Flavonoids purified from Rhus verniciflua Stokes actively inhibit cell growth and induce apoptosis in human osteosarcoma cells

Many studies have suggested that dietary flavonoids are anticancer agents that induce the apoptosis of cancer cells. However, the effects of flavonoids on the induction of apoptosis in osteosarcoma cells are unclear. Previously, a flavonoid fraction, consisting mainly of protocatechuic acid, fustin, fisetin, sulfuretin, and butein, herein named RCMF (the RVS chloroform-methanol fraction), was prepared from a crude acetone extract of Rhus verniciflua Stokes (RVS). This study evaluated the effects of RCMF on the proliferation and apoptosis using human osteosarcoma (HOS) cells. The mechanism of growth inhibition of the HOS cells by the flavonoid fraction, RCMF, was also assessed. The results demonstrated that RCMF exhibited sensitive growth inhibition and induced apoptosis in HOS cells. PARP cleavage was closely associated with the RCMF-induced apoptosis of the HOS cells. Furthermore, the activation of caspase 8 and Bax, the inhibition of Bcl-2 expression, and the release of cytochrome c are believed to be involved in the RCMF-mediated apoptosis. Collectively, these findings suggest that RCMF is an agent which may be capable of inducing sensitive growth inhibition and apoptosis in HOS cells.     

p53 signalling controls cell cycle arrest and caspase‐independent apoptosis in macrophages infected with pathogenic Leptospira species

Pathogenic Leptospira species, the causative agents of leptospirosis, have been shown to induce macrophage apoptosis through caspase‐independent, mitochondrion‐related apoptosis inducing factor (AIF) and endonuclease G (EndoG), but the signalling pathway leading to AIF/EndoG‐based macrophage apoptosis remains unknown. Here we show that infection of Leptospira interrogans caused a rapid increase in reactive oxygen species (ROS), DNA damage, and intranuclear foci of 53BP1 and phosphorylation of H2AX (two DNAdamage indicators) in wild‐type p53‐containing mouse macrophages and p53‐deficient human macrophages. Most leptospire‐infected cells stayed at the G₁ phase, whereas depletion or inhibition of p53 caused a decrease of the G₁‐phase cells and the early apoptotic ratios. Infection with spirochaetes stimulated a persistent activation of p53 and an early activation of Akt through phosphorylation. The intranuclear translocation of p53, increased expression of p53‐dependent p21^Cip1/WAF1 and pro‐apoptotic Bcl‐2 family proteins (Bax, Noxa and Puma), release of AIF and EndoG from mitochondria, and membrane translocation of Fas occurred during leptospire‐induced macrophage apoptosis. Thus, our study demonstrated that ROS production and DNA damage‐dependent p53‐Bax/Noxa/Puma‐AIF/EndoG signalling mediates the leptospire‐induced cell cycle arrest and caspase‐independent apoptosis of macrophages.     

4-hydroperoxy-cyclophosphamide mediates caspase-independent T-cell apoptosis involving oxidative stress-induced nuclear relocation of mitochondrial apoptogenic factors AIF and EndoG

Apoptosis is a major mechanism of treatment-induced T-cell depletion in leukemia and autoimmune diseases. While 'classical' apoptosis is considered to depend on caspase activation, caspase-independent death is increasingly recognized as an alternative pathway. Although the DNA-damaging drug cyclophosphamide (CY) is widely used for therapy of hematological malignancies and autoimmune disorders, the molecular mechanism of apoptosis induction remains largely unknown. Here, we report that treatment of Jurkat, cytotoxic, and primary leukemic T cells with an activated analog of CY, 4-hydroperoxy-cyclophosphamide (4-OOH-CY), induces caspase activation and typical features of apoptosis, although cell death was not prevented by caspase inhibition. Also depletion of murine thymocytes and splenocytes after CY treatment in vivo was not inhibited by Z-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD.fmk). Caspase-8 and receptor-induced protein (RIP) were dispensable for 4-OOH-CY-mediated apoptosis, while overexpression of Bcl-2 was partially protective. 4-OOH-CY treatment induced reactive oxygen species production, upregulation of Bax, and nuclear relocation of the mitochondrial factors apoptosis-inducing factor (AIF) and endonuclease G (EndoG). The antioxidant N-acetyl-L-cysteine substantially inhibited conformational changes of Bax, loss of mitochondrial membrane potential, nuclear relocation of mitochondrial factors, and apoptosis induction in 4-OOH-CY-treated T cells. These results strongly indicate that oxidative damage-induced nuclear translocation of AIF and EndoG in 4-OOH-CY-treated T cells might represent an alternative death pathway in the absence of caspase activity.     

FK-16 Derived from the Anticancer Peptide LL-37 Induces Caspase-Independent Apoptosis and Autophagic Cell Death in Colon Cancer Cells

Host immune peptides, including cathelicidins, have been reported to possess anticancer properties. We previously reported that LL-37, the only cathelicidin in humans, suppresses the development of colon cancer. In this study, the potential anticancer effect of FK-16, a fragment of LL-37 corresponding to residues 17 to 32, on cultured colon cancer cells was evaluated. FK-16 induced a unique pattern of cell death, marked by concurrent activation of caspase-independent apoptosis and autophagy. The former was mediated by the nuclear translocation of AIF and EndoG whereas the latter was characterized by enhanced expression of LC3-I/II, Atg5 and Atg7 and increased formation of LC3-positive autophagosomes. Knockdown of Atg5 or Atg7 attenuated the cytotoxicity of FK-16, indicating FK-16-induced autophagy was pro-death in nature. Mechanistically, FK-16 activated nuclear p53 to upregulate Bax and downregulate Bcl-2. Knockdown of p53, genetic ablation of Bax, or overexpression of Bcl-2 reversed FK-16-induced apoptosis and autophagy. Importantly, abolition of AIF/EndoG-dependent apoptosis enhanced FK-16-induced autophagy while abolition of autophagy augmented FK-16-induced AIF−/EndoG-dependent apoptosis. Collectively, FK-16 induces caspase-independent apoptosis and autophagy through the common p53-Bcl-2/Bax cascade in colon cancer cells. Our study also uncovered previously unknown reciprocal regulation between these two cell death pathways.     

Inhibition of extracellular signal regulated kinase (ERK) leads to apoptosis inducing factor (AIF) mediated apoptosis in epithelial breast cancer cells: the lack of effect of ERK in p53 mediated copper induced apoptosis

Recent studies have shown that MEK/ERK-mediated signals play a major role in regulation of activity of p53 tumor suppressor protein. In this study, we investigated whether or not there is functional interaction between p53 and MEK/ERK pathways in epithelial breast cancer cells exposed to copper or zinc. We demonstrated that expression of wild-type p53 induced by copper or zinc significantly reduced phosphorylation of extracellular signal regulated kinase (ERK) in epithelial breast cancer MCF7 cells. Mutation or suppression of p53 in MDA-MB231 and MCF7-E6 cells, respectively, resulted in a strong ERK phosphorylation in the presence of metals. Weak ERK phosphorylation in MCF7 cells induced by copper or zinc was linked to mitochondrial disruption and apoptosis. Furthermore, inhibition of ERK through addition of PD98059 stimulated p53 activation in MCF7 cells and also led to upregulation of p53 downstream targets, p21 and Bax, which is a proapototic member of Bcl-2 family triggering mitochondrial pore opening. Moreover, blockage of the MEK/ERK pathway caused a breakdown of the mitochondrial membrane potential accompanied by an elevation in the ROS production. Disruption of p53 expression attenuated the depolarization of the mitochondrial membrane and ROS generation. Furthermore, PD98059 initiated apoptosis inducing factor (AIF) translocation from mitochondria to the nucleus in MCF7 cells; which are depleted in caspase 3. Interestingly, repression of MEK/ERK pathway did not intensify the cell stress caused by metal toxicity. Therefore, these findings demonstrate that MEK/ERK pathway plays an important role in downregulation of p53 and cell survival. Inhibition of ERK can lead to apoptosis via nuclear relocation of AIF. However, metal-induced activation of p53 and mitochondrial depolarization appears to be independent of ERK. Our data suggest that copper induces apoptosis through depolarization of mitochondrial membrane with release of AIF, and this process is MEK/ERK independent.     

Spatiotemporal activation of caspase‐dependent and ‐independent pathways in staurosporine‐induced apoptosis of p53wt and p53mt human cervical carcinoma cells

Background information. Caspase‐dependent and ‐independent death mechanisms are involved in apoptosis in a variety of human carcinoma cells treated with antineoplastic compounds. Our laboratory has reported that p53 is a key contributor of mitochondrial apoptosis in cervical carcinoma cells after staurosporine exposure. However, higher mitochondrial membrane potential dissipation and greater DNA fragmentation were observed in p53^wt (wild‐type p53) HeLa cells compared with p53^mt (mutated p53) C‐33A cells. Here, we have studied events linked to the mitochondrial apoptotic pathway. Results. Staurosporine can induce death of HeLa cells via a cytochrome c/caspase‐9/caspase‐3 mitochondrial‐dependent apoptotic pathway and via a delayed caspase‐independent pathway. In contrast with p53^wt cells, p53^mt C‐33A cells exhibit firstly caspase‐8 activation leading to caspase‐3 activation and Bid cleavage followed by cytochrome c release. Attenuation of PARP‐1 [poly(ADP‐ribose) polymerase‐1] cleavage as well as oligonucleosomal DNA fragmentation in the presence of z‐VAD‐fmk points toward a major involvement of a caspase‐dependent pathway in staurosporine‐induced apoptosis in p53^wt HeLa cells, which is not the case in p53^mt C‐33A cells. Meanwhile, the use of 3‐aminobenzamide, a PARP‐1 inhibitor known to prevent AIF (apoptosis‐inducing factor) release, significantly decreases staurosporine‐induced death in these p53^mt carcinoma cells, suggesting a preferential implication of caspase‐independent apoptosis. On the other hand, we show that p53, whose activity is modulated by pifithrin‐α, isolated as a suppressor of p53‐mediated transactivation, or by PRIMA‐1 (p53 reactivation and induction of massive apoptosis), that reactivates mutant p53, causes cytochrome c release as well as mitochondrio—nuclear AIF translocation in staurosporine‐induced apoptosis of cervical carcinoma cells. Conclusions. The present paper highlights that staurosporine engages the intrinsic mitochondrial apoptotic pathway via caspase‐8 or caspase‐9 signalling cascades and via caspase‐independent cell death, as well as through p53 activity.     

Mitochondrial release of AIF and EndoG requires caspase activation downstream of Bax/Bak-mediated permeabilization

Mitochondrial outer-membrane permeabilization by pro-apoptotic Bcl-2 family members plays a crucial role in apoptosis induction. However, whether this directly causes the release of the different mitochondrial apoptogenic factors simultaneously is currently unknown. Here we report that in cells or with isolated mitochondria, pro-apoptotic Bcl-2 proteins cause the release of cytochrome c, Smac/Diablo and HtrA2/Omi but not endonuclease G (EndoG) and apoptosis-inducing factor (AIF). In cells treated with Bax/Bak-dependent pro-apoptotic drugs, neither the caspase inhibitor zVAD-fmk nor loss of Apaf-1 affected the efflux of cytochrome c, Smac/Diablo and HtrA2/Omi, but both prevented the release of EndoG and AIF. Our findings identify the mitochondrial response to pro-apoptotic stimuli as a selective process leading to a hierarchical ordering of the effectors involved in cell death induction. Moreover, as in Caenorhabditis elegans, EndoG and AIF act downstream of caspase activation. Thus EndoG and AIF seem to define a 'caspase-dependent' mitochondria-initiated apoptotic DNA degradation pathway that is conserved between mammals and nematodes.     

Role of HPV E6 proteins in preventing UVB-induced release of pro-apoptotic factors from the mitochondria

Apoptotic elimination of UV-damaged cells from the epidermis is an important step in preventing both the emergence and expansion of cells with carcinogenic potential. A pivotal event in apoptosis is the release of apoptogenic factors from the mitochondria, although the mechanisms by which the different proteins are released are not fully understood. Here we demonstrate that UV radiation induced the mitochondrial to nuclear translocation of apoptosis inducing factor (AIF) in normal skin. The human papillomavirus (HPV) E6 protein prevented release of AIF and other apoptotic factors such as cytochrome c and Omi from mitochondria of UV-damaged primary epidermal keratinocytes and preserved mitochondrial integrity. shRNA silencing of Bak, a target for E6-mediated proteolysis, demonstrated the requirement of Bak for UV-induced AIF release and mitochondrial fragmentation. Furthermore, screening non-melanoma skin cancer biopsies revealed an inverse correlation between HPV status and AIF nuclear translocation. Our results indicate that the E6 activity towards Bak is a key factor that promotes survival of HPV-infected cells that facilitates tumor development.     

Dominant cell death induction by extramitochondrially targeted apoptosis-inducing factor.

The complete AIF cDNA comprising the amino-terminal mitochondrial localization sequence (MLS) and the oxidoreductase domain has been fused in its carboxyl terminus to enhanced green fluorescent protein (GFP), thereby engineering an AIF-GFP fusion protein that is selectively targeted to the mitochondrial intermembrane space. Upon induction of apoptosis, the AIF-GFP protein translocates together with cytochrome c (Cyt-c) to the extramitochondrial compartment. Microinjection of recombinant AIF leads to the release of AIF-GFP and Cyt-c-GFP, indicating that ectopic AIF can favor permeabilization of the outer mitochondrial membrane. These mitochondrial effects of AIF are caspase independent, whereas the Cyt-c-microinjection induced translocation of AIF-GFP and Cyt-c-GFP is suppressed by the pan-caspase inhibitor Z-VAD.fmk. Upon prolonged culture, transfection-enforced overexpression of AIF results in spontaneous translocation of AIF-GFP from mitochondria, nuclear chromatin condensation, and cell death. These effects are caspase independent and do not rely on the oxidoreductase function of AIF. Spontaneous AIF-GFP translocation and subsequent nuclear apoptosis can be retarded by overexpression of a Bcl-2 protein selectively targeted to mitochondria, but not by a Bcl-2 protein targeted to the endoplasmic reticulum. Overexpression of a mutant AIF protein in which the MLS has been deleted (AIF Delta 1-100) results in the primary cytosolic accumulation of AIF. AIF Delta 1-100-induced cell death is suppressed by neither Z-VAD.fmk or by Bcl-2. Thus, extramitochondrially targeted AIF is a dominant cell death inducer.     

Wood smoke extract induces oxidative stress-mediated caspase-independent apoptosis in human lung endothelial cells: role of AIF and EndoG

Although a link between toxic smoke and oxidant lung vascular injury has been indicated, the cellular mechanisms of smoke-induced injury to lung endothelial cells are unknown. We investigated oxidative stress and apoptosis induced by wood smoke extract (SE) in human pulmonary artery endothelial cells (HPAECs) and delineated their relationship. We found that SE increased intracellular reactive oxygen species (ROS), depleted intracellular glutathione, and upregulated Cu/Zn superoxide dismutase and heme oxygenase-1 (2 antioxidant enzymes), but it failed to alter the expression of catalase and glutathione peroxidase. In addition, SE promoted apoptosis as indicated by the external exposure of membrane phosphatidylserine, the loss of mitochondrial membrane potential, an increase in the level of Bax (a proapoptotic protein), and enhanced DNA fragmentation. This apoptosis was associated with mitochondrial-to-nuclear translocation of apoptosis-inducing factor (AIF) and endonuclease G (EndoG) (2 apoptogenic proteins) but was independent of caspase cascade activation. Whereas N-acetylcysteine (an antioxidant) effectively reversed the SE-induced increase in ROS and depletion of glutathione, it also suppressed SE-induced nuclear translocation of either AIF or EndoG and prevented the enhanced DNA fragmentation that would have resulted from this. We conclude that 1) although SE upregulates Cu/Zn superoxide dismutase and heme oxygenase-1, it nevertheless increases intracellular oxidative stress in HPAECs, and 2) SE promotes oxidative stress-mediated caspase-independent HPAEC apoptosis that involves mitochondrial-to-nuclear translocation of AIF and EndoG. Thus modulations of the expression of antioxidant enzymes and the caspase-independent apoptotic pathway are possible target choices for potential therapeutic regimes to treat smoke-induced lung injury.     

Cadmium Induces PC12 Cells Apoptosis via an Extracellular Signal-Regulated Kinase and c-Jun N-Terminal Kinase-Mediated Mitochondrial Apoptotic Pathway

To investigate the role of mitogen-activated protein kinase (MAPK) and downstream events in cadmium (Cd)-induced neuronal apoptosis executed via the mitochondrial apoptotic pathway, this study used the PC-12 cell line as a neuronal model. The result showed that Cd significantly decreased cell viability and the Bcl-2?/?Bax ratio and increased the percentage of apoptotic cells, release of cytochrome c, caspase-3, and poly(ADP-ribose) polymerase cleavage, and nuclear translocation of apoptosis-inducing factor (AIF) and endonuclease G. In addition, exposure to Cd-induced phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. Inhibition of ERK and JNK, but not p38 MAPK, partially protected the cells from Cd-induced apoptosis. ERK and JNK inhibition also blocked alteration of the Bcl-2?/?Bax ratio and cytochrome c release and suppressed caspase-3 and poly(ADP-ribose) polymerase cleavage and AIF and endonuclease G nuclear translocation. Taken together, these data suggest that the ERK- and JNK-mediated mitochondrial apoptotic pathway played an important role in Cd-induced PC12 cells apoptosis.     

The role of mitogen-activated protein kinase in cadmium-induced primary rat cerebral cortical neurons apoptosis via a mitochondrial apoptotic pathway

Cadmium (Cd) is an extremely toxic metal capable of severely damaging several organs, including the brain. Studies have shown that Cd induces neuronal apoptosis partially by activating the mitogen-activated protein kinase (MAPK) pathways. However, the underlying mechanism of MAPK involving the mitochondrial apoptotic pathway in neurons remains unclear. In this study, primary rat cerebral cortical neurons were exposed to Cd, which significantly decreased cell viability and the B-cell lymphoma 2/Bcl-2 associate X protein (Bcl-2/Bax) ratio and increased the percentage of apoptotic cells, release of cytochrome c, cleavages of caspase-3 and poly (ADP-ribose) polymerase (PARP), and nuclear translocation of apoptosis-inducing factor (AIF). In addition, Cd induced phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPK. Inhibition of ERK and JNK, but not p38 MAPK, partially protected the cells from Cd-induced apoptosis. ERK and JNK inhibition also blocked alteration of the Bcl-2/Bax ratio, release of cytochrome c, cleavages of caspase-3 and PARP, and nuclear translocation of AIF. Taken together, these data suggest that the ERK- and JNK-mediated mitochondrial apoptotic pathways play important roles in Cd-induced neuronal apoptosis.     

A role of the mitochondrial apoptosis-inducing factor in granulysin-induced apoptosis

Granulysin is a cytolytic molecule released by CTL via granule-mediated exocytosis. In a previous study we showed that granulysin induced apoptosis using both caspase- and ceramide-dependent and -independent pathways. In the present study we further characterize the biochemical mechanism for granulysin-induced apoptosis of tumor cells. Granulysin-induced death is significantly inhibited by Bcl-2 overexpression and is associated with a rapid (1-5 h) loss of mitochondrial membrane potential, which is not mediated by ceramide generation and is not inhibited by the general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. Ceramide generation induced by granulysin is a slow event, only observable at longer incubation times (12 h). Apoptosis induced by exogenous natural (C(18)) ceramide is truly associated with mitochondrial membrane potential loss, but contrary to granulysin, this event is inhibited by benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. Ceramide-induced apoptosis is also completely prevented by Bcl-2 overexpression. The nuclear morphology of cells dying after granulysin treatment in the presence of caspase inhibitors suggested the involvement of mitochondrial apoptosis-inducing factor (AIF) in granulysin-induced cell death. We demonstrate using confocal microscopy that AIF is translocated from mitochondria to the nucleus during granulysin-induced apoptosis. The majority of Bcl-2 transfectants are protected from granulysin-induced cell death, mitochondrial membrane potential loss, and AIF translocation, while a small percentage are not protected. In this small percentage the typical nuclear apoptotic morphology is delayed, being of the AIF type at 5 h time, while at longer times (12 h) the normal apoptotic morphology is predominant. These and previous results support a key role for the mitochondrial pathway of apoptosis, and especially for AIF, during granulysin-induced tumoral cell death.     

Flavopiridol induces mitochondrial-mediated apoptosis in murine glioma GL261 cells via release of cytochrome c and apoptosis inducing factor

Glioblastoma (GBM) remains one of the most challenging solid cancers to treat due to its highly proliferative, angiogenic and invasive nature. The small molecule CDK inhibitor, flavopiridol, has demonstrated antitumor activity in human xenograft models and is currently in clinical trials showing efficacy in patients with advanced disease. We have developed an experimental animal model using the murine glioma GL261 cells as a novel in vivo system to screen potential therapeutic agents for GBM. Results of in vitro testing demonstrate that flavopiridol has several relevant clinical characteristics such as its ability to: 1. inhibit cell growth; 2. inhibit cell migration; 3. decrease expression of cyclin D1, CDK4 and p21; 4. induce apoptosis in cells with high levels of p27 expression; and 5. decrease the expression of the anti-apoptotic protein Bcl-2. The mechanism by which flavopiridol induces apoptosis is mitochondrial-mediated. We demonstrate by electron microscopy and immunohistochemistry that drug treatment induces mitochondrial damage that was accompanied by the release of cytochrome c into the cytosol together with the translocation of apoptosis inducing factor (AIF) into the nucleus. This finding in murine glioma cells differs from the mechanism of flavopiridolinduced cell death reported by us for human glioma cells (Alonso et al., Mol Cancer Ther 2003; 2:139) where drug treatment induced a caspase- and cytochrome c-independent pathway in the absence of detectable damage to mitochondria. In apoptotic human glioma cells only translocation of AIF into the nucleus occurred. Thus, the same drug kills different types of glioma cells by different mitochondrial-dependent pathways.     

p53-dependent caspase-2 activation in mitochondrial release of apoptosis-inducing factor and its role in renal tubular epithelial cell injury

We demonstrate the role of p53-mediated caspase-2 activation in the mitochondrial release of apoptosis-inducing factor (AIF) in cisplatin-treated renal tubular epithelial cells. Gene silencing of AIF with its small interfering RNA (siRNA) suppressed cisplatin-induced AIF expression and provided a marked protection against cell death. Subcellular fractionation and immunofluorescence studies revealed cisplatin-induced translocation of AIF from the mitochondria to the nuclei. Pancaspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone or p53 inhibitor pifithrin-alpha markedly prevented mitochondrial release of AIF, suggesting that caspases and p53 are involved in this release. Caspase-2 and -3 that were predominantly activated in response to cisplatin provided a unique model to study the role of these caspases in AIF release. Cisplatin-treated caspase-3 (+/+) and caspase-3 (-/-) cells exhibited similar AIF translocation to the nuclei, suggesting that caspase-3 does not affect AIF translocation, and thus, caspase-2 may be involved in the translocation. Caspase-2 inhibitor benzyloxycarbonyl-Val-Asp-Val-Ala-Asp-fluoromethylketone or down-regulation of caspase-2 by its siRNA significantly prevented translocation of AIF. Caspase-2 activation was a critical response from p53, which was markedly induced and phosphorylated in cisplatin-treated cells. Overexpression of p53 not only resulted in caspase-2 activation but also mitochondrial release of AIF. The p53 inhibitor pifithrin-alpha or p53 siRNA prevented both cisplatin-induced caspase-2 activation and mitochondrial release of AIF. Caspase-2 activation was dependent on the p53-responsive gene, PIDD, a death domain-containing protein that was induced by cisplatin in a p53-dependent manner. These results suggest that caspase-2 activation mediated by p53 is an important pathway involved in the mitochondrial release of AIF in response to cisplatin injury.     

Arsenic Trioxide and Paclitaxel Induce Apoptosis by Different Mechanism

Arsenic trioxide (ATO) and paclitaxel (TAXOL) are effective in the treatment of various types of cancers. Both drugs induce G2/M arrest. We have previously shown that ATO is a potent inducer of apoptosis in myeloma cells expressing mutant p53 engaging both the intrinsic and extrinsic apoptotic pathways. Here we compared the effect of ATO and TAXOL on myeloma cells expressing mutant p53 and varying levels of Bcl-2. ATO rapidly induced Apo2/TRAIL, activation of caspase 8, cleavage of BID, depolarization of mitochondrial membrane (MM) and release of AIF from mitochondria in a Bcl-2 independent fashion. Apoptosis was associated with early formation of ring-like perinuclear condensed chromatin co-localized with AIF. In contrast, paclitaxel-induced apoptosis and MM depolarization, cytochrome C release and activation of caspase 9 was blocked by Bcl-2. Apoptosis was associated with a random chromatin condensation and nuclear fragmentation with no early involvement of AIF.