Activation of ERK1/2 protects melanoma cells from TRAIL-induced apoptosis by inhibiting Smac/DIABLO release from mitochondria

We have previously shown that Smac/DIABLO release from mitochondria appears to be the principal pathway by which TRAIL induces apoptosis of human melanoma. We report that TRAIL-induced release of Smac/DIABLO appears to be downregulated by concomitant signaling through the MEK Erk1/2 kinase pathway and that this inhibits TRAIL-induced apoptosis. Inhibition of Erk1/2 signaling by either the MEK inhibitor U0126 or a dominant-negative mutant of MKK1 markedly sensitized melanoma cells to TRAIL-induced apoptosis. The site in the apoptotic pathway acted on by U0126 appeared to be downstream of caspase-8 and Bid but upstream of caspase-3 in that the levels of proteolytic cleavage of caspase-8 and Bid by TRAIL were similar in cells with or without exposure to U0126. Caspase-3 activation and cleavage of its substrates, PARP, ICAD and XIAP, were however increased by cotreatment with U0126. This was associated with a rapid reduction in mitochondrial transmembrane potential (MMP) and increased release of Smac/DIABLO into the cytosol. Exploration of events leading to the changes in MMP revealed an increased translocation of Bax from the cytosol to mitochondria in the presence of U0126. There was also a delayed decrease in the levels of expression of Mcl-1. Bcl-2 and Bcl-X(L). Over expression of Bcl-2 blocked TRAIL-induced apoptosis in the presence of U0126. Cytochrome c appeared not to play a major role in sensitization of melanoma to TRAIL in that caspase-9 activation was not detected in most of the cell lines. These results suggest that Erk1/2 signaling may protect melanoma cells against TRAIL-induced apoptosis by inhibiting the relocation of Bax from the cytosol to mitochondria and that this may reduce TRAIL-mediated release of Smac/DIABLO and induction of apoptosis.     

MnSOD protects colorectal cancer cells from TRAIL-induced apoptosis by inhibition of Smac/DIABLO release

The mitochondrial enzyme manganese superoxide dismutase (MnSOD) has been shown to have two faces with regard to its role in tumor development. On the one side, it is well documented that overexpression of MnSOD slows down cancer cell growth, whereas on the other side MnSOD also has a metastasis-promoting activity. We set out to examine the role of MnSOD in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, thought to be a first-line tumor surveillance mechanism and failure to undergo apoptosis might contribute to metastasis formation. We show that overexpression of MnSOD at moderate levels is able to protect cells from TRAIL-induced apoptosis. While caspase-8 activation and Bid cleavage were not affected by MnSOD, we detected a marked decrease in caspase-3 activation pointing to a mitochondrial resistance mechanism. Indeed, we found that MnSOD-overexpressing cells showed reduced cytochrome c and no Smac/DIABLO release into the cytosol. The resulting lack of X-linked inhibitor of apoptosis (XIAP) inhibition by cytosolic Smac/DIABLO most likely caused the TRAIL resistance as RNAi against XIAP-rescued caspase-3 activity and TRAIL sensitivity. Our results show that reactive oxygen species are involved in TRAIL-induced Smac/DIABLO release and in TRAIL-triggered apoptosis. Hence, high levels of MnSOD, which decompose and neutralize these reactive oxygen species, might contribute to metastasis formation by allowing disseminated tumor cells to escape from TRAIL-mediated tumor surveillance. As part of TRAIL regimens, adjuvant treatment with XIAP inhibitors in the form of Smac/DIABLO mimetics or MnSOD inhibitors might be able to break TRAIL resistance of malignant tumor 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.     

Protein kinase C inhibition and x-linked inhibitor of apoptosis protein degradation contribute to the sensitization effect of luteolin on tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in cancer cells

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is an important member of the TNF superfamily with great potential in cancer therapy. Luteolin is a dietary flavonoid commonly found in some medicinal plants. Here we found that pretreatment with a noncytotoxic concentration of luteolin significantly sensitized TRAIL-induced apoptosis in both TRAIL-sensitive (HeLa) and TRAIL-resistant cancer cells (CNE1, HT29, and HepG2). Such sensitization is achieved through enhanced caspase-8 activation and caspase-3 maturation. Further, the protein level of X-linked inhibitor of apoptosis protein (XIAP) was markedly reduced in cells treated with luteolin and TRAIL, and ectopic expression of XIAP protected against cell death induced by luteolin and TRAIL, showing that luteolin sensitizes TRAIL-induced apoptosis through down-regulation of XIAP. In search of the molecular mechanism responsible for XIAP down-regulation, we found that luteolin and TRAIL promoted XIAP ubiquitination and proteasomal degradation. Next, we showed that protein kinase C (PKC) activation prevented cell death induced by luteolin and TRAIL via suppression of XIAP down-regulation. Moreover, luteolin inhibited PKC activity, and bisindolylmaleimide I, a general PKC inhibitor, simulated luteolin in sensitizing TRAIL-induced apoptosis. Taken together, these results present a novel anticancer effect of luteolin and support its potential application in cancer therapy in combination with TRAIL. In addition, our data reveal a new function of PKC in cell death: PKC activation stabilizes XIAP and thus suppresses TRAIL-induced apoptosis.     

Reactive oxygen species regulate caspase activation in tumor necrosis factor-related apoptosis-inducing ligand-resistant human colon carcinoma cell lines

The effects of reactive oxygen species (ROS) on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in solid cancers have yet to be clearly defined. In this study, we found that the classic uncoupler of oxidative phosphorylation, carbonyl cyanide m-chlorophenylhydrazone (CCCP), induced a reduction in DeltaPsim and generation of ROS. This uncoupling effect enhanced TRAIL-induced apoptosis in TRAIL-resistant human colon carcinoma cell lines (RKO, HT29, and HCT8). Sensitization was inhibited by benzyloxycarbonyl-valine-alanine-aspartate fluoromethylketone, indicating the requirement for caspase activation. CCCP per se did not induce apoptosis or release of proapoptotic factors from mitochondria. Generation of ROS by CCCP was responsible for TRAIL-induced Bax and caspase activation because scavenging ROS completely abrogated apical caspase-8 activation and further downstream events leading to cell death. Overexpression of Bcl-2 did not prevent the initial loss of DeltaPsim and ROS generation following CCCP treatment, but did prevent cell death following TRAIL and CCCP exposure. Uncoupling of mitochondria also facilitated TRAIL-induced release of proapoptotic factors. X-linked inhibitor of apoptosis overexpression abrogated TRAIL-induced apoptosis in the presence of CCCP and decreased initiator procaspase-8 processing, indicating that additional processing of caspase-8 required initiation of a mitochondrial amplification loop via effector caspases. Of interest, depletion of caspase-9 in RKO cells did not protect cells from TRAIL/CCCP-induced apoptosis, indicating that apoptosis occurred via a caspase-9-independent pathway. Data suggest that in the presence of mitochondrial-derived ROS, TRAIL induced mitochondrial release of Smac/DIABLO and inactivation of X-linked inhibitor of apoptosis through caspase-9-independent activation of caspase 3.     

Influence of casein kinase II in tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in human rhabdomyosarcoma cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis via the death receptors DR4 and DR5 in transformed cells in vitro and exhibits potent antitumor activity in vivo with minor side effects. Protein kinase casein kinase II (CK2) is increased in response to diverse growth stimuli and is aberrantly elevated in a variety of human cancers. Rhabdomyosarcoma tumors are the most common soft-tissue sarcoma in childhood. In this investigation, we demonstrate that CK2 is a key survival factor that protects tumor cells from TRAIL-induced apoptosis. We have demonstrated that inhibition of CK2 phosphorylation events by 5,6-dichlorobenzimidazole (DRB) resulted in dramatic sensitization of tumor cells to TRAIL-induced apoptosis. CK2 inhibition also induced rapid cleavage of caspase-8, -9, and -3, as well as the caspase substrate poly(ADP-ribose) polymerase after TRAIL treatment. Overexpression of Bcl-2 protected cells from TRAIL-induced apoptosis in the presence of the CK2 inhibitor. Death signaling by TRAIL in these cells was Fas-associated death domain and caspase dependent because dominant negative Fas-associated death domain or the cowpox interleukin 1beta-converting enzyme inhibitor protein cytokine response modifier A prevented apoptosis in the presence of DRB. Analysis of death-inducing signaling complex (DISC) formation demonstrated that inhibition of CK2 by DRB increased the level of recruitment of procaspase-8 to the DISC and enhanced caspase-8-mediated cleavage of Bid, thereby increasing the release of the proapoptotic factors cytochrome c, HtrA2/Omi, Smac/DIABLO, and apoptosis inducing factor (AIF) from the mitochondria, with subsequent degradation of X-linked inhibitor of apoptosis protein (XIAP). To further interfere with CK2 function, JR1 and Rh30 cells were transfected with either short hairpin RNA targeted to CK2alpha or kinase-inactive CK2alpha (K68M) or CK2alpha' (K69M). Data show that the CK2 kinase activity was abrogated and that TRAIL sensitivity in both cell lines was increased. Silencing of CK2alpha expression with short hairpin RNA was also associated with degradation of XIAP. These findings suggest that CK2 regulates TRAIL signaling in rhabdomyosarcoma by modulating TRAIL-induced DISC formation and XIAP expression.     

Mechanisms of TRAIL and gemcitabine induction of pancreatic cancer cell apoptosis

The aim of this study was to investigate induction of apoptosis by the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and gemcitabine in the pancreatic cancer cell line SW1990. The sensitivity of SW1990 cells to TRAIL and/or gemcitabine-induced apoptosis and the rate of apoptosis were assessed by MTT assay and flow cytometry, respectively. We used Hoechst 33342 staining to observe apoptotic morphology and expression levels of proteins were analyzed by Western blottin. Growth inhibition and apoptosis rates on treatment with the combination of TRAIL and gemcitabine were significantly higher than with each drug alone (p<0.05). Pancreatic cancer cells exhibited a typical apoptosis morphology after treatment with TRAIL or gemcitabine. The levels of cellular apoptosis-associated proteins such as Smac/DIABLO, Cyto C, and the activated fragment of caspase-3 (P17) increased, but the expression of XIAP was significantly decreased after 24 h (p<0.05). SW1990 cells responded to TRAIL and/or gemcitabine-induction of apoptosis in a time and concentration-dependent manner. The mechanism of the apoptosis-sensitization effect appeared associated with significant up-regulation of Smac/DIABLO and cytochrome C, down-regulation of XIAP, and activation of caspase-3.     

Targeting XIAP bypasses Bcl-2-mediated resistance to TRAIL and cooperates with TRAIL to suppress pancreatic cancer growth in vitro and in vivo

Resistance to apoptosis is a hallmark of pancreatic cancer, a leading cause of cancer deaths. Therefore, novel strategies are required to target apoptosis resistance. Here, we report that the combination of X-linked inhibitor of apoptosis (XIAP) inhibition and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an effective approach to trigger apoptosis despite Bcl-2 overexpression and to suppress pancreatic cancer growth in vitro and in vivo. Knockdown of XIAP by RNA interference cooperates with TRAIL to induce caspase activation, loss of mitochondrial membrane potential, cytochrome c release, and apoptosis in pancreatic carcinoma cells. Loss of mitochondrial membrane potential and cytochrome c release are extensively inhibited by a broad range or caspase-3 selective caspase inhibitor and by RNAi-mediated silencing of caspase-3, indicating that XIAP inhibition enhances TRAIL-induced mitochondrial damage in a caspase-3-dependent manner. XIAP inhibition combined with TRAIL even breaks Bcl-2-imposed resistance by converting type II cells that depend on the mitochondrial contribution to the death receptor pathway to type I cells in which TRAIL-induced activation of caspase-3 and caspase-9 and apoptosis proceeds irrespective of high Bcl-2 levels. Most importantly, XIAP inhibition potentiates TRAIL-induced antitumor activity in two preclinical models of pancreatic cancer in vivo. In the chicken chorioallantoic membrane model, XIAP inhibition significantly enhances TRAIL-mediated apoptosis and suppression of tumor growth. In a tumor regression model in xenograft-bearing mice, XIAP inhibition acts in concert with TRAIL to cause even regression of established pancreatic carcinoma. Thus, this combination of XIAP inhibition plus TRAIL is a promising strategy to overcome apoptosis resistance of pancreatic cancer that warrants further investigation.     

Casein kinase I attenuates tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by regulating the recruitment of fas-associated death domain and procaspase-8 to the death-inducing signaling complex

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a wide variety of malignant cell lines, in contrast to normal cells, but with considerable heterogeneity in response. Death receptor-mediated apoptosis may be attenuated by a variety of different mechanisms, including phosphorylation-based signaling pathways. We have demonstrated that casein kinase I can attenuate TRAIL-induced apoptosis in human cell lines derived from colon adenocarcinoma (HT29 and HCT8) and pediatric rhabdomyosarcoma (JR1). Inhibition of casein kinase I (CKI) phosphorylation events in HT29, HCT8, and JR1 cells by CKI-7 dramatically increased apoptosis after exposure to TRAIL, in the absence of apoptosis induced by TRAIL treatment alone. CKI inhibition enhanced the recruitment of Fas-associated death domain and procaspase-8 to the death-inducing signaling complex after TRAIL treatment and enhanced cleavage of procaspase-8 at the death-inducing signaling complex. In HT29 cells studied further, rapid cleavage of caspase-8, caspase-3, Bid, and the caspase substrate poly(ADP-ribose) polymerase occurred when CKI-7 and TRAIL were combined. Overexpression of Bcl-2, Bcl-xL, or mutant DN-Fas-associated death domain protected HT29 cells from TRAIL-induced apoptosis in the presence of the CKI inhibitor. In addition, TRAIL combined with CKI-7 promoted the release of cytochrome c, Smac/DIABLO, HtrA2/Omi, and AIF from the mitochondria and down-regulated the expression of XIAP and c-IAP1. Small hairpin RNAs directed against CKI revealed that the CKIalpha isoform contributed significantly to the inhibition of TRAIL-induced apoptosis. These findings suggest that CKIalpha plays an antiapoptotic role through the generation of phosphorylated sites at the level of the death-inducing signaling complex, thereby conferring resistance to caspase cleavage mediated by TRAIL.     

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.     

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.     

Rottlerin sensitizes colon carcinoma cells to tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis via uncoupling of the mitochondria independent of protein kinase C

Signaling pathways involved in survival responses may attenuate the apoptotic response to the cytotoxic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in human colon carcinomas. In six lines examined, three were sensitive (GC(3)/c1, VRC(5)/c1, HCT116), HT29 demonstrated intermediate sensitivity, and RKO and HCT8 were resistant to TRAIL-induced apoptosis. Calphostin c [an inhibitor of classic and novel isoforms of protein kinase C (PKC)] sensitized five of six cell lines to TRAIL, whereas Go6976, (inhibitor of classic PKC isoforms), did not influence TRAIL sensitivity. Rottlerin, an inhibitor of novel isoforms of PKC, specifically PKC delta, sensitized five of six cell lines to TRAIL-induced apoptosis, suggesting that PKC delta may be involved in the mechanism of TRAIL resistance. Transfection of HCT116 with a proapoptotic cleaved fragment of PKC delta or an antiapoptotic full-length PKC delta did not influence the sensitivity of HCT116 to TRAIL. Furthermore, the incubation of HCT116 or RKO with phorbol myristate acetate for 16 h, which down-regulated the expression of novel PKC isoforms, also did not influence sensitivity to TRAIL either in the absence or presence of rottlerin. However, after 15-min incubation with rottlerin, mitochondrial membrane potential (Delta psi m) was dramatically reduced in RKO cells, and, in cells subsequently treated with TRAIL, rapid apoptosis was evident within 8 h. Calphostin c, but not Go6976, also caused a decrease in Delta psi m. In RKO, rottlerin induced the release of cytochrome c, HtrA2/Omi, Smac/DIABLO, and AIF from the mitochondria, potentiated in combination with TRAIL, with concomitant caspase activation and down-regulation of XIAP. In HT29, the release of proapoptotic factors was demonstrated only when rottlerin and TRAIL were combined, and Bcl-2 overexpression inhibited this release and the induction of apoptosis. TRAIL-induced apoptosis was not influenced by rottlerin or Bcl-2 overexpression in type I (GC(3)/c1) cells. Data suggest that rottlerin affects mitochondrial function independent of PKC delta, thereby sensitizing cells to TRAIL, and that mitochondria constitute an important target in overcoming inherent resistance to TRAIL in colon carcinomas.     

Role of Smac in human leukaemic cell apoptosis and proliferation

Smac (or DIABLO) is a recently identified, novel proapoptotic molecule, which is released from mitochondria into the cytosol during apoptosis. Smac functions by eliminating the caspase-inhibitory properties of the inhibitors of apoptosis proteins (IAP), particularly XIAP. In this study, we stably transfected both full-length (FL) and mature (MT) Smac genes into the K562 and CEM leukaemic cell lines. Both FL and MT Smac transfectants increased the sensitivity of leukaemic cells to UV light-induced apoptosis and the activation of caspase-9 and caspase-3. Purified cytosol from the mature Smac transfectants, or the addition of human recombinant Smac protein or N-7 peptide into nontransfected cytosol, showed an increased sensitivity to cytochrome c-induced activation of caspase-3. The mature Smac enhanced the susceptibility of both K562 and CEM cells to TRAIL-induced apoptosis. Overexpression of the mature Smac protein also inhibited proliferation, as detected by reduced colony formation and Ki-67 expression in leukaemic cells. Cell cycle analysis revealed that Smac transfectants displayed significant G0/G1 arrest and reduction in 5-bromo-2'-deoxyuridine (BrdU) incorporation. Smac sensitized human acute myeloid leukaemia blasts to cytochrome c-induced activation of caspase-3. However, Smac failed to overcome Apaf-1-deficiency-mediated resistance to cytochrome c in primary leukaemic blasts. In summary, this study reveals that Smac/DIABLO exhibits a potential role in increasing apoptosis and suppressing proliferation in human leukaemic cells. Importantly, it also indicates that it is crucial to evaluate the levels of Apaf-1 and XIAP proteins in patient samples before using Smac peptide therapy in the treatment of human leukaemia.     

A mitochondrial block and expression of XIAP lead to resistance to TRAIL-induced apoptosis during progression to metastasis of a colon carcinoma

A pair of isogenic colon carcinoma cells, SW480 and 620, was used to investigate the mechanisms of acquired tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-resistance during tumour progression. Whereas primary tumour SW480 cells are sensitive to TRAIL-induced apoptosis, metastatic SW620 cells are resistant. The apoptotic signalling activated by TRAIL in SW480 cells is a type II pathway. We show that in SW620 cells, although caspase-8 is recruited and activated at the death-inducing-signalling complex and Bid is cleaved, this does not lead to caspase-9 activation. Comparison of Bcl-2, Bcl-xL and Mcl-1 levels in both cell lines showed no difference. In SW620 cells transfected with a tBid-GFP construct, tBid-GFP was correctly localized to the mitochondria. Thus, the resistance of SW620 cells is at the level of the mitochondria that can withstand large amounts of tBid. Although caspase-3 was directly cleaved by caspase-8 in SW620 cells to yield the p20 fragment, no further autocatalytic maturation into the p17 fragment was observed. We show that, in contrast to SW480 cells, the SW620 cell line expresses high amounts of X-linked inhibitor of apoptosis (XIAP). Downregulation of XIAP with bortezomib or small-interfering RNA was sufficient to restore the sensitivity of SW620 cells to TRAIL-induced apoptosis in the absence of SMAC/Diablo or cytochrome c release from the mitochondria. Thus, SW620 cells have developed a dual resistance to TRAIL-induced apoptosis: a block at the level of the mitochondria and, after a conversion to a type I pathway, an increased expression of XIAP which inhibits this pathway.     

Aurora B confers cancer cell resistance to TRAIL-induced apoptosis via phosphorylation of survivin

Tumor necrosis factor-related apoptosis-induced ligand (TRAIL) induces apoptosis selectively in cancer cells while sparing normal cells. However, many cancer cells are resistant to TRAIL-induced cell death. In this study, we examined whether Aurora B, which is frequently overexpressed in cancer cells, is associated with TRAIL resistance. The protein levels of Aurora B were higher in TRAIL-resistant cancer cell lines than in TRAIL-sensitive cancer cell lines. Exogenously expressed Aurora B attenuated TRAIL-induced apoptosis in the tested TRAIL-sensitive cancer cell lines, whereas the small interfering RNA-mediated suppression of Aurora B expression stimulated TRAIL-mediated apoptosis in the tested TRAIL-resistant cancer cell lines. Furthermore, combined treatment with TRAIL and ZM447439, a specific inhibitor of Aurora B, synergistically induced apoptosis in various TRAIL-resistant cancer cells, suggesting that this combined regimen may represent an attractive strategy for effectively treating TRAIL-resistant malignant cancers. Mechanistically, the inhibition of Aurora B activity in various cancer cells commonly downregulated survivin protein levels and potentiated the activation of caspase-3. In addition, Aurora B inhibition induced mitotic catastrophe, which also contributed to the sensitization of cells to TRAIL-mediated apoptosis. Interestingly, forced overexpression of Aurora B increased the protein levels of survivin, but not those of a non-phosphorylatable survivin mutant in which threonine 117 was replaced by alanine, indicating that phosphorylation of survivin is required for this effect. Furthermore, TRAIL-induced apoptosis in MDA-MB-435S cells was attenuated by wild-type survivin but not by the non-phosphorylatable survivin mutant. Collectively, our results demonstrate that Aurora B confers TRAIL resistance to cancer cells via phosphorylation of survivin.     

Chetomin induces degradation of XIAP and enhances TRAIL sensitivity in urogenital cancer cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is one of the most promising anti-cancer agents, but some tumor types develop resistance to TRAIL. Here, we report that chetomin, an inhibitor of hypoxia-inducible factors, is a potent enhancer of TRAIL-induced apoptosis. TRAIL or chetomin alone weakly induced apoptosis, but the combination of chetomin and TRAIL synergistically induced apoptosis in prostate cancer PC-3 cells. The combination of chetomin and TRAIL induces the activation of caspase-3, -8, -9 and -10. Among the apoptotic factors related to the TRAIL pathway, chetomin markedly decreased the X-linked inhibitor of apoptosis (XIAP) protein levels in a dose-dependent manner, but other IAP family members, TRAIL receptors and Bcl-2 family members were not altered by chetomin. Using XIAP siRNA instead of chetomin, down-regulation of XIAP sensitized PC-3 cells to TRAIL-induced apoptosis. Conversely, transient transfection of XIAP reduced the apoptotic response to combined treatment with chetomin and TRAIL. Treatment with chetomin induced a rapid decrease in XIAP protein levels but had no effect on XIAP mRNA levels. Since chetomin-mediated XIAP down-regulation was completely prevented by proteasome inhibitors, it was suggested that chetomin induces the degradation of the XIAP protein in a proteasome-dependent manner. Additionally, chetomin also sensitized renal cancer Caki-1 cells and bladder cancer UM-UC-3 cells to TRAIL-induced apoptosis via down-regulation of XIAP. Co-treatment of chetomin and TRAIL did not enhance apoptosis in normal peripheral blood mononuclear cells (PBMC). Taken together, these findings suggest that TRAIL and chetomin synergistically induce apoptosis in human urogenital cancer cells through a mechanism that involves XIAP down-regulation by chetomin.     

Disturbed balance of expression between XIAP and Smac/DIABLO during tumour progression in renal cell carcinomas

Dysregulation of apoptosis plays an important role in tumour progression and resistance to chemotherapy. The X-linked inhibitor of apoptosis (XIAP) is considered to be the most potent caspase inhibitor of all known inhibitor of apoptosis-family members. Only recently, an antagonist of XIAP has been identified, termed Smac/DIABLO. To explore the relevance of antiapoptotic XIAP and proapoptotic Smac/DIABLO for tumour progression in renal cell carcinomas (RCCs), we analysed XIAP and Smac/DIABLO mRNA and protein expression in the primary tumour tissue from 66 RCCs of all major histological types by quantitative real-time PCR, Western blot and ELISA. X-linked inhibitor of apoptosis and Smac/DIABLO mRNA expression was found in all RCCs. Importantly, the relative XIAP mRNA expression levels significantly increased from early (pT1) to advanced (pT3) tumour stages (P=0.0002) and also with tumour dedifferentiation (P=0.04). Western blot analysis confirmed the tumour stage-dependent increase of XIAP expression on the protein level. In contrast, mRNA and protein expression levels of Smac/DIABLO did not significantly change between early and advanced tumour stages or between low and high tumour grades. Consequently, the mRNA expression ratio between antiapoptotic XIAP and proapoptotic Smac/DIABLO markedly increased during progression from early (pT1) to advanced (pT3) tumour stages. Moreover, RCCs confined within the organ capsule (pT1 and pT2) exhibited a significantly lower XIAP to Smac/DIABLO expression ratio when compared with RCCs infiltrating beyond the kidney (pT3; P=0.01). Thus, our investigation demonstrates that the delicate balance between XIAP and Smac/DIABLO expression is gradually disturbed during progression of RCCs, resulting in a relative increase of antiapoptotic XIAP over proapoptotic Smac/DIABLO, thereby probably contributing to the marked apoptosis resistance of RCC.