Contribution of membrane localization to the apoptotic activity of PUMA

The BH3-only protein PUMA plays an important role in the activation of apoptosis in response to p53. In different studies, PUMA has been described to function by either directly activating the pro-apoptotic proteins Bax and Bak, or by neutralizing anti-apoptotic members of the Bcl2 family. We have examined the contribution of regions of PUMA other than the BH3 domain to its localization and function. Although the hydrophobic domain in the C-terminus of PUMA is necessary for efficient mitochondrial localization of PUMA itself, PUMA proteins lacking this region can still induce apoptosis and localize to the mitochondria through binding to Bcl2. Even a nuclear localization signal (NLS)-tagged PUMA protein retains apoptotic activity and can be efficiently relocalized from the nucleus after interaction with ectopically expressed Bcl2, underscoring the efficiency of this interaction. Interestingly, unlike the Bcl2 interaction, the binding of PUMA to Bax is severely compromised by the loss of the C-terminal domain of PUMA. However, since the loss of the C-terminus does not compromise the ability of PUMA to induce cell death, our results indicate that the key apoptotic function of PUMA is through interaction with anti-apoptotic proteins such as Bcl2.     

Modulation of Bax mitochondrial insertion and induced cell death in yeast by mammalian protein kinase Cα

Protein kinase Cα (PKCα) is a classical PKC isoform whose involvement in cell death is not completely understood. Bax, a major member of the Bcl-2 family, is required for apoptotic cell death and regulation of Bax translocation and insertion into the outer mitochondrial membrane is crucial for regulation of the apoptotic process. Here we show that PKCα increases the translocation and insertion of Bax c-myc (an active form of Bax) into the outer membrane of yeast mitochondria. This is associated with an increase in cytochrome c (cyt c) release, reactive oxygen species production (ROS), mitochondrial network fragmentation and cell death. This cell death process is regulated, since it correlates with an increase in autophagy but not with plasma membrane permeabilization. The observed increase in Bax c-myc translocation and insertion by PKCα is not due to Bax c-myc phosphorylation, and the higher cell death observed is independent of the PKCα kinase activity. PKCα may therefore have functions other than its kinase activity that aid in Bax c-myc translocation and insertion into mitochondria. Together, these results give a mechanistic insight on apoptosis regulation by PKCα through regulation of Bax insertion into mitochondria.     

Apoptosis and the conformational change of Bax induced by proteasomal inhibition of PC12 cells are inhibited by bcl-xL and bcl-2

The function of the proteasome has been linked to various pathologies, including cancer and neurodegeneration. Proteasomal inhibition can lead to death in a variety of cell types, however the manner in which this occurs is unclear, and may depend on the particular cell type. In this work we have extended previous findings pertaining to the effects of pharmacological proteasomal inhibitors on PC12 cells, by examining in more detail the induced death pathway. We find that cell death is apoptotic by ultrastructural criteria. Caspase 9 and 3 are processed, cytochrome c is released from the mitochondria and a dominant negative form of caspase 9 prevents death. Furthermore, Bax undergoes a conformational change and is translocated to the mitochondria in a caspase-independent fashion. Total cell levels of Bax however do not change, whereas levels of the BH3-only protein Bim increase with proteasomal inhibition. Transient overexpression of bcl-xL or, to a lesser extent, of bcl-2, significantly decreased apoptotic death and prevented Bax conformational change. We conclude that death elicited by proteasomal inhibition of PC12 cells follows a classical “intrinsic” pathway. Significantly, antiapoptotic bcl-2 family members prevent apoptosis by inhibiting Bax conformational change. Increased levels of Bim may contribute to cell death in this model.     

BH3-only protein Bim inhibits activity of antiapoptotic members of Bcl-2 family when expressed in yeast

Proteins of the Bcl-2 family regulate programmed cell death in mammals by promoting the release of cytochrome c from mitochondria in response to various proapoptotic stimuli. The mechanism by which BH3-only members of the family activate multidomain proapoptotic proteins Bax and Bak to form a pore in mitochondrial membranes remains under dispute. We report that cell death promoting activity of BH3-only protein Bim can be reconstituted in yeast when both Bax and antiapoptotic protein Bcl-X(L) are present, suggesting that Bim likely activates Bax indirectly by inhibiting antiapoptotic proteins.     

Bax translocation to mitochondria subsequent to a rapid loss of mitochondrial membrane potential

Bax, a pro-apoptotic member of the Bcl-2 family, is a cytosolic protein that inserts into mitochondrial membranes upon induction of cell death. Using the green fluorescent protein fused to Bax (GFP-Bax) to quantitate mitochondrial binding in living cells we have investigated the cause of Bax association with mitochondria and the time course relative to endogenous and induced changes in mitochondrial membrane potential (DeltaPsi(m)). We have found that staurosporine (STS) induces a loss in DeltaPsi(m) before GFP-Bax translocation can be measured. The onset of the DeltaPsi(m) loss is followed by a rapid and complete collapse of DeltaPsi(m) which is followed by Bax association with mitochondria. The mitochondria uncoupler FCCP, in the presence of the F(1)-F(0) ATPase inhibitor oligomycin, can trigger Bax translocation to mitochondria suggesting that when ATP levels are maintained a collapse of DeltaPsi(m) induces Bax translocation. Neither FCCP nor oligomycin alone alters Bax location. Bax association with mitochondria is also triggered by inhibitors of the electron transport chain, antimycin and rotenone, compounds that collapse DeltaPsi(m) without inducing rapid ATP hydrolysis that typically occurs with uncouplers such as FCCP. Taken together, our results suggest that alterations in mitochondrial energization associated with apoptosis can initiate Bax docking to mitochondria.     

Acidification induces Bax translocation to the mitochondria and promotes ultraviolet light-induced apoptosis

It has been suggested that Bax translocation to the mitochondria is related to apoptosis, and that cytosol acidification contributes to apoptosis events. However, the mechanisms remain obscure. We investigated the effect of acidification on Bax translocation and on ultraviolet (UV) light-induced apoptosis. The Bax translocation assay in vitro showed that Bax translocated to the mitochondria at pH 6.5, whereas no Bax translocation was observed at pH 7.4. VHDBB cells expressing the GFP-Bax fusion protein were treated for 12 h with a pH 6.5 DMEM medium, nigericin (5 μg/ml) and UV light (50 J/cm²), separately or in combination, and Bax translocation to the mitochondria was determined by SDS-PAGE and Western blot, and apoptotic cell death was detected by flow cytometry. The results showed that some of the Bax translocated to the mitochondria in the cells treated with the normal medium, nigericin and UV in combination, whereas all of the Bax translocated to the mitochondria in the cells treated with the pH 6.5 medium, nigericin and UV in combination. In VHDBB cells treated for 12 h with nigericin, UV alone, and UV and nigericin in combination, the respective rates of apoptotic cell death were 25.08%, 33.25% and 52.88%. In cells treated with pH 6.5 medium and nigericin, pH 6.5 medium and UV, and pH 6.5 medium, nigericin and UV in combination, the respective rates of apoptotic cell death increased to 37.19%, 41.42% and 89.44%. Our results indicated that acidification induces Bax translocation from the cytosol to the mitochondria, and promotes UV lightmediated apoptosis. This suggests that there is a possibility of improving cancer treatment by combining acidification with irradiation or chemotherapeutic drugs.     

Proapoptotic BCL-2 family members and mitochondrial dysfunction during ischemia/reperfusion injury, a study employing cardiac HL-1 cells and GFP biosensors

The objective of this study was to evaluate mitochondrial alterations in a cell-based model of myocardial ischemia/reperfusion (I/R) injury. Using GFP-biosensors and fluorescence deconvolution microscopy, we investigated mitochondrial morphology in relation to Bax and Bid activation in the HL-1 cardiac cell line. Mitochondria underwent extensive fragmentation during ischemia. Bax translocation from cytosol to mitochondria was initiated during ischemia and proceeded during reperfusion. However, Bax translocation was not sufficient to induce cell death or mitochondrial dysfunction. Bid processing was caspase-8 dependent, and Bid translocation to mitochondria occurred after Bax translocation and clustering, and minutes before cell death. Clustering of Bax into distinct regions on mitochondria could be prevented by CsA, an inhibitor of the mitochondrial permeability transition pore, and also by SB203580, an inhibitor of p38 MAPK. Surprisingly, mitochondrial fragmentation which occurred during ischemia and before Bax translocation could be reversed by the addition of the p38 inhibitor SB203580 at reperfusion. Taken together, these results implicate p38 MAPK in the mitochondrial remodeling response to I/R that facilitates Bax recruitment to mitochondria.     

Regulation of intracellular pH mediates Bax activation in HeLa cells treated with staurosporine or tumor necrosis factor-alpha

Induction of apoptosis in HeLa cells with staurosporine produced a rise in the intracellular pH (pH(i)). Intracellular alkalinization was accompanied by translocation of Bax to the mitochondria, cytochrome c release, and cell death. The chloride channel inhibitor furosemide prevented intracellular alkalinization, Bax translocation, cytochrome c release, and cell death. Translocation of full-length Bid to the mitochondria was also prevented by furosemide. The cleavage product of Bid degradation (truncated Bid, tBid) was not detectable in the mitochondria. Its accumulation in the cytosol was prevented by furosemide. Apoptosis induced by tumor necrosis factor-alpha (TNF) lowered pH(i), an effect also accompanied by Bax translocation, cytochrome c release, and cell killing. Furosemide prevented all of these events. TNF induced a depletion of full-length Bid from the mitochondria and the cytosol but induced an accumulation of mitochondrial tBid. Furosemide only delayed full-length Bid depletion and tBid accumulation. The caspase 8 inhibitor IETD did not prevent the translocation of Bax. Although IETD did inhibit the cleavage of Bid and the accumulation of tBid, cell killing was reduced only slightly. It is concluded that with either staurosporine or TNF a furosemide-sensitive change in pH(i) is linked to Bax translocation, cytochrome c release, and cell killing. With TNF Bax translocation occurs as Bid is depleted and can be dissociated from the accumulation of tBid. With staurosporine a role for full-length Bid in Bax translocation cannot be excluded but is not necessary as evidenced by the data with TNF.     

Postnatal brain development and neural cell differentiation modulate mitochondrial Bax and BH3 peptide-induced cytochrome c release

Bax mediates cytochrome c release and apoptosis during neurodevelopment. Brain mitochondria that were isolated from 8-day, 17-day, and adult rats displayed decreasing levels of mitochondrial Bax. The amount of cytochrome c released from brain mitochondria by a peptide containing the BH3 cell death domain decreased with increasing age. However, approximately 60% of cytochrome c in adult brain mitochondria could be released by the BH3 peptide in the presence of exogenous human recombinant Bax. Mitochondrial Bax was downregulated in PC12S neural cells differentiated with nerve growth factor, and mitochondria isolated from these cells demonstrated decreased sensitivity to BH3-peptide-induced cytochrome c release. These results demonstrate that immature brain mitochondria and mitochondria from undifferentiated neural cells are particularly sensitive to cytochrome c release mediated by endogenous Bax and a BH3 death domain peptide. Postnatal developmental changes in mitochondrial Bax levels may contribute to the increased susceptibility of neurons to pathological apoptosis in immature animals.     

Bax function in the absence of mitochondria in the primitive protozoan Giardia lamblia

Bax-induced permeabilization of the mitochondrial outer membrane and release of cytochrome c are key events in apoptosis. Although Bax can compromise mitochondria in primitive unicellular organisms that lack a classical apoptotic machinery, it is still unclear if Bax alone is sufficient for this, or whether additional mitochondrial components are required. The protozoan parasite Giardia lamblia is one of the earliest branching eukaryotes and harbors highly degenerated mitochondrial remnant organelles (mitosomes) that lack a genome. Here we tested whether human Bax expressed in Giardia can be used to ablate mitosomes. We demonstrate that these organelles are neither targeted, nor compromised, by Bax. However, specialized compartments of the regulated secretory pathway are completely ablated by Bax. As a consequence, maturing cyst wall proteins that are sorted into these organelles are released into the cytoplasm, causing a developmental arrest and cell death. Interestingly, this ectopic cargo release is dependent on the carboxy-terminal 22 amino acids of Bax, and can be prevented by the Bax-inhibiting peptide Ku70. A C-terminally truncated Bax variant still localizes to secretory organelles, but is unable to permeabilize these membranes, uncoupling membrane targeting and cargo release. Even though mitosomes are too diverged to be recognized by Bax, off-target membrane permeabilization appears to be conserved and leads to cell death completely independently of mitochondria.     

Detailing the role of Bax translocation, cytochrome c release, and perinuclear clustering of the mitochondria in the killing of HeLa cells by TNF

Induction of cell death in HeLa cells with TNF and cycloheximide (CHX) required an adequate ATP supply and was accompanied by decrease in intracellular pH, translocation of Bax, perinuclear clustering of the mitochondria, and cytochrome c release. The chloride channel inhibitor furosemide prevented the intracellular acidification, the translocation of Bax and the cell death. Cyclosporin A (CyA) or bongkrekic acid (BK) inhibited the induction of the MPT, the release of cytochrome c and the cell death without affecting the perinuclear clustering of the mitochondria or the translocation of Bax. Energy depletion with the ATP synthase inhibitor oligomycin or the uncoupler FCCP in the presence of 2-deoxy-glucose prevented the perinuclear clustering of the mitochondria and the cell killing. However, mitochondrial translocation of Bax was still observed. By contrast, cytochrome c was released in the oligomycin-treated cells but not in the same cells treated with FCCP. The data demonstrate that apoptosis in HeLa cells is ATP dependent and requires the translocation of Bax. The movement of Bax to the mitochondria occurs before and during the perinuclear clustering of these organelles and does not require the presence of ATP. The release of cytochrome c depends on the induction of the mitochondrial permeability transition but not ATP content.     

Protection against experimental malaria associated with AMA-1 peptide analogue structures

We have identified, using site-directed mutagenesis, a proline located at position 13 of Baxalpha (Bax) as crucial for the maintenance of its cytosolic conformation. The substitution of this proline by a valine results in a strong binding of Bax to mitochondria and to conformational changes monitored by a decreased sensitivity of Bax to mild proteolysis and the enhancement of its oligomerization state. Deletion of the C-terminus of Bax does not modify its intracellular localization. On the other hand, the pro-apoptotic activity of Bax is enhanced by a deletion of the C-terminus in the absence of the N-terminus but is decreased in its presence. These results suggest that both extremities functionally interact to control the activity but not the subcellular localization of Bax.     

Bax-induced cell death of Arabidopsisis meditated through reactive oxygen-dependent and -independent processes

An Arabidopsisprotoplast system was developed for dissecting plant cell death in individual cells. Bax, a mammalian pro-apoptotic member of the Bcl-2 family, induces apoptotic-like cell death in Arabidopsis. Bax accumulation in Arabidopsismesophyll protoplasts expressing murine BaxcDNA from a glucocorticoid-inducible promoter results in cytological characteristics of apoptosis, namely DNA fragmentation, increased vacuolation, and loss of plasma membrane integrity. In vivotargeting analysis monitored using jellyfish green fluorescent protein (GFP) reporter indicated full-length Bax was localized to the mitochondria, as it does in animal cells. Deletion of the carboxyl-terminal transmembrane domain of Bax completely abolished targeting to mitochondria. Bax expression was followed by reactive oxygen species (ROS) accumulation. Treatment of protoplasts with the antioxidant N-acetyl- -cysteine (NAC) during induction of Bax expression strongly suppressed Bax-mediated ROS production and the cell death phenotype. However, some population of the ROS depleted cells still induced cell death, indicating that there is a process that Bax-mediated plant cell death is independent of ROS accumulation. Accordingly, suppression of Bax-mediated plant cell death also takes place in two different processes. Over-expression of a key redox-regulator, Arabidopsisnucleoside diphosphate kinase 2 (AtNDPK2) down-regulated ROS accumulation and suppressed Bax-mediated cell death and transient expression of ArabidopsisBax inhibitor-1 (AtBI-1) substantially suppressed Bax-induced cell death without altering cellular ROS level. Taken together, our results collectively suggest that the Bax-mediated cell death and its suppression in plants is mediated by ROS-dependent and -independent processes.     

The role of p38 MAPK and JNK in Arsenic trioxide-induced mitochondrial cell death in human cervical cancer cells

Previously, we have shown that the release of AIF from mitochondria is required for As2O3-induced cell death in human cervical cancer cells, and that reactive oxygen species (ROS) is necessary for AIF release from mitochondria. In this study, we further investigated the role of MAPKs in ROS-mediated mitochondrial apoptotic cell death triggered by As2O3. As2O3-induced apoptotic cell death in HeLa cells was associated with activation and mitochondrial translocation of Bax, a marked phosphorylation of Bcl-2, reduction of Bcl-2 and Bax interaction, dissipation of mitochondrial membrane potential. Using small interfering RNA, reduced Bax expression effectively attenuated As2O3-induced mitochondrial membrane potential loss and apoptotic cell death. Moreover, the phosphorylation of Bcl-2 induced by As2O3 diminished its ability to bind to Bax. Treatment of cells with As2O3 activated both the p38 MAPK and JNK pathways. Mitochondrial translocation of Bax was completely suppressed in the presence of p38 MAPK inhibitor PD169316 or si-p38 MAPK. The As2O3-induced Bcl-2 phosphorylation was attenuated largely by JNK inhibition using SP600125 or si-JNK and to some extent by p38 MAPK inhibition with PD169316 or si-p38 MAPK. In addition, N-acetyl-L-cystein (NAC), a thiol-containing anti-oxidant, completely blocked As2O3-induced p38 MAPK and JNK activations, mitochondria translocation of Bax, and phosphorylation of Bcl-2. These results support a notion that ROS-mediated activations of p38 MAPK and JNK in response to As2O3 treatment signals activation of Bax and phosphorylation of Bcl-2, resulting in mitochondrial apoptotic cell death in human cervical cancer cells.     

Inhibiting the mitochondrial fission machinery does not prevent Bax/Bak-dependent apoptosis

Apoptosis, induced by a number of death stimuli, is associated with a fragmentation of the mitochondrial network. These morphological changes in mitochondria have been shown to require proteins, such as Drp1 or hFis1, which are involved in regulating the fission of mitochondria. However, the precise role of mitochondrial fission during apoptosis remains elusive. Here we report that inhibiting the fission machinery in Bax/Bak-mediated apoptosis, by down-regulating of Drp1 or hFis1, prevents the fragmentation of the mitochondrial network and partially inhibits the release of cytochrome c from the mitochondria but fails to block the efflux of Smac/DIABLO. In addition, preventing mitochondrial fragmentation does not inhibit cell death induced by Bax/Bak-dependent death stimuli, in contrast to the effects of Bcl-xL or caspase inhibition. Therefore, the fission of mitochondria is a dispensable event in Bax/Bak-dependent apoptosis.     

p38 MAP kinase mediates bax translocation in nitric oxide-induced apoptosis in neurons

Nitric oxide is a chemical messenger implicated in neuronal damage associated with ischemia, neurodegenerative disease, and excitotoxicity. Excitotoxic injury leads to increased NO formation, as well as stimulation of the p38 mitogen-activated protein (MAP) kinase in neurons. In the present study, we determined if NO-induced cell death in neurons was dependent on p38 MAP kinase activity. Sodium nitroprusside (SNP), an NO donor, elevated caspase activity and induced death in human SH-SY5Y neuroblastoma cells and primary cultures of cortical neurons. Concomitant treatment with SB203580, a p38 MAP kinase inhibitor, diminished caspase induction and protected SH-SY5Y cells and primary cultures of cortical neurons from NO-induced cell death, whereas the caspase inhibitor zVAD-fmk did not provide significant protection. A role for p38 MAP kinase was further substantiated by the observation that SB203580 blocked translocation of the cell death activator, Bax, from the cytosol to the mitochondria after treatment with SNP. Moreover, expressing a constitutively active form of MKK3, a direct activator of p38 MAP kinase promoted Bax translocation and cell death in the absence of SNP. Bax-deficient cortical neurons were resistant to SNP, further demonstrating the necessity of Bax in this mode of cell death. These results demonstrate that p38 MAP kinase activity plays a critical role in NO-mediated cell death in neurons by stimulating Bax translocation to the mitochondria, thereby activating the cell death pathway.     

Beta-phenylethyl isothiocyanate mediated apoptosis; contribution of Bax and the mitochondrial death pathway

The initiating events that lead to the induction of apoptosis mediated by the chemopreventative agent beta-phenyethyl isothiocyanate (PEITC) have yet to be elucidated. In the present investigation, we examined the effects of PEITC on mitochondrial function and apoptotic signaling in hepatoma HepG2 cells and isolated rat hepatocyte mitochondria. PEITC induced a conformational change in Bax leading to its translocation to mitochondria in HepG2 cells. Bax accumulation was associated with a rapid loss of mitochondrial membrane potential (Deltapsim), impaired respiratory chain enzymatic activity, release of mitochondrial cytochrome c and the activation of caspase-dependent cell death. Caspase inhibition did not prevent Bax translocation, the release of cytochrome c or the loss of Deltapsim, but blocked caspase-mediated DNA fragmentation and cell death. To determine whether PEITC dependent Bax translocation caused loss of Deltapsim by the activation of the mitochondrial permeability transition (MPT), we examined the effects of PEITC in isolated rat hepatocyte mitochondria. Interestingly, PEITC did not induce MPT in isolated rat mitochondria. Accordingly, using pharmacological inhibitors of MPT namely cyclosporine A, trifluoperazine and Bongkrekic acid we were unable to block PEITC mediated apoptosis in HepG2 cells, this suggesting that mitochondrial permeablisation is a likely consequence of Bax dependent pore formation. Taken together, our data suggest that mitochondria are a key target in PEITC induced apoptosis in HepG2 cells via the pore forming ability of pro-apoptotic Bax.     

Bax and Bak coalesce into novel mitochondria-associated clusters during apoptosis

Bax is a member of the Bcl-2 family of proteins known to regulate mitochondria-dependent programmed cell death. Early in apoptosis, Bax translocates from the cytosol to the mitochondrial membrane. We have identified by confocal and electron microscopy a novel step in the Bax proapoptotic mechanism immediately subsequent to mitochondrial translocation. Bax leaves the mitochondrial membranes and coalesces into large clusters containing thousands of Bax molecules that remain adjacent to mitochondria. Bak, a close homologue of Bax, colocalizes in these apoptotic clusters in contrast to other family members, Bid and Bad, which circumscribe the outer mitochondrial membrane throughout cell death progression. We found the formation of Bax and Bak apoptotic clusters to be caspase independent and inhibited completely and specifically by Bcl-X(L), correlating cluster formation with cytotoxic activity. Our results reveal the importance of a novel structure formed by certain Bcl-2 family members during the process of cell death.     

Bax translocates from cytosol to mitochondria in cardiac cells during apoptosis: development of a GFP-Bax-stable H9c2 cell line for apoptosis analysis

The proapoptotic protein Bax plays an important role in cardiomyocytic cell death. Ablation of this protein has been shown to diminish cardiac damage in Bax-knockout mice during ischemia-reperfusion. Presently, studies of Bax-mediated cardiac cell death examined primarily the expression levels of Bax and its prosurvival factor Bcl-2 rather than the localization of this protein, which dictates its function. Using immunofluorescence labeling, we have shown that in neonatal rat cardiomyocytes and in H9c2 cardiomyoblasts, Bax translocates from cytosol to mitochondria upon the induction of apoptosis by hypoxia-reoxygenation-serum withdrawal and by the presence of the free-radical inducer menadione. Also, we found that Bax translocation to mitochondria was associated with the exposure of an NH2-terminal epitope, and that this translocation could be partially blocked by the prosurvival factors Bcl-2 and Bcl-XL. To visualize the translocation of Bax in living cells, we have developed an H9c2 cell line that stably expresses green fluorescent protein (GFP)-tagged Bax. This cell line has GFP-Bax localized primarily in the cytosol in the absence of apoptotic inducers. Upon induction of apoptosis by a number of stimuli, including menadione, staurosporine, sodium nitroprusside, and hypoxia-reoxygenation-serum withdrawal, we could observe the translocation of Bax from cytosol to mitochondria. This translocation was not affected by retinoic acid-induced differentiation of H9c2 cells. Additionally, this translocation was associated with loss of mitochondrial membrane potential, release of cytochrome c, and fragmentation of nuclei. Finally, using a tetramethylrhodamine-based dye, we have shown that a rapid screening process based on the loss of mitochondrial membrane potential could be developed to monitor GFP-Bax translocation to mitochondria. Overall, the GFP-Bax-stable H9c2 cell line that we have developed represents a unique tool for examining Bax-mediated apoptosis, and it could be of great importance in screening therapeutic compounds that could block Bax translocation to mitochondria to attenuate apoptosis.     

Caspase-2 triggers Bax-Bak-dependent and -independent cell death in colon cancer cells treated with resveratrol

Polyphenol phytoalexin (resveratrol), found in grapes and red wine is a strong chemopreventive agent with promising safety records with human consumption and unique forms of cell death induction in a variety of tumor cells. However, the mechanism of resveratrol-induced apoptosis upstream of mitochondria is still not defined. The results from this study suggest that caspase-2 activation occurs upstream of mitochondria in resveratrol-treated cells. The upstream activation of caspase-2 is not dependent on its antioxidant property or NF-kappaB inhibition. The activated caspase-2 triggers mitochondrial apoptotic events by inducing conformational changes in Bax/Bak with subsequent release of cytochrome c, apoptosis-inducing factor, and endonuclease G. Caspase-8 activation seems to be independent of these events and does not appear to be mediated by classical death receptor processing or downstream caspases. Both caspase-2 and caspase-8 contribute toward the mitochondrial translocation of Bid, since neither caspase-8 inhibition nor caspase-2 inhibition could prevent translocation of Bid DsRed into mitochondria. Caspase-2 inhibitors or antisense silencing of caspase-2 prevented cell death induced by resveratrol and partially prevented processing of downstream caspases, including caspase-9, caspase-3, and caspase-8. Studies using mouse embryonic fibroblasts deficient for both Bax and Bak indicate the contribution of both Bax and Bak in mediating cell death induced by resveratrol and the existence of Bax/Bak-independent cell death possibly through caspase-8- or caspase-2-mediated mitochondria-independent downstream caspase processing.