Taxol-induced apoptosis in human SKOV3 ovarian and MCF7 breast carcinoma cells is caspase-3 and caspase-9 independent

Taxol is used in chemotherapy regimens against breast and ovarian cancer. Treatment of tumor model cell lines with taxol induces apoptosis, but exact mechanism is not sufficiently understood. Our results demonstrate that in response to taxol, various cell types differentially utilize distinct apoptotic pathways. Using MCF7 breast carcinoma cells transfected with caspase-3 gene, we showed that taxol-induced apoptosis occurred in the absence of caspase-3 and caspase-9 activation. Similar results were obtained with ovarian SKOV3 carcinoma cells, expressing high level of endogenous caspase-3. In contrast, staurosporine-induced apoptosis in these cells was accompanied by proteolytic cleavage of pro-caspase-3 and induction of caspase-3 enzymatic activity. The effect of taxol appears to be cell type-specific, since taxol-induced apoptosis in leukemia U937 cells involved caspase-3 activation step. We conclude that a unique caspase-3 and caspase-9 independent pathway is elicited by taxol to induce apoptosis in human ovarian and breast cancinoma cells.     

Beclin 1 augmented cis-diamminedichloroplatinum induced apoptosis via enhancing caspase-9 activity

Beclin 1, identified as a Bcl-2-interacting protein, is known to enhance autophagy. However, the effect of Beclin 1 on apoptotic signaling has remained unclear. Here, we show that overexpression of Beclin 1 in MKN28 human gastric cancer cells augmented cis-diamminedichloroplatinum (CDDP)-induced apoptosis. Conversely, "knockdown" of Beclin 1 by a small inhibitory RNA in MKN 1 cells attenuated this cytotoxicity. Furthermore, not only caspase-3/7 activities, but also caspase-9 activity was increased in Beclin 1 gene transfectants treated with CDDP, and caspase-9 inhibitor completely abolished augmentation of CDDP-induced apoptosis by Beclin 1 as did a caspase-3 inhibitor. Thus, Beclin 1 augments CDDP-induced apoptosis through enhancing caspase-9 activity and functions as a pro-apoptotic molecule.     

UV-induced apoptosis is mediated independent of caspase-9 in MCF-7 cells: a model for cytochrome c resistance

The importance of the mitochondria in UV-induced apoptosis has become increasingly apparent. Following DNA damage cytochrome c and other pro-apoptotic factors are released from the mitochondria, allowing for formation of the apoptosome and subsequent cleavage and activation of caspase-9. Active caspase-9 then activates downstream caspases-3 and/or -7, which in turn cleave poly(ADP)-ribose polymerase (PARP) and other down-stream targets, resulting in apoptosis. In an effort to understand the mechanisms of Akt-mediated cell survival in breast cancer, we studied the effects of insulin-like growth factor (IGF)-I treatment on UV-treated MCF-7 human breast cancer cells. Apoptosis was induced in MCF-7 cells after UV treatment, as measured by caspase-7 and PARP cleavage, and IGF-I co-treatment protected against this response. Surprisingly caspase-9 cleavage was unchanged with UV and/or IGF-I treatment. Using MCF-7 cells overexpressing caspase-3 we have shown that resistance of caspase-9 to cleavage was not altered by the expression of caspase-3. Furthermore, overexpression of caspase-9 did not enhance PARP or caspase-7 cleavage after UV treatment. Because caspase-8 was activated with UV treatment alone, we believe that UV-induced apoptosis in MCF-7 cells occurs independently of cytochrome c and caspase-9, supporting the existence of a cytoplasmic inhibitor of cytochrome c in MCF-7 cells. We anticipate that such inhibitors may be overexpressed in cancer cells, allowing for treatment resistance.     

Antiapoptotic property of human alpha-synuclein in neuronal cell lines is associated with the inhibition of caspase-3 but not caspase-9 activity

Abnormalities of alpha-synuclein (alpha-Syn) are mechanistically linked to Parkinson's disease (PD) and other alpha-synucleinopathies. To gain additional insights into the relationships between alpha-Syn expression and cell death, we examined the effects of expressing human alpha-Syn (Hualpha-Syn) variants on the cellular vulnerability to apoptotic stimuli. We show that the expression of wild-type (WT) and A30P mutant, but not A53T mutant, Hualpha-Syn leads to the protection of neuronal cell lines from apoptosis but not necrosis. Significantly, Hualpha-Syn did not protect non-neuronal cell lines from apoptosis. We also show that A53T mutant is a loss of function in regards to the antiapoptotic property since the expression of WT Hualpha-Syn with an excess of A53T mutant Hualpha-Syn leads to protection of the cells from apoptosis. The antiapoptotic property is specific to human alpha-Syn as neither beta-Syn nor mouse alpha-Syn protected cells from apoptosis, and the carboxy-terminal 20 amino acids are required for the antiapoptotic property. Analyses of capase-3 and caspase-9 activation reveal that the antiapoptotic property of Hualpha-Syn in neuronal cell lines is associated with the attenuation of caspase-3 activity without affecting the caspase-9 activity or the levels of cleaved, active caspase-3. We conclude that Hualpha-Syn modulates the activity of cleaved caspase-3 product in neuronal cell lines.     

Transforming growth factor beta-dependent sequential activation of Smad, Bim, and caspase-9 mediates physiological apoptosis in gastric epithelial cells

Transforming growth factor beta (TGF-beta) has been implicated in the maintenance of homeostasis in various organs, including the gastric epithelium. In particular, TGF-beta-induced signaling was shown to be required for the differentiation-associated physiological apoptosis of gastric epithelial cells, but its mechanism has not been well understood. In this study, the molecular mechanism of TGF-beta-induced apoptosis was analyzed in a human gastric epithelial cell line, SNU16, as an in vitro model. Expression of Smad7 and Bcl-X(L), but not viral FLIP, was shown to prevent TGF-beta-induced apoptosis, indicating an exclusive requirement of the activation of Smad signaling pathway and mitochondrial dysfunction followed by activation of caspase-9. In addition, treatment with TGF-beta induced binding of Bim, a proapoptotic Bcl-2 homology domain 3 (BH3)-only protein, to Bcl-X(L), which is dependent on the activation of Smad, and reduction in the expression of Bim by RNA interference decreased the sensitivity to TGF-beta-induced apoptosis. Moreover, we found abnormalities in the gastric epithelium of both Bim and caspase-9 knockout mice; these abnormalities were associated with a defect of physiological apoptosis in gastric epithelial cells. These results indicate for the first time that TGF-beta is involved in the physiological loss of gastric epithelial cells by activating apoptosis mediated by Smad, Bim, and caspase-9.     

Caspase-2-induced apoptosis is dependent on caspase-9, but its processing during UV- or tumor necrosis factor-dependent cell death requires caspase-3

Mammalian caspases are a family of cysteine proteases that plays a critical role in apoptosis. We have analyzed caspase-2 processing in human cell lines containing defined mutations in caspase-3 and caspase-9. Here we demonstrate that caspase-2 processing, during cell death induced by UV irradiation, depends both on caspase-9 and caspase-3 activity, while, during TNF-alpha-dependent apoptosis, capase-2 processing is independent of caspase-9 but still requires caspase-3. In vitro procaspase-2 is the preferred caspase cleaved by caspase-3, while caspase-7 cleaves procaspase-2 with reduced efficiency. We have also demonstrated that caspase-2-mediated apoptosis requires caspase-9 and that cells co-expressing caspase-2 and a dominant negative form of caspase-9 are impaired in activating a normal apoptotic response and release cytochrome c into the cytoplasm. Our findings suggest a role played by caspase-2 as a regulator of the mitochondrial integrity and open questions on the mechanisms responsible for its activation during cell death.     

PQ1, a quinoline derivative, induces apoptosis in T47D breast cancer cells through activation of caspase-8 and caspase-9

Apoptosis, a programmed cell death, is an important control mechanism of cell homeostasis. Deficiency in apoptosis is one of the key features of cancer cells, allowing cells to escape from death. Activation of apoptotic signaling pathway has been a target of anti-cancer drugs in an induction of cytotoxicity. PQ1, 6-methoxy-8-[(3-aminopropyl)amino]-4-methyl-5-(3-trifluoromethylphenyloxy)quinoline, has been reported to decrease the viability of cancer cells and attenuate xenograft tumor growth. However, the mechanism of the anti-cancer effect is still unclear. To evaluate whether the cytotoxicity of PQ1 is related to induction of apoptosis, the effect of PQ1 on apoptotic pathways was investigated in T47D breast cancer cells. PQ1-treated cells had an elevation of cleaved caspase-3 compared to controls. Studies of intrinsic apoptotic pathway showed that PQ1 can activate the intrinsic checkpoint protein caspase-9, enhance the level of pro-apoptotic protein Bax, and release cytochrome c from mitochondria to cytosol; however, PQ1 has no effect on the level of anti-apoptotic protein Bcl-2. Further studies also demonstrated that PQ1 can activate the key extrinsic player, caspase-8. Pre-treatment of T47D cells with caspase-8 or caspase-9 inhibitor suppressed the cell death induced by PQ1, while pre-treatment with caspase-3 inhibitor completely counteracted the effect of PQ1 on cell viability. This report provides evidence that PQ1 induces cytotoxicity via activation of both caspase-8 and caspase-9 in T47D breast cancer cells.     

Involvement of caspase-9 in autophagy-mediated cell survival pathway

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been considered for use in the prevention and treatment of cancer malignancy. FR122047 (FR) is known to have an anti-inflammatory effect, but the anticancer activity of the chemical has not yet been identified. In the present study, we could find that treatment of breast cancer MCF-7 cells with FR led to apoptosis accompanying with apparent activation of caspases. Treatment of caspase-specific inhibitors revealed that FR-induced apoptosis was caspase-8-dependent and inhibition of caspase-9 activity resulted in unexpected, marked enhancement of cell death. Knockdown of caspase-9 expression by specific siRNA caused increased susceptibility to FR-induced cell death, consistent with the results obtained with treatment of caspase-9 inhibitor. Inhibition of caspase-9 blocked the autophagic process by modulating lysosomal pH and acid-dependent cathepsin activities and augmented cell death due to blockage of cytoprotective autophagy. MCF-7 cells treated with sulforaphane, an autophagy-inducing drug, also showed marked accumulation of LC3-II, and co-treatment with caspase-9 inhibitor brought about increased susceptibility to sulforaphane-induced cell death. Different from the cases with FR or sulforaphane, etoposide- or doxorubicin-induced cell death was suppressed with co-treatment of caspase-9 inhibitor, and the drugs failed to induce significant autophagy in MCF-7 cells. Taken together, our data originally suggest that inhibition of caspase-9 may block the autophagic flux and enhance cell death due to blockage of cytoprotective autophagy.     

Characterization of cell clones stably transfected with short form caspase-9: Apoptotic resistance and Bcl-XL expression

Caspases play important roles in the initiation and progression of apoptosis. In experimental models of ATP depletion, we have demonstrated the activation of caspase-9, -8, and -3, which is followed by the development of apoptotic morphology. To determine the specific contribution of caspase-9 to ATP depletion-induced apoptosis, we transfected renal epithelial cells with its endogenous dominant-negative inhibitor caspase-9S. Two cell clones with stable transfection were obtained. These clones expressed caspase-9S, and the cytosol isolated from these cells was resistant to cytochrome c-induced caspase activation in vitro. The clones were then examined for ATP depletion-induced apoptosis. Compared with the wild-type cells, the caspase-9S clones were markedly resistant to apoptosis in this model. Caspase activation was also inhibited. Surprisingly, these clones also showed significantly less cytochrome c release during ATP-depletion. Moreover, Bax translocation to mitochondria was inhibited, suggesting that these clones were resistant to apoptosis not only at the cytosolic caspase activation level but also at the upstream mitochondrial level. To gain insights into the mitochondrial resistance, we analyzed the expression of Bcl-2 family proteins. While the expression of Bax, Bak, and Bcl-2 was comparable to the wild-type cells, the selected clones showed specific up-regulation of Bcl-XL, an anti-apoptotic protein. We conclude that the selected clones were resistant to apoptosis at two levels. In the cytosol, they expressed dominant negative caspase-9, and at the mitochondria they up-regulated Bcl-XL.     

Targeting colorectal cancer stem cells with inducible caspase-9

Colorectal cancer stem cells (CSCs) drive tumor growth and are suggested to initiate distant metastases. Moreover, colon CSCs are reportedly more resistant to conventional chemotherapy, which is in part due to upregulation of anti-apoptotic Bcl-2 family members. To determine whether we could circumvent this apoptotic blockade, we made use of an inducible active caspase-9 (iCasp9) construct to target CSCs. Dimerization of iCasp9 with AP20187 in HCT116 colorectal cancer cells resulted in massive and rapid induction of apoptosis. In contrast to fluorouracil (5-FU)-induced apoptosis, iCasp9-induced apoptosis was independent of the mitochondrial pathway as evidenced by Bax/Bak double deficient HCT116 cells. Dimerizer treatment of colon CSCs transduced with iCasp9 (CSC-iCasp9) also rapidly induced high levels of apoptosis, while these cells were unresponsive to 5-FU in vitro. More importantly, injection of the dimerizer into mice that developed a colon CSC-iCasp9-induced tumor resulted in a strong decrease in tumor size, an increase in tumor cell apoptosis and a clear loss of CD133⁺ CSCs. Taken together, our data indicate that dimerization of iCasp9 circumvents the apoptosis block in CSCs, which results in effective tumor regression in vivo.     

Thymoquinone inhibits growth and augments 5-fluorouracil-induced apoptosis in gastric cancer cells both in vitro and in vivo

Thymoquinone (TQ), a component derived from the bioactive constituent of black seed (Nigella sativa), has been shown to exert biological activity on various types of human cancers. However, there are few studies addressing its effects on gastric cancer. Here, we present the first report describing the chemosensitizing effect of thymoquinone and 5-fluorouracil (5-FU) on gastric cancer cells both in vitro and in vivo. Studies have shown that pretreatment with TQ significantly increased the apoptotic effects induced by 5-FU in gastric cancer cell lines in vitro. Moreover, we found that TQ enhanced the 5-FU-induced killing of gastric cancer cells by mediating the downregulation of the anti-apoptotic protein bcl-2, the upregulation of the pro-apoptotic protein bax, and the activation of both caspase-3 and caspase-9. In addition to the in vitro results, it has been shown that the combined treatment of TQ with 5-FU represents a significantly more effective antitumor agent than either agent alone in a xenograft tumor mouse model. These data suggest that the TQ/5-FU combined treatment induces apoptosis by enhancing the activation of both caspase-3 and caspase-9 in gastric cancer cells. These results, which provide molecular evidence both in vitro and in vivo, support our conclusion that thymoquinone can activate caspase-3 and caspase-9 and thus result in the chemosensitisation of gastric cancer cells to 5-FU-induced cell death.     

Immunohistochemical localization of caspase-3, caspase-9 and Bax in U87 glioblastoma xenografts

Development of new therapies for glioblastoma requires animal models that mimic the biological characteristics of human brain tumors. On the other hand, potential antitumoral effects of a new therapeutic strategy are often established by evaluation of tumor cells apoptosis. Caspases are key mediators in the regulation and execution of apoptosis. Caspase-9 is activated during the intrinsic pathway downstream of mitochondria while caspase-3 is an effector caspase that initiates degradation of the cell in the final stages of apoptosis. Bax is a pro-apoptotic member of the Bcl-2 family that play key roles in the regulation of intrinsic apoptotic signaling. In the present study we investigated the immunohistochemical distribution of caspase 3, 9 and Bax in intracranial U87 glioblastoma xenograft. Immunohistochemistry showed that the glioblastoma xenografts contain cells positive for caspase-3, caspase-9, and Bax.     

Signaling defect in the activation of caspase-3 and PKCdelta in human TUR leukemia cells is associated with resistance to apoptosis

Exposure of the two related human leukemic cell lines U937 and TUR to chemotherapeutic compounds resulted in opposite effects on induction and resistance to apoptosis. Incubation of U937 cells with 1-beta-d-arabinofuranosylcytosine or the etoposide VP-16 was accompanied by growth arrest in G0/G1 of the cell cycle and an accumulation of a population in the sub-G1 phase which exhibited characteristics typical for the apoptotic pathway. In contrast, human TUR leukemia cells demonstrated no significant effects after a similar treatment with Ara-C and VP-16. Thus, TUR cells continued to proliferate in the presence of these anti-cancer drugs and the number of apoptotic cells as evaluated by propidium iodide staining and the detection of internucleosomal DNA fragmentation was significantly reduced when compared to the parental U937 cells. Similar effects were observed upon serum-starvation demonstrating resistance to apoptosis in TUR cells. Whereas induction of apoptosis is regulated by a network of distinct factors including the activation of proteolytically active caspases, we investigated these pathways in both cell lines. U937 cells demonstrated activation of the 32-kDa caspase-3 upon drug treatment by cleavage into the 20-kDa activated form. However, there was no 20-kDa caspase-3 fragment detectable in TUR cells. Simultaneously, the enzymatic activity of caspase-3 was significantly increased in drug-treated U937 cells as measured in vitro by enhanced metabolization of a fluorescence substrate and in vivo by cleavage of an appropriate substrate for caspase-3, namely, protein kinase Cdelta. In contrast, there was little if any caspase-3 activation detectable in drug-treated TUR cells. Taken together, these data suggest a signaling defect in the activation of the caspase-3 proteolytic system in TUR cells upon treatment with chemotherapeutic compounds which is associated with resistance to apoptosis in these human leukemia cells.     

A novel thiazolidine compound induces caspase-9 dependent apoptosis in cancer cells

The forward chemogenomics strategy allowed us to identify a potent cytotoxic thiazolidine compound as an apoptosis-inducing agent. Chemical structures were designed around a thiazolidine ring, a structure already noted for its anticancer properties. Initially, we evaluated these novel compounds on liver, breast, colon and endometrial cancer cell lines. The compound 3 (ALC67) showed the strongest cytotoxic activity (IC(50) ～5μM). Cell cycle analysis with ALC67 on liver cells revealed SubG1/G1 arrest bearing apoptosis. Furthermore we demonstrated that cytotoxicity of this compound was due to the activation of caspase-9 involved apoptotic pathway, which is death receptor independent.     

Resveratrol induces mitochondria-mediated AIF and to a lesser extent caspase-9-dependent apoptosis in human lung adenocarcinoma ASTC-a-1 cells

Resveratrol (RV), a natural plant polyphenol widely present in foods such as grapes, wine, and peanuts, has an ability to inhibit various stages of carcinogenesis in vitro and in vivo. In this report, we explored the roles of intrinsic and extrinsic apoptotic pathways during RV-induced apoptosis in human lung adenocarcinoma (ASTC-a-1) cells. After exposure of cells to different concentrations of RV, we found that RV induced concentration-dependent apoptosis. Fluorometric substrates assay and western blotting (WB) analysis showed that caspase-8 was not activated, which was further verified by monitoring the cleavage of Bid to tBid using fluorescence resonance energy transfer (FRET) microscopy imaging inside single living cells, indicating that extrinsic apoptotic pathway was not involved in RV-induced apoptosis. In addition, inhibition of caspases-3 or -9 but not caspase-8 using the specific inhibitors of caspases modestly but significantly attenuated RV-induced apoptosis. Moreover, flow cytometry (FCM) analysis showed that RV treatment induced time-dependent loss of mitochondrial membrane potential (?ψ(m)), in combination with the activation of caspases-3 and -9; we therefore concluded that RV-induced apoptosis involved the intrinsic apoptotic pathway. It is noteworthy that RV treatment induced translocation of AIF from mitochondria to nucleus in a time dependent manner, and that knockdown of AIF remarkably attenuated RV-induced apoptosis. Collectively, our findings demonstrate that RV induces caspase-8-independent apoptosis via AIF and to a lesser extent caspase-9-dependent mitochondrial pathway in ASTC-a-1 cells.     

Activation of caspase-9 is required for UV-induced apoptosis of human keratinocytes

UV radiation from the sun activates both the membrane death receptor and the intrinsic or mitochondrial apoptotic signaling pathways in epidermal keratinocytes, triggering apoptosis and affording protection against skin cancer formation. We have investigated the involvement of caspase-9 in the UV death effector pathway in human keratinocytes, since this is the initiating caspase in the mitochondrial pathway required for UV-induced apoptosis in some, but not all, cell types. UV radiation triggered activation of caspase-3, caspase-9, and caspase-8 with similar kinetics, although the rank order of activation was caspase-3 > caspase-9 > caspase-8. Inhibition of caspase-9 with either the peptide inhibitor benzyloxycarbonyl-Leu-Glu(OCH(3))-His-Asp(OCH(3))-fluoromethyl ketone, or expression of a catalytically inactive caspase-9 by retroviral transduction, protected normal keratinocytes from UV-induced apoptosis. HaCaT keratinocytes harboring mutant p53 alleles were also protected from UV-induced apoptosis by the dominant negative caspase-9. The dominant negative caspase-9 blocked UV-induced activation of caspase-3, caspase-9, and caspase-8, and also protected cells from the loss of mitochondrial membrane potential. In contrast, the dominant negative caspase-9 did not protect from anti-Fas-induced apoptosis or caspase activation. These results identify caspase-9 as the critical upstream caspase initiating apoptosis by UV radiation in human keratinocytes, the relevant cell type for this important environmental carcinogen.     

Role of XIAP in inhibiting cisplatin-induced caspase activation in non-small cell lung cancer cells: a small molecule Smac mimic sensitizes for chemotherapy-induced apoptosis by enhancing caspase-3 activation

X-linked IAP (XIAP) suppresses apoptosis by binding to initiator caspase-9 and effector caspases-3 and -7. Smac/DIABLO that is released from mitochondria during apoptosis can relieve its inhibitory activity. Here we investigated the role of XIAP in the previously found obstruction of chemotherapy-induced caspase-9 activation in non-small cell lung cancer (NSCLC) cells. Endogenously expressed XIAP bound active forms of both caspase-9 and caspase-3. However, downregulation of XIAP using shRNA or disruption of XIAP/caspase-9 interaction using a small molecule Smac mimic were unable to significantly induce caspase-9 activity, indicating that despite a strong binding potential of XIAP to caspase-9 it is not a major determinant in blocking caspase-9 in NSCLC cells. Although unable to revert caspase-9 blockage, the Smac mimic was able to enhance cisplatin-induced apoptosis, which was accompanied by increased caspase-3 activity. Additionally, a more detailed analysis of caspase activation in response to cisplatin indicated a reverse order of activation, whereby caspase-3 cleaved caspase-9 yielding an inactive form. Our findings indicate that the use of small molecule Smac mimic, when combined with an apoptotic trigger, may have therapeutic potential for the treatment of NSCLC.     

Ceramide induces neuronal apoptosis through the caspase-9/caspase-3 pathway

C(2)-ceramide, a cell-permeable analog of ceramide, caused cell death in cultured rat cortical neuronal cells. C(2)-ceramide-induced neuronal loss was accompanied by upregulation of caspase-3 activity, measured by cleavage of its fluorogenic substrate Ac-DEVD-AMC. Similar results were obtained when cortical neuronal cultures were treated with sphingomyelinase, an enzyme responsible for ceramide formation in the cell. Morphological evaluation of C(2)-ceramide-treated cortical neurons showed nuclear condensation and fragmentation as visualized by Hoechst 33258 staining. Co-administration of the selective caspase-3 inhibitor z-DEVD-fmk or caspase-9 inhibitor z-LEHD-fmk significantly reduced C(2)-ceramide-induced cell death, while co-application of the caspase-8, inhibitor z-IETD-fmk, was without effect. Immunoblot analysis of protein extracts from C(2)-ceramide-treated cortical neuronal cultures revealed upregulation of active caspase-9 and caspase-3 protein levels, whereas presence of active caspase-8 immunoreactivity was undetectable in this system. Administration of C(2)-ceramide to SH-SY5Y human neuroblastoma cells also caused apoptotic cell death. Moreover, ceramide-induced cell death was significantly decreased in caspase-9 dominant-negative SH-SY5Y cells, while both caspase-8 dominant-negative cultures and mock-transfected cells showed equally high levels of cell death following C(2)-ceramide treatment. Taken together, these data suggest that neuronal death induced by ceramide may be linked to the caspase-9/caspase-3 regulated intrinsic pathway of cellular apoptosis.     

Stobadine inhibits doxorubicin-induced apoptosis through a caspase-9 dependent pathway in P815 mastocytoma cells

Doxorubicin (DOXO), a widely used chemotherapeutic agent, induces apoptosis in transformed and non-transformed cells. The apoptotic effect of DOXO has been linked to the generation of reactive oxygen species (ROS). Antioxidants may be effective in the prevention of DOX-induced apoptosis. In the present study we investigated the effects of stobadine, a pyridoindole antioxidant in a DOXO-induced apoptosis model of P815 cells by flow cytometric analyses and by measuring caspase-3 and caspase-9 activities. Pretreating cells with stobadine significantly increased cell viability and decreased apoptosis rate. Inhibition in apoptosis was observed at maximum levels following treatment of cells with 10(-7)M stobadine as evident from flow cytometric analyses. The antiapoptotic effect of stobadine was further confirmed by inhibition of caspase-3 and caspase-9 activities. We found that the antioxidative effects of stobadine were comparable to the effects of a well known antioxidant, N-acetyl l-cysteine (NAC).