Activation of caspase-9, but not caspase-2 or caspase-8, is essential for heat-induced apoptosis in Jurkat cells

Exposure of cells to hyperthermia is known to induce apoptosis, although the underlying mechanisms are only partially understood. Here, we examine the molecular requirements necessary for heat-induced apoptosis using genetically modified Jurkat T-lymphocytes. Cells stably overexpressing Bcl-2/Bcl-x(L) or stably depleted of Apaf-1 were completely resistant to heat-induced apoptosis, implicating the involvement of the mitochondria-mediated pathway. Pretreatment of wild-type cells with the cell-permeable biotinylated general caspase inhibitor b-VAD-fmk (biotin-Val-Ala-Asp(OMe)-CH(2)F) both inhibited heat-induced apoptosis and affinity-labeled activated initiator caspase-2, -8, and -9. Despite this finding, however, cells engineered to be deficient in caspase-8, caspase-2, or the caspase-2 adaptor protein RAIDD (receptor-interacting protein (RIP)-associated Ich-1/CED homologous protein with death domain) remained susceptible to heat-induced apoptosis. Additionally, b-VAD-fmk failed to label any activated initiator caspase in Apaf-1-deficient cells exposed to hyperthermia. Cells lacking Apaf-1 or the pro-apoptotic BH3-only protein Bid exhibited lower levels of heat-induced Bak activation, cytochrome c release, and loss of mitochondrial membrane potential, although cleavage of Bid to truncated Bid (tBid) occurred downstream of caspase-9 activation. Combined, the data suggest that caspase-9 is the critical initiator caspase activated during heat-induced apoptosis and that tBid may function to promote cytochrome c release during this process as part of a feed-forward amplification loop.     

Doxorubicin treatment in vivo activates caspase-12 mediated cardiac apoptosis in both male and female rats

We investigated in vivo the chemotherapeutic anthracycline agents doxorubicin and its ability to activate mitochondrial-mediated, receptor-mediated and endoplasmic/sarcoplasmic reticulum-mediated apoptosis transduction pathways in cardiac tissue from male and female rats. We administered a single low dose of doxorubicin (10 mg/kg of body weight, i.p.) and then isolated mitochondrial and cytosolic proteins one and four days later from the heart. Caspase-3 protein content and caspase-3 activity were significantly increased after day four of doxorubicin treatment in both male and female rats. However, while males had DNA fragmentation at day one but not day four following doxorubicin administration, females showed no significant increase in DNA fragmentation at either time. Caspase-12, localized in the SR, is considered a central caspase, and its activation by cleavage via calpain indicates activation of the SR-mediated pathway of apoptosis. Cleaved caspase-12 content and calpain activity significantly increased after day four of doxorubicin treatment in both sexes. In the mitochondrial-mediated pathway, there were no significant treatment effects observed in cytosolic cytochrome c and cleaved (active) caspase-9 in either sex. In control rats (saline injection), glutathione peroxidase (GPX) activity and hydrogen peroxide (H2O2) production were lower in females compared to males. Doxorubicin treatment did not significantly affect H2O2, GPX activity or ATP production in isolated mitochondria in either sex. Female rats produced significantly lower levels of H2O2 production one day after doxorubicin treatment, whereas male rats produced significantly less mitochondrial H2O2 four days after doxorubicin treatment. The receptor-mediated pathway (caspase-8 and c-FLIP) showed no evidence of being significantly activated by doxorubicin treatment. Hence, doxorubicin-induced apoptosis in vivo is mediated by the SR to a greater extent than other apoptotic pathways and should therefore be considered for targeted therapeutic interventions. Moreover, no major sex differences exist in apoptosis signaling transduction cascade due to doxorubicin treatment.     

Mitochondria-dependent caspase-9 activation is necessary for antigen receptor-mediated effector caspase activation and apoptosis in WEHI 231 lymphoma cells

Engagement of the B cell Ag receptor (BCR) on immature B cells leads to growth arrest followed by apoptosis. Concomitant signaling through CD40 sustains proliferation and rescues the cells from apoptosis. Previously, we have shown that cross-linking CD40 on B cells stimulates the expression of A1, an antiapoptotic member of the Bcl-2 family, and that transduction of the murine B lymphoma line WEHI 231, a model for immature B cells, with A1 protected the cells against BCR-induced apoptosis. Here we demonstrate that A1 strongly interferes with activation of caspase-7, the major effector caspase activated after BCR cross-linking on WEHI 231 lymphoma cells. The pathway leading to activation of the effector caspase cascade including caspase-7 is unclear. Using retrovirally transduced WEHI 231 cell populations, we show that a catalytically inactive mutant of caspase-7 is cleaved almost as efficiently as the wild-type form, arguing against autocatalysis as the sole activating process. In contrast, overexpression of catalytically inactive caspase-9 strongly interferes with caspase-7 processing, poly(ADP-ribose) polymerase cleavage, and DNA laddering, suggesting a role for caspase-9 and hence for the mitochondrial pathway. The importance of the mitochondrial/caspase-9 pathway for BCR-triggered apoptosis is highlighted by our finding that both A1 and the mutant caspase-9 attenuate BCR-induced apoptosis. Thus, our data suggest that the BCR-mediated apoptotic signal in immature B cells spreads via a mitochondrial/caspase-9 pathway.     

An endoplasmic reticulum stress-specific caspase cascade in apoptosis. Cytochrome c-independent activation of caspase-9 by caspase-12

Activation of caspase-12 from procaspase-12 is specifically induced by insult to the endoplasmic reticulum (ER) (Nakagawa, T., Zhu, H., Morishima, N., Li, E., Xu, J., Yankner, B. A., and Yuan, J. (2000) Nature 403, 98-103), yet the functional consequences of caspase-12 activation have been unclear. We have shown that recombinant caspase-12 specifically cleaves and activates procaspase-9 in cytosolic extracts. The activated caspase-9 catalyzes cleavage of procaspase-3, which is inhibitable by a caspase-9-specific inhibitor. Although cytochrome c released from mitochondria has been believed to be required for caspase-9 activation during apoptosis (Zou, H., Henzel, W. J., Liu, X., Lutschg, A., and Wang, X. (1997) Cell 90, 405-413, Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S. M., Ahmad, M., Alnemri, E. S., and Wang, X. (1997) Cell 91, 479-489), caspase-9 as well as caspase-12 and -3 are activated in cytochrome c-free cytosols in murine myoblast cells under ER stress. These results suggest that caspase-12 can activate caspase-9 without involvement of cytochrome c. To examine the role of caspase-12 in the activation of downstream caspases, we used a caspase-12-binding protein, which we identified in a yeast two-hybrid screen, for regulation of caspase-12 activation. The binding protein protects procaspase-12 from processing in vitro. Stable expression of the binding protein renders procaspase-12 insensitive to ER stress, thereby suppressing apoptosis and the activation of caspase-9 and -3. These data suggest that procaspase-9 is a substrate of caspase-12 and that ER stress triggers a specific cascade involving caspase-12, -9, and -3 in a cytochrome c-independent manner.     

Pramanicin induces apoptosis in Jurkat leukemia cells: A role for JNK,p38 and caspase activation

Pramanicin is a novel anti-fungal drug with a wide range of potential application against human diseases. It has been previously shown that pramanicin induces cell death and increases calcium levels in vascular endothelial cells. In the present study, we showed that pramanicin induced apoptosis in Jurkat T leukemia cells in a dose- and time-dependent manner. Our data reveal that pramanicin induced the release of cytochrome c and caspase-9 and caspase-3 activation, as evidenced by detection of active caspase fragments and fluorometric caspase assays. Pramanicin also activated c-jun N-terminal kinase (JNK), p38 and extracellular signal-regulated kinases (ERK 1/2) with different time and dose kinetics. Treatment of cells with specific MAP kinase and caspase inhibitors further confirmed the mechanistic involvement of these signalling cascades in pramanicin-induced apoptosis. JNK and p38 pathways acted as pro-apoptotic signalling pathways in pramanicin-induced apoptosis, in which they regulated release of cytochrome c and caspase activation. In contrast the ERK 1/2 pathway exerted a protective effect through inhibition of cytochrome c leakage from mitochondria and caspase activation, which were only observed when lower concentrations of pramanicin were used as apoptosis-inducing agent and which were masked by the intense apoptosis induction by higher concentrations of pramanicin. These results suggest pramanicin as a potential apoptosis-inducing small molecule, which acts through a well-defined JNK- and p38-dependent apoptosis signalling pathway in Jurkat T leukemia cells.     

alpha-tocopheryl succinate-induced apoptosis in Jurkat T cells involves caspase-3 activation, and both lysosomal and mitochondrial destabilisation

Alpha-Tocopheryl succinate (alpha-TOS), but not a-tocopherol, triggered apoptosis in Jurkat T cells. Apoptosis was induced by alpha-TOS in a time- and concentration-dependent mode, and signs of apoptosis were visible at concentrations of alpha-TOS as low as 30 microM, and within 3-5 h after addition of the ester. Employing a specific fluorogenic substrate, caspase-3 was found to be activated rapidly in response to alpha-TOS at 50 microM. We also found that Jurkat T cells challenged with alpha-TOS, when exposed to the lysosomotropic weak base acridine orange, showed decreased lysosomal uptake of the dye. This is suggestive of the involvement of lysosomal destabilisation in apoptosis of the cells. Apoptosis of Jurkat T cells induced with alpha-TOS also involved a drop in the mitochondrial membrane potential, although this phenomenon occurred after the initiation of lysosomal rupture. All apoptotic features observed with alpha-TOS were very similar to those found when cross-linking of the Fas receptor triggered apoptosis. These findings are consistent with the recent idea that vitamin E can contribute to elimination of malignant cells by the induction of apoptosis, and can be of (patho)physiological significance.     

Ordering of ceramide formation and caspase-9 activation in CD95L-induced Jurkat leukemia T cell apoptosis

Ceramide, a biologically active sphingolipid in cell death signaling, accumulates upon CD95L treatment, concomitantly to apoptosis induction in Jurkat leukemia T cells. Herein, we show that ceramide did not increase in caspase-8 and -10-doubly deficient Jurkat cells in response to CD95L, indicating that apical caspases are essential for CD95L-triggered ceramide formation. Jurkat cells are typically defined as type 2 cells, which require the activation of the mitochondrial pathway for efficient apoptosis induction in response to CD95L. Caspase-9-deficient Jurkat cells significantly resisted CD95L-induced apoptosis, despite ceramide accumulation. Knock-down of sphingomyelin synthase 1, which metabolizes ceramide to sphingomyelin, enhanced (i) CD95L-triggered ceramide production, (ii) cytochrome c release from the mitochondria and (iii) caspase-9 activation. Exogenous ceramide-induced caspase-3 activation and apoptosis were impaired in caspase-9-deficient Jurkat cells. Conversely, caspase-9 re-expression in caspase-9-deficient Jurkat cells restored caspase-3 activation and apoptosis upon exogenous ceramide treatment. Collectively, our data provide genetic evidence that CD95L-triggered endogenous ceramide increase in Jurkat leukemia T cells (i) is not a mere consequence of cell death and occurs mainly in a caspase-9-independent manner, (ii) is likely involved in the pro-apoptotic mitochondrial pathway leading to caspase-9 activation.     

Tissue transglutaminase is a caspase substrate during apoptosis. Cleavage causes loss of transamidating function and is a biochemical marker of caspase 3 activation

Tissue transglutaminase (tTG) is a Ca2+-dependent cross-linking enzyme that participates in the apoptotic machinery by irreversibly assembling a protein scaffold that prevents the leakage of intracellular components. In the present study a single-chain antibody fragment (scFv) detecting tTG is described. We demonstrate that TG/F8 scFv, selected from a phase display library of human V-gene segments by binding to guinea-pig liver tTG, can react with human tTG both in Western blot and in immunohistochemistry. The specific detection of tTG by TG/F8 in human thymocytes is verified by mass spectrometric analysis of the purified protein. Furthermore, we demonstrate that in lymphoid cells tTG is cleaved by caspase 3 during the late phase of apoptotic death, concomitant to DNA fragmentation, and that such cleavage causes loss of cross-linking function. We propose tTG cleavage as a valuable biochemical marker of caspase 3 activation during the late execution phase of apoptosis.     

BMP-dependent activation of caspase-9 and caspase-8 mediates apoptosis in pulmonary artery smooth muscle cells

Germ line mutations in the bone morphogenetic protein (BMP) receptor type II (BMPRII) gene have been found in >50% of familial idiopathic pulmonary arterial hypertension (IPAH) patients and in 30% of sporadic cases of IPAH. Mutations of BMPRII occur in the extracellular ligand-binding domain, in the cytoplasmic serine/threonine kinase domain, or in the long carboxy terminus domain of unknown function. In this study, we demonstrate that BMPs promote apoptotic cell death in normal human pulmonary artery smooth muscle cells (PASMCs) by activation of caspases-3, -8, and -9, cytochrome c release, and downregulation of Bcl-2. Normal PASMCs expressing a kinase domain mutant or a carboxy-terminal domain deletion mutant of BMPRII identified in IPAH patients are resistant to BMP-mediated apoptosis. This dominant-negative effect may act in heterozygous patients and lead to the development of the pulmonary vascular medial hypertrophy found in IPAH patients. Our study also demonstrates an essential role of the carboxy terminus domain of BMPRII in the activation of the apoptotic signaling cascade.     

TRAIL causes cleavage of bid by caspase-8 and loss of mitochondrial membrane potential resulting in apoptosis in BJAB cells.

A new member of the TNF family, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), has been shown to induce apoptosis. However, the mechanism for TRAIL-induced apoptosis remains to be clarified. SDS-PAGE and Western blot analysis showed that cleavage of Bid was induced by a 1-h incubation of BJAB cells with TRAIL and was blocked by a caspase-8 inhibitor. Flow cytometry demonstrated that loss of mitochondrial membrane potential in BJAB cells began about 1.5 h after the treatment with TRAIL and was apparent at 2 h in comparison with the control. DNA ladder formation, which is characteristic for apoptosis, in the cells treated with TRAIL was detected at 2 h and observed most effectively at 3 h. The time course study suggests that TRAIL causes cleavage of Bid via activation of caspase-8, subsequently the loss of mitochondrial membrane potential, resulting in apoptosis in BJAB cells.     

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.     

Effect of glutathione depletion on caspase-3 independent apoptosis pathway induced by curcumin in Jurkat cells

Curcumin, a yellow pigment from Curcuma longa, exhibits anti-inflammatory, antitumor, and antioxidative properties. Although its precise mode of action has not been elucidated so far, numerous studies have shown that curcumin may induce apoptosis in normal and cancer cells. Previously, we showed that in Jurkat cells curcumin induced nontypical apoptosis-like pathway, which was independent of mitochondria and caspase-3. Now we show that the inhibition of caspase-3 by curcumin, which is accompanied by attenuation of internucleosomal DNA fragmentation, may be due to elevation of glutathione, which increased in curcumin-treated cells to 130% of control. We have demonstrated that glutathione depletion does not itself induce apoptosis in Jurkat cells; though, it can release cytochrome c from mitochondria and caspase-3 from inhibition by curcumin, as shown by Western blot. The level of Bcl-2 protein was not affected by glutathione depletion even upon curcumin treatment. Altogether, our results show that in Jurkat cells curcumin prevents glutathione decrease, thus protecting cells against caspase-3 activation and oligonucleosomal DNA fragmentation. On the other hand, it induces nonclassical apoptosis via a still-unrecognized mechanism, which leads to chromatin degradation and high-molecular-weight DNA fragmentation.     

Glutathione selectively inhibits Doxorubicin induced phosphorylation of p53Ser, caspase dependent ceramide production and apoptosis in human leukemic cells

Glutathione (GSH) is the most abundant non-protein antioxidant in mammalian cells. It has been implicated in playing an important role in different signal transduction pathways, and its depletion is an early hallmark in the progression of apoptosis in response to a number of proapoptotic stimuli. We have selectively investigated the role of GSH in cytotoxic response of Jurkat and Molt-4 human leukemic cells to the anti-cancer drug Doxorubicin. In this study, we have shown that extracellular supplementation of GSH to human leukemic cells renders them a resistant phenotype to Doxorubicin treatment. Glutathione pre-treatment inhibits Doxorubicin-induced p53Ser¹⁵ phosphorylation, caspase dependent ceramide (Cer) generation, Poly (ADP-ribose) polymerase (PARP) cleavage, and DNA fragmentation. Taken together, these results indicate that the major cellular antioxidant GSH influences the chemotherapeutic efficacy of Doxorubicin towards human leukemic cells.     

Zearalenone induces apoptosis and necrosis in porcine granulosa cells via a caspase-3- and caspase-9-dependent mitochondrial signaling pathway

Zearalenone is a mycotoxin produced mainly by Fusarium. There are numerous incidences of mycotoxicosis in laboratory and domestic animals, especially in pigs. However, little is known about molecular mechanisms of zearalenone toxicity. Granulosa cells are the maximal cell population in follicles, and they play an essential role in the development and maturation of follicles. The objective of this study was to explore the effect of zearalenone at high concentrations on proliferation and apoptosis of porcine granulosa cells and uncover signaling pathway underlying the cytotoxicity of zearalenone. We found that zearalenone reduced the proliferation of porcine granulosa cells in a dose-dependent manner as shown by the MTT assay and zearalenone resulted in an obvious apoptosis and necrosis in porcine granulosa cells as determined by the TUNEL analysis and flow cytometry. In addition, TUNEL assay with caspase inhibitors showed that zearalenone triggered a caspase-3- and caspase-9-dependent apoptotic process in porcine granulosa cells. Fluorescence spectrophotometer displayed that zearalenone led to a loss of mitochondrial transmembrane potential of porcine granulosa cells but enhanced reactive oxygen species (ROS) levels of the cells. Notably, Western blots revealed that caspase-3 and caspase-9 were activated by zearalenone in porcine granulosa cells. Collectively, our results suggest that zearalenone induces apoptosis and necrosis of porcine granulosa cells in a dose-dependent manner via a caspase-3- and caspase-9-dependent mitochondrial pathway. This study thus offers a novel insight into molecular mechanisms by which zearalenone has adverse cytotoxicity on reproduction.     

Caspase-resistant vimentin suppresses apoptosis after photodynamic treatment with a silicon phthalocyanine in Jurkat cells

Oxidative stress, such as photodynamic therapy, is an apoptosis inducer. Apoptosis, as well as photosensitization, have been associated with disruption of the cytoskeletal network. The purpose of the present study was to assess the role of vimentin, a major cytoskeletal protein, in apoptosis after photodynamic treatment (PDT) with the silicon phthalocyanine Pc 4 in human Jurkat T cells. Here we show for the first time that photosensitization with Pc 4 initiates vimentin cleavage and that this event precedes poly(ADP-ribose) polymerase (PARP) degradation. Similar findings were obtained in the presence of C2-ceramide, an inducer of oxidative stress and apoptosis. In the presence of benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoromethylketone, a pan-caspase inhibitor, Pc 4-PDT-induced vimentin and PARP cleavage were abolished. In Jurkat cells transfected with a caspase-resistant vimentin apoptosis was partly suppressed and delayed post-Pc 4-PDT. We suggest that the full-length vimentin confers resistance to nuclear apoptosis after PDT with Pc 4.     

Saturated free fatty acids and apoptosis in microvascular mesangial cells: palmitate activates pro-apoptotic signaling involving caspase 9 and mitochondrial release of endonuclease G

Background

In type 2 diabetes, free fatty acids (FFA) accumulate in microvascular cells, but the phenotypic consequences of FFA accumulation in the microvasculature are incompletely understood. Here we investigated whether saturated FFA induce apoptosis in human microvascular mesangial cells and analyzed the signaling pathways involved.

Methods

Saturated and unsaturated FFA-albumin complexes were added to cultured human mesangial cells, after which the number of apoptotic cells were quantified and the signal transduction pathways involved were delineated.

Results

The saturated FFA palmitate and stearate were apoptotic unlike equivalent concentrations of the unsaturated FFA oleate and linoleate. Palmitate-induced apoptosis was potentiated by etomoxir, an inhibitor of mitochondrial β-oxidation, but was prevented by an activator of AMP-kinase, which increases fatty acid β-oxidation. Palmitate stimulated an intrinsic pathway of pro-apoptotic signaling as evidenced by increased mitochondrial release of cytochrome-c and activation of caspase 9. A caspase 9-selective inhibitor blocked caspase 3 activation but incompletely blocked apoptosis in response to palmitate, suggesting an additional caspase 9-independent pathway. Palmitate stimulated mitochondrial release of endonuclease G by a caspase 9-independent mechanism, thereby implicating endonuclease G in caspase 9-indpendent regulation of apoptosis by saturated FFA. We also observed that the unsaturated FFA oleate and linoleate prevented palmitate-induced mitochondrial release of both cytochrome-c and endonuclease G, which resulted in complete protection from palmitate-induced apoptosis.

Conclusions

Taken together, these results demonstrate that palmitate stimulates apoptosis by evoking an intrinsic pathway of proapoptotic signaling and identify mitochondrial release of endonuclease G as a key step in proapoptotic signaling by saturated FFA and in the anti-apoptotic actions of unsaturated FFA.     

Cytochrome c-mediated caspase-9 activation triggers apoptosis in Streptococcus pyogenes-infected epithelial cells

Epithelial cells are the initial sites of host invasion by group A Streptococcus pyogenes (GAS), and their infection of epithelial cells has been suggested to induce apoptosis. However, the mechanism responsible for bacteria–host interaction and the induction of apoptosis has not been clearly understood. We demonstrate here that human pharyngeal epithelial HEp-2 cells became apoptotic with DNA fragmentation by invasion of GAS strains JRS4 (M6⁺, F1⁺) and JRS145 (M6⁻, F1⁺ mutant of JRS4), whereas apoptotic cellular changes were not observed in SAM1 (M6⁺, F1⁻ mutant) or SAM2 (M6⁻, F1⁻ mutant) infected HEp-2 cells. Confocal microscopy revealed that Bax translocation to mitochondria and cytochrome c release occurred after 4 h of infection. Western blot analyses showed that the amounts of Bcl-2 and Bcl-x_L were decreased in the mitochondria of infected cells. In addition, we demonstrated that the release of nuclear histone from infected cells was prevented by the addition of caspase-9 inhibitor (Ac-LEHD-CHO). We conclude that the internalization of GAS in epithelial cells is necessary and sufficient for the induction of apoptosis, which is initiated by mitochondrial dysfunction, and the mechanism of GAS-induced apoptosis is clearly different from that induced by other intracellular invasive bacteria, e.g. Shigella and Salmonella species.     

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.     

HIV-1 Vpr induces apoptosis through caspase 9 in T cells and peripheral blood mononuclear cells

Human immunodeficiency virus, type 1 (HIV-1), vpr gene encodes a 14-kDa virion-associated protein, which exhibits significant effects on human cells. One important property of Vpr is its ability to induce apoptosis during infection. Apoptotic induction is likely to play a role in the pathogenesis of AIDS. However, the pathway of apoptosis is not clearly defined. In this report we investigate the mechanism of apoptosis induced by HIV-1 Vpr using a Vpr pseudotype viral infection system or adeno delivery of Vpr in primary human lymphoid cells and T-cells. With either vector, HIV-1 Vpr induced cell cycle arrest at the G(2)/M phase and apoptosis in lymphoid target cells. Furthermore, we observed that with both vectors, caspase 9, but not caspase 8, was activated following infection of human peripheral blood mononuclear cell with either Vpr-positive HIV virions or adeno-delivered Vpr. Activation of the caspase 9 pathway resulted in caspase 3 activation and apoptosis in human primary cells. These effects were coincident with the disruption of the mitochondrial transmembrane potential and induction of cytochrome c release by Vpr. The Vpr-induced signaling pathway did not induce CD95 or CD95L expression. Bcl-2 overexpressing cells succumb to Vpr-induced apoptosis. These studies illustrate that Vpr induces a mitochondria-dependent apoptotic pathway that is distinct from apoptosis driven by the Fas-FasL pathway.     

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).