Paclitaxel-induced apoptosis in BJAB cells proceeds via a death receptor-independent,caspases-3/-8-driven mitochondrial amplification loop

Caspase-8 is a key effector of death-receptor-triggered apoptosis. In a previous study, we demonstrated, however, that caspase-8 can also be activated in a death receptor-independent manner via the mitochondrial apoptosis pathway, downstream of caspase-3. Here, we show that caspases-3 and -8 mediate a mitochondrial amplification loop that is required for the optimal release of cytochrome c, mitochondrial permeability shift transition, and cell death during apoptosis induced by treatment with the microtubule-damaging agent paclitaxel (Taxol). In contrast, Smac release from mitochondria followed a different pattern, and therefore seems to be regulated independently from cytochrome c release. Taxol-induced cell death was inhibited by the use of synthetic, cell-permeable caspase-3- (zDEVD-fmk) or caspase-8-specific (zIETD-fmk) inhibitors. Apoptosis signaling was not affected by a dominant-negative FADD mutant (FADD-DN), thereby excluding a role of death receptor signaling in the amplification loop and drug-induced apoptosis. The inhibitor experiments were corroborated by the use of BJAB cells overexpressing the natural serpin protease inhibitor, cytokine response modifier A. These data demonstrate that the complete activation of mitochondria, release of cytochrome c, and execution of drug-induced apoptosis require a mitochondrial amplification loop that depends on caspases-3 and -8 activation. In addition, this is the first report to demonstrate death receptor-independent caspase-8 autoprocessing in vivo.     

Molecular determinants of apoptosis induced by the cytotoxic ribonuclease onconase: evidence for cytotoxic mechanisms different from inhibition of protein synthesis

Cytotoxic endoribonucleases (RNases) possess a potential for use in cancer therapy. However, the molecular determinants of RNase-induced cell death are not well understood. In this work, we identify such determinants of the cytotoxicity induced by onconase, an amphibian cytotoxic RNase. Onconase displayed a remarkable specificity for tRNA in vivo, leaving rRNA and mRNA apparently undamaged. Onconase-treated cells displayed apoptosis-associated cell blebbing, nuclear pyknosis and fragmentation (karyorrhexis), DNA fragmentation, and activation of caspase-3-like activity. The cytotoxic action of onconase correlated with inhibition of protein synthesis; however, we present evidence for the existence of a mechanism of onconase-induced apoptosis that is independent of inhibition of protein synthesis. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe) fluoromethyl ketone (zVADfmk), at concentrations that completely prevent apoptosis and caspase activation induced by ligation of the death receptor Fas, had only a partial protective effect on onconase-induced cell death. The proapoptotic activity of the p53 tumor suppressor protein and the Fas ligand/Fas/Fas-associating protein with death domain (FADD)/caspase-8 proapoptotic cascade were not required for onconase-induced apoptosis. Procaspases-9, -3, and -7 were processed in onconase-treated cells, suggesting the involvement of the mitochondrial apoptotic machinery in onconase-induced apoptosis. However, the onconase-induced activation of the caspase-9/caspase-3 cascade correlated with atypically little release of cytochrome c from mitochondria. In turn, the low levels of cytochrome c released from mitochondria correlated with a lack of detectable translocation of proapoptotic Bax from the cytosol onto mitochondria in response to onconase. This suggests the possibility of involvement of a different, potentially Bax- and cytochrome c-independent mechanism of caspase-9 activation in onconase-treated cells. As one possible mechanism, we demonstrate that procaspase-9 is released from mitochondria in onconase-treated cells. A detailed understanding of the molecular determinants of the cytotoxic action of onconase could provide means of positive or negative therapeutic modulation of the activity of this potent anticancer agent.     

Drug-induced Myc-mediated apoptosis of cancer cells is inhibited by stress protein Hsp70

The Myc oncoprotein serves a dual function by stimulating cells both towards growth and apoptosis. The latter functions are often abrogated during tumor development. The Hsp70 stress protein is a potent anti-apoptotic molecule, but its potential role in protecting cells from Myc-mediated apoptosis has not been investigated. Our results show that activated Myc potentiated apoptosis induced by the cancer drugs etoposide (ETO) and camptothecin (CAMP) in v-Myc-expressing human U-937 monoblastic cells and in Rat1 cells containing a conditionally active Myc/estrogen receptor (MycER) fusion protein. However, both heat shock and ectopic Hsp70 expression protected the cells from Myc-mediated apoptosis after drug treatment in both systems. The increased susceptibility to the anti-tumor drugs by activated Myc was enhanced by siRNA-mediated knockdown of Hsp70 expression in U-937 cells. Addressing the mechanisms by which Myc and Hsp70 promotes and inhibits drug-induced apoptosis, respectively, we found that v-Myc stimulated cytochrome c release and activation of effector caspase-9, -3 and -7, but not of initiator caspase-8. Inhibition of caspase-9 specifically reduced v-Myc-stimulated apoptosis, whereas inhibition of caspase-8 and -3/7 reduced apoptosis both in v-myc-expressing and parental ETO-treated U-937 cells. Interestingly, Myc-stimulated activation of effector caspases was inhibited, but cytochrome c release was not affected by Hsp70 expression, suggesting that Hsp70 interferes with the proapoptotic function of Myc downstream of mitochondria, at the level of caspase-9 and downstream caspases. In conclusion, Hsp70 seems to have key function in inhibition of apoptosis mediated by Myc and may therefore play an important role in Myc-driven oncogenesis.     

Cell type specific involvement of death receptor and mitochondrial pathways in drug-induced apoptosis

Apoptosis in response to cellular stress such as treatment with cytotoxic drugs is mediated by effector caspases (caspase-3) which can be activated by different initiator pathways. Here, we report on a cell type specific triggering of death receptor and/or mitochondrial pathways upon drug treatment. In type I cells (BJAB), both the receptor and the mitochondrial pathway were activated upon drug treatment, since blockade of either the receptor pathway by overexpression of dominant negative FADD (FADD-DN) or of the mitochondrial pathway by overexpression of Bcl-X(L) only partially inhibited apoptosis. Drug treatment induced formation of a FADD- and caspase-8-containing CD95 death-inducing signaling complex (DISC) in type I cells resulting in activation of caspase-8 as the most apical caspase. In contrast, in type II cells (Jurkat), apoptosis was predominantly controlled by mitochondria, since overexpression of Bcl-2 completely blocked drug-induced apoptosis, while overexpression of FADD-DN had no protective effect. In these cells, caspases including caspase-8 were activated by mitochondria-driven signaling events and no DISC was detected despite expression levels of CD95, FADD and caspase-8 proteins comparable to type I cells. Likewise, drug-induced CD95 aggregation was predominantly found in type I cells. Bid was cleaved prior to mitochondrial alterations in type I cells providing a molecular link between caspase-8 activation and mitochondrial perturbations, whereas in type II cells, Bid was cleaved downstream of mitochondria. Our findings of a cell type specific response to cytotoxic drugs have implications for the identification of molecular parameters for chemosensitivity or resistance in different tumor cells.     

Regulation of the mitochondrial checkpoint in p53-mediated apoptosis confers resistance to cell death

The p53 tumor suppressor protein inhibits tumor formation, in part by inducing apoptosis, which is inhibited by anti-apoptotic Bcl-2 family members Bcl-2 and adenovirus E1B 19K. We have identified p53-apoptotic signaling events which are targeted for inhibition by E1B 19K. Apoptotic signaling by p53 induced a Bid-independent conformational change in Bax, a Bax-Bak interaction, release of cytochrome c and Smac/DIABLO from mitochondria, caspase-9 and -3 activation, cleavage of known caspase substrates, and apoptosis. When p53-dependent apoptosis was blocked by E1B 19K expression, E1B 19K bound Bak, and the Bax-Bak interaction was inhibited. Cytochrome c and Smac/DIABLO release from mitochondria was also inhibited in E1B 19K expressing cells and cells remained viable. After a prolonged p53 death stimulus, the inhibition of the mitochondrial death checkpoint by E1B 19K failed, and cytochrome c and Smac/DIABLO were released from mitochondria, and became degraded. Despite this eventual failure to inhibit the mitochondrial checkpoint, caspase-9 and -3 were not activated, and cells remained viable even upon treatment with an exogenous death stimulus. Thus, p53 induces apoptosis in part through Bax and Bak, and even an incomplete inhibition of this mitochondrial checkpoint may be sufficient to confer resistance to cell death.     

Current status of the molecular mechanisms of anticancer drug-induced apoptosis. The contribution of molecular-level analysis to cancer chemotherapy

Apoptosis is an important phenomenon in cytotoxicity induced by anticancer drugs. Here, we review the current status of the molecular mechanisms of anticancer drug-induced apoptosis in order to assess the contribution of molecular-level analysis to cancer chemotherapy. It is apparent that the molecular mechanisms by which anticancer drugs induce apoptosis are mediated by death receptor-dependent and -independent pathways, which are related to the release of cytochrome c through voltage-dependent anion channels in the mitochondrial inner membrane. The release of cytochrome c is the central gate in turning on/off apoptosis, and is regulated by the interaction of proapoptotic proteins, including Bid, Bax and Bak, and antiapoptotic proteins including Bcl-2 and Bcl-X(L), and a specific class of inhibitors of apoptosis proteins (IAPs) including Akt, survivin, and heat-shock proteins. The caspase cascade is activated by the release of cytochrome c, which is initiated by the formation of apoptosomes consisting of procaspase-9, Apaf-1 and cytochrome c in the presence of dATP, and results in the activation of caspase-9 and caspase-3, thereby leading to apoptosis. Drug sensitivity can be enhanced by the introduction of proapoptotic genes and the inhibition of antiapoptotic proteins. The latter process is mediated by antisense oligonucleotides and is associated with apoptosis. The signal transduction pathways that are triggered by the central gate in mitochondria play a critical role in anticancer drug-induced apoptosis. The modulation of signal transduction pathways targeting the proteins involved in these signal transduction pathways using antisense IAPs, and growth factor antibodies may be a good strategy for enhancing therapeutic efficacy of anticancer drugs in cancer chemotherapy.     

Growth inhibitory effect of green tea extract in Ehrlich ascites tumor cells involves cytochrome c release and caspase activation

We reported previously that the mechanism by which Green tea extract (GTE) elicited growth-inhibitory effects in Ehrlich ascites tumor cells involved a decrease in ornithine decarboxylase (ODC) activity and in cell viability. Decrease in ODC activity has been associated with apoptotic cell death and we therefore studied changes in cytochrome c release and caspase activation, which characterize apoptosis. GTE caused a dose- and time-dependent increase in caspase-3-like protease activation, preceded by a release of cytochrome c from the mitochondria. Inhibiting the activation of caspase-3 with acetyl-Asp-Glu-Val-Asp-alpha-aldehyde (caspase inhibitor) caused a reversal in the effect on cell viability.     

PRIMA-1MET induces mitochondrial apoptosis through activation of caspase-2

p53 mutations occur frequently in human tumors. The low-molecular-weight compound PRIMA-1(MET) reactivates mutant p53, induces apoptosis in human tumor cells and inhibits tumor xenograft growth in vivo. Here, we show that PRIMA-1(MET) induces mutant p53-dependent mitochondria-mediated apoptosis through activation of caspase-2 with subsequent cytochrome c release and further activation of downstream caspase-9 and caspase-3. Inhibition of caspase-2 by a selective inhibitor and/or siRNA prevents cytochrome c release on PRIMA-1(MET) treatment and causes a significant reduction in PRIMA-1(MET)-induced cell death. Our findings highlight a chain of cellular events triggered by PRIMA-1(MET) that lead to apoptotic cell death. This should facilitate further development and optimization of efficient PRIMA-1(MET)-based anticancer drugs.     

Helenalin triggers a CD95 death receptor-independent apoptosis that is not affected by overexpression of Bcl-x(L) or Bcl-2

Apoptosis is required for proper tissue homeostasis. Defects in apoptosis signaling pathways, thus, contribute to carcinogenesis and chemoresistance. A major goal in chemotherapy is, therefore, to find cytotoxic agents that restore the ability of tumor cells to undergo apoptosis. We show here that the sesquiterpene lactone helenalin (10-50 microM) induces apoptosis in leukemia Jurkat T cells even if they lack the CD95 death receptor or overexpress the antiapoptotic proteins Bcl-x(L) or Bcl-2. Activated peripheral blood mononuclear cells, however, are not affected (10-50 microM helenalin). Helenalin led to a time-dependent (0-24 h) cleavage of the specific caspase-3-like substrate Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin as well as to the proteolytic processing of procaspase-3 and -8. Caspase activation was a necessary requirement for apoptosis because the broad-spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk, 50 microM) completely abrogated helenalin-induced DNA fragmentation as well as phosphatidylserin translocation. Although the initiator caspase-8 was activated, the helenalin-induced signaling pathway did not require the CD95 death receptor as shown using cells without or with an antibody (ZB4)-blocked CD95 receptor. Helenalin also did not induce CD95 or CD95-ligand expression. On the other hand, helenalin was found to induce the release of cytochrome c from mitochondria that was not inhibited by the caspase inhibitor zVAD-fmk, which indicated that cytochrome c release precedes caspase activation. Cytochrome c release was accompanied by dissipation of the mitochondrial transmembrane potential (DeltaPsi(m)), which was partly inhibited by zVAD-fmk, which suggests that caspases are involved in loss of DeltaPsi(m). Most importantly, overexpression of the mitochondria protecting proteins Bcl-x(L) or Bcl-2 failed to confer resistance to helenalin-induced apoptosis, although the data presented here suggest that helenalin induces a mitochondria-dependent pathway. Thus, helenalin is a promising experimental cytotoxic agent that possibly points to new strategies to overcome apoptosis resistance attributable to overexpression of antiapoptotic Bcl-2 proteins.     

MT-21 is a synthetic apoptosis inducer that directly induces cytochrome c release from mitochondria

We reported previously that a synthetic compound, MT-21, induced apoptosis by activating c-Jun-NH2-terminal kinase via the Krs/MST protein, which is activated by caspase-3 cleavage dependent on reactive oxygen species production. Here we examine the activation mechanism of caspase-3, an important cysteine aspartic protease, during MT-21-induced apoptosis. We found that MT-21 activated caspase-3 via caspase-9, but not via caspase-8. In addition, MT-21 induced the release of cytochrome c from the mitochondria that is necessary to activate caspase-9, and this release occurred before a change in membrane potential. This initiation process of MT-21-induced apoptosis was suppressed by overexpression of Bcl-2, which is known to prevent cells from undergoing apoptosis in response to a variety of stimuli. Moreover, when we treated mitochondria isolated from the cells with MT-21, the direct release of cytochrome c from the mitochondria was observed, whereas this effect was not observed in the mitochondria isolated from cells that overexpressed Bcl-2. Other apoptosis-inducing agents known to induce apoptosis via cytochrome c release from the mitochondria failed to release cytochrome c directly from isolated mitochondria. These findings indicate that MT-21 is a possible candidate antitumor agent that is able to induce apoptosis via the direct release of cytochrome c from the mitochondria.     

Matrix detachment induces caspase-dependent cytochrome c release from mitochondria: inhibition by PKB/Akt but not Raf signalling

Detachment of epithelial cells from extracellular matrix results in induction of apoptosis ('anoikis') which can be blocked by expression of activated Ras or PKB/Akt. Here we show that detachment causes release of cytochrome c from mitochondria in MDCK cells. This is blocked by caspase inhibitors, suggesting a role for caspases upstream of mitochondria in the initiation of anoikis, in accord with the ability of dominant negative FADD to inhibit this form of cell death. Bulk activation of caspase-8 following detachment lags behind cytochrome c release, and is likely the result of a mitochondrial positive feed back loop. Matrix detachment also induces Bax translocation to mitochondria in a caspase-dependent manner. Expression of activated Ras or PKB/Akt blocks all the detectable events on the detachment-induced apoptosis signalling pathway, suggesting that PKB/Akt acts at an early point in the pathway, providing the signal normally generated by matrix attachment. Strong activation of Raf can also protect MDCK cells from detachment induced apoptosis, but this occurs at a point downstream of cytochrome c release from mitochondria, and is clearly distinct from the effect of PKB/Akt. Oncogene (2000) 19, 4461 - 4468.     

Interferon-gamma treatment elevates caspase-8 expression and sensitizes human breast tumor cells to a death receptor-induced mitochondria-operated apoptotic program

In this report, we have assessed the role of IFN-gamma as a sensitizing agent in apoptosis mediated by activation of death receptor CD95 in breast tumor cells. Treatment of the tumor cell lines MCF-7 and MDA-MB231 with IFN-gamma significantly facilitated apoptosis induced by CD95 receptor ligation at the plasma membrane, independently of p53 status. In contrast, IFN-gamma treatment did not enhance the apoptotic effect of the DNA-damaging drug, doxorubicin. Analysis of apoptosis regulators indicated that caspase-8 mRNA and protein levels were up-regulated in both of the cell lines after treatment with IFN-gamma. Furthermore, IFN-gamma sensitized MCF-7 and MDA-MB231 cells to CD95-mediated activation of caspase-8, induction of cytochrome c release from mitochondria, and processing of caspase-9. Release of cytochrome c, caspases activation, and apoptosis were prevented in MCF-7 cells overexpressing Bcl-2. Altogether these results indicate that IFN-gamma, maybe through the elevation of caspase-8 levels, sensitizes human breast tumor cells to a death receptor-mediated, mitochondria-operated pathway of apoptosis.     

Calpain-mediated pathway dominates cisplatin-induced apoptosis in human lung adenocarcinoma cells as determined by real-time single cell analysis

Cisplatin is an efficient anticancer agent. Cisplatin-based chemotherapy is believed to involve different signal transduction pathways, among which calpain activation has been proposed as an important factor in the induced apoptosis. In our study, based on real-time single cell analysis, we investigated the molecular involvement of calpain in cisplatin-induced apoptosis in living human lung adenocarcinoma cells. After cisplatin treatment, calpain was activated, resulting in Bid cleavage at 4-5 hr, followed by Bid translocation and cytochrome c release, leading to cell death. Calpeptin and PD150606, specific inhibitors of calpain, blocked Bid activation completely; however, cytochrome c release was delayed by more than 2 hr, which was associated with the delay of caspase-3 activation and cell death. Remarkably, calpain-mediated release of cytochrome c and cell death was significantly compromised in the Bid knockdown cells. Z-IETD-fmk and Z-VDVAD-fmk were used to block the activation of caspase-8 and caspase-2, respectively; however, the progression of apoptosis were not affected, suggesting that caspase-8 and caspase-2 were not involved in this experimental model. Taken together, the data demonstrate that calpain mediated cisplatin-induced apoptosis in human lung adenocarcinoma cells through activating Bid, which then regulated the mitochondrial apoptotic pathway. The delays of cytochrome c release, caspase-3 activation and subsequent cell death by inactivating calpain or silencing Bid exclude other earlier or parallel pathways, strongly suggesting that the calpain-mediated pathway is the kinetically earliest one, which dominates the cisplatin-induced apoptosis.     

Smac induces cytochrome c release and apoptosis independently from Bax/Bcl-x(L) in a strictly caspase-3-dependent manner in human carcinoma cells

The mitochondrial apoptosis pathway mediates cell death through the release of various pro-apoptotic factors including cytochrome c and Smac, the second mitochondrial activator of caspases, into the cytosol. Smac was shown previously to inhibit IAP proteins and to facilitate initiation of the caspase cascade upon cytochrome c release. To investigate Smac function during apoptosis and to explore Smac as an experimental cancer therapeutic, we constructed an expression system based on a single adenoviral vector containing Smac under control of the Tet-off system supplied in cis. Conditional expression of Smac induced apoptosis in human HCT116 and DU145 carcinoma cells regardless of the loss of Bax or overexpression of Bcl-x(L). Nevertheless, apoptosis induced by Smac was associated with cytochrome c release and breakdown of the mitochondrial membrane potential. This indicates that Smac acts independently of Bax and Bcl-x(L) during initiation of apoptosis and triggers a positive feedback loop that results in Bax/Bcl-x(L)-independent activation of mitochondria. In caspase-proficient cells, Smac-induced apoptosis could be inhibited partially by cell-permeable LEHD (caspase-9 inhibitor) and DEVD (caspase-3 inhibitor) peptides. Furthermore, loss of caspase-3 expression in MCF-7 cells carrying a caspase-3 null mutation completely abrogated the sensitivity for Smac-induced apoptotic or nonapoptotic, necrosis-like cell death, while re-expression of caspase-3 conferred sensitivity. Altogether, caspase-3 but not caspase-9 activation was necessary for execution of Smac-induced cell death. Notably, Smac did not induce caspase-9 processing in the absence of caspase-3. Thus, caspase-9 processing occurs secondary to caspase-3 activation during Smac-induced apoptosis. Altogether, Smac is capable of circumventing defects in mitochondrial apoptosis signaling such as loss of Bax or overexpression of Bcl-x(L) that are frequently observed in tumor cells resistant to anticancer therapy. Consequently, Smac appears to be a promising therapeutic target in anticancer treatment.     

Actinomycin D-mediated sensitization of AIDS-Kaposi's sarcoma cells to Fas-mediated apoptosis: involvement of the mitochondrion-dependent pathway

Fas engagement rapidly induces formation of the death-inducing signaling complex (DISC) that consists of Fas, FADD and pro-caspase-8. Activated caspase-8 at the DISC directly activates downstream caspases, resulting in induction of apoptosis of the independent mitochondria. In this study, we have obtained evidence demonstrating that Fas-mediated apoptosis in AIDS-KS cells takes place in a mitochondria-dependent manner. FADD and pro-caspase-8 were detected in immunoprecipitates with anti-Fas antibody in anti-Fas mAb (CH-11)-treated Hut 78, a typical Fas-sensitive cell line. On the other hand, DISC formation by CH-11 was markedly reduced in AIDS-KS cells. In addition, CH-11-induced activation of caspase-8-like protease in AIDS-KS cells was much less pronounced compared with that in Hut 78; however, a caspase-8 inhibitor, zIETD-fmk, completely blocked the apoptosis. Further, a caspase-9 inhibitor, zLEHD-fmk, markedly inhibited Fas-mediated apoptosis in AIDS-KS cells. Several apoptotic stimuli induce mitochondria activation allowing cytochrome c release from the mitochondria. In the apoptosome, cytochrome c and Apaf-1 activate caspase-9 which subsequently leads to the activation of caspase-3. In AIDS-KS cells, CH-11 triggered cytochrome c release, an event which was inhibited by zIETD-fmk. Further, a caspase-3 inhibitor, zDEVD-fmk completely inhibited the apoptosis. Altogether, the present data provide evidence that the Fas signal in AIDS-KS cells is preferentially transduced through the mitochondria-dependent pathway, which is initiated by caspase-8 activation.     

Defects in apoptotic signal transduction in cisplatin-resistant non-small cell lung cancer cells

Non-small cell lung cancer (NSCLC) often shows intrinsic multidrug resistance, which is one of the most serious problems in cisplatin-based adjuvant chemotherapy. Anticancer drugs exert at least part of their cytotoxic effect by triggering apoptosis. In order to understand the molecular alterations leading to heterogeneous cisplatin sensitivity and apoptosis inducibility in NSCLC cells, we analyzed various apoptotic pathways, including the activation of caspase-8, -9 and -3, the release of cytochrome c from mitochondria and the expression levels of pro- and anti-apoptotic proteins such as Bax, Bad, Bcl-2, Bcl-xL, Fas and p53 using heterogeneously apoptosis-sensitive cells (Ma-10, Ma-31 and Ma-46). Cisplatin treatment induced the activation of caspase-8, -9 and -3 and the release of cytochrome c in apoptosis-sensitive Ma-46. The expression of Bcl-xL was the highest and p53 was not expressed in apoptosis-resistant Ma-31, and Fas was not expressed in Ma-46. These expression levels were not correlated with the apoptosis inducibility of the three cell lines. These results suggest that blockage of the apoptotic signal from mitochondria is responsible for apoptosis resistance in NSCLC cell lines. Our findings also indicate that anti-apoptotic Bcl-xL and pro-apoptotic p53 are necessary but not sufficient for resistance to cisplatin-induced apoptosis in NSCLC 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.     

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.     

Augmentation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis by the synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) through up-regulation of TRAIL receptors in human lung cancer cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis via the death receptors DR4 and DR5 in different transformed cells in vitro and exhibits potent antitumor activity in vivo with minor side effects. The synthetic retinoid CD437 is a potent inducer of apoptosis in cancer cells through increased levels of death receptors. We demonstrate that treatment of human lung cancer cells with a combination of suboptimal concentrations of CD437 and TRAIL enhanced induction of apoptosis in tumor cell lines with wild-type p53 but not in normal lung epithelial cells. CD437 up-regulated DR4 and DR5 expression. The CD437 and TRAIL combination enhanced activation of caspase-3, caspase-7, caspase-8, and caspase-9 and the subsequent cleavage of poly(ADP-ribose) polymerase and DNA fragmentation factor 45. Caspase inhibitors blocked the induction of apoptosis by this combination. Moreover, this combination induced Bid cleavage and increased cytochrome c release from mitochondria. These results suggest that the mechanism of enhanced apoptosis by this combination involves p53-dependent increase of death receptors by CD437, activation of these receptors by TRAIL, enhanced Bid cleavage, release of cytochrome c, and activation of caspase-3, caspase-7, caspase-8, and caspase-9. These findings suggest a novel strategy for the prevention and treatment of human lung cancer with the CD437 and TRAIL combination.     

Cytochrome c is involved in Fas-mediated apoptosis of prostatic carcinoma cell lines

We have shown previously that the pathways leading to Fas-mediated apoptosis in prostatic carcinoma cell lines are intact, because apoptosis can be triggered either by Fas ligation alone in the Fas-sensitive cell lines PC3 and ALVA31 or by rendering the Fas-resistant cell lines DU145 and JCA1 Fas-sensitive by combined treatment with anti-Fas monoclonal antibody and cycloheximide (O. W. Rokhlin et al., Cancer Res., 57: 1758-1768, 1997). In this study, we demonstrate that two of the early events after Fas ligation are the release of cytochrome c from the mitochondria and activation of caspase-9. We also found that Bid is processed after Fas ligation and thus might activate the mitochondria-dependent apoptotic cascade. In a cell-free system, cytochrome c induced caspase-3-like activity in cytoplasmic extracts from all four cell lines studied, although differences in the level of enzymatic activity were observed. Western blot analysis revealed that caspase-7 is activated by cytochrome c at the same level in all extracts, whereas expression and activation of caspase-3 varied considerably. Cytochrome c-activated extracts displayed different abilities in the induction of apoptotic features in isolated nuclei such as morphological changes and DNA fragmentation. However, differences in nuclear apoptotic activity induced by cytochrome c did not correlate with the level of caspase-3 like activity in the different extracts. These results suggest that the mitochondrial pathway is involved in Fas-mediated apoptosis in prostatic carcinoma cell lines and that, in addition to caspase-7 and caspase-3, there are other factors that confer nuclear apoptotic activity.