apoptosis in chronic lymphocytic leukaemic cells and activates cleavage of Mcl-1.

New agents are required for the treatment of chronic lymphocytic leukaemia (CLL). We show here that a protein kinase C inhibitor, bisindolylmaleimide IX, is a potent inducer of apoptosis in CLL cells, and investigate the mechanisms by which this is induced. Bisindolylmaleimide IX induced a conformational change and subcellular redistribution of Bax from the cytosol to the mitochondria, resulting in the release of the proapoptotic mediators cytochrome c, Smac and Omi/HtrA2 from the mitochondrial inner membrane space. This was followed by the activation of caspase-9 as the apical caspase and subsequent activation of effector caspases. CLL cells undergoing apoptosis showed a rapid caspase-mediated cleavage of Mcl-1, an antiapoptotic member of the Bcl-2 family implicated in CLL survival and poor prognosis. This cleavage was mediated primarily by caspase-3. Cleavage of Mcl-1 may provide a feed-forward amplification loop, resulting in the rapid induction of apoptosis. Bisindolylmaleimide IX or a related derivative may be of clinical use in the treatment of CLL.