Resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in neuroblastoma cells correlates with a loss of caspase-8 expression

Disruption of apoptotic pathways may be involved in tumor formation, regression, and treatment resistance of neuroblastoma (NB). Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in cancer cell lines, whereas normal cells are not sensitive to TRAIL-mediated apoptosis. In this study we analyzed the expression and function of TRAIL and its agonistic and antagonistic receptors as well as expression of cellular FLICE-like inhibitory protein and caspase-2, -3, -8, -9, and -10 in 18 NB cell lines. Semiquantitative RT-PCR revealed that TRAIL-R2 and TRAIL-R3 are the main TRAIL-receptors used by NB cells. Sensitivity to TRAIL-induced apoptosis did not correlate with mRNA expression of TRAIL receptors or cellular FLICE-like inhibitory protein. Surprisingly, caspase-8 and caspase-10 mRNA expression was detected in only 5 of 18 NB cell lines. Interestingly, only these five NB cell lines were susceptible to TRAIL-induced apoptosis in a time- and dose-dependent manner. Treatment with 5-aza-2'-deoxycytidine restored mRNA and protein expression of caspase-8 and TRAIL sensitivity of resistant cell lines, suggesting that gene methylation is involved in caspase inactivation. The TRAIL system seems to be functional in NB cells expressing caspase-8 and/or caspase-10. Because many cytotoxic drugs induce caspase-dependent apoptosis, failure to express caspase-8 and/or caspase-10 might be an important mechanism of resistance to chemotherapy in NB.     

Tunicamycin enhances tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in human prostate cancer cells

Death receptor 5 (DR5/TRAIL-R2) is an apoptosis-inducing membrane receptor for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L). In this study, we showed that tunicamycin, a naturally occurring antibiotic, is a potent enhancer of TRAIL-induced apoptosis through up-regulation of DR5 expression. Tunicamycin significantly sensitized PC-3, androgen-independent human prostate cancer cells, to TRAIL-induced apoptosis. The tunicamycin-mediated enhancement of TRAIL-induced apoptosis was markedly blocked by a recombinant human DR5/Fc chimeric protein. Tunicamycin and TRAIL cooperatively activated caspase-8, -10, -9, and -3 and Bid cleavage and this activation was also blocked in the presence of the DR5/Fc chimera. Tunicamycin up-regulated DR5 expression at the mRNA and protein levels in a dose-dependent manner. Furthermore, the tunicamycin-mediated sensitization to TRAIL was efficiently reduced by DR5 small interfering RNA, suggesting that the sensitization was mediated through induction of DR5 expression. Tunicamycin increased DR5 promoter activity and this enhanced activity was diminished by mutation of a CHOP-binding site. In addition, suppression of CHOP expression by small interfering RNA reduced the tunicamycin-mediated induction of DR5. Of note, tunicamycin-mediated induction of CHOP and DR5 protein expression was not observed in normal human peripheral blood mononuclear cells. Moreover, tunicamycin did not sensitize the cells to TRAIL-induced apoptosis. Thus, combined treatment with tunicamycin and TRAIL may be a promising candidate for prostate cancer therapy.     

Anticancer agents sensitize tumor cells to tumor necrosis factor-related apoptosis-inducing ligand-mediated caspase-8 activation and apoptosis

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a new cytokine that was proposed to specifically induce apoptosis of cancer cells. In tumor cells that are resistant to the cytokine, subtoxic concentrations of chemotherapeutic drugs can restore the response to TRAIL. The present study further explores the mechanisms that determine tumor cell sensitivity to TRAIL by comparing four human colon carcinoma cell lines We show that colon cancer cell sensitivity to TRAIL-induced apoptosis and cytotoxicity correlates with the expression of the death receptors TRAIL-R1 and TRAIL-R2 at the cell surface, as determined by now cytometry, whereas the two decoy receptors TRAIL-R3 and TRAIL-R4 can be detected only in permeabilized cells. Clinically relevant concentrations of cisplatin and doxorubicin sensitize the most resistant colon cancer cell lines to TRAIL-induced cell death without modifying the expression nor the localization of TRAIL receptors in these cells. TRAIL induces the activation of procaspase-8 and triggers caspase-dependent apoptosis off colon cancer cells. Cytotoxic drugs lower the signaling threshold required for TRAIL-induced procaspase-8 activation. In turn, caspase-8 cleaves Bid, a BH3 domain-containing proapoptotic molecule of the Bcl-2 family and activates effector caspases. Together, these data indicate that chemotherapeutic drugs sensitize colon tumor cells to TRAIL-mediated caspase-8 activation and apoptosis.     

Adenovirus-mediated gene transfer of caspase-8 sensitizes human adenocarcinoma cells to tumor necrosis factor-related apoptosis-inducing ligand

Resistance to apoptosis has been implicated in the poor response of cancer cells to various anti-tumor agents. Caspase-8 is a family member of executioner caspases associated with tumor necrosis factor (TNF) family death receptors-mediated apoptotic signaling cascade. In this study, to specify caspase-8-mediated apoptotic activity, we examined the anti-tumor effect of adenoviral vector expressing caspase-8 (Adv-caspase-8) in combination with TNF-related apoptosis-inducing ligand (TRAIL) which induces specifically caspase-8 activation. First, we demonstrated that expression procaspase-8 is related to apoptosis sensitivity to TRAIL in pancreatic and colonic cancer cells. In human pancreatic cancer cell line Panc1 which demonstrates low expression of procaspase-8, Adv-caspase-8 transfection strongly augmented TRAIL-induced apoptosis. Adv-caspase-8 similarly enhanced the susceptibility of human colonic cancer cell line Colo320DM to TRAIL. These results suggest that Adv-caspase-8 may be a good combination partner of TRAIL and enables TRAIL to be a more potent anticancer agent in a wide range of adenocarcinoma cells which demonstrate low expression of caspase-8.     

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.     

Galectin-3 inhibits tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by activating Akt in human bladder carcinoma cells

The antiapoptotic molecule galectin-3 was previously shown to regulate CD95, a member of the tumor necrosis factor (TNF) family of proteins in the apoptotic signaling pathway. Here, we question the generality of the phenomenon by studying a different member of this family of proteins [e.g., TNF-related apoptosis-inducing ligand (TRAIL), which induces apoptosis in a wide variety of cancer cells]. Overexpression of galectin-3 in J82 human bladder carcinoma cells rendered them resistant to TRAIL-induced apoptosis, whereas phosphatidylinositol 3-kinase (PI3K) inhibitors (wortmannin and LY-294002) blocked the galectin-3 protecting effect. Because Akt is a major downstream PI3K target reported to play a role in TRAIL-induced apoptosis, we questioned the possible relationship between galectin-3 and Akt. Parental J82 and the control vector-transfected J82 cells (barely detectable galectin-3) exhibit low level of constitutively active Akt, resulting in sensitivity to TRAIL. On the other hand, J82 cells overexpressing galectin-3 cells expressed a high level of constitutively active Akt and were resistant to TRAIL. Moreover, the blockage of TRAIL-induced apoptosis in J82 cells seemed to be mediated by Akt through the inhibition of BID cleavage. These results suggest that galectin-3 involves Akt as a modulator molecule in protecting bladder carcinoma cells from TRAIL-induced apoptosis.     

Hypoxia inhibits tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by blocking Bax translocation

The hypoxic environment in solid tumors results from oxygen consumption by rapid proliferation of tumor cells. Hypoxia has been shown to facilitate the survival of tumor cells and to be a cause of malignant transformation. Hypoxia also is well known to attenuate the therapeutic activity of various therapies in cancer management. These observations indicate that hypoxia plays a critical role in tumor biology. However, little is known about the effects of hypoxia on apoptosis, especially on apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a potent apoptosis inducer that has been shown to specifically limit tumor growth without damaging normal cells and tissues in vivo. To address the effects of hypoxia on TRAIL-induced apoptosis, HCT116 human colon carcinoma cells were exposed to hypoxic or normoxic conditions and treated with soluble TRAIL protein. Hypoxia dramatically inhibited TRAIL-induced apoptosis in HCT116 cells, which are highly susceptible to TRAIL in normoxia. Hypoxia increased antiapoptotic Bcl-2 family member proteins and inhibitors of apoptosis proteins. Interestingly, these hypoxia-increased antiapoptotic molecules were decreased by TRAIL treatment to the levels lower than those of the untreated conditions, suggesting that hypoxia inhibits TRAIL-induced apoptosis via other mechanisms rather than up-regulation of these antiapoptotic molecules. Additional characterization revealed that hypoxia significantly inhibits TRAIL-induced translocation of Bax from the cytosol to the mitochondria in HCT116 and A549 cells, with the concomitant inhibition of cytochrome c release from the mitochondria. Bax-deficient HCT116 cells were completely resistant to TRAIL regardless of oxygen content, demonstrating a pivotal role of Bax in TRAIL-induced apoptotic signaling. Thus, our data indicate that hypoxia inhibits TRAIL-induced apoptosis by blocking Bax translocation to the mitochondria, thereby converting cells to a Bax-deficient state.     

Downmodulation of dimethyl transferase activity enhances tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in prostate cancer cells

One of the major obstacles in curing prostate cancer is the development of drug resistance. It is not only imperative to discover the molecular basis of resistance but also to find therapeutic agents that can disrupt the resistant pathways. Tumor necrosis factor TNF-related apoptosis-inducing ligand TRAIL-like ligands or agonist TRAIL-receptor monoclonal antibodies have entered phase I and II clinical trials with a very limited cytotoxic profile when used systemically in a variety of cancers. Therefore, TRAIL-receptor agonists are new proapoptotic pharmaceutical agents with great potential as new cancer therapeutic agents. Although many cancer cells undergo TRAIL-mediated apoptosis, some are resistant to TRAIL. Therefore, we have been investigating mechanisms to overcome TRAIL resistance in cancer cells so that TRAIL-associated compounds can be used effectively in clinical trials. Epigenetic inactivation of proapoptotic genes, or activation of survival signaling, can cause cross-resistance to several anti-tumor therapies and to immune cytotoxic lymphocytes. We hypothesize that 5-aza-2 deoxycytidine aza-dCR, decitabine may render TRAIL-resistant prostate cancer cells sensitive to caspase-8-mediated apoptosis and may, therefore, be therapeutically efficient. We evaluated the antiproliferative effects of decitabine on the following four prostate cancer cell lines: well-differentiated AR positive LnCaP p53(+), PTEN- and 22rv1 p53(+) and PTEN(+)]; poorly-differentiated AR negative PC3 p53-, PTEN- and DU145 p53 mutant, PTEN(+). Here, we provide evidence that treatment with sub-optimal concentrations of decitabine are additive to TRAIL effects in well-differentiated PCa cells whereas the same treatment shows synergistic effects in poorly-differentiated PCa cells through increased caspase-8 expression, down-modulation of Akt activation and through the expression of certain anti-apoptotic molecules including FLIP, PED/PEA-15, survivin and c-IAP-1. Our findings demonstrate that decitabine at relatively low concentrations restores caspase-8 expression and sensitises resistant PCa cells to TRAIL-induced apoptosis leading to important implications in novel therapeutic strategies targeting defective apoptosis pathways in advanced prostate tumors.     

Oncogenic Ras sensitizes normal human cells to tumor necrosis factor-alpha-related apoptosis-inducing ligand-induced apoptosis

Tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) is a cytotoxic cytokine that induces apoptosis in tumor cells but rarely kills normal ones. To determine how normal human cells acquire TRAIL-sensitive phenotype during the process of malignant transformation, we used an experimental system that allows for controlled conversion of human cells from normal to cancerous by introduction of several genes. Human embryonic kidney cells and foreskin fibroblasts were first immortalized by combination of the early region of simian virus 40 and telomerase and then were transformed with oncogenic Ras. Both normal and immortalized cells were resistant to TRAIL-induced apoptosis, whereas Ras-transformed cells were susceptible. Ras transformation enhanced TRAIL-induced activation of caspase 8 by increasing its recruitment to TRAIL receptors. The proapoptotic effects of Ras could be reversed by mutations in its effector loop or by inhibitors of either farnesyl transferase or mitogen-activated protein kinase kinase. The expression of constitutively activated mitogen-activated protein kinase kinase 1 enhanced caspase 8 recruitment and sensitized immortalized human embryonic kidney cells to TRAIL-induced death. These results indicate that in normal human cells the TRAIL-induced apoptotic signal is blocked at the level of caspase 8 recruitment and that this block can be eliminated by Ras transformation, involving activation of the mitogen-activated protein kinase pathway.     

Bortezomib sensitizes primary human astrocytoma cells of WHO grades I to IV for tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis.

PURPOSE: Malignant gliomas are the most aggressive human brain tumors without any curative treatment. The antitumor effect of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in gliomas has thus far only been thoroughly established in tumor cell lines. In the present study, we investigated the therapeutic potential of TRAIL in primary human glioma cells. EXPERIMENTAL DESIGN: We isolated primary tumor cells from 13 astrocytoma and oligoastrocytoma patients of all four WHO grades of malignancy and compared the levels of TRAIL-induced apoptosis induction, long-term tumor cell survival, caspase, and caspase target cleavage. RESULTS: We established a stable culture model for isolated primary human glioma cells. In contrast to cell lines, isolated primary tumor cells from all investigated glioma patients were highly TRAIL resistant. Regardless of the tumor heterogeneity, cotreatment with the proteasome inhibitor bortezomib efficiently sensitized all primary glioma samples for TRAIL-induced apoptosis and tremendously reduced their clonogenic survival. Due to the pleiotropic effect of bortezomib-enhanced TRAIL DISC formation upon TRAIL triggering, down-regulation of cFLIP(L) and activation of the intrinsic apoptosis pathway seem to cooperatively contribute to the antitumor effect of bortezomib/TRAIL cotreatment. CONCLUSION: TRAIL sensitivity of tumor cell lines is not a reliable predictor for the behavior of primary tumor cells. The widespread TRAIL resistance in primary glioma cells described here questions the therapeutic clinical benefit of TRAIL as a monotherapeutic agent. Overcoming TRAIL resistance by bortezomib cotreatment might, however, provide a powerful therapeutic option for glioma patients.     

Sensitization for tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by the chemopreventive agent resveratrol

Survivin is a member of the inhibitor of apoptosis proteins that is expressed at high levels in most human cancers and may facilitate evasion from apoptosis and aberrant mitotic progression. Naturally occurring dietary compounds such as resveratrol have gained considerable attention as cancer chemopreventive agents. Here, we discovered a novel function of the chemopreventive agent resveratrol: resveratrol is a potent sensitizer of tumor cells for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis through p53-independent induction of p21 and p21-mediated cell cycle arrest associated with survivin depletion. Concomitant analysis of cell cycle, survivin expression, and apoptosis revealed that resveratrol-induced G(1) arrest was associated with down-regulation of survivin expression and sensitization for TRAIL-induced apoptosis. Accordingly, G(1) arrest using the cell cycle inhibitor mimosine or induced by p21 overexpression reduced survivin expression and sensitized cells for TRAIL treatment. Likewise, resveratrol-mediated cell cycle arrest followed by survivin depletion and sensitization for TRAIL was impaired in p21- deficient cells. Also, down-regulation of survivin using survivin antisense oligonucleotides sensitized cells for TRAIL-induced apoptosis. Importantly, resveratrol sensitized various tumor cell lines, but not normal human fibroblasts, for apoptosis induced by death receptor ligation or anticancer drugs. Thus, this combined sensitizer (resveratrol)/inducer (e.g., TRAIL) strategy may be a novel approach to enhance the efficacy of TRAIL-based therapies in a variety of human cancers.     

Bcl-2 inhibitors sensitize tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by uncoupling of mitochondrial respiration in human leukemic CEM cells

Previous studies have shown that the lymphoblastic leukemia CEM cell line is resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis because of a low expression of caspase-8. Bcl-2 inhibitors, BH3I-2' and HA14-1, are small cell-permeable nonpeptide compounds, are able to induce apoptosis by mediating cytochrome c release, and also lead to dissipation of the mitochondrial membrane potential (DeltaPsim). This study aimed to use the Bcl-2 inhibitors to sensitize CEM cells to TRAIL-induced apoptosis by switching on the mitochondrial apoptotic pathway. We found that a low dose of BH3I-2' or HA14-1, which did not induce cytochrome c release, greatly sensitized CEM cells to TRAIL-induced apoptosis. In a similar manner to the classical uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP), both BH3I-2' and HA14-1 induced a reduction in DeltaPsim, a generation of reactive oxygen species (ROS), an increased mitochondrial respiration, and a decreased ATP synthesis. This uncoupling function of the Bcl-2 inhibitors was responsible for the synergy with TRAIL-induced apoptosis. CCCP per se did not induce apoptosis but again sensitized CEM cells to TRAIL-induced apoptosis by uncoupling mitochondrial respiration. The uncoupling effect facilitated TRAIL-induced Bax conformational change and cytochrome c release from mitochondria. Inhibition of caspases failed to block TRAIL-mediated cell death when mitochondrial respiration was uncoupled. We observed that BH3I-2', HA14-1, or CCCP can overcome resistance to TRAIL-induced apoptosis in TRAIL-resistant cell lines, such as CEM, HL-60, and U937. Our results suggest that the uncoupling of mitochondrial respiration can sensitize leukemic cells to TRAIL-induced apoptosis. However, caspase activation per se does not represent an irreversible point of commitment to TRAIL-induced cell death when mitochondrial respiration is uncoupled.     

Mcl-1 mediates tumor necrosis factor-related apoptosis-inducing ligand resistance in human cholangiocarcinoma cells

Cholangiocarcinomas are usually fatal neoplasms originating from bile duct epithelia. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent for cancer therapy, including cholangiocarcinoma. However, many cholangiocarcinoma cells are resistant to TRAIL-mediated apoptosis. Thus, our aim was to examine the intracellular mechanisms responsible for TRAIL resistance in human cholangiocarcinoma cell lines. Three TRAIL-resistant human cholangiocarcinoma cell lines were identified. All of the cell lines expressed TRAIL receptor 1/death receptor 4 (TRAIL-R1/DR4) and TRAIL-R2/DR5. Expression of TRAIL decoy receptors and the antiapoptotic cellular FLICE-inhibitory protein (cFLIP) was inconsistent across the cell lines. Of the antiapoptotic Bcl-2 family of proteins profiled (Bcl-2, Bcl-x(L), and Mcl-1), Mcl-1 was uniquely overexpressed by the cell lines. When small-interfering-RNA (siRNA) technology was used to knock down expression of Bcl-2, Bcl-x(L), and Mcl-1, only the Mcl-1-siRNA sensitized the cells to TRAIL-mediated apoptosis. In a cell line stably transfected with Mcl-1-small-hairpin-RNA (Mcl-1-shRNA), Mcl-1 depletion sensitized cells to TRAIL-mediated apoptosis despite Bcl-2 expression. TRAIL-mediated apoptosis in the stably transfected cells was associated with mitochondrial depolarization, Bax activation, cytochrome c release from mitochondria, and caspase activation. Finally, flavopiridol, an anticancer drug that rapidly down-regulates Mcl-1, also sensitized cells to TRAIL cytotoxicity. In conclusion, these studies not only demonstrate that Mcl-1 mediates TRAIL resistance in cholangiocarcinoma cells by blocking the mitochondrial pathway of cell death but also identify two strategies for circumventing this resistance.     

Targeting the tumor necrosis factor-related apoptosis-inducing ligand path in neuroblastoma

The identification of the tumor necrosis factor (TNF) superfamily member TNF-related apoptosis-inducing ligand (TRAIL) a few years ago generated considerable enthusiasm for it as a potential cancer therapeutic agent. This is because TRAIL shows potent apoptosis inducing activity in a wide spectrum of transformed cell lines but not in cell lines derived from normal tissue origin. As the details in the signal transduction pathway of TRAIL-induced apoptosis are clarified, various defects of TRAIL pathway have been identified in TRAIL resistant cancer cells. Neuroblastoma is the most common extracranial solid tumor in children and those with a poor prognosis require more sensitive therapies. Unlike other cancer cells, most neuroblastoma cell lines are resistant to TRAIL induced apoptosis and the resistance correlates with caspase 8 deficiency, which is attributed to the methylation of the gene. Interferon (IFN)-gamma induces caspase 8 expression in most neuroblastoma cell lines regardless of the methylation status but fails to sensitize most NB to TRAIL. Further analysis indicates a TRAIL receptor deficiency contributes to TRAIL resistance in NB. Multiple lesions suggest that this path may play an important role in tumorigenesis and/ or evasion from therapies. Furthermore it indicates that the clinical application of TRAIL in NB will require a multi-modality approach. Important questions remain unanswered: How does IFN-gamma induce caspase 8 and why is the induction heterogeneous? How to stimulate the caspase 8 induction in cells that fail to respond to IFN-gamma? How to target other TRAIL pathway lesions with the clinically feasible approaches?     

Tumor necrosis factor-related apoptosis-inducing ligand-mediated proliferation of tumor cells with receptor-proximal apoptosis defects

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) might represent a future cytotoxic drug to treat cancer as it induces apoptosis in tumor cells without toxicity in animal trials. We recently described that in contrast to apoptosis, TRAIL mediates tumor cell survival and proliferation in certain tumor cells. Here we studied the effect of TRAIL on 18 cell lines and 53 primary leukemia cells and classified these tumor cells into four groups: TRAIL, anti-DR4 or anti-DR5 induced apoptosis in group A cells, whereas they had no effect on group 0 cells and mediated proliferation in group P cells. To our surprise, TRAIL induced simultaneous apoptosis and proliferation in group AP cells. More than 20% of all cells tested belonged to group P and showed TRAIL-mediated proliferation even in the presence of certain cytotoxic drugs but not inhibitors of nuclear factor-kappaB. Transfection with B-cell leukemia/lymphoma protein 2 transformed group A cells into group 0 cells, whereas transfection with Fas-associated polypeptide with death domain (FADD)-like interleukin-1-converting enzyme-inhibitory protein (FLIP) transformed them into group AP cells. Loss of caspase-8 or transfection of dominant-negative FADD transformed group A cells into group P cells. Taken together, our data suggest that proliferation is a frequent effect of TRAIL on tumor cells, which is related to receptor-proximal apoptosis defects at the level of the death-inducing signaling complex and should be prevented during antitumor therapy with TRAIL.     

Role of tumor necrosis factor-related apoptosis-inducing ligand in interferon-induced apoptosis in human bladder cancer cells

Immunomodulators such as Bacillus Calmette-Guerin and interferon are clinically active in transitional cell carcinoma of the bladder, but their mechanisms of action remain unclear. Here we investigated the effects of IFNalpha on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression and apoptosis in a panel of 20 human bladder cancer cell lines. Six (30%) displayed significant DNA fragmentation in response to increasing concentrations of IFNalpha (10-100,000 units/mL). In these lines IFNalpha induced early activation of caspase-8, and DNA fragmentation was blocked by a caspase-8-selective inhibitor (IETDfmk), consistent with the involvement of death receptor(s) in cell death. IFNalpha stimulated marked increases in TRAIL mRNA and protein in the majority of IFN-sensitive and IFN-resistant cell lines. A blocking anti-TRAIL antibody significantly inhibited IFN-induced DNA fragmentation in four of six IFN-sensitive cell lines, confirming that TRAIL played a direct role in cell death. Bortezomib (PS-341, Velcade), a potent TRAIL-sensitizing agent, increased sensitivity to IFNalpha in two of the IFN-resistant cell lines that produced large amounts of TRAIL in response to IFN treatment. Our data show that IFN-induced apoptosis in bladder cancer cells frequently involves autocrine TRAIL production. Combination therapy strategies aimed at overcoming TRAIL resistance may be very effective in restoring IFN sensitivity in a subset of human bladder tumors.     

Epidermal growth factor protects epithelial-derived cells from tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by inhibiting cytochrome c release.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in combination with chemotherapeutic drugs induces a synergistic apoptotic response in cancer cells. TRAIL death receptors have also been implicated in chemotherapeutic drug-induced apoptosis. This has lead to TRAIL being proposed as a potential cancer treatment. In nude mice injected with human tumors, TRAIL reduces the size of these tumors without toxic side effects. We demonstrate that the epidermal growth factor (EGF) stimulation of human embryonic kidney cells HEK 293 and breast cancer cell line MDA MB 231 effectively protects these cells from TRAIL-induced apoptosis in a dose-dependent manner. This stimulation blocks apoptosis by inhibiting TRAIL-mediated cytochrome c release from the mitochondria and caspase 3-like activation. EGF survival response involves the activation of AKT. Expression of activated AKT was sufficient to block TRAIL-induced apoptosis, and kinase-inactive AKT expression blocked EGF-protective response. In contrast, inhibition of EGF stimulation of extracellular-regulated kinase (ERK) activity did not affect EGF protection. These findings indicate that EGF receptor activation provides a survival response against TRAIL-induced apoptosis by inhibiting mitochondrial cytochrome c release that is mediated by AKT activation in epithelial-derived cells.