E1A sensitizes cancer cells to TRAIL-induced apoptosis through enhancement of caspase activation

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to induce apoptosis of cancer cells. Sensitization of cancer cells to TRAIL, particularly TRAIL-resistant cancer cells, could improve the effectiveness of TRAIL as an anticancer agent. The adenovirus type 5 E1A that associates with anticancer activities including sensitization to apoptosis induced by tumor necrosis factor is currently being tested in clinical trials. In this study, we investigated the sensitivity to TRAIL in the E1A transfectants ip1-E1A2 and 231-E1A cells and the parental TRAIL-resistant human ovarian cancer SKOV3.ip1 and TRAIL-sensitive human breast cancer MDA-MB-231 cells. The results indicated that the percentage of TRAIL-induced apoptotic cells was significantly higher in the E1A transfectants of both cell lines than it was in the parental cell lines. To further investigate the cellular mechanism of this effect, we found that E1A enhances TRAIL-induced activation of caspase-8, caspase-9, and caspase-3. Inhibition of caspase-3 activity by a specific inhibitor, Z-DEVD-fmk, abolished TRAIL-induced apoptosis. In addition, E1A enhanced TRAIL expression in ip1-E1A2 cells, but not in 231-E1A cells, and the anti-TRAIL neutralizing antibody N2B2 blocked the E1A-mediated bystander effect in vitro. Taken together, these results suggest that E1A sensitizes both TRAIL-sensitive and TRAIL-resistant cancer cells to TRAIL-induced apoptosis, which occurs through the enhancement of caspase activation; activation of caspase-3 is required for TRAIL-induced apoptosis; and E1A-induced TRAIL expression is involved in the E1A-mediated bystander effect. Combination of E1A and TRAIL could be an effective treatment for cancer.     

Celastrol, a Triterpene, Enhances TRAIL-induced Apoptosis through the Down-regulation of Cell Survival Proteins and Up-regulation of Death Receptors

Whether celastrol, a triterpene from traditional Chinese medicine, can modulate the anticancer effects of TRAIL, the cytokine that is currently in clinical trial, was investigated. As indicated by assays that measure plasma membrane integrity, phosphatidylserine exposure, mitochondrial activity, and activation of caspase-8, caspase-9, and caspase-3, celastrol potentiated the TRAIL-induced apoptosis in human breast cancer cells, and converted TRAIL-resistant cells to TRAIL-sensitive cells. When examined for its mechanism, we found that the triterpene down-regulated the expression of cell survival proteins including cFLIP, IAP-1, Bcl-2, Bcl-xL, survivin, and XIAP and up-regulated Bax expression. In addition, we found that celastrol induced the cell surface expression of both the TRAIL receptors DR4 and DR5. This increase in receptors was noted in a wide variety of cancer cells including breast, lung, colorectal, prostate, esophageal, and pancreatic cancer cells, and myeloid and leukemia cells. Gene silencing of the death receptor abolished the effect of celastrol on TRAIL-induced apoptosis. Induction of the death receptor by the triterpenoid was found to be p53-independent but required the induction of CAAT/enhancer-binding protein homologous protein (CHOP), inasmuch as gene silencing of CHOP abolished the induction of DR5 expression by celastrol and associated enhancement of TRAIL-induced apoptosis. We found that celastrol also induced reactive oxygen species (ROS) generation, and ROS sequestration inhibited celastrol-induced expression of CHOP and DR5, and consequent sensitization to TRAIL. Overall, our results demonstrate that celastrol can potentiate the apoptotic effects of TRAIL through down-regulation of cell survival proteins and up-regulation of death receptors via the ROS-mediated up-regulation of CHOP pathway.     

Inhibition of RIP and c-FLIP enhances TRAIL-induced apoptosis in pancreatic cancer cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has recently emerged as a cancer therapeutic agent because it is capable of preferentially inducing apoptosis in human cancer over normal cells. The majority of human pancreatic cancers, unfortunately, are resistant to TRAIL treatment. Here, we show that the inhibition of caspase-8 cleavage is the most upstream event in TRAIL resistance in pancreatic cancers. TRAIL treatment led to the cleavage of caspase-8 and downstream caspase-9, caspase-3, and DNA fragmentation factor 45 (DFF45) in TRAIL-sensitive pancreatic cancer cell lines (BXPC-3, PACA-2). This caspase-8-initiated caspase cascade, however, was inhibited in TRAIL-resistant pancreatic cancer cell lines (PANC-1, ASPC-1, CAPAN-1, CAPAN-2). The long and short forms of cellular Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein (c-FLIP(L), c-FLIP(S)) were highly expressed in the TRAIL-resistant as compared to the sensitive cells; knockdown of c-FLIP(L) and c-FLIP(S) by a short hairpin RNA (shRNA) rendered the resistant cells sensitive to TRAIL-induced apoptosis through the cleavage of caspase-8 and activation of the mitochondrial pathway. Receptor-interacting protein (RIP) has been reported in TRAIL-induced activation of NF-kappaB and we show here that knockdown of RIP sensitized the resistant cells to TRAIL-induced apoptosis. These results indicate the role of c-FLIP and RIP in caspase-8 inhibition and thus TRAIL resistance. Treatment of the resistant cells with camptothecin, celecoxib and cisplatin resulted in the downregulation of c-FLIP and caused a synergistic apoptotic effect with TRAIL. These studies therefore suggest that combination treatment with chemotherapy can overcome TRAIL resistance and enhance TRAIL therapeutic efficacy in treating pancreatic cancers.     

Inhibition of CaMKII-mediated c-FLIP expression sensitizes malignant melanoma cells to TRAIL-induced apoptosis

TRAIL can induce apoptosis in melanoma cells and thus may offer new hope for melanoma therapy. However, many melanoma cells are resistant to TRAIL. To examine molecular mechanisms in cell resistance, we analyzed TRAIL-induced DISC in TRAIL-sensitive melanoma cells and showed that apoptosis-initiating caspase-8 and caspase-10 were recruited to the DISC where they became activated through autocatalytical cleavage, leading to apoptosis through cleavage of downstream substrates such as caspase-3 and DFF45. In TRAIL-resistant melanoma cells, however, c-FLIP proteins were recruited to the DISC, resulting in the inhibition of caspase-8 and caspase-10 cleavage in the DISC. Both calmodulin-dependent protein kinase II (CaMKII) protein and enzymatic activity were upregulated in resistant cells and CaMKII inhibitor KN-93 downregulated expression of c-FLIP proteins, thus sensitizing resistant cells to TRAIL-induced apoptosis. Transfection of CaMKII cDNA in sensitive melanoma cells resulted in cell resistance to TRAIL, where transfection of CaMKII dominant-negative cDNA in resistant cells restored TRAIL sensitivity in cells. These results indicate that the CaMKII-mediated pathway for c-FLIP upregulation protects melanoma cells from TRAIL-induced apoptosis and targeting this pathway may provide novel therapeutic strategies in treatment of melanomas.     

Selective inhibition of FLICE-like inhibitory protein expression with small interfering RNA oligonucleotides is sufficient to sensitize tumor cells for TRAIL-induced apoptosis

BACKGROUND: Most tumors express death receptors and their activation represents a potential selective approach in cancer treatment. The most promising candidate for tumor selective death receptor-activation is tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo2L, which activates the death receptors TRAIL-R1 and TRAIL-R2, and induces apoptosis preferentially in tumor cells but not in normal tissues. However, many cancer cells are not or only moderately sensitive towards TRAIL and require cotreatment with irradiation or chemotherapy to yield a therapeutically reasonable apoptotic response. Because chemotherapy can have a broad range of unwanted side effects, more specific means for sensitizing tumor cells for TRAIL are desirable. The expression of the cellular FLICE-like inhibitory protein (cFLIP) is regarded as a major cause of TRAIL resistance. We therefore analyzed the usefulness of targeting FLIP to sensitize tumor cells for TRAIL-induced apoptosis. MATERIALS AND METHODS: To selectively interfere with expression of cFLIP short double-stranded RNA oligonucleotides (small interfering RNAs [siRNAs]) were introduced in the human cell lines SV80 and KB by electroporation. Effects of siRNA on FLIP expression were analyzed by Western blotting and RNase protection assay and correlated with TRAIL sensitivity upon stimulation with recombinant soluble TRAIL and TRAIL-R1- and TRAIL-R2-specific agonistic antibodies. RESULTS: FLIP expression can be inhibited by RNA interference using siRNAs, evident from reduced levels of FLIP-mRNA and FLIP protein. Inhibition of cFLIP expression sensitizes cells for apoptosis induction by TRAIL and other death ligands. In accordance with the presumed function of FLIP as an inhibitor of death receptor-induced caspase-8 activation, down-regulation of FLIP by siRNAs enhanced TRAIL-induced caspase-8 activation. CONCLUSION: Inhibition of FLIP expression was sufficient to sensitize tumor cells for TRAIL-induced apoptosis. The combination of TRAIL and FLIP-targeting siRNA could therefore be a useful strategy to attack cancer cells, which are resistant to TRAIL alone.     

The small heat shock protein alpha B-crystallin is a novel inhibitor of TRAIL-induced apoptosis that suppresses the activation of caspase-3

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor alpha family of cytokines that preferentially induces apoptosis in transformed cells, making it a promising cancer therapy. However, many neoplasms are resistant to TRAIL-induced apoptosis by mechanisms that are poorly understood. We demonstrate that the expression of the small heat shock protein alpha B-crystallin (but not other heat shock proteins or apoptosis-regulating proteins) correlates with TRAIL resistance in a panel of human cancer cell lines. Stable expression of wild-type alpha B-crystallin, but not a pseudophosphorylation mutant impaired in its assembly and chaperone function, protects cancer cells from TRAIL-induced caspase-3 activation and apoptosis in vitro. Furthermore, selective inhibition of alpha B-crystallin expression by RNA interference sensitizes cancer cells to TRAIL. In addition, wild-type alpha B-crystallin promotes xenograft tumor growth and inhibits TRAIL-induced apoptosis in vivo in nude mice, whereas a pseudophosphorylation alpha B-crystallin mutant impaired in its anti-apoptotic function inhibits xenograft tumor growth. Collectively, these findings indicate that alpha B-crystallin is a novel regulator of TRAIL-induced apoptosis and tumor growth. Moreover, these results demonstrate that targeted inhibition of alpha B-crystallin promotes TRAIL-induced apoptosis, thereby suggesting a novel strategy to overcome TRAIL resistance in cancer.     

PTHrP Overexpression Increases Sensitivity of Breast Cancer Cells to Apo2L/TRAIL

Parathyroid hormone-related protein (PTHrP) is a key component in breast development and breast tumour biology. PTHrP has been discovered as a causative agent of hypercalcaemia of malignancy and is also one of the main factors implicated in breast cancer mediated osteolysis. Clinical studies have determined that PTHrP expression by primary breast cancers was an independent predictor of improved prognosis. Furthermore, PTHrP has been demonstrated to cause tumour cell death both in vitro and in vivo. Apo2L/TRAIL is a promising new anti-cancer agent, due to its ability to selectively induce apoptosis in cancer cells whilst sparing most normal cells. However, some cancer cells are resistant to Apo2L/TRAIL-induced apoptosis thus limiting its therapeutic efficacy. The effects of PTHrP on cell death signalling pathways initiated by Apo2L/TRAIL were investigated in breast cancer cells. Expression of PTHrP in Apo2L/TRAIL resistant cell line MCF-7 sensitised these cells to Apo2L/TRAIL-induced apoptosis. The actions of PTHrP resulted from intracellular effects, since exogenous treatment of PTHrP had no effect on Apo2L/TRAIL-induced apoptosis. Apo2L/TRAIL-induced apoptosis in PTHrP expressing cells occurred through the activation of caspase-10 resulting in caspase-9 activation and induction of apoptosis through the effector caspases, caspase-6 and -7. PTHrP increased cell surface expression of Apo2L/TRAIL death receptors, TRAIL-R1 and TRAIL-R2. Antagonistic antibodies against the death receptors demonstrated that Apo2L/TRAIL mediated its apoptotic signals through activation of the TRAIL-R2 in PTHrP expressing breast cancer cells. These studies reveal a novel role for PTHrP with Apo2L/TRAIL that maybe important for future diagnosis and treatment of breast cancer.     

The effect of epigenetic regulation of fucosylation on TRAIL-induced apoptosis

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in many cancer cells but not in normal ones. Recombinant TRAIL and agonistic antibodies to its cognate receptors are currently being studied as promising anticancer drugs. However, preclinical and clinical studies have shown that many types of human cancers are resistant to TRAIL agonists. We previously reported that a deficiency of fucosylation, which is one of the most common oligosaccharide modifications, leads to resistance to TRAIL-induced apoptosis. In contrast, DNA methylation is associated with silencing of various tumor suppressor genes and resistance of cancer cells to anticancer drugs. The aim of this study is to clarify the involvement of DNA methylation in the regulation of cellular fucosylation and the susceptibility to TRAIL-induced apoptosis. When nineteen cancer cell lines with relatively low fucosylation levels were treated with a novel methyltransferase inhibitor, zebularine, an increase in the fucosylation level was observed in many cancer cell lines. The expression of fucosylation-related genes, such as the FX, GDP-fucose transporter, and Fut4 genes, was significantly increased after the treatment with zebularine. Moreover, a synergistic effect of zebularine on TRAIL-induced apoptosis was observed in several cancer cell lines, in which fucosylation was increased by treatment with zebularine. This synergistic effect was independent of the expression of TRAIL receptors and caspase-8. These results indicate that cellular fucosylation is regulated through DNA methylation in many cancer cells. Moreover, zebularine might be useful as a combination drug with TRAIL-based therapies in patients with TRAIL-resistant cancer.     

The Oncogene Metadherin Modulates the Apoptotic Pathway Based on the Tumor Necrosis Factor Superfamily Member TRAIL (Tumor Necrosis Factor-related Apoptosis-inducing Ligand) in Breast Cancer

Metadherin (MTDH), the newly discovered gene, is overexpressed in more than 40% of breast cancers. Recent studies have revealed that MTDH favors an oncogenic course and chemoresistance. With a number of breast cancer cell lines and breast tumor samples, we found that the relative expression of MTDH correlated with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) sensitivity in breast cancer. In this study, we found that knockdown of endogenous MTDH cells sensitized the MDA-MB-231 cells to TRAIL-induced apoptosis both in vitro and in vivo. Conversely, stable overexpression of MTDH in MCF-7 cells enhanced cell survival with TRAIL treatment. Mechanically, MTDH down-regulated caspase-8, decreased caspase-8 recruitment into the TRAIL death-inducing signaling complex, decreased caspase-3 and poly(ADP-ribose) polymerase-2 processing, increased Bcl-2 expression, and stimulated TRAIL-induced Akt phosphorylation, without altering death receptor status. In MDA-MB-231 breast cancer cells, sensitization to TRAIL upon MTDH down-regulation was inhibited by the caspase inhibitor Z-VAD-fmk (benzyloxycarbonyl-VAD-fluoromethyl ketone), suggesting that MTDH depletion stimulates activation of caspases. In MCF-7 breast cancer cells, resistance to TRAIL upon MTDH overexpression was abrogated by depletion of Bcl-2, suggesting that MTDH-induced Bcl-2 expression contributes to TRAIL resistance. We further confirmed that MTDH may control Bcl-2 expression partly by suppressing miR-16. Collectively, our results point to a protective function of MTDH against TRAIL-induced death, whereby it inhibits the intrinsic apoptosis pathway through miR-16-mediated Bcl-2 up-regulation and the extrinsic apoptosis pathway through caspase-8 down-regulation.     

C-FLIP(L) contributes to TRAIL resistance in HER2-positive breast cancer

Breast cancers with HER2 amplification have a poorer prognosis than the luminal phenotypes. TRAIL activates apoptosis upon binding its receptors in some but not all breast cancer cell lines. Herein, we investigated the expression pattern of c-FLIP(L) in a cohort of 251 invasive breast cancer tissues and explored its potential role in TRAIL resistance. C-FLIP(L) was relatively high-expressed in HER2-positive breast cancer in comparison with other molecular subtypes, co-expressed with TRAIL death receptors, and inversely correlated with the apoptosis index. Downregulation of c-FLIP(L) sensitized SKBR3 cells to TRAIL-induced apoptosis in a concentration- and time-dependent manner and enhanced the activities and cleavages of caspase-8 and caspase-3, without altering the surface expression of death receptors. Together, our results indicate that c-FLIP(L) promotes TRAIL resistance and inhibits caspase-3 and caspase-8 activation in HER2-positive breast cancer.     

ZD1839 (Gefitinib, 'Iressa'), an epidermal growth factor receptor-tyrosine kinase inhibitor, enhances the anti-cancer effects of TRAIL in human esophageal squamous cell carcinoma

The EGF (epidermal growth factor) receptor-tyrosine kinase inhibitor ZD1839 (Gefitinib, 'Iressa') blocks the cell signaling pathways involved in cell proliferation, survival, and angiogenesis in various cancer cells. TNF-related death apoptosis inducing ligand (TRAIL) acts as an anticancer agent. We investigated the antitumor effects of ZD1839 alone or in combination with TRAIL against human esophageal squamous cell cancer (ESCC) lines. Although all ESCC cells expressed EGF receptor at a protein level, the effect of ZD1839 on cell growth did not correlate with the level of EGFR expression and phosphorylation of EGF receptor protein in ESCC lines. ZD1839 caused a dose-dependent growth arrest at G0-G1 phase associated with increased p27 expression. As TE8 cells are resistant to TRAIL, we tested whether ZD1839 combined with TRAIL induced apoptosis of TE8 cells via the inhibition of EGF receptor signaling by ZD1839. ZD1839 inhibited the phosphorylation of Akt, and enhanced TRAIL-induced apoptosis via activation of caspase-3 and caspase-9, and inactivation of Bcl-xL. Our results indicated that ZD1839 has anti-cancer properties against human esophageal cancer cells. ZD1839 also augmented the anti-cancer activity of TRAIL, even in TRAIL-resistant tumors. These results suggest that treatment with ZD1839 and TRAIL may have potential in the treatment of ESCC patients.     

Mitogen-activated protein kinase/extracellular signal-regulated kinase signaling in activated T cells abrogates TRAIL-induced apoptosis upstream of the mitochondrial amplification loop and caspase-8

Fas ligand and TNF-related apoptosis-inducing ligand (TRAIL) induce apoptosis in many different cell types. Jurkat T cells die rapidly by apoptosis after treatment with either ligand. We have previously shown that mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) can act as a negative regulator of apoptosis mediated by the Fas receptor. In this study we examined whether MAPK/ERK can also act as a negative regulator of apoptosis induced by TRAIL. Activated Jurkat T cells were efficiently protected from TRAIL-induced apoptosis. The protection was shown to be MAPK/ERK dependent and independent of protein synthesis. MAPK/ERK suppressed TRAIL-induced apoptosis upstream of the mitochondrial amplification loop because mitochondrial depolarization and release of cytochrome c were inhibited. Furthermore, caspase-8-mediated relocalization and activation of Bid, a proapoptotic member of the Bcl family, was also inhibited by the MAPK/ERK signaling. The protection occurred at the level of the apoptotic initiator caspase-8, as the cleavage of caspase-8 was inhibited but the assembly of the death-inducing signaling complex was unaffected. Both TRAIL and Fas ligand have been suggested to regulate the clonal size and persistence of different T cell populations. Our previous results indicate that MAPK/ERK protects recently activated T cells from Fas receptor-mediated apoptosis during the initial phase of an immune response before the activation-induced cell death takes place. The results of this study show clearly that MAPK/ERK also participates in the inhibition of TRAIL-induced apoptosis after T cell activation.     

Caspase-8 dependent trail-induced apoptosis in cancer cell lines is inhibited by vitamin C and catalase

TNF-related apoptosis-inducing ligand (TRAIL/ Apo-2L) is a member of the TNF family of apoptosis-inducing proteins that initiates apoptosis in a variety of neoplastic cells while displaying minimal or absent cytotoxicity to most normal cells. Therefore, TRAIL is currently considered a promising target to develop anti-cancer therapies. TRAIL-receptor ligation recruits and activates pro-caspase-8, which in turn activates proteins that mediate disruption of the mitochondrial membranes. These events lead to the nuclear and cytosolic damage characteristic of apoptosis. Here we report that TRAIL-induced apoptosis is mediated by oxidative stress and that vitamin C (ascorbic acid), a potent nutritional antioxidant, protects cancer cell lines from apoptosis induced by TRAIL. Vitamin C impedes the elevation of reactive oxygen species (ROS) levels induced by TRAIL and impairs caspase-8 activation. We found that the removal of hydrogen peroxide by extracellular catalase during TRAIL-induced apoptosis also impairs caspase-8 activation. These data suggest that hydrogen peroxide is produced during TRAIL-receptor ligation, and that the increase of intracellular ROS regulates the activation of caspase-8 during apoptosis. Additionally we propose a mechanism by which cancer cells might resist apoptosis via TRAIL, by the intake of the nutritional antioxidant vitamin C. This work was supported by grants from the National Institutes of Health (CA 30388), the New York State Department of Health (M020113) and the Lebensfeld Foundation.     

Snake venom toxin from Vipera lebetina turanica sensitizes cancer cells to TRAIL through ROS- and JNK-mediated upregulation of death receptors and downregulation of survival proteins

We investigated whether snake venom toxin (SVT) from Vipera lebetina turanica enhances the apoptosis ability of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) in cancer cells. TRAIL inhibited HCT116 cell growth in a dose-dependent manner; however, this reduction did not occur in TRAIL resistant HT-29, A549 and HepG2 cells with an even higher dose of TRAIL. SVT, but not TRAIL enhanced expression of cell death receptor (DR) in TRAIL resistant cancer cells in a dose-dependent manner. A combination of SVT with TRAIL significantly inhibited cell growth of TRAIL resistant HT-29, A549 and HepG2 cells. Consistent with cell growth inhibition, the expression of TRAIL receptors; DR4 and DR5 was significantly increased as well as apoptosis related proteins such as cleaved caspase-3, -8, -9 and Bax. However, the expression of survival proteins (e.g., cFLIP, survivin, XIAP and Bcl2) was suppressed by the combination treatment of SVT and TRAIL. Depletion of DR4 or DR5 by small interfering RNA significantly reversed the cell growth inhibitory and apoptosis blocking effects of SVT in HCT116 and HT-29 cells. Pretreatment with the c-Jun N-terminal kinase (JNK) inhibitor SP600125 and the reactive oxygen species (ROS) scavenger N-acetylcysteine reduced the SVT and TRAIL-induced upregulation of DR4 and DR5 expression, expression of the apoptosis related protein such as caspase-3 and-9, as well as cell growth inhibitory effects. The collective results suggest that SVT facilitates TRAIL-induced apoptosis in cancer cells through up-regulation of the TRAIL receptors; DR4 and DR5 via ROS/JNK pathway signals.     

The adaptor protein FADD and the initiator caspase-8 mediate activation of NF-��B by TRAIL

Besides inducing apoptosis, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) activates NF-κB. The apoptosis signaling pathway of TRAIL is well characterized involving TRAIL receptors, Fas-associated protein with death domain (FADD) and caspase-8. In contrast, the molecular mechanism of TRAIL signaling to NF-κB remains controversial. Here, we characterized the receptor–proximal mediators of NF-κB activation by TRAIL. Deletion of the DD of TRAIL receptors 1 and 2 revealed that it is essential in NF-κB signaling. Because FADD interacts with the TRAIL receptor DD, FADD was tested. RNAi-mediated knockdown of FADD or FADD deficiency in JURKAT T-cell leukemia cells decreased or disabled NF-κB signaling by TRAIL. In contrast, TRAIL-induced activation of NF-κB was maintained upon loss of receptor interacting protein 1 (RIP1) or knockdown of FLICE-like inhibitory protein (FLIP). Exogenous expression of FADD rescued TRAIL-induced NF-κB signaling. Loss-of-function mutations of FADD within the RHDLL motif of the death effector domain, which is required for TRAIL-induced apoptosis, abrogated FADD''s ability to recruit caspase-8 and mediate NF-κB activation. Accordingly, deficiency of caspase-8 inhibited TRAIL-induced activation of NF-κB, which was rescued by wild-type caspase-8, but not by a catalytically inactive caspase-8 mutant. These data establish the mechanism of TRAIL-induced NF-κB activation involving the TRAIL receptor DD, FADD and caspase-8, but not RIP1 or FLIP. Our results show that signaling of TRAIL-induced apoptosis and NF-κB bifurcates downstream of caspase-8.     

Reconstitution of caspase-8 sensitizes JB6 cells to TRAIL

TRAIL induces apoptosis in various tumor cells. We report here that caspase-8 is required in TRAIL-induced cell death. Western blot analyses and enzyme assays showed that exposing Jurkat cells to TRAIL resulted in activation of caspases-8 followed by caspase-3 and -9. Acetyl-IETD-fluoromethylketone, a caspase-8 inhibitor, potently suppressed TRAIL-induced cell death compared to acetyl-DEVD-fluoromethylketone and acetyl-LEHD-fluoromethylketone, inhibitors of caspase-3 and caspase-9, respectively. JB6 cells, a caspase-8-deficient Jurkat variant, were completely resistant to TRAIL. However, reconstitution with a caspase-8, but not with caspase-2 or -3, sensitized JB6 cells to subsequent exposure to TRAIL. These results are indicative of the crucial function of caspase-8 in TRAIL-induced apoptosis in Jurkat cells.     

The Deubiquitinase Inhibitor PR-619 Sensitizes Normal Human Fibroblasts to Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL)-mediated Cell Death

TNF-related apoptosis-inducing ligand (TRAIL) is a potential cancer therapy that selectively targets cancer cell death while non-malignant cells remain viable. Using a panel of normal human fibroblasts, we characterized molecular differences in human foreskin fibroblasts and WI-38 TRAIL-resistant cells and marginally sensitive MRC-5 cells compared with TRAIL-sensitive human lung and colon cancer cells. We identified decreased caspase-8 protein expression and protein stability in normal fibroblasts compared with cancer cells. Additionally, normal fibroblasts had incomplete TRAIL-induced caspase-8 activation compared with cancer cells. We found that normal fibroblasts lack the ubiquitin modification of caspase-8 required for complete caspase-8 activation. Treatment with the deubiquitinase inhibitor PR-619 increased caspase-8 ubiquitination and caspase-8 enzymatic activity and sensitized normal fibroblasts to TRAIL-mediated apoptosis. Therefore, posttranslational regulation of caspase-8 confers resistance to TRAIL-induced cell death in normal cells through blockade of initiation of the extrinsic cell death pathway.     

Roscovitine sensitizes breast cancer cells to TRAIL-induced apoptosis through a pleiotropic mechanism

The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/APO2L) is a member of the TNF gene superfamily that induces apoptosis upon engagement of cognate death receptors. While TRAIL is relatively non-toxic to normal cells, it selectively induces apoptosis in many transformed cells. Nevertheless, breast tumor cells are particularly resistant to the effects of TRAIL. Here we report that, in combination with the cyclin-dependent kinase inhibitor roscovitine, exposure to TRAIL induced marked apoptosis in the majority of TRAIL-resistant breast cancer cell lines examined. Roscovitine facilitated TRAIL death-inducing signaling complex formation and the activation of caspase-8. The cFLIP(L) and cFLIP(S) FLICE-inhibitory proteins were significantly down-regulated following exposure to roscovitine and, indeed, the knockdown of cFLIP isoforms by siRNA sensitized breast tumor cells to TRAIL-induced apoptosis. In addition, we demonstrate that roscovitine strongly suppressed Mcl-1 expression and up-regulated E2F1 protein levels in breast tumor cells. Significantly, the silencing of Mcl-1 by siRNA sensitized breast tumor cells to TRAIL-induced apoptosis. Furthermore, the knockdown of E2F1 protein by siRNA reduced the sensitizing effect of roscovitine in TRAIL-induced apoptosis. In summary, our results reveal a pleitropic mechanism for the pro-apoptotic influence of roscovitine, highlighting its potential as an antitumor agent in breast cancer in combination with TRAIL.     

Inhibition of TRAIL-induced apoptosis by IL-8 is mediated by the p38-MAPK pathway in OVCAR3 cells

Introduction: TRAIL (TNF-Related Apoptosis Inducing Ligand) is a member of the TNF superfamily of cell death inducing ligands. Interestingly, while malignant cells are responsive to TRAIL-induced cell death when used alone or in combination with other agents, normal cells do not appear to be sensitive to this ligand, making it a desirable therapeutic compound against many cancers, including many ovarian carcinomas. Interleukin-8 (IL-8), a member of the C-X-C chemokine family, has been found to be at significantly higher level in the ascites from patients with ovarian cancer. We have previously demonstrated a role for IL-8 in blocking TRAIL's ability to induce apoptosis in the ovarian cancer cell line, OVCAR3, possibly by repressing the DR4 TRAIL receptor expression and blocking caspase-8 cleavage. In addition, we showed a member of the mitogen-activated protein kinase (MAPK) superfamily, p38γ, is among the genes regulated in OVCAR3 cells by TRAIL and IL-8. The present study further investigates involvement of the p38 MAPK pathway in IL-8's ability to block TRAIL-induced apoptosis in the ovarian surface epithelial cancer cell line, OVCAR3. Results: In this study we demonstrate that p38γ as well as p38α play a significant role in IL-8's ability to block TRAIL-induced apoptosis. Through array analysis, as well as confirmation with other methods, we detected regulation of p38γ and p38α following treatment of the cancer cell line with IL-8 or TRAIL. We also tested two other isoforms of p38 MAPK, p38β and p38δ, but did not find significant regulation by IL-8 or TRAIL. We also examined activation of the p38 MAPK pathway, up-stream as well as down-stream, and noticed activation of the pathway following treatment with TRAIL and decreased activity when IL-8 was introduced. With the use of specific inhibitors, we were able to further confirm the role of this pathway in TRAIL-induced apoptosis, and IL-8's ability to block this apoptosis, in ovarian cancer cell lines. Conclusion: Taken together, these results further solidify the role of IL-8 in blocking the TRAIL-induced apoptosis in these ovarian carcinoma cells and provide new molecular insight into this potentially important therapeutic target.     

Pro-survival function of Akt/protein kinase B in prostate cancer cells. Relationship with TRAIL resistance.

Tumor necrosis factor superfamily member TRAIL/Apo-2L has recently been shown to induce apoptosis in transformed and cancer cells. Some prostate cancer cells express constitutively active Akt/protein kinase B due to a complete loss of lipid phosphatase PTEN gene, a negative regulator of phosphatidylinositol 3-kinase pathway. Constitutively active Akt promotes cellular survival and resistance to chemotherapy and radiation. We have recently noticed that some human prostate cancer cells are resistant to TRAIL. We therefore examined the intracellular mechanisms of cellular resistance to TRAIL. The cell lines expressing the highest level of constitutively active Akt were more resistant to undergo apoptosis by TRAIL than those expressing the lowest level. Down-regulation of constitutively active Akt by phosphatidylinositol 3-kinase inhibitors, wortmannin and LY294002, reversed cellular resistance to TRAIL. Treatment of resistant cells with cycloheximide (a protein synthesis inhibitor) rendered cells sensitive to TRAIL. Transfecting dominant negative Akt decreased Akt activity and increased TRAIL-induced apoptosis in cells with high Akt activity. Conversely, transfecting constitutively active Akt into cells with low Akt activity increased Akt activity and attenuated TRAIL-induced apoptosis. Inhibition of TRAIL sensitivity occurs at the level of BID cleavage, as caspase-8 activity was not affected. Enforced expression of anti-apoptotic protein Bcl-2 or Bcl-X(L) inhibited TRAIL-induced mitochondrial dysfunction and apoptosis. We therefore identify Akt as a constitutively active kinase that promotes survival of prostate cancer cells and demonstrate that modulation of Akt activity, by pharmacological or genetic approaches, alters the cellular responsiveness to TRAIL. Thus, TRAIL in combination with agents that down-regulate Akt activity can be used to treat prostate cancer.