Bid, a critical mediator for apoptosis induced by the activation of Fas/TNF-R1 death receptors in hepatocytes

The Bcl-2 family proteins consist of both antiapoptosis and pro-apoptosis members that regulate apoptosis typically at the mitochondrial level, mainly by controlling the release of cytochrome c and other mitochondrial apoptotic events. However, death signals mediated by Fas/TNF-R1 receptors can usually activate caspases directly, bypassing the need for mitochondria and escaping the regulation by Bcl-2 family proteins. Bid is a novel pro-apoptosis Bcl-2 family protein that is activated by Caspase 8 in response to Fas/TNF-R1 death receptor activation. Activated Bid is translocated to mitochondria and induces cytochrome c release, which in turn activates the downstream caspases. This Bid-mediated pathway is critical in hepatocyte apoptosis induced by Fas/TNF-R1 engagement, where direct activation of cytosolic caspase cascade seems inefficient. The dependence on Bid, and thus on the mitochondrial cytochrome c release, of hepatocyte apoptosis induced by the death receptors also renders it sensitive to the inhibitory regulation by the anti-apoptosis members of the Bcl-2 family proteins, such as Bcl-2 and Bcl-xL. Moreover, the revealing of this death pathway in hepatocytes is important to the understanding of the pathogenesis of a number of hepatic diseases such as hepatitis or endotoxemia-related hepatic failure.     

Lysosome-Mediated Apoptosis is Associated with Cathepsin D-Specific Processing of Bid at Phe24, Trp48, and Phe183

Bax-mediated permeabilization of the outer mitochondrial membrane and release of apoptogenic factors into the cytosol are key events that occur during apoptosis. Likewise, apoptosis is associated with permeabilization of the lysosomal membrane and release of lysosomal cathepsins into the cytosol. This report identifies proteolytically active cathepsin D as an important component of apoptotic signaling following lysosomal membrane permeabilization in fibroblasts. Lysosome-mediated cell death is associated with degradation of Bax sequestering 14-3-3 proteins, cleavage of the Bax activator Bid, and translocation of Bax to mitochondria, all of which were cathepsin D-dependent. Processing of Bid could be reproduced by enforced lysosomal membrane permeabilization, using the lysosomotropic detergent O-methyl-serine dodecylamine hydrochloride (MSDH). We identified three cathepsin D-specific cleavage sites in Bid, Phe24, Trp48, and Phe183. Cathepsin D-cleaved Bid induced Bax-mediated release of cytochrome c from purified mitochondria, indicating that the fragments generated are functionally active. Moreover, apoptosis was associated with cytosolic acidification, thereby providing a more favorable environment for the cathepsin D-mediated cleavage of Bid. Our study suggests that cytosolic cathepsin D triggers Bax-mediated cytochrome c release by proteolytic activation of Bid.     

Mitochondrial Release of Apoptosis-Inducing Factor and Cytochrome c During Smooth Muscle Cell Apoptosis

Photodynamic therapy (PDT) is under investigation for the treatment of intimal hyperplastia in conditions such as atherosclerosis and restenosis. Although smooth muscle cells (SMCs) may be a key target for treatment, the effects of PDT on these cells are poorly characterized. In the present study, apoptosis was induced in primary human aortic SMCs by the combination of the photosensitizer verteporfin and visible light. After PDT, an increase in mitochondrial cytochrome c (cyt c) and apoptosis-inducing factor (AIF) levels were detected in the cytosol immediately and their levels increased steadily up to 2 hours. Cytosolic levels of the pro-apoptotic Bcl-2 family member Bax decreased reciprocally throughout this period, but this change did not occur before cyt c release. Confocal microscopy revealed a diffuse staining pattern of cyt c within apoptotic cells as compared to a distinct mitochondrial staining in normal cells. AIF translocated from mitochondria to the nucleus during the progression of apoptosis. After cyt c release, caspase-9 and caspase-3 processing was visible by 1 hour and caspase-6, -7, and -8 processing was apparent by 2 hours after PDT. In summary, these results demonstrate for the first time the cellular redistribution of mitochondrial AIF during SMC apoptosis, as well as the early release of cyt c and the subsequent activation of multiple caspases during PDT-induced SMC apoptosis.     

Chlamydia trachomatis infection inhibits both Bax and Bak activation induced by staurosporine

We have previously shown that Chlamydia trachomatis inhibits host cell apoptosis and blocks mitochondrial cytochrome c release. We now report that activation of both Bax and Bak, two proapoptotic members of the Bcl-2 family that regulate mitochondrial cytochrome c release, was inhibited in chlamydia-infected cells. This observation has provided new information on the mechanisms of chlamydial antiapoptotic activity.     

Role of Bcl-2 family proteins in apoptosis: apoptosomes or mitochondria?

Apoptosis is an essential physiological process for the selective elimination of cells, which is involved in a variety of biological events. The Bcl-2 family is the best characterized protein family involved in the regulation of apoptotic cell death, consisting of anti-apoptotic and pro-apoptotic members. The anti-apoptotic members of this family, such as Bcl-2 and Bcl-x_L, prevent apoptosis either by sequestering proforms of death-driving cysteine proteases called caspases (a complex called the apoptosome) or by preventing the release of mitochondrial apoptogenic factors such as cytochrome c and AIF (apoptosis-inducing factor) into the cytoplasm. After entering the cytoplasm, cytochrome c and AIF directly activate caspases that cleave a set of cellular proteins to cause apoptotic changes. In contrast, pro-apoptotic members of this family, such as Bax and Bak, trigger the release of caspases from death antagonists via heterodimerization and also by inducing the release of mitochondrial apoptogenic factors into the cytoplasm via acting on mitochondrial permeability transition pore, thereby leading to caspase activation. Thus, the Bcl-2 family of proteins acts as a critical life–death decision point within the common pathway of apoptosis.     

Involvement of NF-kappaB and caspases in silibinin-induced apoptosis of endothelial cells

Silibinin, the flavonoid found in the milk thistle, has been shown to suppress cell growth and exhibit anti-cancer effects. Some flavonoids were reported to inhibit angiogenesis which is essential for tumor growth and metastasis. In this study, to clarify the underlying mechanisms for the anti-cancer effect of silibinin, we examined the effects of silibinin on human endothelial ECV304 cells. Silibinin was found to suppress the growth and induce the apoptosis of ECV304 cells. The induction of apoptosis by silibinin was confirmed by ladder-patterned DNA fragmentation, cleaved and condensed nuclear chromatin and DNA hypoploidy. Silibinin could effectively inhibit constitutive NF-kappaB activation as revealed by electrophoretic mobility shift assay and NF-kappaB-dependent luciferase reporter study. Consistent with this, silibinin treatment resulted in a significant decrease in the nuclear level of p65 subunit of NF-kappaB. In addition, silibinin treatment caused a change in the ratio of Bax/Bcl-2 in a manner that favors apoptosis. Silibinin also induced the cytochrome c release, activation of caspase-3 and caspase-9 and cleavage of PARP. These results suggest that silibinin may exert, at least partly, its anti-cancer effect by inhibiting angiogenesis through induction of endothelial apoptosis via modulation of NF-kappaB, Bcl-2 family and caspases.     

Caspase activation by granzyme B is indirect,and caspase autoprocessing requires the release of proapoptotic mitochondrial factors

Apoptosis in response to granzyme B involves activation of caspase-dependent target cell death pathways. Herein, we show that granzyme B initiates caspase processing but cannot fully process procaspase-3 in intact Jurkat T leukemia or NT2 neuronal cells. Rather, the release from mitochondria of proapoptotic mediators cytochrome c, Smac/Diablo, and HtrA2/Omi facilitates full activation of caspases that results from autoprocessing. Bcl-2 overexpression in mitochondria suppresses the release of these proapoptotic molecules, resulting in cell survival despite partial procaspase processing by granzyme B. We propose that binding of inhibitor of apoptosis (IAP) proteins to partially processed procaspases inhibits cell death unless mitochondrial disruption also occurs in response to granzyme B or activated BH3-domain proteins such as truncated Bid.     

Degradation of the proapoptotic proteins Bik, Puma, and Bim with Bcl-2 domain 3 homology in Chlamydia trachomatis-infected cells

We have previously correlated Chlamydia trachomatis antiapoptotic activity with the blockade of mitochondrial cytochrome c release and the inhibition of Bax and Bak activation. We now report that C. trachomatis infection leads to degradation of Bik, Puma, and Bim, three upstream proapoptotic BH3-only proteins of the Bcl-2 family that can transmit death signals to mitochondria by inhibiting the Bcl-2 antiapoptotic proteins and/or activating the Bcl-2 proapoptotic members, such as Bax and Bak. This observation has provided new information on the chlamydial antiapoptosis mechanisms.     

Antiproliferative effect of alpinetin in BxPC-3 pancreatic cancer cells

Alpinetin is a novel plant flavonoid derived from Alpinia katsumadai Hayata, found to possess strong anticancer effects. However, the antitumor effect of alpinetin on pancreatic cancer cells and the detailed mechanism remain unclear. The aim of this study was to investigate alpinetin's beneficial effect on pancreatic cancer and the possible molecular mechanism involved. Pancreatic cancer cell lines were treated with alpinetin at various doses and for different times, and the effect of alpinetin on cell growth inhibition, apoptosis and the cell cycle was determined. The expression of Bcl-2, Bcl-xL, XIAP and Bax, the activity of caspases and the levels of cytochrome?c released were measured. The results showed that alpinetin inhibited the viability of three pancreatic cancer cell lines and induced apoptosis of BxPC-3 cells in a dose- and time-dependent manner. This was accompanied by regulation of the expression of Bcl-2, Bcl-xL, Bax and XIAP. Furthermore, alpinetin treatment led to the release of cytochrome?c and activation of caspases-3, -8 and -9 proteins. Taken together, our studies indicate that alpinetin inhibited the proliferation of pancreatic cancer cells possibly through the regulation of the Bcl-2 family and XIAP expression, release of cytochrome?c and the activation of caspases. Alpinetin may serve as a potential agent for the development of pancreatic cancer cell therapies.     

Early release of mitochondrial cytochrome c and expression of mitochondrial epitope 7A6 with a porphyrin-derived photosensitizer: Bcl-2 and Bcl-xL overexpression do not prevent early mitochondrial events but still depress caspase activity.

Certain nonmetallic porphyrins have potent antitumor activity upon visible light irradiation. Treatment of HeLa cells with nanomolar amounts of the photochemo therapeutic agent verteporfin and red light mobilized caspases 2, 3, 6, 7, 8, and 9, caused degradation of specific caspase substrates, and resulted in morphologic changes consistent with apoptosis. Caspase processing was detectable by 1 hour after light irradiation. The mitochondrial 7A6 epitope, recognized by monoclonal antibody APO2.7, became accessible, and cytochrome c was detectable within the cytosolic fraction of cells treated with verteporfin immediately after light irradiation. The general caspase inhibitor benzyloxycarboyl-Val-Ala-Asp-fluoromethylketone did not prevent 7A6 expression produced by photosensitization at peptide concentrations which completely prevented caspase activation and cleavage of caspase-specific substrates. Enforced overexpression of Bcl-2 or Bcl-xL prevented cytochrome c release and 7A6 expression produced by ultraviolet B light treatment, but did not prevent cytochrome c release or 7A6 expression elicited by verteporfin photosensitization. Bcl-2 or Bcl-xL overexpression delayed morphologic changes, depressed caspase activation, and limited substrate degradation, but did not protect against loss of viability after verteporfin photosensitization. This observation indicates that cells overexpressing Bcl-2 or Bcl-xL exhibit resistance to caspase activation even after the appearance of cytochrome c in the cytosol. Porphyrin photosensitizers are effective chemotherapeutic agents that elicit primary proapoptotic mitochondrial events even in the setting of heightened Bcl-2 or Bcl-xL expression.     

Differential involvement of p38 MAP kinase pathway and Bax translocation in the mitochondria-mediated cell death in TCR- and dexamethasone-stimulated thymocytes

Mitochondria play a central role in many apoptotic reactions. Although mitochondrial apoptotic changes and caspase activation have been demonstrated in the apoptotic thymocytes, cell death signal through mitochondria in TCR-stimulated thymocytes has not been fully understood. In this study, we show that TCR stimulation induced disruption of mitochondrial transmembrane potential (Delta Psi(m)), the cytochrome c release from mitochondira, capase-3 activation, and the cell death of thymocytes. Bongkrekic acid, an inhibitor of Delta Psi(m) disruption, blocked the cytochrome c release from mitochondria and the following caspase-3-mediated cell death. Furthermore, a pro-apoptotic Bcl-2 family protein, Bax, but not Bad or Bid, was translocated from cytosol to mitochondria in TCR-stimulated thymocytes. This translocation and the following apoptotic changes were inhibited by SB203580, a p38 kinase inhibitor, in a specific manner. These results suggest that activated p38 kinase pathway by TCR stimulation induces translocation of Bax to mitochondria, causing Delta Psi(m) disruption, and the release of cytochrome c, which finally induces caspase-3-mediated apoptosis in thymocytes.     

A179L, a viral Bcl-2 homologue, targets the core Bcl-2 apoptotic machinery and its upstream BH3 activators with selective binding restrictions for Bid and Noxa

Several large DNA viruses encode Bcl-2 protein homologues involved in the regulation of the cellular apoptosis cascade. This regulation often involves the interaction of these viral proteins with diverse cellular Bcl-2 family members. We have identified the specific interactions of A179L, an African swine fever virus (ASFV) Bcl-2 homologue, with the active forms of the porcine BH3-only Bid protein (truncated Bid p13 and p15). Transient expression of ASFV A179L gene in Vero cells prevented apoptosis induced by these active forms of Bid protein. Interestingly, A179L protein was able to interact, also with the main core Bcl-2 proapoptotic proteins Bax and Bak, and with several BH3-only proteins with selective binding restrictions for full length Bid and Noxa. These results suggest a fine regulation for A179L action in the suppression of apoptosis in infected cells which is essential for efficient virus replication.     

Bax-inhibiting peptide protects glutamate-induced cerebellar granule cell death by blocking Bax translocation

Glutamate-induced excitotoxicity has been implicated in the pathogenesis of various neurological damages and disorders. In the brain damage of immature animals such as neonatal hypoxic-ischemic brain injury, the excitotoxicity appears to be more intimately involved through apoptosis. Bax, a member of the Bcl-2 family proteins, plays a key role in the promotion of apoptosis by translocation from the cytosol to the mitochondria and the release of apoptogenic factors such as cytochrome c. Recently, Bax-inhibiting peptide (BIP), a novel membrane-permeable peptide which can bind Bax in the cytosol and inhibit its translocation to the mitochondria, was developed. To investigate the possibility of a new neuroprotection strategy targeting Bax translocation in glutamate-induced neuronal cell death, cerebellar granule neurons (CGNs) were exposed to glutamate with or without BIP. Pretreatment of CGNs with BIP elicited a dose-dependent reduction of glutamate-induced neuronal cell death as measured by MTT assay. BIP significantly suppressed both the number of TUNEL-positive cells and the increase in caspases 3 and 9 activities induced by glutamate. In addition, immunoblotting after subcellular fractionation revealed that BIP prevented the glutamate-induced Bax translocation to the mitochondria and the release of cytochrome c from the mitochondria. These results suggest that agents capable of inhibiting Bax activity such as BIP might lead to new drugs for glutamate-related diseases in the future.     

The CD95 type I/type II model

CD95 (APO-1/Fas) has become the prototype of a death domain containing receptor and is the best studied member of the death receptors that activate the extrinsic apoptosis pathway. This pathway is initiated by recruitment and activation of caspase-8, an initiator caspase, in the death-inducing signaling complex (DISC) followed by direct cleavage of downstream effector caspases. In contrast, the intrinsic apoptosis pathway starts from within the cell either by direct activation of caspases or through intracellular changes such as DNA damage resulting in the release of a number of pro-apoptotic factors from the intermembrane space of mitochondria. The release of these factors results in the activation of another initiator caspase, caspase-9, and ultimately in the activation of effector caspases in a protein complex called the apoptosome. In recent years, it has become apparent that there is cross talk between the extrinsic and intrinsic pathway. In the death receptor pathway of apoptosis induction, the best characterized connection between the two pathways is the Bcl-2 family member Bid which translocates to mitochondria after cleavage by caspase-8 causing pro-apoptotic changes. Cells that die through CD95 without help from mitochondria are called Type I cells, whereas cells in which CD95-mediated death relies mostly on the intrinsic pathway are called Type II. This review focuses on recent developments in the delineation of the biochemistry and the physiological function of the two CD95 pathways.     

Role of Bcl-2 Family Members in Caspase-Independent Apoptosis during Chlamydia Infection

Infection with an obligate intracellular bacterium, the Chlamydia trachomatis lymphogranuloma venereum (LGV/L2) strain or the guinea pig inclusion conjunctivitis serovar of Chlamydia psittaci, leads to apoptosis of host cells. The apoptosis is not affected by a broad-spectrum caspase inhibitor, and caspase-3 is not activated in infected cells, suggesting that apoptosis mediated by these two strains of Chlamydia is independent of known caspases. Overexpression of the proapoptotic Bcl-2 family member, Bax, was previously shown to induce caspase-independent apoptosis, and we find that Bax is activated and translocates from the cytosol to the mitochondria in C. psittaci-infected cells. C. psittaci-induced apoptosis is inhibited in host cells overexpressing Bax inhibitor-1 and is inhibited through overexpression of Bcl-2, which blocks both caspase-dependent and -independent apoptosis. As Bax and mitochondria are ideally located to sense stress-related metabolic changes emanating from the interior of an infected cell, it is likely that Bax-dependent apoptosis may also be observed in cells infected with other intracellular pathogens.     

Cytotoxic chemotherapy upregulates pro-apoptotic Bax and Bak in the small intestine of rats and humans

AIMS: Small intestinal crypt cells rapidly undergo apoptosis in response to cytotoxic drug treatment that results in gastrointestinal toxicity. The Bcl-2 family have been implicated in both positive and negative regulation of intestinal cell apoptosis. The aim of this study was to examine the effect of cytotoxic treatment on Bcl-2 protein expression in patients and rats with tumours. METHODS: Four pro- and four anti-apoptotic members of the Bcl-2 family, caspase-3 and p53 were examined in small intestinal crypts before and after treatment in rats and humans. Immunohistochemistry identified changes in protein expression over time, while relative RT-PCR was used to investigate mRNA expression in rat small intestine. RESULTS: Cytotoxic treatment increased p53 and caspase-3 which coincided with elevated levels of apoptosis. Bax and Bak protein and mRNA expression also significantly increased at 6 hours following treatment in rats. Bax and Bak protein increased at day 1 after treatment in humans. Anti-apoptotic Mcl-1 protein decreased within 24hours. Other Bcl-2 family members showed only modest changes. CONCLUSION: Increased expression of Bax and Bak but not other Bcl-2 family members is associated with apoptosis in small intestinal crypts and may amplify the sensitivity and susceptibility of crypt cells to chemotherapy-induced enteropathy.     

Tissue-specific Bcl-2 protein partners in apoptosis: An ovarian paradigm

Apoptosis is an essential physiological process by which multicellular organisms eliminate superfluous cells. An expanding family of Bcl-2 proteins plays a pivotal role in the decision step of apoptosis, and the differential expression of Bcl-2 members and their binding proteins allows the regulation of apoptosis in a tissue-specific manner mediated by diverse extra- and intracellular signals. The Bcl-2 proteins can be divided into three subgroups: 1) antiapoptotic proteins with multiple Bcl-2 homology (BH) domains and a transmembrane region, 2) proapoptotic proteins with the same structure but missing the BH4 domain, and 3) proapoptotic ligands with only the BH3 domain. In the mammalian ovary, a high rate of follicular cell apoptosis continues during reproductive life. With the use of the yeast two-hybrid system, the characterization of ovarian Bcl-2 genes serves as a paradigm to understand apoptosis regulation in a tissue-specific manner. We identified Mcl-1 as the main ovarian antiapoptotic Bcl-2 protein, the novel Bok (Bcl-2-related ovarian killer) as the proapoptotic protein, as well as BOD (Bcl-2-related ovarian death agonist) and BAD as the proapoptotic ligands. The activity of the proapoptotic ligand BAD is regulated by upstream follicle survival factors through its binding to constitutively expressed 14-3-3 or hormone-induced P11. In contrast, the channel-forming Mcl-1 and Bok regulate cytochrome c release and, together with the recently discovered Diva/Boo, control downstream apoptosis-activating factor (Apaf)-1 homologs and caspases. Elucidation of the role of Bcl-2 members and their interacting proteins in the tissue-specific regulation of apoptosis could facilitate an understanding of normal physiology and allow the development of new therapeutic approaches for pathological states.     

Rapid apoptosis induced by Shiga toxin in HeLa cells

Apoptosis was induced rapidly in HeLa cells after exposure to bacterial Shiga toxin (Stx1 and Stx2; 10 ng/ml). Approximately 60% of HeLa cells became apoptotic within 4 h as detected by DNA fragmentation, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, and electron microscopy. Stx1-induced apoptosis required enzymatic activity of the Stx1A subunit, and apoptosis was not induced by the Stx2B subunit alone or by the anti-globotriaosylceramide antibody. This activity was also inhibited by brefeldin A, indicating the need for toxin processing through the Golgi apparatus. The intracellular pathway leading to apoptosis was further defined. Exposure of HeLa cells to Stx1 activated caspases 3, 6, 8, and 9, as measured both by an enzymatic assay with synthetic substrates and by detection of proteolytically activated forms of these caspases by Western immunoblotting. Preincubation of HeLa cells with substrate inhibitors of caspases 3, 6, and 8 protected the cells against Stx1-dependent apoptosis. These results led to a more detailed examination of the mitochondrial pathway of apoptosis. Apoptosis induced by Stx1 was accompanied by damage to mitochondrial membranes, measured as a reduced mitochondrial membrane potential, and increased release of cytochrome c from mitochondria at 3 to 4 h. Bid, an endogenous protein known to permeabilize mitochondrial membranes, was activated in a Stx1-dependent manner. Caspase-8 is known to activate Bid, and a specific inhibitor of caspase-8 prevented the mitochondrial damage. Although these data suggested that caspase-8-mediated cleavage of Bid with release of cytochrome c from mitochondria and activation of caspase-9 were responsible for the apoptosis, preincubation of HeLa cells with a specific inhibitor of caspase-9 did not protect against apoptosis. These results were explained by the discovery of a simultaneous Stx1-dependent increase in endogenous XIAP, a direct inhibitor of caspase-9. We conclude that the primary pathway of Stx1-induced apoptosis and DNA fragmentation in HeLa cells is unique and includes caspases 8, 6, and 3 but is independent of events in the mitochondrial pathway.     

Cathepsin-cleaved Bid promotes apoptosis in human neutrophils via oxidative stress-induced lysosomal membrane permeabilization

Lysosomal membrane permeabilization (LMP) is emerging as an important regulator of cell apoptosis. Human neutrophils are highly granulated phagocytes, which respond to pathogens by exhibiting increased production of reactive oxygen species (ROS) and lysosomal degranulation. In a previous study, we observed that intracellular, nonphagosomal generation of ROS triggered by adherent bacteria induced ROS-dependent neutrophil apoptosis, whereas intraphagosomal production of ROS during phagocytosis had no effect. In the present study, we measured lysosomal membrane stability and leakage in human neutrophils and found that adherent, noningested, Type 1-fimbriated Escherichia coli bacteria induced LMP rapidly in neutrophils. Pretreatment with the NADPH oxidase inhibitor diphenylene iodonium markedly blocked the early LMP and apoptosis in neutrophils stimulated with Type 1-fimbriated bacteria but had no effect on the late LMP seen in spontaneously apoptotic neutrophils. The induced lysosomal destabilization triggered cleavage of the proapoptotic Bcl-2 protein Bid, followed by a decrease in the antiapoptotic protein Mcl-1. Involvement of LMP in initiation of apoptosis is supported by the following observations: Bid cleavage and the concomitant drop in mitochondrial membrane potential required activation of cysteine-cathepsins but not caspases, and the differential effects of inhibitors of cysteine-cathepsins and cathepsin D on apoptosis coincided with their ability to inhibit Bid cleavage in activated neutrophils. Together, these results indicate that in microbe-induced apoptosis in neutrophils, ROS-dependent LMP represents an early event in initiation of the intrinsic apoptotic pathway, which is followed by Bid cleavage, mitochondrial damage, and caspase activation.     

Induction of apoptosis by Se-MSC in U937 human leukemia cells through release of cytochrome c and activation of caspases and PKC-delta: mutual regulation between caspases and PKC-delta via a positive feedback mechanism

Se-methylselenocysteine (Se-MSC) has been shown to possess potent chemopreventive and anti-tumor properties. However, its exact mechanism of action is still not well understood. The present study investigated the mechanism of Se-MSC on the induction of apoptosis using U937 human leukemia cells. Se-MSC induced dose- and time-dependent apoptosis of U937 cells as assessed by flow cytometry analysis, DNA fragmentation, and proteolytic cleavage of poly-(ADP-ribose) polymerase (PARP). Se-MSC increased time- and dose-dependent cytochrome c accumulation in the cytosol, which was greatly inhibited by overexpression of Bcl-2, suggesting that the apoptotic effect by Se-MSC in U937 cells is mitochondrial-dependent. Se-MSC also induced activation of caspases, followed by proteolytic cleavage of PKC-delta. The Se-MSC-induced apoptosis required activities of caspases since pretreatment of a pan-caspase inhibitor z-VAD-fmk greatly suppressed the Se-MSC-induced apoptosis as well as proteolytic cleavage of PKC-delta, suggesting activation of caspases is critical for the Se-MSC-induced apoptosis, and caspases lie upstream of PKC-delta. The Se-MSC-induced apoptosis of U937 cells also required activity of PKC-delta because pretreatment of rottlerin, a specific PKC-delta inhibitor greatly blocked the Se-MSC-induced apoptosis as well as processing and activities of caspases, suggesting activation of PKC-delta is also important for the Se-MSC-induced apoptosis of U937 cells, and PKC-delta lies upstream of caspases. Together, our data suggest the apoptotic mechanism by Se-MSC in U937 cells may be related to cytochrome c release from the mitochondria, and mutual activation between caspases and PKC-delta via a positive feedback mechanism, which may potentiate the apoptotic action by Se-MSC in U937 cells.