Rosiglitazone promotes tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by reactive oxygen species-mediated up-regulation of death receptor 5 and down-regulation of c-FLIP

Death receptor 5 (DR5/TRAIL-R2) is an apoptosis-inducing membrane receptor for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). In this study, we show that rosiglitazone sensitizes human renal cancer cells to TRAIL-mediated apoptosis, but not normal human mesangial cells. Furthermore, because rosiglitazone-enhanced TRAIL-mediated apoptosis is induced in various types of cancer cells but is not interrupted by Bcl-2 overexpression, this combinatory treatment may provide an attractive strategy for cancer treatment. We found that treatment with rosiglitazone significantly induces DR5 expression at both its mRNA and its protein levels, accompanying the generation of reactive oxygen species (ROS). Both treatment with DR5/Fc chimeric protein and silencing of DR5 expression using small interfering RNAs attenuated rosiglitazone plus TRAIL-induced apoptosis, showing the critical role of DR5 in this cell death. Pretreatment with GSH significantly inhibited rosiglitazone-induced DR5 up-regulation and the cell death induced by the combined treatment with rosiglitazone and TRAIL, suggesting that ROS mediate rosiglitazone-induced DR5 up-regulation, contributing to TRAIL-mediated apoptosis. However, both DR5 up-regulation and sensitization of TRAIL-mediated apoptosis induced by rosiglitazone are likely PPARgamma-independent, because a dominant-negative mutant of PPARgamma and a potent PPARgamma inhibitor, GW9662, failed to block DR5 induction and apoptosis. Interestingly, we also found that rosiglitazone treatment induced down-regulation of cellular FLICE-inhibitory protein (c-FLIPs), and ectopic expression of c-FLIPs attenuated rosiglitazone plus TRAIL-mediated apoptosis, demonstrating the involvement of c-FLIPs in this apoptosis. Taken together, the results of this study demonstrate that rosiglitazone enhances TRAIL-induced apoptosis in various cancer cells by ROS-mediated DR5 up-regulation and down-regulation of c-FLIPs.     

Pretreatment of acetylsalicylic acid promotes tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by down-regulating BCL-2 gene expression

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to be selective in the induction of apoptosis in cancer cells with minimal toxicity to normal tissues. However, not all cancers are sensitive to TRAIL-mediated apoptosis. Thus, TRAIL-resistant cancer cells must be sensitized first to become responsive to TRAIL. In this study, we observed that pretreatment by acetylsalicylic acid (ASA) augmented TRAIL-induced apoptotic death in human prostate adenocarcinoma LNCaP and human colorectal carcinoma CX-1 cells. Western blot analysis showed that pretreatment of ASA followed by TRAIL treatment activated caspases (8, 9, and 3) and cleaved poly(ADP-ribose) polymerase, the hallmark feature of apoptosis. Most interestingly, at least 12 h of pretreatment with ASA was prerequisite for promoting TRAIL-induced apoptosis and was related to down-regulation of BCL-2. Biochemical analysis revealed that ASA inhibited NF-kappaB activity, which is known to regulate BCL-2 gene expression, by dephosphorylating IkappaB-alpha and inhibiting IKKbeta activity but not by affecting the HER-2/neu phosphatidylinositol 3-kinase-Akt signal pathway. Overexpression of BCL-2 suppressed the promotive effect of ASA on TRAIL-induced apoptosis and changes in mitochondrial membrane potential. Taken together, our studies suggested that ASA-promoted TRAIL cytotoxicity is mediated through down-regulating BCL-2 and by decreasing mitochondrial membrane potential.     

Distinct intracellular signaling in tumor necrosis factor-related apoptosis-inducing ligand- and CD95 ligand-mediated apoptosis

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in tumor cells but not in healthy cells. Similar to CD95 ligand (CD95L), TRAIL signaling requires ligand-receptor interaction; the downstream signaling molecules, such as Fas-associated death domain and caspase-8, also seem similar. Using cells stably expressing TRAIL and CD95L, we show that both TRAIL and CD95L induce apoptosis in the rat colon carcinoma cell line CC531. The mitochondrial damage (loss of mitochondrial membrane potential (MMP) and release of cytochrome c) observed after co-incubation with TRAIL-expressing cells occurs much earlier than that observed with CD95L-expressing cells. The decrease in MMP induced by both ligands was caspase-8-mediated; no difference in caspase-8 activation by TRAIL and CD95L was found. TRAIL, but not CD95L, induced activation of caspase-10. bcl-2 overexpression could not prevent TRAIL-induced mitochondrial dysfunction, whereas it completely prevented CD95L-mediated loss of MMP and cytochrome c release. The selective effect of TRAIL on tumor cells and the apparent inability of bcl-2 to block TRAIL-induced apoptosis suggest that TRAIL may offer a lead for cancer therapy in the future.     

Protein kinase C zeta associates with death inducing signaling complex and regulates Fas ligand-induced apoptosis

Previous studies have shown that Protein kinase C (PKC) stimulation may interfere with Fas signaling pathway and Fas ligand (FasL)-induced apoptosis. In this study, we investigated in Jurkat cells, a FasL-sensitive human T-cell model, whether PKC(zeta) targets apical events of Fas signaling. We describe for the first time that in Jurkat cells, both PKC(zeta) and Prostate apoptosis response-4 (Par-4), one of the major endogenous PKC(zeta) regulators, are components of the death inducing signaling complex (DISC). Using PKC(zeta) overexpressing cells or si-RNA depletion, we demonstrate that PKC(zeta) interferes neither with Fas expression nor Fas clustering in raft microdomains, but negatively regulates FasL-induced apoptosis by interfering with DISC formation and subsequent caspase-8 processing.     

Phenylarsine oxide interferes with the death inducing signaling complex and inhibits tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induced apoptosis

The mechanism by which tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces death is the subject of intense scrutiny due to its preferential targeting of transformed cells for deletion. Based on recent findings that the TRAIL-dependent death inducing signaling complex (DISC) forms and signals at the plasma membrane without being internalized, we investigated the possibility that agents that prevent endocytosis may stabilize the surface bound DISC and thereby enhance TRAIL-dependent signaling. We utilized phenylarsine oxide (PAO), a trivalent arsenical that has been reported to inhibit endocytosis and to induce mitochondrial permeability transition. Therefore PAO could, by two separate and independent activities, enhance TRAIL-induced killing. Paradoxically, we found that rather than synergizing with TRAIL, PAO was an effective inhibitor of TRAIL-induced killing. Recruitment of FADD and caspase-8 to the TRAIL-dependent DISC was diminished in a concentration-dependent manner in cells exposed to PAO. The effects of PAO could not be reversed by washing cells under non-reducing conditions, suggesting covalent linkage of PAO with its cellular target(s); however, 2,3-dimercaptoethanol effectively overcame the inhibitory action of PAO and restored sensitivity to TRAIL-induced apoptosis. PAO inhibited formation of the TRAIL-dependent DISC and therefore prevented all subsequent apoptotic events.     

Receptor-selective mutants of apoptosis-inducing ligand 2/tumor necrosis factor-related apoptosis-inducing ligand reveal a greater contribution of death receptor (DR) 5 than DR4 to apoptosis signaling

Apoptosis-inducing ligand 2 (Apo2L), also called tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), triggers programmed cell death in various types of cancer cells but not in most normal cells. Apo2L/TRAIL is a homotrimeric protein that interacts with five receptors: death receptor 4 (DR4) and DR5 mediate apoptosis activation, whereas decoy receptor 1 (DcR1), DcR2, and osteoprotegerin counteract this function. Many cancer cell lines express both DR4 and DR5, and each of these receptors can initiate apoptosis independently of the other. However, the relative contribution of DR4 and DR5 to ligand-induced apoptosis is unknown. To investigate this question, we generated death receptor-selective Apo2L/TRAIL variants using a novel approach that enables phage display of mutated trimeric proteins. Selective binding to DR4 or DR5 was achieved with three to six-ligand amino acid substitutions. The DR4-selective Apo2L/TRAIL variants examined in this study showed a markedly reduced ability to trigger apoptosis, whereas the DR5-selective variants had minimally decreased or slightly increased apoptosis-inducing activity. These results suggest that DR5 may contribute more than DR4 to Apo2L/TRAIL-induced apoptosis in cancer cells that express both death receptors.     

Hypoxia inhibition of apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)

Hypoxia is a common environmental stress. Particularly, the center of rapidly growing solid tumors is easily exposed to hypoxic conditions. Thus, tumor cell response to hypoxia plays an important role in tumor progression as well as tumor therapy. However, little is known about hypoxic effect on apoptotic cell death. To examine the effects of hypoxia on TRAIL-induced apoptosis, human lung carcinoma A549 cells were exposed to hypoxia and treated with TRAIL protein. Hypoxia significantly protected A549 cells from apoptosis induced by TRAIL. Western blotting analysis demonstrated that hypoxia increased expression of antiapoptotic proteins such as Bcl-2, Bcl-XL, and IAP family members. The increase of these antiapoptotic molecules is believed to play an hypoxia-mediated protective role in TRAIL-induced apoptosis. Our findings suggest that an increase of antiapoptotic proteins induced by hypoxia may regulate the therapeutic activity of TRAIL protein in cancer therapy.     

APRIL, a new member of the tumor necrosis factor family, modulates death ligand-induced apoptosis

APRIL (a proliferation-inducing ligand) is a newly identified member of the tumor necrosis factor (TNF) family. Tumor growth-promoting as well as apoptosis-inducing effects of APRIL have been described. Here, we report that five of 12 human malignant glioma cell lines express APRIL. APRIL gene transfer experiments revealed that malignant glioma cells are refractory to growth-promoting activity of APRIL in vitro and in vivo. Interestingly, ectopic expression of APRIL confers minor protection from apoptotic cell death induced by the death ligands, CD95 ligand (CD95L) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo2 ligand (Apo2L). This antiapoptotic activity is specific for death ligand/receptor-mediated apoptosis since APRIL does not protect glioma cells from the cytotoxicity of the drugs, teniposide, vincristine, lomustine or cisplatin. Ectopic expression of APRIL is associated with the upregulation of X-linked inhibitor of apoptosis protein (XIAP), providing a possible explanation for the antiapoptotic activity observed here. In contrast, APRIL does not regulate the expression levels of the antiapoptotic proteins FLICE-inhibitory protein (FLIP), Bcl-2 or Bcl-X(L). These findings suggest that APRIL is involved in the regulation of death ligand-induced apoptotic signaling in malignant glioma cells.     

Kaempferol sensitizes tumor necrosis factor-related apoptosis-inducing ligand-resistance chronic myelogenous leukemia cells to apoptosis

Molecular Biology Reports - The tumor necrosis factor (TNF)-related apoptosis-inducing ligand, TRAIL, an apoptosis-inducing cytokine, has attracted much attention in the treatment of cancer for its...     

Roles of tyrosine phosphorylation and cleavage of protein kinase Cdelta in its protective effect against tumor necrosis factor-related apoptosis inducing ligand-induced apoptosis

Protein kinase Cdelta (PKCdelta) regulates cell apoptosis in a cell- and stimulus-specific manner. Here, we studied the role of PKCdelta in the apoptotic effect of TRAIL in glioma cells. We found that transfection of the cells with a PKCdelta kinase-dead mutant (K376R) or with a small interfering RNA targeting the PKCdelta mRNA increased the apoptotic effect of tumor necrosis factor-related apoptosis inducing ligand (TRAIL), whereas overexpression of PKCdelta decreased it. PKCdelta acted downstream of caspase 8 and upstream of cytochrome c release from the mitochondria. TRAIL induced cleavage of PKCdelta within 2-3 h of treatment, which was abolished by caspase 3, 8, and 9 inhibitors. The cleavage of PKCdelta was essential for its protective effect because overexpression of a caspase-resistant mutant (PKCdeltaD327A) did not protect glioma cells from TRAIL-induced apoptosis but rather increased it. TRAIL induced translocation of PKCdelta to the perinuclear region and the endoplasmic reticulum and phosphorylation of PKCdelta on tyrosine 155. Using a PKCdeltaY155F mutant, we found that the phosphorylation of PKCdelta on tyrosine 155 was essential for the cleavage of PKCdelta in response to TRAIL and for its translocation to the endoplasmic reticulum. In addition, phosphorylation of PKCdelta on tyrosine 155 was necessary for the activation of AKT in response to TRAIL. Our results indicate that PKCdelta protects glioma cells from the apoptosis induced by TRAIL and implicate the phosphorylation of PKCdelta on tyrosine 155 and its cleavage as essential factors in the anti-apoptotic effect of PKCdelta.     

Role of HER-2/neu signaling in sensitivity to tumor necrosis factor-related apoptosis-inducing ligand: enhancement of TRAIL-mediated apoptosis by amiloride

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to induce apoptosis in numerous transformed cell lines but not in most normal cells. Although this selectivity offers a potential therapeutic application in cancer, not all cancers are sensitive to TRAIL-mediated apoptosis. In this study, we observed that amiloride, a current clinically used diuretic drug, which had little or no cytotoxicity, sensitized TRAIL-resistant human prostate adenocarcinoma LNCaP and human ovarian adenocarcinoma SK-OV-3 cells. The TRAIL-mediated activation of caspase, and PARP cleavage, were promoted in the presence of amiloride. Western blot analysis showed that combined treatment with TRAIL and amiloride did not change the levels of TRAIL receptors (DR4, DR5, and DcR2) and anti-apoptotic proteins (FLIP, IAP, and Bcl-2). However, amiloride dephosphorylated HER-2/neu tyrosine kinase as well as Akt, an anti-apoptotic protein. Interestingly, amiloride also dephosphorylated PI3K and PDK-1 kinases along with PP1alpha phosphatase. In vitro kinase assay revealed that amiloride inhibited phosphorylation of kinase as well as phosphatase by competing with ATP. Taken together, the present studies suggest that amiloride enhances TRAIL-induced cytotoxicity by inhibiting phosphorylation of the HER-2/neu-PI3K-Akt pathway-associated kinases and phosphatase.     

Tumor necrosis factor-related apoptosis-inducing ligand-induced death-inducing signaling complex and its modulation by c-FLIP and PED/PEA-15 in glioma cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can trigger apoptosis in some tumor cells but not other tumor cells. To explore the signal transduction events in TRAIL-triggered apoptosis and its modulation in nontransfected tumor cells, we analyzed TRAIL-induced death-inducing signaling complex (DISC) in TRAIL-sensitive and -resistant glioma cells. Caspase-8 and caspase-10 were recruited to the DISC, where they were proteolytically activated to initiate apoptosis in TRAIL-sensitive glioma cells. Caspase-8 and caspase-10 were also recruited to the DISC in TRAIL-resistant cells, but their further activation was inhibited by two antiapoptotic proteins termed cellular Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein (c-FLIP) and phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes-15kDa (PED/PEA-15). Both long and short forms of c-FLIP were recruited to the DISC, where the long form c-FLIP was cleaved to produce intermediate fragments. Of the three isoforms of PED/PEA-15 proteins, only the doubly phosphorylated form was expressed and recruited to the DISC in TRAIL-resistant cells, indicating that the phosphorylation status of PED/PEA-15 determines its recruitment in the cells. Treatment with calcium/calmodulin-dependent protein kinase inhibitor rescued TRAIL sensitivity in TRAIL-resistant cells, providing a potential new approach to sensitize the cells to TRAIL-induced apoptosis.     

Apoptosis induced in normal human hepatocytes by tumor necrosis factor-related apoptosis-inducing ligand

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been reported to induce apoptosis in various tumor cells but not in nontransformed, normal cells. Preclinical studies in mice and nonhuman primates have shown that administration of TRAIL can induce apoptosis in human tumors, but that no cytotoxicity to normal organs or tissues is found. The susceptibility of tumor cells to TRAIL and an apparent lack of activity in normal cells has lead to a proposal to use TRAIL in cancer therapy. Here, we assessed the sensitivity of hepatocytes from rat, mouse, rhesus monkey and human livers to TRAIL-induced apoptosis. TRAIL induced apoptosis in normal human hepatocytes in culture but not in hepatocytes isolated from the other species. Human hepatocytes showed characteristic features of apoptosis, including cytoplasmic shrinkage, the activation of caspases and DNA fragmentation. Apoptosis and cell death in human hepatocytes was massive and rapid, occurring in more than 60% of the cells exposed to TRAIL within 10 hours. These results indicate that there are species differences in sensitivity to TRAIL, and that substantial liver toxicity might result if TRAIL were used in human cancer therapy.     

Restricted expression of tumor necrosis factor-related apoptosis-inducing ligand receptor 4 in human peripheral blood lymphocytes

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in tumor but not normal cells, thus providing therapeutic possibilities for human cancers. However, it is not fully clear how widespread TRAIL receptors are, or how TRAIL signaling is modulated in normal cells. We characterized cell surface expression of TRAIL receptors in normal healthy donor peripheral blood and report that each of the TRAIL receptors are characteristically expressed on restricted cell populations. TRAIL-R1 is distinctively expressed on B-lymphocytes, TRAIL-R2 on monocytes, TRAIL-R3 on neutrophils and most impressively, CD8+ lymphocytes and NKT lymphocytes but not CD4+ lymphocytes express TRAIL-R4.     

Real time analysis of tumor necrosis factor-related apoptosis-inducing ligand/cycloheximide-induced caspase activities during apoptosis initiation

Employing fluorescence resonance energy transfer (FRET) imaging, we previously demonstrated that effector caspase activation is often an all-or-none response independent of drug choice or dose administered. We here investigated the signaling dynamics during apoptosis initiation via the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor pathway to investigate how variability in drug exposure can be translated into largely kinetically invariant cell death execution pathways. FRET-based microscopy demonstrated dose-dependent responses of caspase-8 activation and activity within individual living HeLa cells. Caspase-8 on average was activated 45-600 min after TRAIL/cycloheximide addition. Caspase-8-like activities persisted for 15-60 min before eventually inducing mitochondrial outer membrane permeabilization. Independent of the TRAIL concentrations used or the resulting caspase-8-like activities, mitochondrial outer membrane permeabilization was induced when 10% of the FRET substrate was cleaved. In contrast, in Bid-depleted cells, caspase-8-like activity persisted for hours without causing immediate cell death. Our findings provide detailed insight into the intracellular signaling kinetics during apoptosis initiation and describe a threshold mechanism controlling the induction of apoptosis execution.