Expression and function of prostate-apoptosis-response-gene-4 in lymphatic cells

Inhibition of apoptosis contributes to the pathogenesis of lymphatic malignancies. In particular, the elevated expression of Bcl-2 is considered to be a marker of poor prognosis, since increased levels of Bcl-2 confer longevity as well as chemoresistance. After demonstrating an inverse expressional pattern of Bcl-2 and prostate-apoptosis-response-4 (Par-4) in ex vivo cells of patients suffering from acute lymphatic leukemia (ALL) as well as a deregulated expression of Par-4 in acute and chronic lymphatic neoplasias, the molecular mechanisms underlying these results were investigated. Thus, it was demonstrated that in neoplastic lymphatic cells Par-4 exerts a proapoptotic role augmenting chemosensitivity by down-regulating Bcl-2, promoting disruption of mitochondrial membrane potential and enforcing caspase-activation. Moreover, Par-4 enables cells to circumvent inhibition of the central executioner caspase-3 by alternative activation of caspases following a decrease in expression levels of inhibitors of apoptosis proteins (IAP).     

Protein expression and intracellular localization of prostate apoptosis response-4 (Par-4) are associated with apoptosis induction in nasopharyngeal carcinoma cell lines

Prostate apoptosis response-4 (Par-4) is a proapoptotic gene that selectively induces cell death in most cancer cells. In addition to the increased percentage of apoptotic cells, caspase-3 activity, and poly (ADP-ribose) polymerase (PARP) cleavage, we demonstrate that elevated expression of Par-4 and nuclear entry resulted in apoptosis of nasopharyngeal carcinoma (NPC) cell lines either in serum deprivation or by ectopic overexpression of Par-4. Moreover, disassociation from the Par-4/Akt complex was correlated with the induced proapoptotic ability of Par-4. Therefore, our data suggest that the cytoplasmic localization and expression level of endogenous Par-4 in NPC cells are not sufficient to augment apoptosis.     

In the erythroleukemic cell line HEL Prostate-apoptosis-response-gene-4 (par-4) fails to down-regulate Bcl-2 and to promote apoptosis

In a variety of malignant cells Prostate-apoptosis-response-gene-4 (Par-4) exhibits a pro-apoptotic influence sensitizing these cells to apoptosis-inducing agents by downregulating expression of Bcl-2. Considering the crucial role of Bcl-2 in the development of chemoresistance of acute myeloid leukemia (AML) cells, we here assessed the potential of Par-4 to down-regulate Bcl-2 and to induce apoptosis in the erythroleukemic cell line HEL. Testing a potential pro-apoptotic role of Par-4 upon incubation with various conventional chemotherapeutic drugs, novel agents such as the signal transduction inhibitor STI 571 and the histone deacetylase (HDAC)- inhibitor trichostatin A (TSA), as well as with the experimental substances Fas and TRAIL, we provide evidence that in the erythroleukemic cell line HEL expression of Par-4 is not sufficient to sensitize to any of these pro-apoptotic stimuli. We further demonstrate that--in contrast to previous reports in non-AML cells--Par-4 expression in HEL cells leads to an upregulation of Bcl-2. Moreover, Par-4-positive HEL cells exhibit a decreased level of the proapoptotic protein Bax as compared to Par-4- negative cells. In addition, Par-4 increases the expression of Daxx--whose downregulation is associated with augmented chemosensitivity--as well as expression of the procaspases-8, -9 and -10, whereas the levels of the procaspases-3 and -7 remain unaltered. In conclusion we here demonstrate that in the erythroleukemic cell line HEL--in contrast to other cell types Par-4 fails to promote apoptosis and outline the underlying molecular mechanisms.     

Novel mechanisms of apoptosis induced by histone deacetylase inhibitors

Histone deacetylase inhibitors (HDACIs) are a new class of chemotherapeutic drugs able to induce tumor cell apoptosis and/or cell cycle arrest; however, the molecular mechanisms underpinning their anticancer effects are poorly understood. Herein, we assessed the apoptotic pathways activated by three HDACIs, suberoylanilide hydroxamic acid, oxamflatin, and depsipeptide. We determined that all three drugs induced the accumulation of cells with a 4n DNA content and apoptosis mediated by the intrinsic apoptotic pathway. HDACI-induced mitochondrial membrane damage and apoptosis were inhibited by overexpression of Bcl-2, but not by the polycaspase inhibitor N-tert-butoxy-carbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk). Moreover, induction of a G(1)-S checkpoint through overexpression of p16(INK4A) or suppression of de novo protein synthesis also inhibited HDACI-induced cell death. Proteolytic cleavage of caspase-2, which is poorly inhibited by zVAD-fmk, was concomitant with HDACI-induced death; however, full processing of caspase-2 to the p19 active form was blocked by Bcl-2. Whereas all three drugs induce the activation of the proapoptotic Bcl-2 protein Bid upstream of mitochondrial membrane disruption, Bid cleavage in response to depsipeptide was significantly attenuated by zVAD-fmk. Suberoylanilide hydroxamic acid and oxamflatin could kill both P-glycoprotein (P-gp)(+) MDR cells and their P-gp(-) counterparts, whereas depsipeptide was shown to be a substrate for P-gp and was less effective in killing P-gp(+) cells. These data provide insight into the functional profile of three HDACIs and are important for the development of more rational approaches to chemotherapy, where information regarding the genetic profile of the tumor is matched with the functional profile of a given chemotherapeutic drug to promote favorable clinical responses.     

Inhibition of TRAIL-induced apoptosis by Bcl-2 overexpression

Primary or acquired resistance to current treatment protocols remains a major concern in clinical oncology and may be caused by defects in apoptosis programs. Since recent data suggest that TRAIL can bypass apoptosis resistance caused by Bcl-2, we further investigated the role of Bcl-2 in TRAIL-induced apoptosis. Here we report that overexpression of Bcl-2 conferred protection against TRAIL in neuroblastoma, glioblastoma or breast carcinoma cell lines. Bcl-2 overexpression reduced TRAIL-induced cleavage of caspase-8 and Bid indicating that caspase-8 was activated upstream and also downstream of mitochondria in a feedback amplification loop. Importantly, Bcl-2 blocked cleavage of caspases-9, -7 and -3 into active subunits and cleavage of the caspase substrates DFF45 or PARP. Also, Bcl-2 blocked cleavage of XIAP and overexpression of XIAP conferred resistance against TRAIL indicating that apoptosis was also amplified through a feedforward loop between caspases and XIAP. In contrast, in SKW lymphoblastoid cells, TRAIL-induced activation of caspase-8 directly translated into full activation of caspases, cleavage of XIAP, DFF45 or PARP and apoptosis independent of Bcl-2 overexpression, although Bcl-2 similarly inhibited loss of mitochondrial membrane potential and the release of cytochrome c, AIF and Smac from mitochondria in all cell types. By demonstrating a cell type dependent regulation of the TRAIL signaling pathway at different level, e.g. by Bcl-2 and by XIAP, these findings may have important clinical implication. Thus, strategies targeting the molecular basis of resistance towards TRAIL may be necessary in some tumors for cancer therapy with TRAIL.     

E2F-1 gene therapy induces apoptosis and increases chemosensitivity in human pancreatic carcinoma cells.

Pancreatic cancer is often resistant to conventional chemotherapy. In this study, we examined the role of adenovirus-mediated overexpression of E2F-1 in inducing apoptosis and increasing the sensitivity of pancreatic cancer cells to chemotherapeutic agents. MIA PaCa-2 pancreatic head exocrine adenocarcinoma cells (mutant p53) were treated by mock infection or adenoviruses expressing beta-galactosidase or E2F-1 (Ad-E2F-1) alone or in combination with sublethal concentrations of each chemotherapeutic drug. Cell growth and viability were assessed at selected time points. Apoptosis was evaluated by flow cytometry, characteristic changes in cell morphology and poly (ADP-ribose) polymerase (PARP) cleavage. Western blot analysis was used to examine the expression of E2F-1 and Bcl-2 family member proteins and PARP cleavage. Western blot analysis revealed marked overexpression of E2F-1 at a multiplicity of infection (MOI) of 20 and 70. By 3 days after infection, Ad-E2F-1 treatment at an MOI of 70 resulted in approximately a 20-fold reduction in cell growth and 60% reduction in cell viability as compared to mock-infected cells. Cell cycle analysis, PARP cleavage and changes in cell morphology supported apoptosis as the mechanism of cell death in response to E2F-1. In order to test the efficacy of treatment with a combination of gene therapy and chemotherapy, we utilized concentrations of Ad-E2F-1 which reduced viability to 50% in combination with each chemotherapeutic agent. Cotreatment of the cells with E2F-1 virus and roscovitine (ROS) or etoposide resulted in an additive effect on cell growth inhibition and induction of apoptosis. Interestingly, 5-fluorouracil did not cooperate with Ad-E2F-1 in the mediation of tumor death or inhibition of cell growth. Immunoblotting for Bcl-2 family members revealed no significant changes in the expression levels of Bcl-2, Bcl X(L), Bax or Bak following gene or 'chemogene' therapy with E2F-1. However, a Bax cleavage product was noted which was substantially increased by cotreatment with ROS or etoposide. E2F-1 overexpression initiates apoptosis and suppresses growth in pancreatic MIA PaCa-2 cells in vitro. E2F-1-mediated apoptosis was not associated with significant changes in the expression of Bcl-2 family member proteins in these pancreatic cancer cells. ROS and etoposide, when combined with E2F-1 overexpression, induce apoptosis in an additive manner. This chemogene combination may provide a potentially useful therapeutic strategy for advanced pancreatic cancer.     

Apoptosis induction in prostate cancer cells and xenografts by combined treatment with Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand and CPT-11

Because apoptosis is deregulated in most cancers, apoptosis-modulating approaches offer an attractive opportunity for clinical therapy of many tumors, including that of the prostate. LNCaP-derived C4-2 human prostate cancer cells are quite resistant to treatment with Apo2 ligand (Apo2L) or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), when using a nontagged, Zn-bound recombinant trimeric version that is devoid of any exogeneous sequences and therefore least likely to be immunogenic in human patients and that has been optimized for maximum efficacy and minimum toxicity. When combined with the topoisomerase I inhibitor CPT-11 (irinotecan), Apo2L/TRAIL exhibits enhanced apoptotic activity in C4-2 cells cultured in vitro as well as xenografted as tumors in vivo. Apoptosis both in vitro and in vivo was characterized by two major molecular events. First, apoptosis induction was accompanied by changes in expression levels of the Bcl-2 family genes and their products. However, whereas combination treatment applied to in vitro cell culture was characterized by a significant up-regulation and activation of Bax and down-regulation of Bcl-xL, the treatment applied to tumors induced Bak and Bcl-xS, whereas Bcl-omega and Bcl-xL were down-regulated. Because there are multiple members of the Bcl-2 family (24 members to date), these data indicate that, under different biological conditions, different proteins may be responsible for activating apoptosis and provide evidence for a differential regulation of the multidomain Bcl-2 protein-encoding genes, bax and bak. Increased Bax expression led to its activation, translocation to the mitochondria, and release of cytochrome c. In addition, this combination treatment induced apoptosis through potent activation of caspase-8 and the proapoptotic protein Bid, resulting in activation of effector caspase-3 and cleavage of its cellular target protein, poly(ADP-ribose) polymerase (PARP), events blocked by the pan-caspase inhibitor N-tert-butoxy-carbonyl-Val-Ala-Asp-fluoro methylketone (zVAD-fmk). Activation of multiple caspases and PARP cleavage were also observed in the C4-2 tumors treated with doses resulting in effective tumor control at 42 days after Apo2L/TRAIL plus CPT-11 treatment. Down-regulation of Bax by small interference (RNA) (siRNA) in C4-2 cells significantly prevented PARP cleavage and apoptosis. Strikingly, similar experiments in cells stably expressing a dominant-negative death receptor DR5 led to complete ablation of PARP cleavage and apoptosis, indicating the essential role of both mitochondrial and receptor-mediated apoptotic pathways. Our data indicate that the combined treatment of Apo2L/TRAIL and CPT-11 achieves tumor control in prostate cancer tumors through regulation of Bcl-2 family proteins and potent activation of caspases.     

RRR-alpha-tocopheryl succinate-induced apoptosis of human breast cancer cells involves Bax translocation to mitochondria

Previous studies have identified RRR-alpha-tocopheryl succinate (vitamin E succinate, VES) as a potential chemotherapeutic agent. VES induces human breast cancer cells to undergo apoptosis in a concentration- and time-dependent manner by restoring transforming growth factor beta (TGF-beta) and Fas (CD95) apoptotic signaling pathways, that contribute to the activation of c-Jun NH(2)-terminal kinase (JNK)-mediated apoptosis. The objective of these studies was to clarify biochemical events involved in VES-induced apoptosis. Data show that VES-induced apoptosis involves: (a) translocation of Bax from the cytosol to the mitochondria and cytochrome c release from the mitochondria to the cytosol as determined by Western immunoblot analyses of mitochondrial- and cytosolic-enriched cellular fractions; (b) increased permeabilization of mitochondrial membranes as determined by confocal and fluorescence-activated cell sorting analyses of loss of a mitochondrial selective fluorescent dye; (c) processing of caspase-9 and -3 but not caspase-8 to active forms and cleavage of poly(ADP-ribose) polymerase (PARP) as determined by Western immunoblot analyses using antibodies capable of detecting both proenzyme and processed enzyme forms or the intact or cleaved forms of PARP. Transient transfection of cells with antisense oligonucleotides to Bax or transient overexpression of Bcl-2 prevented VES-induced mitochondrial permeability transition and apoptosis. The use of cell-permeable caspase inhibitors indicated that caspase-9 and -3 but not caspase-8 are involved in VES-induced apoptosis. JNK inhibitor II blocked VES-induced Bax conformational change, indicating a role for JNK in Bax translocation to the mitochondria. Taken together, these data suggest that the activation of JNK, translocation of Bax to the mitochondria, increased mitochondrial membrane permeability with release of cytochrome c, and activation of caspase-9 and -3 are critical events in VES-induced apoptosis of human MDA-MB-435 breast cancer cells.     

DNA damaging drugs-induced down-regulation of Bcl-2 is essential for induction of apoptosis in high-risk HPV-positive HEp-2 and KB cells

DNA damaging chemotherapeutic agents like carboplatin (Carb) and 5-fluorouracil (5-FU), whose effects are mediated through diverse intracellular targets, induce apoptosis in various cancer cells including human papillomavirus (HPV) positive HEp-2 and KB cells. The present work reports the involvement of Bcl-2 in response to the exposure of HEp-2 and KB cells to Carb or 5-FU. We demonstrate that both these drugs are potent inducers of apoptosis. Apoptosis was preceded by decrease in Bcl-2 protein level accompanied by caspase-9 activation and poly(ADP-ribose) polymerase (PARP) cleavage without altering Bax expression. Further analysis revealed down-regulation of Bcl-2 mRNA as well as protein in drugs treated cells. Ectopic expression of Bcl-2 protected cells against drugs mediated DNA damage-induced apoptosis. Overall, data indicates that genotoxic stress leads to down-regulation of Bcl-2 in HEp-2 and KB cells, which plays a decisive role in the outcome of stress in these cells.     

Tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis is inhibited by Bcl-2 but restored by the small molecule Bcl-2 inhibitor, HA 14-1, in human colon cancer cells.

PURPOSE: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent that induces apoptosis in multiple tumor cell types while sparing most normal cells. We determined the effect of ectopic Bcl-2 expression on TRAIL-induced apoptosis and whether the small molecule Bcl-2 inhibitor, HA14-1, could increase TRAIL sensitivity. EXPERIMENTAL DESIGN: SW480 human colon cancer cells were stably transfected with the PC3-Bcl-2 plasmid or vector alone. Cells were incubated with recombinant human TRAIL +/- HA14-1 or caspase-9 inhibitor (Z-LEHD-FMK). Apoptosis was analyzed by Annexin V-fluorescein isothiocyanate labeling and DNA fragmentation factor 45 (DFF45) cleavage. Clonigenic survival was also studied. Caspase activation was determined by immunoblotting or colorimetric assay. The cytosolic expression of Bid, Bax, and XIAP and release of cytochrome c and Smac/DIABLO were determined by immunoblotting. RESULTS: Bcl-2 overexpression partially protected SW480 cells from a dose-dependent induction of apoptosis by TRAIL, as did a caspase-9 inhibitor, and increased their clonogenic survival. Bcl-2 overexpression attenuated TRAIL-induced cleavage of caspase-8, indicating its activation upstream and downstream of mitochondria, as well as cleavage of Bid and caspase-3. Bcl-2 inhibited TRAIL-induced Bax translocation, cytosolic release of cytochrome c and Smac/DIABLO, and the downstream cleavage of XIAP and DFF45. Coadministration of HA14-1 and TRAIL increased apoptosis in SW480/Bcl-2 cells by restoring Bax redistribution and cytochrome c release. CONCLUSIONS: Bcl-2 confers apoptosis resistance to TRAIL by inhibiting a mitochondrial amplification step and by inactivating downstream XIAP in SW480 cells. HA14-1 reversed Bcl-2-mediated TRAIL resistance, suggesting a novel strategy for increasing TRAIL sensitivity in Bcl-2-overexpressing colon cancers.     

Millimeter wave radiation induces apoptosis via affecting the ratio of Bax/Bcl-2 in SW1353 human chondrosarcoma cells

The efficacy and safety of millimeter wave radiation has been proven for various types of malignant tumors. However, the mechanisms underlying effects of millimeter wave radiation on apoptosis are still unclear. The present study was undertaken to examine the effects of millimeter wave radiation on cell apoptosis and mitochondrial membrane potential, and to determine the molecular mechanism of millimeter wave radiation-induced apoptosis by investigating the expression of Bcl-2 family proteins (Bcl-2, Bax), caspase-9 and caspase-3 in SW1353 cells. We found that millimeter wave radiation suppressed the viability of SW1353 cells, demonstrating that millimeter wave radiation induced cell apoptosis and reduced cell viability in a time-dependent manner. Furthermore, we observed that treatment of cells with millimeter wave radiation significantly induced loss of mitochondrial membrane potential, upregulated proapoptotic Bax, caspase-9 and caspase-3, but did not significantly change levels of antiapoptotic Bcl-2. These data suggested that millimeter wave radiation may induce apoptosis via affecting the ratio of Bax/Bcl-2 in SW1353 cells.     

Tumor necrosis factor-related apoptosis-inducing ligand retains its apoptosis-inducing capacity on Bcl-2- or Bcl-xL-overexpressing chemotherapy-resistant tumor cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor family and has recently been shown to exert tumoricidal activity in vivo in the absence of any observable toxicity. The signaling pathways triggered by TRAIL stimulation and the mechanisms involved in resistance against TRAIL-mediated apoptosis are still poorly defined. We show here that TRAIL-induced apoptosis involves late dissipation of mitochondrial membrane potential (delta psi(m)) and cytochrome c release. These events follow activation of caspase-8 and caspase-3 and induction of DNA fragmentation. In addition, caspase-8-deficient cells are resistant against TRAIL-induced apoptosis, and inhibition of caspase-8 but not caspase-9 prevents mitochondrial permeability transition and apoptosis. In contrast, various Bcl-2- or Bcl-xL-overexpressing tumor cell lines are sensitive to TRAIL-induced apoptosis; however, they show a delay in TRAIL-induced mitochondrial permeability transition compared with control transfectants. This indicates that TRAIL-induced apoptosis depends on caspase-8 activation rather than on the disruption of mitochondrial integrity. Because most chemotherapeutic drugs used in the treatment of malignancies lead to apoptosis primarily by engagement of the mitochondrial proapoptotic machinery, we tested whether drug-resistant tumor cells retain sensitivity for TRAIL-induced apoptosis. Tumor cells overexpressing Bcl-2 or Bcl-xL become resistant to apoptosis induced by the chemotherapeutic drug etoposide. However, these cells are not protected or are only marginally protected against TRAIL-induced apoptosis. Thus, TRAIL may still kill tumors that have acquired resistance to chemotherapeutic drugs by overexpression of Bcl-2 or Bcl-xL. These data will influence future treatment strategies involving TRAIL.     

Bcl-2-mediated potentiation of neocarzinostatin-induced apoptosis: requirement for caspase-3, sulfhydryl groups, and cleavable Bcl-2

Overexpression of antiapoptotic Bcl-2 family members is thought to contribute to chemotherapeutic resistance of neural crest tumors. Paradoxical potentiation by Bcl-2 of apoptosis induced by the antineoplastic prodrug, neocarzinostatin (NCS), has been observed in PC12 pheochromocytoma cells. Prior studies have indicated that the cleavage of Bcl-2 to its proapoptotic counterpart mediated by caspase-3 is responsible for this potentiation of apoptosis. This has led to the hypothesis that induction of caspase-3 expression in bcl-2-transfected, caspase-3-deficient MCF-7 cells, will result in Bcl-2 cleavage and Bcl-2-dependent potentiation of NCS-induced apoptosis. These studies have further led to the hypothesis that both cleavable Bcl-2 and sulfhydryl groups are required for the activity of caspase-3 in this regard. As hypothesized, co-transfection of bcl-2-transfected MCF-7 cells with a caspase-3 expression construct results in cleavage of Bcl-2 and potentiation of dose-dependent, NCS-mediated cell death. Furthermore, PC12 cells transfected with an expression construct for cleavage-resistant Bcl-2 demonstrated attenuated potentiation of apoptosis relative to their counterparts transfected with wild-type bcl-2. Finally, irreversible oxidative titration of sulfhydryl groups resulted in concentration-dependent attenuation of apoptosis in PC12 cells, along with prevention of caspase-3 activation and Bcl-2 cleavage. These results definitively demonstrate the requirement for caspase-3, cleavable Bcl-2, and available sulfhydryl groups (separate from those required for NCS activation) in potentiation of NCS-induced apoptosis by Bcl-2.     

Intracellular mediators of erucylphosphocholine-induced apoptosis

Induction of apoptosis contributes to the cytotoxic action of the intravenously applicable alkylphosphocholine erucylphosphocholine (ErPC). To define molecular requirements for ErPC-induced apoptosis, activation of caspases-8, -9 and -3 and cleavage of the caspase-3 substrates PARP and ICAD were tested in normal Jurkat T cells, Jurkat cells resistant to death receptor (CD95 or TNFalpha-related apoptosis inducing ligand (TRAIL)-induced apoptosis, Jurkat cells lacking caspase-8 or Fas-associated death domain (FADD) Jurkat cells expressing a dominant-negative caspase-9 or overexpressing Bcl-2 as well as BJAB B-lymphoma cells expressing a dominant-negative FADD (FADD-DN). ErPC induced a time- and dose-dependent apoptotic cell death in Jurkat and BJAB cells, which was characterized by breakdown of the phosphatidylserine asymmetry, depolarization of the mitochondrial membrane potential, release of cytochrome c, activation of caspases-9, -8 and -3, cleavage of PARP and ICAD, as well as chromatin condensation. ErPC-induced apoptosis was independent from CD95-receptor signaling and FADD since CD95- and TRAIL-resistant, caspase-8- and FADD-negative Jurkat cells, as well as BJAB cells expressing FADD-DN were sensitive to ErPC-induced apoptosis. In contrast, inhibition of caspase-9 and overexpression of Bcl-2 significantly reduced ErPC-induced caspase activation and apoptosis. Thus, ErPC triggers apoptosis via a Bcl-2-dependent mitochondrial but death receptor-independent pathway.     

Bax overexpression enhances cytochrome c release from mitochondria and sensitizes KATOIII gastric cancer cells to chemotherapeutic agent-induced apoptosis

To evaluate whether overexpression of Bax, an apoptosis-promoting gene, sensitizes KATOIII gastric cancer cells to apoptosis induced by chemotherapeutic agents, three stable cell lines of KATOIII transfected with Bax (KATOIII-Bax), Bcl-2 (KATOIII-Bcl-2), or control pCI-neo expression vector (KATOIII-pCI-neo) were established. The cells were treated with paclitaxel, 5-fluorouracil, or doxorubicin, and the apoptotic response was measured. Our results showed that the sensitivity of the KATOIII-Bax cells to chemotherapeutic agents was enhanced compared with that of the KATOIII-pCI-neo cells, and the KATOIII-Bcl-2 cells were more resistant to these agents. Western blotting revealed that cytochrome c level in the cytosol fraction of the KATOIII-Bax cells was higher than that of the KATOIII-pCI-neo cells. Significant increase of cytochrome c level in the cytosol fraction of the KATOIII-Bax cells was detected 24 h after exposure to chemotherapeutic agents, when apoptotic cells were less than 10%. The cytochrome c level in the cytosol fraction of the KATOIII-Bax cells was higher than that of the KATOIII-pCI-neo cells at all time points examined after exposure to chemotherapeutic agents. Marked activation of caspase-3 in the KATOIII-Bax cells was observed 48 h and 72 h after exposure to chemotherapeutic agents compared with that in the KATOIII-pCI-neo cells. Consistently, zVAD-fmk, a pancaspase inhibitor, repressed the paclitaxel-induced apoptosis. In addition, Bcl-2 overexpression strongly blocked KATOIII cell apoptosis by inhibiting the cytochrome c release from mitochondria and caspase-3 activation. These findings suggest that cytochrome c release is a major mechanism of apoptotic response and Bax overexpression sensitizes KATOIII cells to chemotherapeutic agent-induced apoptosis through enhancing the release of cytochrome c from mitochondria.     

Bcl-XL protects pancreatic adenocarcinoma cells against CD95- and TRAIL-receptor-mediated apoptosis

In this study we sought to clarify the role of the proapoptotic potential of mitochondria in the death pathway emanating from the TRAIL (APO-2L) and CD95 receptors in pancreatic carcinoma cells. We focused on the role of the Bcl-2 family member Bcl-XL, using three pancreatic carcinoma cell lines as a model system, two of which have high (Panc-1, PancTuI) and one has low (Colo357) Bcl-XL expression. In these cell lines, the expression of Bcl-XL correlated with sensitivity to apoptosis induced by TRAIL or anti-CD95. Flow cytometric analysis revealed cell surface expression of TRAIL-R1 and TRAIL-R2 on PancTuI and Colo357, and TRAIL-R2 on Panc-1 cells. In Colo357 cells retrovirally transduced with Bcl-XL, caspase-8 activation in response to treatment with TRAIL or anti-CD95 antibody was not different from parental cells and EGFP-transfected controls, however, apoptosis was completely suppressed as measured by the mitochondrial transmembrane potential deltapsim, caspase-3 activity (PARP cleavage) and DNA-fragmentation. Inhibition of Bcl-XL function by overexpression of Bax or administration of antisense oligonucleotides against Bcl-XL mRNA resulted in sensitization of Panc-1 cells to TRAIL and PancTuI cells to anti-CD95 antibody-induced cell death. The results show that Bcl-XL can protect pancreatic cancer cells from CD95- and TRAIL-mediated apoptosis. Thus, in these epithelial tumour cells the mitochondrially mediated 'type II' pathway of apoptosis induction is not only operative regarding the CD95 system but also regarding the TRAIL system.     

Bax mediates the apoptosis-sensitizing effect of maspin

Maspin, a serine protease inhibitor (serpin), can suppress tumor growth and metastasis in vivo and tumor cell motility and invasion in vitro. This may occur through maspin-mediated inhibition of pericellular proteolysis. In a recent report, we provided evidence that maspin may also suppress tumor progression by enhancing cellular sensitivity to apoptotic stimuli. To our knowledge, maspin is the only proapoptotic serpin among all of the serpins implicated thus far in apoptosis regulation. The goal of the present study is to identify the specific target molecule(s), the modification of which by maspin renders tumor cells sensitive to chemotherapeutic agents. Our cellular, molecular, and biochemical studies demonstrate an essential role of Bax in the proapoptotic effect of maspin. First, Bax was up-regulated in maspin-transfected prostate and breast tumor cells, whereas the levels of other Bcl-2 family members including Bcl-2, Bcl-xl, and Bak remained unchanged. Second, on apoptosis induction, a greater amount of Bax was translocated from cytosol to mitochondria in maspin-transfected cells. After treatment with a Bax-silencing small interfering RNA, maspin-transfected cells became significantly more resistant to drug-induced apoptosis. Consistently, the release of cytochrome c and Smac/DIABLO from mitochondria was more responsive to apoptosis stimuli in maspin-transfected cells than in the mock-transfected cells. Third, the apoptosis induction of maspin-transfected cells was associated with increased activation of both caspase-8 and caspase-9. However, a caspase-9-specific inhibitor blocked the sensitization effect of maspin in a dose-dependent and time-dependent manner, demonstrating a rate-limiting role for caspase-9. In line with the central role of the Bax-mediated mitochondrial apoptotic pathway, maspin sensitized the apoptotic response of breast and prostate carcinoma cells to various drugs, ranging from death ligands to endoplasmic reticulum stress. The link between maspin and Bax up-regulation explains the loss of maspin-expressing tumor cells in invasive breast and prostate carcinomas. Our data reveal a novel mechanism for tumor suppressive maspin and suggest that maspin may be used as a modifier for apoptosis-based cancer therapy.     

Doxorubicin pretreatment sensitizes prostate cancer cell lines to TRAIL induced apoptosis which correlates with the loss of c-FLIP expression

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL/Apo2L) can induce receptor-mediated apoptosis in prostate cancer cell lines that have been co-treated with the chemotherapeutic agent doxorubicin (Voelkel-Johnson C, et al. Cancer Gene Therapy 2002; 9:164-172). In this study, we report that pretreatment with doxorubicin is sufficient to sensitize cells to TRAIL. To identify possible targets of doxorubicin, we analyzed levels of several Bcl-2 family members, TRAIL receptors and the anti-apoptotic protein c-FLIP. Doxorubicin did not affect steady state levels of Bax, Bcl-2 and Bcl-X(L) in the majority of the prostate cancer cell lines. TRAIL receptor mRNAs (DR4, DR5, and DcR2) were induced by doxorubicin but these changes were not reflected at the protein level. In contrast, in response to doxorubicin, levels of c-FLIP, particularly FLIP(S), decreased in all cell lines tested. The decrease in c-FLIP(S) correlated with onset and magnitude of caspase-8 and PARP cleavage in PC3 cells. In two TRAIL resistant cell lines, DU145 and LNCaP, treatment with TRAIL alone resulted in processing of c-FLIP(L) and initiated abortive caspase-8 proteolysis. TRAIL treatment did not affect levels of c-FLIP(S) in Du145 and LNCaP cells and did not result in PARP cleavage. Therefore, our results suggest that doxorubicin- mediated down regulation of c-FLIP(S) predisposes cells to TRAIL-induced apoptosis.     

Bax plays a pivotal role in thapsigargin-induced apoptosis of human colon cancer HCT116 cells by controlling Smac/Diablo and Omi/HtrA2 release from mitochondria

Bax is a crucial mediator of the mitochondrial pathway for apoptosis, and loss of this proapoptotic Bcl-2 family protein contributes to drug resistance in human cancers. We report here that the endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin (THG) induces apoptosis of human colon cancer HCT116 cells through a Bax-dependent signaling pathway controlling the cytosolic release of mitochondrial apoptogenic molecules. Treating HCT116 cells with THG results in caspase-8 activation; Bid cleavage; Bax conformational change and mitochondrial translocation; the release of cytochrome c, Smac/Diablo, and Omi/HtrA2 into the cytosol; caspase-3 activation; and apoptosis. In contrast, knockout of Bax completely abrogates the full processing/activation of caspase-3 but has no effect on the processing of caspase-8 and the initial cleavage of caspase-3 to p24 fragment after THG treatment. The caspase-8-specific inhibitor z-IETD-fmk, as well as pan-caspase inhibitor z-VAD-fmk, but not the calpain inhibitor E-64d, prevents Bid cleavage, Bax conformational change, and subsequent caspase-3 processing and apoptosis. Caspase-8 processing is dependent on de novo protein synthesis; DR5 expression is strongly up-regulated by THG treatment. Moreover, the absence of Bax blocks THG-induced Omi and Smac release from mitochondria, and expression of cytosolic Omi (GFP-IETD-Omi) or Smac (GFP-IETD-Smac) restores the sensitivity of Bax-knockout HCT116 cells to apoptosis in response to THG treatment. Taken together, our results indicate that Bax-dependent Smac and Omi release plays an essential role in caspase-3 activation and apoptosis induced by THG in human colon cancer HCT116 cells.     

SCP,a polysaccharide from Schisandra chinensis,induces apoptosis in human renal cell carcinoma Caki-1 cells through mitochondrial-dependent pathway via inhibition of ERK activation

This study is the first to investigate the anticancer effect of Schisandra chinensis polysaccharide (SCP) in renal cell carcinoma (RCC) cells. The results revealed that SCP treatment showed high cytotoxic potency in Caki-1 cells by inducing apoptosis, which is associated with the disruption of mitochondrial membrane potential (MMP), release of cytochrome c into the cytosol, increase of Bax/Bcl-2 ratio, activation of caspase-3/9, and cleavage of poly(ADP-ribose) polymerase (PARP). Furthermore, pan-caspase inhibitor (z-VAD-fmk) significantly blocked SCP-induced apoptosis and PARP cleavage in Caki-1 cells. As well, we also observed that SCP inhibited the phosphorylation of ERK1/2, whereas it had no significant inhibition effect on the phospho-p38 and phospho-JNK activity. All the above parameters provided scientific evidence that SCP induced mitochondrial-mediated apoptosis in Caki-1 cells through the inactivation of ERK pathways, which may shed further light on its potential application as a cancer chemopreventive agent against RCC.