Involvement of proapoptotic molecules Bax and Bak in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced mitochondrial disruption and apoptosis: differential regulation of cytochrome c and Smac/DIABLO release

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo2L induces apoptosis in a wide variety of cancer and transformed cells. Activation of BID, a "BH3-domain-only" Bcl-2 family member, triggers the oligomerization of proapoptotic family members Bak or Bax, resulting in the release of mitochondrial proteins to cytosol. In this study, we have shown the importance of Bax and Bak in TRAIL-induced apoptosis by studying in murine embryonic fibroblasts (MEFs) from Bax(-/-) and Bak(-/-) animals. TRAIL induced cytochrome c release and apoptosis in wild-type, Bid(-/-), Bax(-/-), or Bak(-/-) MEFs, but not in Bax(-/-) Bak(-/-) double knockout (DKO) MEFs. Bid, which functions upstream of cytochrome c release, was cleaved in all of the knockout cells except in Bid(-/-) MEFs. The release of cytochrome c was correlated with caspase-9 activity. TRAIL increased caspase-3 activity in all of the cells except in DKO cells. TRAIL-induced drop in mitochondrial membrane potential was not observed in DKO MEFs. Unlike cytochrome c release, TRAIL-induced Smac/DIABLO release was blocked in Bid(-/-), Bax(-/-), Bak(-/-), or DKO MEFs, suggesting the differential regulation of these mitochondrial proteins during apoptosis. The apoptotic events downstream of mitochondria were intact in DKO MEFs, because microinjection of cytochrome c, or ectopic expression of mature Smac/DIABLO or pretreatment of Smac N7 peptide completely restored TRAIL sensitivity. In conclusion, the data suggest that Bax and Bak differentially regulate the release of cytochrome c and Smac/DIABLO from mitochondria, and Smac/DIABLO can be used to sensitize cells that are deficient in Bax and Bak genes, or resistant to TRAIL.     

Two distinct Fas-activated signaling pathways revealed by an antitumor drug D609

During the process of death receptor-mediated apoptosis, Bid is cleaved by activated caspase-8, and then cleaved Bid conveys apoptotic signals to the mitochondria by activating Bax/Bak. In the present study, we found that D609 (an antitumor drug with multiple activities) blocks Fas-induced apoptosis. D609 did not interfere with activation of caspase-8 and cleavage of Bid, whereas it blocked cytochrome c release from the mitochondria by inhibiting the activation of Bax and Bak. D609 had no protective effect against apoptosis of SKW6.4 cells, which are typical type I cells. Studies using permeabilized cells revealed that in addition to activation of caspase-8, Fas activated a distinct and D609-sensitive signaling pathway that transmitted signal(s) sensitizing the mitochondria to apoptotic stimuli, and that D609 itself promoted mitochondrial resistance to apoptotic stimuli.     

BH3 mimetic ABT-737 potentiates TRAIL-mediated apoptotic signaling by unsequestering Bim and Bak in human pancreatic cancer cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to induce mitochondrial apoptotic signaling that can be negatively regulated by prosurvival Bcl-2 proteins. ABT-737 is a small-molecule BH3 mimetic that binds to and antagonizes Bcl-2/Bcl-x(L) but not Mcl-1. We show that ABT-737 can synergistically enhance TRAIL-mediated cytotoxicity in human pancreatic cancer cell lines. ABT-737 was shown to enhance TRAIL-induced apoptosis as shown by DNA fragmentation, activation of caspase-8 and Bid, and cleavage of caspase-3 and poly(ADP-ribose) polymerase. A Bax conformational change induced by TRAIL was enhanced by ABT-737. ABT-737 disrupted the interaction of Bak with Bcl-x(L) in both cell lines. Furthermore, ABT-737 untethered the proapoptotic BH3-only protein Bim from its sequestration by Bcl-x(L) or Bcl-2. Bim small hairpin RNA (shRNA) was shown to attenuate caspase-3 cleavage and to reduce the cytotoxic effects of TRAIL plus ABT-737 compared with shRNA control cells. Finally, Mcl-1 shRNA potentiated caspase-3 cleavage by ABT-737 and enhanced its cytotoxic effects. Taken together, ABT-737 augments TRAIL-induced cell killing by unsequestering Bim and Bak and enhancing a Bax conformational change induced by TRAIL. These findings suggest a novel strategy to enhance cross-talk between the extrinsic and intrinsic apoptotic pathways to improve therapeutic efficacy against pancreatic cancer.     

Bax and Bak genes are essential for maximum apoptotic response by curcumin, a polyphenolic compound and cancer chemopreventive agent derived from turmeric, Curcuma longa

Curcumin, an active ingredient of turmeric (Curcuma longa), inhibits proliferation and induces apoptosis in cancer cells, but the sequence of events leading to cell death is poorly defined. The objective of this study was to examine the molecular mechanisms by which multidomain pro-apoptotic Bcl-2 family members Bax and Bak regulate curcumin-induced apoptosis using mouse embryonic fibroblasts (MEFs) deficient in Bax, Bak or both genes. Curcumin treatment resulted an increase in the protein levels of both Bax and Bak, and mitochondrial translocation and activation of Bax in MEFs to trigger drop in mitochondrial membrane potential, cytosolic release of apoptogenic molecules [cytochrome c and second mitochondria-derived activator of caspases (Smac)/direct inhibitor of apoptosis protein-binding protein with low isoelectric point], activation of caspase-9 and caspase-3 and ultimately apoptosis. Furthermore, MEFs derived from Bax and Bak double-knockout (DKO) mice exhibited even greater protection against curcumin-induced release of cytochrome c and Smac, activation of caspase-3 and caspase-9 and induction of apoptosis compared with wild-type MEFs or single-knockout Bax(-/-) or Bak(-/-) MEFs. Interestingly, curcumin treatment also caused an increase in the protein level of apoptosis protease-activating factor-1 in wild-type MEFs. Smac N7 peptide enhanced curcumin-induced apoptosis, whereas Smac siRNA inhibited the effects of curcumin on apoptosis. Mature form of Smac sensitized Bax and Bak DKO MEFs to undergo apoptosis by acting downstream of mitochondria. The present study demonstrates the role of Bax and Bak as a critical regulator of curcumin-induced apoptosis and over-expression of Smac as interventional approaches to deal with Bax- and/or Bak-deficient chemoresistant cancers for curcumin-based therapy.     

Differential involvement of Bax and Bak in TRAIL-mediated apoptosis of leukemic T cells.

TRAIL-induced apoptosis has been considered a promising therapeutic approach for tumors that are resistant to chemotherapy, which is usually mediated via mitochondrial apoptotic cascades. Recent studies have shown that in certain cancer cells, TRAIL-mediated apoptosis is also dependent on mitochondrial involvement, suggesting that similar mechanisms of resistance to chemotherapy might be implicated in the resistance of tumor cells to TRAIL. We have used TRAIL-resistant leukemic cells that are deficient in both Bax and Bak to determine the roles of these Bcl-2 members in TRAIL-mediated apoptosis. Exposure of these cells to TRAIL did not have an impact on cell viability, although it induced the processing of caspase-3 to its active p20 subunit. The activity of the p20 caspase-3 appeared to be inhibited as no autoprocessing of this p20 subunit or cleavage of known caspase-3 substrates were detected. Also, in the absence of Bax and Bak, no release of mitochondrial apoptogenic proteins was observed following TRAIL treatment. Adenoviral transduction of the Bax, but not the Bak gene, to the Bax/Bak-deficient leukemic cells rendered them TRAIL-sensitive as assessed by enhanced apoptotic death and caspase-3 processing. These findings demonstrate preferential utilization of Bax over Bak in leukemic cell response to specific apoptotic stimulation.     

Bax plays a pivotal role in thapsigargin-induced apoptosis of human colon cancer HCT116 cells by controlling Smac/Diablo and Omi/HtrA2 release from mitochondria

Bax is a crucial mediator of the mitochondrial pathway for apoptosis, and loss of this proapoptotic Bcl-2 family protein contributes to drug resistance in human cancers. We report here that the endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin (THG) induces apoptosis of human colon cancer HCT116 cells through a Bax-dependent signaling pathway controlling the cytosolic release of mitochondrial apoptogenic molecules. Treating HCT116 cells with THG results in caspase-8 activation; Bid cleavage; Bax conformational change and mitochondrial translocation; the release of cytochrome c, Smac/Diablo, and Omi/HtrA2 into the cytosol; caspase-3 activation; and apoptosis. In contrast, knockout of Bax completely abrogates the full processing/activation of caspase-3 but has no effect on the processing of caspase-8 and the initial cleavage of caspase-3 to p24 fragment after THG treatment. The caspase-8-specific inhibitor z-IETD-fmk, as well as pan-caspase inhibitor z-VAD-fmk, but not the calpain inhibitor E-64d, prevents Bid cleavage, Bax conformational change, and subsequent caspase-3 processing and apoptosis. Caspase-8 processing is dependent on de novo protein synthesis; DR5 expression is strongly up-regulated by THG treatment. Moreover, the absence of Bax blocks THG-induced Omi and Smac release from mitochondria, and expression of cytosolic Omi (GFP-IETD-Omi) or Smac (GFP-IETD-Smac) restores the sensitivity of Bax-knockout HCT116 cells to apoptosis in response to THG treatment. Taken together, our results indicate that Bax-dependent Smac and Omi release plays an essential role in caspase-3 activation and apoptosis induced by THG in human colon cancer HCT116 cells.     

Type I and type II reactions in TRAIL-induced apoptosis -- results from dose-response studies

Death receptor-induced apoptosis is paradigmatically mediated via the recruitment of FADD adapter molecule to the ligand/receptor complex and subsequent activation of caspase-8. However, several observations provided evidence that components of the mitochondrial apoptosis pathway are involved in death receptor-mediated apoptosis. In this regard, caspase-8-mediated activation of Bid induces the release of cytochrome c from the mitochondria, which, in turn, triggers the formation of the apoptosome protein complex, resulting in the activation of caspase-9. Whereas Bax or Bak were shown to be required for the proapoptotic effect of Bid, Bcl-2 was described to interfere with its action. Up to now, contradictory results regarding the role of Bcl-2 in TRAIL-induced apoptosis have been published. In order to study the influence of Bcl-2 on TRAIL-induced cell death more detailed, we utilized a tetracycline-regulated Bcl-2 expression system in Jurkat T cells. After having analysed the dose response for TRAIL-induced activation of caspase-8, -9, -3, breakdown of the mitochondrial membrane potential, and changes in the apoptotic morphology in cells expressing different Bcl-2 levels, we conclude that overexpression of Bcl-2 mediates a partial resistance towards lower doses of TRAIL that can be overcome when higher doses of TRAIL are applied. Thus, the requirement of the mitochondrial pathway for death receptor-induced apoptosis in type II cells should be reconsidered, since the protective effect of Bcl-2 is limited to lower TRAIL doses or early observation time points.     

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.     

Hierarchical involvement of Bak, VDAC1 and Bax in cisplatin-induced cell death

Following the screening of a battery of distinct small-interfering RNAs that target various components of the apoptotic machinery, we found that knockdown of the voltage-dependent anion channel 1 (VDAC1) was particularly efficient in preventing cell death induced by cisplatin (CDDP) in non-small cell lung cancer cells. Both the downregulation of VDAC1 and its chemical inhibition with 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid reduced the apoptosis-associated modifications induced by CDDP, including mitochondrial transmembrane potential dissipation and plasma membrane permeabilization. VDAC1 inhibition strongly reduced the CDDP-induced conformational activation of Bax, yet had no discernible effect on the activation of Bak, suggesting that VDAC1 acts downstream of Bak and upstream of Bax. Accordingly, knockdown of Bak abolished the activation of Bax, whereas Bax downregulation had no effect on Bak activation. In VDAC1-depleted cells, the failure of CDDP to activate Bax could be reversed by means of the Bcl-2/Bcl-X(L) antagonist ABT-737, which concomitantly restored CDDP cytotoxicity. Altogether, these results delineate a novel pathway for the induction of mitochondrial membrane permeabilization (MMP) in the course of CDDP-induced cell death that involves a hierarchical contribution of Bak, VDAC1 and Bax. Moreover, our data suggest that VDAC1 may act as a facultative regulator/effector of MMP, depending on the initial cytotoxic event.     

Ionizing radiation but not anticancer drugs causes cell cycle arrest and failure to activate the mitochondrial death pathway in MCF-7 breast carcinoma cells

There is considerable evidence that ionizing radiation (IR) and chemotherapeutic drugs mediate apoptosis through the intrinsic death pathway via the release of mitochondrial cytochrome c and activation of caspases -9 and -3. Here we show that MCF-7 cells that lack caspase-3 undergo a caspase-dependent apoptotic cell death in the absence of DNA fragmentation and alpha-fodrin cleavage following treatment with etoposide or doxorubicin, but not after exposure to IR. Re-expression of caspase-3 restored DNA fragmentation and alpha-fodrin cleavage following drug treatment, but it did not alter the radiation-resistant phenotype of these cells. In contrast to the anticancer drugs, IR failed to induce the intrinsic death pathway in MCF-7/casp-3 cells, an event readily observed in IR-induced apoptosis of HeLa cells. Although IR-induced DNA double-strand breaks were repaired with similar efficiencies in all cell lines, cell cycle analyses revealed a persistent G2/M arrest in the two MCF-7 cell lines, but not in HeLa cells. Together, our data demonstrate that caspase-3 is required for DNA fragmentation and alpha-fodrin cleavage in drug-induced apoptosis and that the intrinsic death pathway is fully functional in MCF-7 cells. Furthermore, they show that the radiation-resistant phenotype of MCF-7 cells is not due to the lack of caspase-3, but is caused by the failure of IR to activate the intrinsic death pathway. We propose (1) different signaling pathways are induced by anticancer drugs and IR, and (2) IR-induced G2/M arrest prevents the generation of an apoptotic signal required for the activation of the intrinsic death pathway.     

Activation of the caspase-8/Bid and Bax pathways in aspirin-induced apoptosis in gastric cancer

Aspirin-induced apoptosis is one of the important mechanisms for its antitumour effect against gastric cancer. We aimed at investigating the involvement of bcl-2 family members in the apoptotic pathway in gastric cancer. Gastric cancer cell line AGS and MKN-45 were observed as to cell growth inhibition and induction of apoptosis in response to treatment with aspirin. Cell proliferation was measured by MTT assay. Apoptosis was determined by 4'-6-diamidino-2-phenylindole staining. Protein expression was determined by western blotting. We showed that aspirin activated caspase-8, caspase-9 and capase-3, cleaved and translocated Bid, induced a conformational change in and translocation of Bax and cytochrome c release. In addition, suppression of caspase-8 with the specific inhibitor z-IETD-fmk, as well as the pan-caspase inhibitor z-VAD-fmk, prevented Bid cleavage and subsequent apoptosis. The caspase inhibitors failed to abolish the effects on Bax activation. In conclusion, our results identify a role of caspase-8/Bid and activation of Bax as a novel mechanism for aspirin-induced apoptosis in gastric cancer.     

The role of Apaf-1, caspase-9, and bid proteins in etoposide- or paclitaxel-induced mitochondrial events during apoptosis

Ectopic overexpression of Apaf-1 (2.5-fold) in human acute myelogenous leukemia HL-60 cells (HL-60/Apaf-1 cells) induced apoptosis and sensitized HL-60/Apaf-1 cells to etoposide- and paclitaxel-induced apoptosis (C. Perkins et al., Cancer Res., 58: 4561-4566, 1998). In this report, we demonstrate that in HL-60/Apaf-1 cells, the activity of caspase-9 and -3 induced by Apaf-1 overexpression was associated with a significant increase (5-fold) in the cytosolic accumulation of cytochrome c (cyt c), loss of mitochondrial membrane potential (deltapsim), and an increase in the reactive oxygen species. These were also associated with the processing of procaspase-8 and Bid (cytosolic, proapoptotic BH3 domain containing protein). Transient transfection of Apaf-1 into the Apaf-1-containing mouse embryogenic fibroblasts (MEFs; Apaf-1+/- MEFs) or Apaf-1-/- MEFs also induced the processing of procaspase-9 and procaspase-8, Bid cleavage, and apoptosis. These events were secondary to the activity of the downstream caspases induced by Apaf-1. This conclusion is supported by the observation that in HL-60/Apaf-1 cells, ectopic expression of dominant negative caspase-9, its inhibitory short isoform caspase-9b, or XIAP or treatment with the caspase inhibitor zVAD (50 microM) inhibited Apaf-1-induced caspase-8 and Bid cleavage, mitochondrial deltapsim, release of cyt c, and apoptosis. In contrast, a transient transfection of dominant negative caspase-8 or CrmA or exposure to caspase-8 inhibitor zIETD-fmk inhibited the processing of procaspase-8 and Bid but did not inhibit the cytosolic accumulation of cyt c in either the untreated HL-60/Apaf-1 cells or the etoposide-treated HL-60/Apaf-1 and HL-60/neo cells. These results indicate that Apaf-1 overexpression lowers the apoptotic threshold by activating caspase-9 and caspase-3. This triggers the mitochondrial deltapsim and cyt c release into the cytosol through a predominant mechanism other than cleavage of caspase-8 and/or Bid. This mechanism may involve a cytosolic mitochondrial permeability transition factor, which may be processed and activated by the downstream effector caspases, thereby completing an amplifying feedback loop, which triggers the mitochondrial events during apoptosis.     

Caspase-dependent and -independent cell death pathways after DNA damage (Review)

Apoptosis is known to be an important phenomenon in exerting antitumor response to cancer therapy, which is regulated by Bcl-2 family proteins through mitochondrial permeability transition (MPT). Insertion by the activated Bax/Bak in response to DNA damage induces mitochondrial membrane permeabilization (MMP) via an anion channel, VDAC in mitochondrial outer membrane that plays a crucial role in releasing small molecules such as cytochrome c, Smac/DIABLO, Omi/HtrA2, AIF, and endonuclease G leading to cell death. The released small molecules are involved in caspase-dependent and -independent cell death pathway that is inhibited by Bcl-2/xL. Despite the fact that the pancaspase inhibitor, zVAD-fmk inhibited the caspase cascade, cell death mediated by caspase-independent pathway was not blocked. Similarly, although etoposide induced-apoptosis was inhibited in Bax(-/-)/Bak(-/-)mouse embryonic fibroblasts, autophagy was not inhibited, which was regulated by Bcl-xL. It appears that the cross-talk between caspase-dependent and -independent apoptotic cell death including autophagic cell death that was mediated by MPT affects overall tumor response to anticancer treatment. In this review, to assist a comprehensive understanding of MPT-mediated cell death pathway for exploring appropriate targets in cancer therapy, role of the caspase-dependent and -independent cell death pathway in the interaction of these pathways is discussed.     

Activation of the mitochondrial death pathway is commonly mediated by a preferential engagement of Bak

Among the members of the Bcl-2 family, the multidomain proteins Bax and Bak are crucial for the activation of mitochondria. However, it is still unclear whether they act in a unique and distinct manner or whether they exhibit redundant functions. To systematically investigate their activation on a single-cell level, we established MCF-7 cell lines stably expressing GFP-fusion variants of these proteins. We found that MCF-7/GFP-Bak cells showed an increased sensitivity to apoptosis induction by staurosporine, actinomycin D, TRAIL and overexpression of Puma compared to GFP-Bax-expressing cells. Independently of the death stimulus used, oligomerization of endogenous and exogenous Bak was mostly detected prior to an activation of Bax, whereas cells displaying oligomerized Bax in the absence of Bak clusters were not observed. In addition, activation of Bax but not Bak was attenuated by a caspase inhibitor. Consistent with this, caspase-3-deficient MCF-7 cells displayed a significantly reduced activation of endogenous Bax than caspase-3-proficient MCF-7 cells. Thus, our data strongly suggest that diverse apoptotic stimuli preferentially engage the Bak pathway, whereas the triggering of Bax occurs, at least partially, downstream of mitochondrial caspase activation, most likely constituting a positive feedback loop for the amplification of the death signal.     

The multikinase inhibitor sorafenib potentiates TRAIL lethality in human leukemia cells in association with Mcl-1 and cFLIPL down-regulation

Interactions between the multikinase inhibitor sorafenib and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) were examined in malignant hematopoietic cells. Pretreatment (24 h) of U937 leukemia cells with 7.5 micromol/L sorafenib dramatically increased apoptosis induced by sublethal concentrations of TRAIL/Apo2L (75 ng/mL). Similar interactions were observed in Raji, Jurkat, Karpas, K562, U266 cells, primary acute myelogenous leukemia blasts, but not in normal CD34+ bone marrow cells. Sorafenib/TRAIL-induced cell death was accompanied by mitochondrial injury and release of cytochrome c, Smac, and AIF into the cytosol and caspase-9, caspase-3, caspase-7, and caspase-8 activation. Sorafenib pretreatment down-regulated Bcl-xL and abrogated Mcl-1 expression, whereas addition of TRAIL sharply increased Bid activation, conformational change of Bak (ccBak) and Bax (ccBax), and Bax translocation. Ectopic Mcl-1 expression significantly attenuated sorafenib/TRAIL-mediated lethality and dramatically reduced ccBak while minimally affecting levels of ccBax. Similarly, inhibition of the receptor-mediated apoptotic cascade with a caspase-8 dominant-negative mutant significantly blocked sorafenib/TRAIL-induced lethality but not Mcl-1 down-regulation or Bak/Bax conformational change, indicating that TRAIL-mediated receptor pathway activation is required for maximal lethality. Sorafenib/TRAIL did not increase expression of DR4/DR5, or recruitment of procaspase-8 or FADD to the death-inducing signaling complex (DISC), but strikingly increased DISC-associated procaspase-8 activation. Sorafenib also down-regulated cFLIP(L), most likely through a translational mechanism, in association with diminished eIF4E phosphorylation, whereas ectopic expression of cFLIP(L) significantly reduced sorafenib/TRAIL lethality. Together, these results suggest that in human leukemia cells, sorafenib potentiates TRAIL-induced lethality by down-regulating Mcl-1 and cFLIP(L), events that cooperate to engage the intrinsic and extrinsic apoptotic cascades, culminating in pronounced mitochondrial injury and apoptosis.     

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.     

Changes in mitochondrial membrane potential and mitochondrial mass occur independent of the activation of caspase-8 and caspase-3 during CD95-mediated apoptosis in peripheral blood T cells

Although death receptor pathway and mitochondrial pathways of apoptosis are connected through Bid cleavage, such activation of mitochondrial pathway by death receptor signaling is observed in type II cells and not in type I cells (peripheral blood T cells). Furthermore, activation of mitochondria via Bid is associated with release of cytochrome c and caspase-9 activation. In this study we demonstrate that anti-CD95-induced apoptosis in T cells is associated with both depolarization of mitochondrial membrane potential (Delta(psi)m) and increase in mitochondrial mass and activation of caspase-8 and caspase-3 but without caspase-9 activation. Furthermore, changes in mitochondrial membrane potential and mitochondrial mass by anti-CD95 monoclonal antibodies were unaffected by inhibitors of caspase-8 and caspase-3, suggesting that anti-CD95-induced changes in Delta(psi)m and mitochondrial mass are independent of caspase-8 and caspase-3 activation.     

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.     

Caspase-2 is required for cell death induced by cytoskeletal disruption

Caspase-2 is one of the most conserved caspases, yet its biological function remains a matter of controversy. In the present article we analysed mouse embryonic fibroblasts (MEFs) from caspase-2 knockout mice for their sensitivity to various apoptosis inducing agents. We found that cell death induced by drugs that disrupt cytoskeleton is significantly inhibited in Casp2(-/-) MEFs. These drugs included zoledronic acid, vincristine, cytochalasin D and paclitaxel. We demonstrate that MEFs lacking Casp2 show clonogenic survival following drug treatment, whereas all Casp2(+/+) MEFs die, indicating that caspase-2 is required for apoptosis induced by cytoskeletal disruption. We further found that caspase-2 mediates apoptosis via Piddosome, Bid and Bax activation, and cytochrome c release. In the absence of caspase-2, Bid and Bax activation, and cytochrome c release are significantly delayed following drug treatment. Our data provide strong support for a context-dependent function of caspase-2 in apoptosis.     

Activation of prodeath Bcl-2 family proteins and mitochondrial apoptosis pathway by sanguinarine in immortalized human HaCaT keratinocytes.

Sanguinarine, derived from the root of Sanguinaria canadensis and other poppy fumaria species, possesses strong antimicrobial, anti-inflammatory, and antioxidant properties. We earlier showed that sanguinarine kills human epidermoid carcinoma A431 cells via an induction of apoptosis [N. Ahmad et al., Clin. Cancer Res., 6: 1524-1528, 2000]. In this study, using immortalized human keratinocytes (HaCaT cells), we provide information about mechanism of the antiproliferative effect of sanguinarine. Sanguinarine [0.1 (M-2 (M)] treatment to HaCaT cells was found to inhibit in a dose-dependent manner the cell proliferation and induce apoptosis, as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and ELISA, respectively. Sanguinarine treatment also resulted in a significant cleavage of poly(ADP-ribose) polymerase in HaCaT cells. Because mitochondrial pathway is critical for the regulation of apoptosis, we studied the involvement and regulation of mitochondrial events in sanguinarine-mediated apoptosis of HaCaT cells. As shown by the immunoblot analysis, our data clearly demonstrated that sanguinarine treatment to HaCaT cells resulted in a dose-dependent (a) increase in the level of Bax with a concomitant decrease in Bcl-2 levels and (b) increase in Bax/Bcl-2 ratio. Sanguinarine also resulted in significant increases in the proapoptotic members of Bcl-2 family proteins, i.e., Bak and Bid. This was accompanied by increase in (a) protein expression of cytochrome c and apoptotic protease-activating factor-1 and (b) activity and protein expression of caspase-3, caspase-7, caspase-8, and caspase-9. Taken together, our data showed the involvement of mitochondrial pathway and Bcl-2 family proteins during sanguinarine-mediated apoptosis of immortalized keratinocytes. We suggest that sanguinarine could be developed as a drug for the management of hyperproliferative skin disorders, including skin cancer.