Caspase-8/FLICE functions as an executioner caspase in anticancer drug-induced apoptosis

Caspase-8 plays an essential role in apoptosis triggered by death receptors. Through the cleavage of Bid, a proapoptotic Bcl-2 member, it further activates the mitochondrial cytochrome c/Apaf-1 pathway. Because caspase-8 can be processed also by anticancer drugs independently of death receptors, we investigated its exact role and order in the caspase cascade. We show that in Jurkat cells either deficient for caspase-8 or overexpressing its inhibitor c-FLIP apoptosis mediated by CD95, but not by anticancer drugs was inhibited. In the absence of active caspase-8, anticancer drugs still induced the processing of caspase-9, -3 and Bid, indicating that Bid cleavage does not require caspase-8. Overexpression of Bcl-x(L) prevented the processing of caspase-8 as well as caspase-9, -6 and Bid in response to drugs, but was less effective in CD95-induced apoptosis. Similar responses were observed by overexpression of a dominant-negative caspase-9 mutant. To further determine the order of caspase-8 activation, we employed MCF7 cells lacking caspase-3. In contrast to caspase-9 that was cleaved in these cells, anticancer drugs induced caspase-8 activation only in caspase-3 transfected MCF7 cells. Thus, our data indicate that, unlike its proximal role in receptor signaling, in the mitochondrial pathway caspase-8 rather functions as an amplifying executioner caspase.     

'Loop' domain deletional mutant of Bcl-xL is as effective as p29Bcl-xL in inhibiting radiation-induced cytosolic accumulation of cytochrome c (cyt c), caspase-3 activity, and apoptosis

PURPOSE/OBJECTIVE: To investigate the effect of the enforced expression of p29Bcl-xL or its loop deletional mutant, p18Bcl-xLdelta, on irradiation-induced apoptosis and cell-cycle distribution of HL-60 cells. MATERIALS & METHODS: We compared the irradiation-induced molecular cascade of apoptosis in control human AML HL-60/neo versus Bcl-xL overexpressing (approximately 8-fold) (HL-60/Bcl-xL) and HL-60/Bcl-XLdelta cells that express the loop domain deletional mutant construct (delta26-83 AA) of Bcl-xL. The three cell lines were irradiated with 6MV photons to varying doses up to 20 Gy. Following this, cytosolic cyt c levels, caspase-3 activity, and the Bcl-2 family of proteins were evaluated utilizing Western blot analysis (whole cell lysate or cytosolic S-100 fraction). Apoptosis was assessed by internucleosomal DNA fragmentation, Annexin-V staining and FACS analysis, as well as by morphologic criteria. The cell-cycle effects of radiation were analyzed by flow cytometry. RESULTS: Eight hours following irradiation (12 Gy) of HL-60/neo cells, a marked increase (approximately 8-fold) in the cytosolic accumulation of cyt c in the S-100 fraction was observed. This was associated with the cleavage of caspase-3, as well as the generation of its poly (ADP-ribose) polymerase (PARP) and DFF (DNA fragmentation factor)-45 cleavage activity. Twenty-four to forty-eight hours after irradiation, internucleosomal DNA fragmentation and positive Annexin-V staining (32.3+/-3.3%) was detected in HL-60/neo cells. In contrast, in both HL-60/Bcl-xL and HL-60/Bcl-xLdelta cells, a significantly lower percentage of apoptotic cells (p<0.05) were detected and internucleosomal DNA fragmentation was not induced. Following irradiation, Western analysis neither demonstrated any significant alteration in Bcl-2, p29Bcl-xL, p18Bcl-xLdelta, or Bax; nor induced CD95 (Fas receptor) or Fas ligand expression in any cell type. However, in all cell types, irradiation produced approximately a 2-fold increase in the percentage of cells in the G2/M phase of the cell cycle. CONCLUSION: These results demonstrate that an intact loop domain is not necessary for the full antiapoptotic function of Bcl-xL against irradiation-induced cytosolic accumulation of cyt c, caspase activation, and apoptosis of HL-60 cells. Additionally, the cell-cycle effects of ionizing radiation in HL-60 cells are not affected by enforced expression of Bcl-xL or Bcl-xLdelta.     

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.     

Synergistic induction of mitochondrial damage and apoptosis in human leukemia cells by flavopiridol and the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA).

Interactions between the histone deacetylase inhibitor SAHA (suberoylanilide hydroxamic acid) and the cyclin-dependent kinase (CDK) inhibitor flavopiridol (FP) were examined in human leukemia cells. Simultaneous exposure (24 h) of myelomonocytic leukemia cells (U937) to SAHA (1 microM) and FP (100 nM), which were minimally toxic alone (1.5 +/- 0.5% and 16.3 +/- 0.5% apoptosis respectively), produced a dramatic increase in cell death (ie 63.2 +/- 1.9% apoptotic), reflected by morphology, procaspase-3 and -8 cleavage, Bid activation, diminished DeltaPsi(m), and enhanced cytochrome c release. FP blocked SAHA-mediated up-regulation of p21(CIP1) and CD11b expression, while inducing caspase-dependent Bcl-2 and pRb cleavage. Similar interactions were observed in HL-60 and Jurkat leukemic cells. Enhanced apoptosis in SAHA/FP-treated cells was accompanied by a marked reduction in clonogenic surivival. Ectopic expression of either dominant-negative caspase-8 (C8-DN) or CrmA partially attenuated SAHA/FP-mediated apoptosis (eg 45 +/- 1.5% and 38.2 +/- 2.0% apoptotic vs 78 +/- 1.5% in controls) and Bid cleavage. SAHA/FP induced-apoptosis was unaffected by the free radical scavenger L-N-acetyl cysteine or the PKC inhibitor GFX. Finally, ectopic Bcl-2 expression marginally attenuated SAHA/FP-related apoptosis/cytochrome c release, and failed to restore clonogenicity in cells exposed to these agents. Together, these findings indicate that SAHA and FP interact synergistically to induce mitochondrial damage and apoptosis in human leukemia cells, and suggest that this process may also involve engagement of the caspase-8-dependent apoptotic cascade.     

Pretreatment with paclitaxel enhances apo-2 ligand/tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis of prostate cancer cells by inducing death receptors 4 and 5 protein levels

We have demonstrated that Apo-2 ligand (Apo-2L)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis of human prostate cancer PC-3, DU145, and LNCaP cells in a dose-dependent manner, with PC-3 cells displaying the greatest sensitivity to Apo-2L/TRAIL. Susceptibility of the prostate cancer cell types to Apo-2L/TRAIL-induced apoptosis did not appear to correlate with the levels of the Apo-2L/TRAIL receptors death receptor (DR) 4 (TRAIL receptor 1) or DR5 (TRAIL receptor 2), decoy receptor (DcR) 1 and DcR2, Flame-1, or the inhibitors of apoptosis proteins family of proteins. Apo-2L/TRAIL-induced apoptosis of PC-3 cells was associated with the processing of caspase-8, caspase-10, and the proapoptotic Bid protein, resulting in the cytosolic accumulation of cytochrome c as well as the processing of procaspase-9 and procaspase-3. Cotreatment with the caspase-8 inhibitor z-IETD-fmk or DR4:Fc significantly inhibited Apo-2L/TRAIL-induced apoptosis. Treatment with paclitaxel or taxotere increased DR4 and/or DR5 protein levels (up to 8-fold) without affecting the protein levels of DcR1 and DcR2, Apo-2L/TRAIL, Fas, or Fas ligand. Up-regulation of DR4 and DR5 was not preceded by the induction of their mRNA levels but was inhibited by cotreatment with cycloheximide. Importantly, sequential treatment of PC-3, DU145, and LNCaP cells with paclitaxel followed by Apo-2L/TRAIL induced significantly more apoptosis than Apo-2L/TRAIL treatment alone (P < 0.01). This was also associated with greater processing of procaspase-8 and Bid, as well as greater cytosolic accumulation of cytochrome c and the processing of caspase-3. These findings indicate that up-regulation of DR4 and DR5 protein levels by treatment with paclitaxel enhances subsequent Apo-2L/TRAIL-induced apoptosis of human prostate cancer cells.     

Hsp27 functions as a negative regulator of cytochrome c-dependent activation of procaspase-3

The release of mitochondrial cytochrome c by genotoxic stress induces the formation of a cytosolic complex with Apaf-1 (mammalian CED4 homolog) and thereby the activation of procaspase-3 (cas-3) and procaspase-9 (cas-9). Here we demonstrate that heat-shock protein 27 (Hsp27) inhibits cytochrome c (cyt c)-dependent activation of cas-3. Hsp27 had no effect on cyt c release, Apaf-1 and cas-9 activation. By contrast, our results show that Hsp27 associates with cas-3, but not Apaf-1 or cas-9, and inhibits activation of cas-3 by cas-9-mediated proteolysis. Furthermore, the present results demonstrate that immunodepletion of Hsp27 depletes cas-3. Importantly, treatment of cells with DNA damaging agents dissociates the Hsp27/cas-3 complex and relieves inhibition of cas-3 activation. These findings define a novel function for Hsp27 and provide the first evidence that a heat shock protein represses cas-3 activation.     

Echinocystic acid induces apoptosis in HL-60 cells through mitochondria-mediated death pathway

Echinocystic acid (EA) is a natural triterpone enriched in various herbs and used for medicinal purpose in many Asian countries. In the present study, we reported that EA can induce apoptosis in human promyelocytic leukemia cells (HL-60), as characterized by DNA fragmentation, poly (ADP) ribose polymerase cleavage. The efficacious induction of apoptosis was observed at 100 microM for 6 h. Further molecular analysis showed that EA induced the cleavage of Bid protein, the loss of mitochondrial membrane potential (DeltaPsim) cytochrome c release from mitochondria into cytosol, and activation of caspase-3, -8 and -9. However, EA did not generate reactive oxygen species (ROS), and antioxidants including N-acetyl cysteine and catalase could not block EA-induced apoptosis in the HL-60 cells. These data suggest that EA induces apoptosis in HL-60 cells through ROS-independent mitochondrial dysfunction pathway.     

Cotreatment with STI-571 enhances tumor necrosis factor alpha-related apoptosis-inducing ligand (TRAIL or apo-2L)-induced apoptosis of Bcr-Abl-positive human acute leukemia cells.

Bcr-Abl tyrosine kinase inhibitor STI-571 induces differentiation and apoptosis of HL-60/Bcr-Abl (with ectopic expression of p190 Bcr-Abl) and K562 (with endogenous expression of p210 Bcr-Abl) cells (Blood, 96: 2246-2253, 2000). Cotreatment with STI-571 partially overcomes the resistance to antileukemic drug-induced apoptosis of HL-60/Bcr-Abl and K562 cells. Tumor necrosis factor (TNF) alpha-related apoptosis-inducing ligand (Apo-2L/TRAIL), after binding with its signaling death receptors (DR4 and DR5), triggers the intrinsic "mitochondrial" pathway of apoptosis more efficiently in the cancer than do normal cells. In the present studies, we compared the apoptotic effects of Apo-2L/TRAIL, with or without cotreatment with STI-571, in HL-60/neo, HL-60/Bcr-Abl, and K562 cells. As compared with HL-60/neo, HL-60/Bcr-Abl and K562 cells are relatively resistant to Apo-2L/TRAIL-induced apoptosis. In HL-60/Bcr-Abl and K562 versus HL-60/neo cells, Apo-2L/TRAIL caused less cytosolic accumulation of cytochrome c and the processing of caspase-9 and -3. This was also associated with decreased processing of caspase-8, c-FLIP(L) and Bid. Reduced effects of Apo-2L/TRAIL in Bcr-Abl-positive leukemic cells were not attributable to diminished expression of DR4 and DR5, or higher expressions of the decoy receptors DcR1 and -2 or c-FLIP(L). Cotreatment with STI-571 significantly enhanced Apo-2L/TRAIL-induced apoptosis (P < 0.01) as well as increased the processing of caspase-9 and -3 and XIAP, without affecting the levels of DR4, DR5, decoy receptors, or c-FLIP(L). Cotreatment with STI-571 did not enhance Apo-2L/TRAIL-induced apoptosis of HL-60/neo cells. These studies suggest that a combined treatment with STI-571 may be an effective strategy to selectively sensitize Bcr-Abl-positive leukemic blasts to Apo-2L/TRAIL-induced apoptosis.     

Dysfunctional apoptosome activation in ovarian cancer: implications for chemoresistance

Alterations in the regulation of apoptosis may contribute to the pathogenesis of cancer and resistance of tumor cells to chemotherapy. In mammalian cells, nonreceptor-mediated apoptosis occurs predominantly via assembly of a cytochrome c-dependent apoptosome complex containing caspase-9 and apoptotic protease-activating factor-1 (Apaf-1). We show here that cytosolic extracts from human ovarian carcinoma cell lines and primary ovarian tumor samples are deficient in their ability to activate procaspase-9 in the presence of cytochrome c and dATP when compared with control extracts. SKOV3, a human ovarian carcinoma cell line with diminished apoptosome activity, was significantly more resistant to chemotherapy-induced apoptosis than cell lines with functional Apaf-1 activity. This dysfunctional apoptosome activity was not explained by reduced expression levels of caspase-9 or Apaf-1. Moreover, expression levels of known inhibitors of the apoptosome, including heat shock protein 70, heat shock protein 90, or X-linked inhibitor of apoptosis, did not correlate with functional activity of the apoptosome. SKOV3, an ovarian cancer cell line with dysfunctional apoptosome activity, retains the ability to form the Apaf-1 oligomer; however, there is a diminished amount of caspase-9 in the apoptosome. The reduction in the amount of caspase-9 in the apoptosome in the SKOV3 cell line was associated with diminished caspase-3 activity. Dysfunctional apoptosome activation may contribute both to the pathogenesis of ovarian carcinoma and to chemoresistance.     

Bid can mediate a pro-apoptotic response to etoposide and ionizing radiation without cleavage in its unstructured loop and in the absence of p53

BH3-only protein Bid is a key player in death receptor-induced apoptosis, because it provides the link with the mitochondrial route for caspase activation. In this pathway, Bid is activated upon cleavage by caspase-8. Its BH3 domain-containing carboxy-terminal fragment subsequently provokes mitochondrial outer membrane permeabilization by Bak/Bax activation. Bid has also been implicated in the apoptotic response to ionizing radiation (IR) and the topoisomerase inhibitor etoposide, anti-cancer regimens that cause double-strand (ds)DNA breaks. We confirm the existence of this pathway and show that it is p53-independent. However, the degree of Bid participation in the apoptotic response to dsDNA breaks depends on the nature of cell transformation. We used Bid-deficient mouse embryonic fibroblast (MEF) lines that were reconstituted with Bid to control the cellular background and demonstrated that the Bid-dependent apoptotic pathway induced by IR and etoposide operates in MEFs that are transformed by SV40, but is not evident in E1A/Ras-transformed MEFs. The Bid-dependent apoptotic response in p53-deficient SV40-transformed MEFs contributed to clonogenic execution of the cells, implying relevance for treatment outcome. In these cells, Bid acted in a conventional manner in that it required its BH3 domain to mediate apoptosis in response to IR and etoposide, and triggered apoptotic execution by indirect activation of Bak/Bax, mitochondrial permeabilization and caspase-9 activation. However, the mechanism of Bid activation was unconventional, because elimination of all known or suspected cleavage sites for caspases or other proteolytic enzymes and even complete elimination of its unstructured cleavage loop left Bid's pro-apoptotic role in the response to IR and etoposide unaffected.     

The newly synthesized 2-(3-hydroxy-5-methoxyphenyl)-6,7-methylenedioxyquinolin-4-one triggers cell apoptosis through induction of oxidative stress and upregulation of the p38 MAPK signaling pathway in HL-60 human leukemia cells

The aim of the present study was to discover the signaling pathways associated with 2-(3-hydroxy-5-methoxy-phenyl)-6,7-methylenedioxyquinolin-4-one (YYK1)-induced apoptosis in HL-60 human leukemia cells. YYK1 induced cytotoxic effects, cell morphological changes, decreased the cell number and increased reactive oxygen species (ROS) production and loss of mitochondrial membrane potential (ΔΨm) in HL-60 cells. YYK1-induced apoptosis was confirmed by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Results from colorimetric assays and western blot analysis indicated that activities of caspase-7/-3, caspase-8 and caspase-9 were increased in YYK1-treated HL-60 cells. Western blot analysis showed that the protein levels of extrinsic apoptotic proteins (Fas/CD95, FasL and FADD), intrinsic related proteins (cytochrome c, Apaf-1, AIF and Endo G), the ratio of Bax/Bcl-2 and phosphorylated p38 MAPK were increased in HL-60 cells after YYK1 treatment. Cell apoptosis was significantly reduced after pre-treatment with N-acetylcysteine (NAC; a ROS scavenger) or diphenyleneiodonium chloride (DPI; a NADPH oxidase inhibitor). Blockage of p38 MAPK signaling by SB202190 abolished YYK1-induced Fas/CD95 upregulation and apoptosis in HL-60 cells. We conclude that YYK1 induces both of extrinsic and intrinsic apoptotic pathways via ROS-mediated activation of p38 MAPK signaling in HL-60 human leukemia cells in?vitro.     

Bax and Bak are required for apoptosis induction by sulforaphane, a cruciferous vegetable-derived cancer chemopreventive agent

Sulforaphane, a constituent of many edible cruciferous vegetables, including broccoli, effectively suppresses proliferation of cancer cells in culture and in vivo by causing apoptosis induction, but the sequence of events leading to cell death is poorly defined. Here, we show that multidomain proapoptotic Bcl-2 family members Bax and Bak play a critical role in apoptosis induction by sulforaphane. This conclusion is based on the following observations: (a) sulforaphane treatment caused a dose- and time-dependent increase in the protein levels of both Bax and Bak and conformational change and mitochondrial translocation of Bax in SV40-transformed mouse embryonic fibroblasts (MEF) derived from wild-type mice to trigger cytosolic release of apoptogenic molecules (cytochrome c and Smac/DIABLO), activation of caspase-9 and caspase-3, and ultimately cell death; (b) MEFs derived from Bax or Bak knockout mice resisted cell death by sulforaphane, and (c) MEFs derived from Bax and Bak double knockout mice exhibited even greater protection against sulforaphane-induced cytochrome c release, caspase activation, and apoptosis compared with wild-type or single knockout cells. Interestingly, sulforaphane treatment also caused a dose- and time-dependent increase in the protein level of Apaf-1 in wild-type, Bax-/-, and Bak-/- MEFs but not in double knockout, suggesting that Bax and Bak might regulate sulforaphane-mediated induction of Apaf-1 protein. A marked decline in the protein level of X-linked inhibitor of apoptosis on treatment with sulforaphane was also observed. Thus, it is reasonable to postulate that sulforaphane-induced apoptosis is amplified by a decrease in X-linked inhibitor of apoptosis level, which functions to block cell death by inhibiting activities of caspases. In conclusion, the results of the present study indicate that Bax and Bak proteins play a critical role in initiation of cell death by sulforaphane.     

5-aza-2'-deoxycytidine upregulates caspase-9 expression cooperating with p53-induced apoptosis in human lung cancer cells

Treating lung cancer cell lines using low-dose 5-aza-2'-deoxycytidine (DAC) caused an accumulation of procaspase-9 through mRNA upregulation, but the cells did not undergo apoptosis. However, when cells were treated with DAC and infected with a low dose of a recombinant wild-type p53 adenovirus vector (Ad-p53), a synergistic growth inhibitory effect was observed. Combination treatment induced Apaf-1 and procaspase-9 expression in which cytochrome c releases by Ad-p53 triggered the mitochondrial pathway of apoptosis. Selective blockage of caspase-9 activities by Z-LEHD-FMK completely attenuated DAC-induced enhancement of apoptosis mediated by Ad-p53 infection, and ectopic overexpression of procaspase-9 sensitized cells to Ad-p53-induced apoptosis in p53-null cells. In addition, DAC sensitized lung cancer cells to cisplatin and paclitaxel. Induction of the mitochondrial pathway of apoptosis using a slightly toxic dose of DAC may therefore be a strategy for treating lung cancer, and DAC treatment may have clinical implications when combined with chemotherapy or apoptosis-inducing gene therapy.     

Induction of apoptosis by apigenin and related flavonoids through cytochrome c release and activation of caspase-9 and caspase-3 in leukaemia HL-60 cells

The aim of this study was to investigate the mechanism of flavonoid-induced apoptosis in HL-60 leukaemic cells. Thus, the effect of structurally related flavonoids on cell viability, DNA fragmentation and caspase activity was assessed. Loss of membrane potential and reactive oxygen species generation were also monitored by flow cytometry. The structurally related flavonoids, such as apigenin, quercetin, myricetin, and kaempferol were able to induce apoptosis in human leukaemia HL-60 cells. Treatment with flavonoids (60 microM) caused a rapid induction of caspase-3 activity and stimulated proteolytic cleavage of poly-(ADP-ribose) polymerase (PARP). Furthermore, these flavonoids induced loss of mitochondrial transmembrane potential, elevation of reactive oxygen species (ROS) production, release of mitochondrial cytochrome c into the cytosol, and subsequent induction of procaspase-9 processing. The potency of these flavonoids on these features of apoptosis were in the order of: apigenin > quercetin > myricetin > kaempferol in HL-60 cells treated with 60 microM flavonoids. These results suggest that flavonoid-induced apoptosis is stimulated by the release of cytochrome c to the cytosol, by procaspase-9 processing, and through a caspase-3-dependent mechanism. The induction of apoptosis by flavonoids may be attributed to their cancer chemopreventive activity. Furthermore, the potency of flavonoids for inducing apoptosis may be dependent on the numbers of hydroxyl groups in the 2-phenyl group and on the absence of the 3-hydroxyl group. This provides new information on the structure-activity relationship of flavonoids.     

Involvement of proapoptotic molecules Bax and Bak in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced mitochondrial disruption and apoptosis: differential regulation of cytochrome c and Smac/DIABLO release

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo2L induces apoptosis in a wide variety of cancer and transformed cells. Activation of BID, a "BH3-domain-only" Bcl-2 family member, triggers the oligomerization of proapoptotic family members Bak or Bax, resulting in the release of mitochondrial proteins to cytosol. In this study, we have shown the importance of Bax and Bak in TRAIL-induced apoptosis by studying in murine embryonic fibroblasts (MEFs) from Bax(-/-) and Bak(-/-) animals. TRAIL induced cytochrome c release and apoptosis in wild-type, Bid(-/-), Bax(-/-), or Bak(-/-) MEFs, but not in Bax(-/-) Bak(-/-) double knockout (DKO) MEFs. Bid, which functions upstream of cytochrome c release, was cleaved in all of the knockout cells except in Bid(-/-) MEFs. The release of cytochrome c was correlated with caspase-9 activity. TRAIL increased caspase-3 activity in all of the cells except in DKO cells. TRAIL-induced drop in mitochondrial membrane potential was not observed in DKO MEFs. Unlike cytochrome c release, TRAIL-induced Smac/DIABLO release was blocked in Bid(-/-), Bax(-/-), Bak(-/-), or DKO MEFs, suggesting the differential regulation of these mitochondrial proteins during apoptosis. The apoptotic events downstream of mitochondria were intact in DKO MEFs, because microinjection of cytochrome c, or ectopic expression of mature Smac/DIABLO or pretreatment of Smac N7 peptide completely restored TRAIL sensitivity. In conclusion, the data suggest that Bax and Bak differentially regulate the release of cytochrome c and Smac/DIABLO from mitochondria, and Smac/DIABLO can be used to sensitize cells that are deficient in Bax and Bak genes, or resistant to TRAIL.     

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.     

Adriamycin Sensitizes Adriamycin-Resistant HL-60/ADR Cells to TRAIL-Mediated Apoptosis

OBJECTIVE To study whether an adriamycin-resistant cell line(HL-60/ADR) can be sensitized by adriamycin(ADR) to TRAIL-mediated apoptosis.METHODS The mRNA levels of the TRAIL receptor and apoptosis-related signaling molecules involved in the TRAIL-mediated apoptotic pathway were measured by RT-PCR.The protein levels of apoptotic-related signaling molecules involved in the TRAIL-mediated apoptotic pathway and processed caspase-3,caspase-9,and caspase-8 were measured by Western blots.Apoptosis was assessed by flow cytometry.Mitochondrial membrane potential was analyzed by DiOC6(3) staining.Cytotoxicity was determined by the colorimetric MTT viability/ proliferation assay.RESULTS Treatment with a combination of TRAIL and subtoxic concentrations of ADR resulted in synergistic cytotoxicity and apoptosis for both the parental HL-60 and the HL-60/ADR cells.For HL-60,there was a 5-fold potentiation and synergy in cytotoxicity for TRAIL and for HL-60/ADR,cytotoxicity to TRAIL was potentiated 6-fold with ADR.Adriamycin treatment modestly up-regulated TRAIL-R2(DR5),but had no effect on the expression of Fas-associated death domain,c-FLIP,Bcl-2,Bcl-xL,Bax,and IAP family members(cIAP-1,cIAP-2,XIAP,and survivin).The protein levels of pro-caspase-8 and pro-caspase-3 were not affected by ADR,whereas pro-caspase-9 and Apaf-1 were up-regulated.Combined treatment with TRAIL and ADR resulted in activation of caspase-9 and caspase-3,but there was no detectable processing of caspase-8 beyond the background levels.There was signif icant depolarization of the mitochondrial membrane by the combined treatment of both cell lines and it was more pronounced in the parental HL-60 cell line.The combined treatment with TRAIL and ADR resulted in 42.6% of the HL-60/ADR cells undergoing DNA fragmentation,whereas treatment with either ADR or TRAIL alone resulted in 5.46% and 21.3% DNA fragmented cells,respectively.Similar results were obtained with the HL-60 cells.CONCLUSION These fi ndings demonstrate that ADR can still signal ADR-resistant tumor cells,resulting in the modifi cation of the TRAIL-mediated signaling pathway and apoptosis.     

IFN-alpha and bortezomib overcome Bcl-2 and Mcl-1 overexpression in melanoma cells by stimulating the extrinsic pathway of apoptosis

We hypothesized that IFN-alpha would enhance the apoptotic activity of bortezomib on melanoma cells. Combined treatment with bortezomib and IFN-alpha induced synergistic apoptosis in melanoma and other solid tumor cell lines. Apoptosis was associated with processing of procaspase-3, procaspase-7, procaspase-8, and procaspase-9 and with cleavage of Bid and poly(ADP-ribose) polymerase. Bortezomib plus IFN-alpha was effective at inducing apoptosis in melanoma cells that overexpressed Bcl-2 or Mcl-1, suggesting that this treatment combination can overcome mitochondrial pathways of cell survival and resistance to apoptosis. The proapoptotic effects of this treatment combination were abrogated by a caspase-8 inhibitor, led to increased association of Fas and FADD before the onset of cell death, and were significantly reduced in cells transfected with a dominant-negative FADD construct or small interfering RNA targeting Fas. These data suggest that bortezomib and IFN-alpha act through the extrinsic pathway of apoptosis via FADD-induced caspase-8 activation to initiate cell death. Finally, bortezomib and IFN-alpha displayed statistically significant antitumor activity compared with either agent alone in both the B16 murine model of melanoma and in athymic mice bearing human A375 xenografts. These data support the future clinical development of bortezomib and IFN-alpha for malignant melanoma.     

Thymoquinone induces apoptosis through activation of caspase-8 and mitochondrial events in p53-null myeloblastic leukemia HL-60 cells

Thymoquinone (TQ), the major biologically active component isolated from a traditional medicinal herb, Nigella sativa Linn, is a potential chemopreventive and chemotherapeutic compound. Despite the promising antineoplastic activities of TQ, the molecular mechanism of its pharmacologic effects is poorly understood. Here, we report that TQ exhibits antiproliferative effect, induces apoptosis, disrupts mitochondrial membrane potential and triggers the activation of caspases 8, 9 and 3 in myeloblastic leukemia HL-60 cells. The apoptosis induced by TQ was inhibited by a general caspase inhibitor, z-VAD-FMK; a caspase-3-specific inhibitor, z-DEVD-FMK; as well as a caspase-8-specific inhibitor, z-IETD-FMK. Moreover, the caspase-8 inhibitor blocked the TQ-induced activation of caspase-3, PARP cleavage and the release of cytochrome c from mitochondria into the cytoplasm. In addition, TQ treatment of HL-60 cells caused a marked increase in Bax/Bcl2 ratios due to upregulation of Bax and downregulation of Bcl2 proteins. These results indicate that TQ-induced apoptosis is associated with the activation of caspases 8, 9 and 3, with caspase-8 acting as an upstream activator. Activated caspase-8 initiates the release of cytochrome c during TQ-induced apoptosis. Overall, these results offer a potential mechanism for TQ-induced apoptosis in p53-null HL-60 cancer cells.     

Bax and Bak genes are essential for maximum apoptotic response by curcumin, a polyphenolic compound and cancer chemopreventive agent derived from turmeric, Curcuma longa

Curcumin, an active ingredient of turmeric (Curcuma longa), inhibits proliferation and induces apoptosis in cancer cells, but the sequence of events leading to cell death is poorly defined. The objective of this study was to examine the molecular mechanisms by which multidomain pro-apoptotic Bcl-2 family members Bax and Bak regulate curcumin-induced apoptosis using mouse embryonic fibroblasts (MEFs) deficient in Bax, Bak or both genes. Curcumin treatment resulted an increase in the protein levels of both Bax and Bak, and mitochondrial translocation and activation of Bax in MEFs to trigger drop in mitochondrial membrane potential, cytosolic release of apoptogenic molecules [cytochrome c and second mitochondria-derived activator of caspases (Smac)/direct inhibitor of apoptosis protein-binding protein with low isoelectric point], activation of caspase-9 and caspase-3 and ultimately apoptosis. Furthermore, MEFs derived from Bax and Bak double-knockout (DKO) mice exhibited even greater protection against curcumin-induced release of cytochrome c and Smac, activation of caspase-3 and caspase-9 and induction of apoptosis compared with wild-type MEFs or single-knockout Bax(-/-) or Bak(-/-) MEFs. Interestingly, curcumin treatment also caused an increase in the protein level of apoptosis protease-activating factor-1 in wild-type MEFs. Smac N7 peptide enhanced curcumin-induced apoptosis, whereas Smac siRNA inhibited the effects of curcumin on apoptosis. Mature form of Smac sensitized Bax and Bak DKO MEFs to undergo apoptosis by acting downstream of mitochondria. The present study demonstrates the role of Bax and Bak as a critical regulator of curcumin-induced apoptosis and over-expression of Smac as interventional approaches to deal with Bax- and/or Bak-deficient chemoresistant cancers for curcumin-based therapy.