Characterization of 4-O-methyl-ascochlorin-induced apoptosis in comparison with typical apoptotic inducers in human leukemia cell lines

Apoptosis can be induced by various stimuli such as the ligands of death receptors, chemotherapeutic drugs and irradiation. It is generally believed that chemotherapeutic drugs induce mitochondrial damage, cytochrome c release and activation of caspase-9, leading to apoptosis. Here, we found that an isoprenoid antibiotic, 4-O-methyl ascochlorin, significantly induces typical apoptotic events in Jurkat cells including the degradation of poly (ADP-ribose) polymerase, DNA fragmentation, activation of caspase-3, -9 and -8, and cytochrome c release from mitochondria. Similar to Fas stimulation, 4-O-methyl ascochlorin but not staurosporine, cycloheximide and actinomycin D, induced apoptosis in SKW6.4 cells, in which apoptosis is strongly dependent on death-inducing signaling-complex. Bcl-2 overexpression in Jurkat cells completely suppressed the apoptosis, but procaspase-9 processing was partially induced. A caspase-8 inhibitor, IETD-fmk, effectively suppressed poly (ADP-ribose) polymerase cleavage and cytochrome c release. However, 4-O-methyl ascochlorin induced apoptosis in Jurkat cells deficient of caspase-8 or Fas-associated death domain protein. These results suggest that 4-O-methyl ascochlorin induces apoptosis through the mechanism distinct from conventional apoptosis inducers.     

Interleukin-11 antagonizes Fas ligand-mediated apoptosis in IEC-18 intestinal epithelial crypt cells: role of MEK and Akt-dependent signaling

Interleukin-11 (IL-11) displays epithelial cytoprotective effects during intestinal injury. Antiapoptotic effects of IL-11 have been described, yet mechanisms remain unclear. Fas/CD95 death receptor signaling is upregulated in ulcerative colitis, leading to mucosal breakdown. We hypothesized that IL-11 inhibits Fas ligand (FasL)-mediated apoptosis in intestinal epithelia. Cell death was monitored in IEC-18 cells by microscopy, caspase and poly(ADP-ribose) polymerase cleavage, mitochondrial release of cytochrome c, and abundance of cytoplasmic oligonucleosomal DNA. RT-PCR was used to monitor Fas, cIAP1, cIAP2, XIAP, cFLIP, survivin, and Bcl-2 family members. Fas membrane expression was detected by immunoblot. Inhibitors of JAK2, phosphatidylinositol 3-kinase (PI3-kinase), Akt 1, MEK1 and MEK2, and p38 MAPK were used to delineate IL-11's antiapoptotic mechanisms. IL-11 did not alter Fas expression. Pretreatment with IL-11 for 24 h before FasL reduced cytoplasmic oligonucleosomal DNA by 63.2%. IL-11 also attenuated caspase-3, caspase-9, and poly(ADP-ribose) polymerase cleavage without affecting expression of activated caspase-8 p20 or cytochrome c release. IL-11 did not affect mRNA expression of the candidate antiapoptotic genes. The MEK1 and MEK2 inhibitors U-0126 and PD-98059 significantly attenuated the protection of IL-11 against caspase-3 and caspase-9 cleavage and cytoplasmic oligonucleosomal DNA accumulation. Although Akt inhibition reversed IL-11-mediated effects on caspase cleavage, it did not reverse the protective effects of IL-11 by DNA ELISA. We conclude that IL-11-dependent MEK1 and MEK2 signaling inhibits FasL-induced apoptosis. The lack of reversal of the IL-11 effect on DNA cleavage by Akt inhibition, despite antagonism of caspase cleavage, suggests that IL-11 inhibits caspase-independent cell death signaling by FasL in a MEK-dependent manner.     

Swainsonine activates mitochondria-mediated apoptotic pathway in human lung cancer A549 cells and retards the growth of lung cancer xenografts

Swainsonine (1, 2, 8-trihyroxyindolizidine, SW), a natural alkaloid, has been reported to exhibit anti-cancer activity on several mouse models of human cancer and human cancers in vivo. However, the mechanisms of SW-mediated tumor regression are not clear. In this study, we investigated the effects of SW on several human lung cancer cell lines in vitro. The results showed that SW significantly inhibited these cells growth through induction of apoptosis in different extent in vitro. Further studies showed that SW treatment up-regulated Bax, down-regulated Bcl-2 expression, promoted Bax translocation to mitochondria, activated mitochondria-mediated apoptotic pathway, which in turn caused the release of cytochrome c, the activation of caspase-9 and caspase-3, and the cleavage of poly (ADP-ribose) polymerase (PARP), resulting in A549 cell apoptosis. However, the expression of Fas, Fas ligand (FasL) or caspase-8 activity did not appear significant changes in the process of SW-induced apoptosis. Moreover, SW treatment inhibited Bcl-2 expression, promoted Bax translocation, cytochrome c release and caspase-3 activity in xenograft tumor cells, resulting in a significant decrease of tumor volume and tumor weight in the SW-treated xenograft mice groups in comparison to the control group. Taken together, this study demonstrated for the first time that SW inhibited A549 cancer cells growth through a mitochondria-mediated, caspase-dependent apoptotic pathway in vitro and in vivo.     

N-terminal cleavage of bax by calpain generates a potent proapoptotic 18-kDa fragment that promotes bcl-2-independent cytochrome C release and apoptotic cell death

Upon apoptosis induction, the proapoptotic protein Bax is translocated from the cytosol to mitochondria, where it promotes release of cytochrome c, a caspase-activating protein. However, the molecular mechanisms by which Bax triggers cytochrome c release are unknown. Here we report that before the initiation of apoptotic execution by etoposide or staurosporin, an active calpain activity cleaves Bax at its N-terminus, generating a potent proapoptotic 18-kDa fragment (Bax/p18). Both the calpain-mediated Bax cleavage activity and the Bax/p18 fragment were found in the mitochondrial membrane-enriched fraction. Cleavage of Bax was followed by release of mitochondrial cytochrome c, activation of caspase-3, cleavage of poly(ADP-ribose) polymerase, and fragmentation of DNA. Unlike the full-length Bax, Bax/p18 did not interact with the antiapoptotic Bcl-2 protein in the mitochondrial fraction of drug-treated cells. Pretreatment with a specific calpain inhibitor calpeptin inhibited etoposide-induced calpain activation, Bax cleavage, cytochrome c release, and caspase-3 activation. In contrast, transfection of a cloned Bax/p18 cDNA into multiple human cancer cell lines targeted Bax/p18 to mitochondria, which was accompanied by release of cytochrome c and induction of caspase-3-mediated apoptosis that was not blocked by overexpression of Bcl-2 protein. Therefore, Bax/p18 has a cytochrome c-releasing activity that promotes cell death independent of Bcl-2. Finally, Bcl-2 overexpression inhibited etoposide-induced calpain activation, Bax cleavage, cytochrome c release, and apoptosis. Our results suggest that the mitochondrial calpain plays an essential role in apoptotic commitment by cleaving Bax and generating the Bax/p18 fragment, which in turn mediates cytochrome c release and initiates the apoptotic execution.     

Carbon monoxide modulates Fas/Fas ligand,caspases, and Bcl-2 family proteins via the p38alpha mitogen-activated protein kinase pathway during ischemia-reperfusion lung injury

Carbon monoxide is protective in ischemia-reperfusion organ injury, but the precise mechanisms remain elusive. We have recently shown that low levels of exogenous carbon monoxide (CO) utilize p38 MAPK and attenuate caspase 3 activity to exert an antiapoptotic effect during lung ischemia-reperfusion injury. Our current data demonstrate that CO activates the p38alpha MAPK isoform and the upstream MAPK kinase MKK3 to modulate Fas/Fas ligand expression; caspases 3, 8, and 9; mitochondrial cytochrome c release; Bcl-2 proteins; and poly(ADP-ribose) polymerase cleavage. We correlate our in vitro findings with in vivo studies using MKK3-deficient and Fas-deficient mice. Taken together, our data are the first to demonstrate that CO has an antiapoptotic effect by inhibiting Fas/Fas ligand, caspases, proapoptotic Bcl-2 proteins, and cytochrome c release via the MKK3/p38alpha MAPK pathway.     

Sulfuretin from heartwood of Rhus verniciflua triggers apoptosis through activation of Fas, Caspase-8, and the mitochondrial death pathway in HL-60 human leukemia cells

Sulfuretin, a flavonoid isolated from heartwood of Rhus verniciflua, has been reported to have anti-cancer activities but the underlying molecular mechanism was not clear. In this study, sulfuretin induced apoptosis by activating caspases-8, -9, and -3 as well as cleavage of poly(ADP-ribose) polymerase. Furthermore, treatment with sulfuretin caused mitochondrial dysfunctions, including the loss of mitochondrial membrane potential (ΔΨ(m)), the release of cytochrome c to the cytosol, and the translocations of Bax and tBid. Sulfuretin also activated the extrinsic apoptosis pathway, that is, it increased the expressions of Fas and FasL, the activation of caspase-8, and the cleavage of Bid. Furthermore, blocking the FasL-Fas interaction with NOK-1 monoclonal antibody prevented the sulfuretin-induced apoptosis. The therapeutical effect of sulfuretin in leukemia is due to its potent apoptotic activity through the extrinsic pathway driven by a Fas-mediated caspase-8-dependent pathway.     

CD47 augments Fas/CD95-mediated apoptosis

Fas (CD95) mediates apoptosis of many cell types, but the susceptibility of cells to killing by Fas ligand and anti-Fas antibodies is highly variable. Jurkat T cells lacking CD47 (integrin-associated protein) are relatively resistant to Fas-mediated death but are efficiently killed by Fas ligand or anti-Fas IgM (CH11) upon expression of CD47. Lack of CD47 impairs events downstream of Fas activation including caspase activation, poly-(ADP-ribose) polymerase cleavage, cytochrome c release from mitochondria, loss of mitochondrial membrane potential, and DNA cleavage. Neither CD47 signaling nor raft association of CD47 is required to enable Fas apoptosis. CH11 induces association of Fas and CD47. Primary T cells from CD47-null mice are also protected from Fas-mediated killing relative to wild type T cells. Thus CD47 associates with Fas upon its activation and augments Fas-mediated apoptosis.     

Staurosporine-induced death of MCF-7 human breast cancer cells: a distinction between caspase-3-dependent steps of apoptosis and the critical lethal lesions

To test the role of caspase 3 in apoptosis and in overall cell lethality caused by the protein kinase inhibitor staurosporine, we compared the responses of MCF-7c3 cells that express a stably transfected CASP-3 gene to parental MCF-7:WS8 cells transfected with vector alone and lacking procaspase-3 (MCF-7v). Cells were exposed to increasing doses (0.15-1 microM) of staurosporine for periods up to 19 h. Apoptosis was efficiently induced in MCF-7c3 cells, as demonstrated by cytochrome c release, processing of procaspase-3, procaspase-8, and Bid, increase in caspase-3-like DEVDase activity, cleavage of the enzyme poly(ADP-ribose) polymerase, DNA fragmentation, changes in nuclear morphology, and TUNEL assay and flow cytometry. For all of these measures except cytochrome c release, little or no activity was detected in MCF-7v cells, confirming that caspase-3 is essential for efficient induction of apoptosis by staurosporine, but not for mitochondrial steps that occur earlier in the pathway. MCF-7c3 cells were more sensitive to staurosporine than MCF-7v cells when assayed for loss of viability by reduction of a tetrazolium dye. However, the two cell lines were equally sensitive to killing by staurosporine when evaluated by a clonogenic assay. A similar distinction between apoptosis and loss of clonogenicity was observed for the cancer chemotherapeutic agent VP-16. These results support our previous conclusions with photodynamic therapy: (a) assessing overall reproductive death of cancer cells requires a proliferation-based assay, such as clonogenicity; and (b) the critical staurosporine-induced lethal event is independent of those mediated by caspase-3.     

Asparanin A induces G2/M cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells

We recently established that asparanin A, a steroidal saponin extracted from Asparagus officinalis L., is an active cytotoxic component. The molecular mechanisms by which asparanin A exerts its cytotoxic activity are currently unknown. In this study, we show that asparanin A induces G₂/M phase arrest and apoptosis in human hepatocellular carcinoma HepG2 cells. Following treatment of HepG2 cells with asparanin A, cell cycle-related proteins such as cyclin A, Cdk1 and Cdk4 were down-regulated, while p21^WAF1/Cip1 and p-Cdk1 (Thr14/Tyr15) were up-regulated. Additionally, we observed poly (ADP-ribose) polymerase (PARP) cleavage and activation of caspase-3, caspase-8 and caspase-9. The expression ratio of Bax/Bcl-2 was increased in the treated cells, where Bax was also up-regulated. We also found that the expression of p53, a modulator of p21^WAF1/Cip1 and Bax, was not affected in asparanin A-treated cells. Collectively, our findings demonstrate that asparanin A induces cell cycle arrest and triggers apoptosis via a p53-independent manner in HepG2 cells. These data indicate that asparanin A shows promise as a preventive and/or therapeutic agent against human hepatoma.     

Surfactin from Bacillus subtilis displays anti-proliferative effect via apoptosis induction, cell cycle arrest and survival signaling suppression

The effect of surfactin on the proliferation of LoVo cells, a human colon carcinoma cell line, was examined. Surfactin strongly blocked the proliferation of LoVo cells by inducing pro-apoptotic activity and arresting the cell cycle, according to several lines of evidence on DNA fragmentation, Annexin V staining, and altered levels of poly (ADP-ribose) polymerase, caspase-3, p21(WAF1/Cip1), p53, CDK2 and cyclin E. The anti-proliferative activity of surfactin was mediated by inhibiting extracellular-related protein kinase and phosphoinositide 3-kinase/Akt activation, as assessed by phosphorylation levels. Therefore, our data suggest that surfactin may have anti-cancer properties as a result of its ability to downregulate the cell cycle and suppress its survival.     

HSP27 inhibits cytochrome c-dependent activation of procaspase-9.

We have previously shown that the small heat shock protein HSP27 inhibited apoptotic pathways triggered by a variety of stimuli in mammalian cells. The present study demonstrates that HSP27 overexpression decreases U937 human leukemic cell sensitivity to etoposide-induced cytotoxicity by preventing apoptosis. As observed for Bcl-2, HSP27 overexpression delays poly(ADP-ribose)polymerase cleavage and procaspase-3 activation. In contrast with Bcl-2, HSP27 overexpression does not prevent etoposide-induced cytochrome c release from the mitochondria. In a cell-free system, addition of cytochrome c and dATP to cytosolic extracts from untreated cells induces the proteolytic activation of procaspase-3 in both control and bcl-2-transfected U937 cells but fails to activate procaspase-3 in HSP27-overexpressing cells. Immunodepletion of HSP27 from cytosolic extracts increases cytochrome c/dATP-mediated activation of procaspase-3. Overexpression of HSP27 also prevents procaspase-9 activation. In the cell-free system, immunodepletion of HSP27 increases LEDH-AFC peptide cleavage activity triggered by cytochrome c/dATP treatment. We conclude that HSP27 inhibits etoposide-induced apoptosis by preventing cytochrome c and dATP-triggered activity of caspase-9, downstream of cytochrome c release.     

SMT-A07, a 3-(Indol-2-yl) indazole derivative, induces apoptosis of leukemia cells in vitro

N-(2-(1H-indazol-3-yl)-1H-pyrrolo[3,2-b]pyridin-5-yl)-4-chloro-N-methylbenzamide (SMT-A07) is a novel 3-(Indol-2-yl) indazole derivative. The anticancer activities in vitro and the cell apoptosis-induction abilities of SMT-A07 on human leukemia HL60 and NB4 cell lines were investigated in this study. The results of MTT assay showed SMT-A07 was a potential and highly efficient antitumor compound with IC₅₀ values ranging from 0.09 to 1.19 μM in five leukemia cell lines. SMT-A07 treatment for 24 h caused the increment of apoptosis rate from 6.88 to 49.72% in HL60 cells and from 8.72 to 56.28% in NB4 cells by flow cytometry analysis. Agarose gel electrophoresis showed DNA fragmentation that appeared after cells were exposed to SMT-A07. After SMT-A07 incubation, DAPI staining revealed the presence of DNA fragmentation, and perinuclear apoptotic body. SMT-A07 also resulted in a loss of ΔΨm in both HL60 and NB4 cells by JC-1 staining. Moreover, apoptosis-related proteins were examined by western blotting to explore the mechanism of its cytotoxicity. SMT-A07 exposure caused down-regulation and cleavage of procaspase-8, procaspase-3, Bid, PARP and up-regulation of cleaved caspase-8, cleaved caspase-3, PARP (Cleaved Fragment). In addition, the presence of pan-caspase inhibitor BOC-D-FMK prevented cells from caspase-3 activation, PARP cleavage, and subsequent apoptosis. Our study demonstrates that SMT-A07 displays an apparent antitumor activity with extensive anti-leukemia spectrum, and SMT-A07 can induce the apoptosis of HL60 and NB4 cells activation of the caspase cascade, which deserves further development.     

Vibrio vulnificus cytolysin induces superoxide anion-initiated apoptotic signaling pathway in human ECV304 cells

Previous studies showed that exposure to Vibrio vulnificus cytolysin (VVC) caused characteristic morphologic changes and dysfunction of vascular structures in lung. VVC showed cytotoxicity for mammalian cells in culture and acted as a vascular permeability factor. In this study, the underlying mechanisms of VVC-induced cytotoxicity was investigated on ECV304 cell, a human vascular endothelial cell line. When cells were exposed to 0.4 hemolytic units (HU) of VVC, consecutive apoptotic events were observed; the elevation of superoxide anion (O (-.)(2)), the release of cytochrome c, the activation of caspase-3, the cleavage of poly(ADP-ribose) polymerase, and the DNA fragmentation. The pretreatment with 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO), O(-.) 2) scavenger, completely abolished O(-.)(2) levels and downstream apoptotic events. Moreover, pretreatment with cyclosporin A (CsA), a mitochondrial permeability transition inhibitor, was capable of attenuating O(-.)(2)-mediated cytochrome c release and caspase-3 activation, and consequent apoptosis. Apoptosis, as demonstrated by oligonucleosomal DNA fragmentation and fluorescence microscopy, was induced 24 h after VVC treatment, which was also prevented by caspase-3 inhibitor, Ac-DEVD-CHO. Caspase-1 inhibitor, Ac-YVAD-CHO, did not protect ECV 304 cells from apoptosis. These results suggest a scenario where VVC-induced apoptosis is triggered by the generation of O(-.)(2), release of cytochrome c from mitochondria, activation of caspase-3, degradation of poly(ADP-ribose) polymerase, and DNA fragmentation. The induction of apoptosis in endothelial cells by VVC may provide a pivotal mechanism for understanding the pathophysiology of septicemia.     

Hypersensitivity of mtDNA-depleted cells to staurosporine-induced apoptosis: roles of Bcl-2 downregulation and cathepsin B

We show that mitochondrial DNA (mtDNA)-depleted 143B cells are hypersensitive to staurosporine-induced cell death as evidenced by a more pronounced DNA fragmentation, a stronger activation of caspase-3, an enhanced poly(ADP-ribose) polymerase-1 (PARP-1) cleavage, and a more dramatic cytosolic release of cytochrome c. We also show that B-cell CLL/lymphoma-2 (Bcl-2), B-cell lymphoma extra large (Bcl-X(L)), and myeloid cell leukemia-1 (Mcl-1) are constitutively less abundant in mtDNA-depleted cells, that the inhibition of Bcl-2 and Bcl-X(L) can sensitize the parental cell line to staurosporine-induced apoptosis, and that overexpression of Bcl-2 or Bcl-X(L) can prevent the activation of caspase-3 in ρ(0)143B cells treated with staurosporine. Moreover, the inactivation of cathepsin B with CA074-Me significantly reduced cytochrome c release, caspase-3 activation, PARP-1 cleavage, and DNA fragmentation in mtDNA-depleted cells, whereas the pan-caspase inhibitor failed to completely prevent PARP-1 cleavage and DNA fragmentation in these cells, suggesting that caspase-independent mechanisms are responsible for cell death even if caspases are activated. Finally, we show that cathepsin B is released in the cytosol of ρ(0) cells in response to staurosporine, suggesting that the absence of mitochondrial activity leads to a facilitated permeabilization of lysosomal membranes in response to staurosporine.     

Granzyme B activates procaspase-3 which signals a mitochondrial amplification loop for maximal apoptosis

Granzyme B (GrB), acting similar to an apical caspase, efficiently activates a proteolytic cascade after intracellular delivery by perforin. Studies here were designed to learn whether the physiologic effector, GrB-serglycin, initiates apoptosis primarily through caspase-3 or through BH3-only proteins with subsequent mitochondrial permeabilization and apoptosis. Using four separate cell lines that were either genetically lacking the zymogen or rendered deficient in active caspase-3, we measured apoptotic indices within whole cells (active caspase-3, mitochondrial depolarization [DeltaPsim] and TUNEL). Adhering to these conditions, the following were observed in targets after GrB delivery: (a) procaspase-3-deficient cells fail to display a reduced DeltaPsim and DNA fragmentation; (b) Bax/Bak is required for optimal DeltaPsim reduction, caspase-3 activation, and DNA fragmentation, whereas BID cleavage is undetected by immunoblot; (c) Bcl-2 inhibits GrB-mediated apoptosis (reduced DeltaPsim and TUNEL reactivity) by blocking oligomerization of caspase-3; and (d) in procaspase-3-deficient cells a mitochondrial-independent pathway was identified which involved procaspase-7 activation, PARP cleavage, and nuclear condensation. The data therefore support the existence of a fully implemented apoptotic pathway initiated by GrB, propagated by caspase-3, and perpetuated by a mitochondrial amplification loop but also emphasize the presence of an ancillary caspase-dependent, mitochondria-independent pathway.     

Transforming growth factor-beta 1 induces apoptosis through Fas ligand-independent activation of the Fas death pathway in human gastric SNU-620 carcinoma cells

To date, two major apoptotic pathways, the death receptor and the mitochondrial pathway, have been well documented in mammalian cells. However, the involvement of these two apoptotic pathways, particularly the death receptor pathway, in transforming growth factor-beta 1 (TGF-beta 1)-induced apoptosis is not well understood. Herein, we report that apoptosis of human gastric SNU-620 carcinoma cells induced by TGF-beta 1 is caused by the Fas death pathway in a Fas ligand-independent manner, and that the Fas death pathway activated by TGF-beta 1 is linked to the mitochondrial apoptotic pathway via Bid mediation. We showed that TGF-beta 1 induced the expression and activation of Fas and the subsequent caspase-8-mediated Bid cleavage. Interestingly, expression of dominant negative FADD and treatment with caspase-8 inhibitor efficiently prevented TGF-beta 1-induced apoptosis, whereas the treatment with an activating CH11 or a neutralizing ZB4 anti-Fas antibody, recombinant Fas ligand, or Fas-Fc chimera did not affect activation of Fas and the subsequent induction of apoptosis by TGF-beta 1. We further demonstrated that TGF-beta 1 also activates the mitochondrial pathway showing Bid-mediated loss of mitochondrial membrane potential and subsequent cytochrome c release associated with the activations of caspase-9 and the effector caspases. Moreover, all these apoptotic events induced by TGF-beta 1 were found to be effectively inhibited by Smad3 knockdown and also completely abrogated by Smad7 expression, suggesting the involvement of the Smad3 pathway upstream of the Fas death pathway by TGF-beta 1.     

Akt inhibition upregulates FasL, downregulates c-FLIPs and induces caspase-8-dependent cell death in Jurkat T lymphocytes

In T lymphocytes, the role of Akt in regulating Fas/Fas ligand (FasL)-mediated apoptotic signaling and death is not clearly understood. In this study, we observed that inhibition of Akt causes enhanced expression of FasL mRNA and protein and increased death-inducing signaling complex (DISC) formation with Fas-associated death domain (FADD) and procaspase-8 recruitment. Also, caspase-8 was activated at the DISC with accompanying decrease in c-FLIPs expression. FasL neutralizing antibody significantly decreased apoptotic death in the Akt-inhibited T cells. Additionally, Akt inhibition-induced Fas signaling was observed to link to the mitochondrial pathway via Bid cleavage. Further, inhibition of caspase-8 activity effectively blocked the loss of mitochondrial membrane potential and DNA fragmentation, suggesting that DISC formation and subsequent caspase-8 activation are critical initiating events in Akt inhibition-induced apoptotic death in T lymphocytes. These data demonstrate yet another important survival function governed by Akt kinase in T lymphocytes, which involves the regulation of FasL expression and consequent apoptotic signaling.     

Bax is activated during rotavirus-induced apoptosis through the mitochondrial pathway

Rotaviruses are the leading cause of infantile viral gastroenteritis worldwide. Mature enterocytes of the small intestine infected by rotavirus undergo apoptosis, and their replacement by less differentiated dividing cells probably leads to defective absorptive function of the intestinal epithelium, which, in turn, contributes to osmotic diarrhea and rotavirus pathogenesis. Here we show that infection of MA104 cells by the simian rhesus rotavirus strain RRV induced caspase-3 activation, DNA fragmentation, and cleavage of poly(ADP-ribose) polymerase; all three phenomena are features of apoptosis. RRV induced the release of cytochrome c from mitochondria to the cytosol, indicating that the mitochondrial apoptotic pathway was activated. RRV infection of MA104 cells activated Bax, a proapoptotic member of the Bcl-2 family, as revealed by its conformational change. Most importantly, Bax-specific small interfering RNAs partially inhibited cytochrome c release in RRV-infected cells. Thus, mitochondrial dysfunction induced by rotavirus is Bax dependent. Apoptosis presumably leads to impaired intestinal functions, so our findings contribute to improving our understanding of rotavirus pathogenesis at the cellular level.