Identification of inhibitors of TRAIL-induced death (ITIDs) in the TRAIL-sensitive colon carcinoma cell line SW480 using a genetic approach

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in tumor cell lines, whereas normal cells appear to be protected from its cytotoxic effects. Therefore TRAIL holds promise as a potential therapeutic agent against cancer. To elucidate some of the critical factors that contribute to TRAIL resistance, we performed a genetic screen in the human colon carcinoma cell line SW480 by infecting this TRAIL-sensitive cell line with a human placental cDNA retroviral library and isolating TRAIL-resistant clones. Characterization of the resulting clones for inhibitors of TRAIL-induced death (ITIDs) led to the isolation of c-FLIP(S), Bax inhibitor 1, and Bcl-XL as candidate suppressors of TRAIL signaling. We have demonstrated that c-FLIP(S) and Bcl-XL are sufficient when overexpressed to convey resistance to TRAIL treatment in previously sensitive cell lines. Furthermore both c-FLIP(S) and Bcl-XL protected against overexpression of the TRAIL receptors DR4 and KILLER/DR5. When c-FLIP(S) and Bcl-XL were overexpressed together in SW480 and HCT 116, an additive inhibitory effect was observed after TRAIL treatment suggesting that these two molecules function in the same pathway in the cell lines tested. Furthermore, we have demonstrated for the first time that a proapoptotic member of the Bcl-2 family, Bax, is required for TRAIL-mediated apoptosis in HCT 116 cells. Surprisingly, we have found that the serine/threonine protein kinase Akt, which is an upstream regulator of both c-FLIP(S) and Bcl-XL, is not sufficient when overexpressed to protect against TRAIL in the cell lines tested. These results suggest a key role for c-FLIP(S), Bcl-XL, and Bax in determining tumor cell sensitivity to TRAIL.     

Ionizing radiation can overcome resistance to TRAIL in TRAIL-resistant cancer cells

Although the majority of cancer cells are killed by TRAIL (tumor necrosis factor-related apoptosis-inducing ligand treatment), certain types show resistance to it. Ionizing radiation also induces cell death in cancer cells and may share common intracellular pathways with TRAIL leading to apoptosis. In the present study, we examined whether ionizing radiation could overcome TRAIL resistance in the variant Jurkat clones. We first selected TRAIL-resistant or -sensitive Jurkat clones and examined cross-responsiveness of the clones between TRAIL and radiation. Treatment with gamma-radiation induced significant apoptosis in all the clones, indicating that there seemed to be no cross-resistance between TRAIL and radiation. Combined treatment of radiation with TRAIL synergistically enhanced killing of TRAIL-resistant cells, compared to TRAIL or radiation alone. Apoptosis induced by combined treatment of TRAIL and radiation in TRAIL-resistant cells was associated with cleavage of caspase-8 and the proapoptotic Bid protein, resulting in the activation of caspase-9 and caspase-3. No changes in the expressions of TRAIL receptors (DR4 and DR5) and Bcl-2 or Bax were found after treatment. The caspase inhibitor z-VAD-fmk completely counteracted the synergistic cell killing induced by combined treatment of TRAIL and gamma-radiation. These results demonstrated that ionizing radiation in combination with TRAIL could overcome resistance to TRAIL in TRAIL-resistant cells through TRAIL receptor-independent synergistic activation of the cascades of the caspase-8 pathway, suggesting a potential clinical application of combination treatment of TRAIL and ionizing radiation to TRAIL-resistant cancer cells.     

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.     

The Herbal Compound Cryptotanshinone Restores Sensitivity in Cancer Cells That Are Resistant to the Tumor Necrosis Factor-related Apoptosis-inducing Ligand

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis and kills cancer cells but not normal cells. However, TRAIL resistance due to low level of TRAIL receptor expression is widely found in cancer cells and hampers its development for cancer treatment. Thus, the agents that can sensitize the tumor cells to TRAIL-mediated apoptosis are urgently needed. We investigated whether tanshinones, the major bioactive compounds of Salvia miltiorrhiza (danshen), can up-regulate TRAIL receptor expression. Among the major tanshinones being tested, cryptotanshinone (CT) showed the best ability to induce TRAIL receptor 2 (DR5) expression. We further showed that CT was capable of promoting TRAIL-induced cell death and apoptosis in A375 melanoma cells. CT-induced DR5 induction was not cell type-specific, as DR5 induction was observed in other cancer cell types. DR5 knockdown abolished the enhancing effect of CT on TRAIL responses. Mechanistically, induction of the DR5 by CT was found to be p53-independent but dependent on the induction of CCAAT/enhancer-binding protein-homologous protein (CHOP). Knockdown of CHOP abolished CT-induced DR5 expression and the associated potentiation of TRAIL-mediated cell death. In addition, CT-induced ROS production preceded up-regulation of CHOP and DR5 and consequent sensitization of cells to TRAIL. Interestingly, CT also converted TRAIL-resistant lung A549 cancer cells into TRAIL-sensitive cells. Taken together, our results indicate that CT can potentiate TRAIL-induced apoptosis through up-regulation of DR5.     

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.     

GDP-mannose-4,6-dehydratase (GMDS) deficiency renders colon cancer cells resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor- and CD95-mediated apoptosis by inhibiting complex II formation

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis through binding to TRAIL receptors, death receptor 4 (DR4), and DR5. TRAIL has potential therapeutic value against cancer because of its selective cytotoxic effects on several transformed cell types. Fucosylation of proteins and lipids on the cell surface is a very important posttranslational modification that is involved in many cellular events. Recently, we found that a deficiency in GDP-mannose-4,6-dehydratase (GMDS) rendered colon cancer cells resistant to TRAIL-induced apoptosis, resulting in tumor development and metastasis by escape from tumor immune surveillance. GMDS is an indispensable regulator of cellular fucosylation. In this study, we investigated the molecular mechanism of inhibition of TRAIL signaling by GMDS deficiency. DR4, but not DR5, was found to be fucosylated; however, GMDS deficiency inhibited both DR4- and DR5-mediated apoptosis despite the absence of fucosylation on DR5. In addition, GMDS deficiency also inhibited CD95-mediated apoptosis but not the intrinsic apoptosis pathway induced by anti-cancer drugs. Binding of TRAIL and CD95 ligand to their cognate receptors primarily leads to formation of a complex comprising the receptor, FADD, and caspase-8, referred to as the death-inducing signaling complex (DISC). GMDS deficiency did not affect formation of the primary DISC or recruitment to and activation of caspase-8 on the DISC. However, formation of secondary FADD-dependent complex II, comprising caspase-8 and cFLIP, was significantly inhibited by GMDS deficiency. These results indicate that GMDS regulates the formation of secondary complex II from the primary DISC independent of direct fucosylation of death receptors.     

Evaluating the Effect of Therapeutic Stem Cells on TRAIL Resistant and Sensitive Medulloblastomas

Mesenchymal stem cells (MSC) are emerging as novel cell-based delivery agents; however, a thorough investigation addressing their therapeutic potential in medulloblastomas (MB) has not been explored to date. In this study, we engineered human MSC to express a potent and secretable variant of a tumor specific agent, tumor necrosis factor-apoptosis-inducing ligand (S-TRAIL) and assessed the ability of MSC-S-TRAIL mediated MB killing alone or in combination with a small molecule inhibitor of histone-deacetylase, MS-275, in TRAIL-sensitive and -resistant MB in vitro and in vivo. We show that TRAIL sensitivity/resistance correlates with the expression of its cognate death receptor (DR)5 and MSC-S-TRAIL induces caspase-3 mediated apoptosis in TRAIL-sensitive MB lines. In TRAIL-resistant MB, we show upregulation of DR4/5 levels when pre-treated with MS-275 and a subsequent sensitization to MSC-S-TRAIL mediated apoptosis. Using intracranially implanted MB and MSC lines engineered with different combinations of fluorescent and bioluminescent proteins, we show that MSC-S-TRAIL has significant anti-tumor effects in mice bearing TRAIL-sensitive and MS-275 pre-treated TRAIL-resistant MBs. To our knowledge, this is the first study that explores the use of human MSC as MB-targeting therapeutic-vehicles in vivo in TRAIL-sensitive and resistant tumors, and has implications for developing effective therapies for patients with medulloblastomas.     

Targeting c-Met Receptor Overcomes TRAIL-Resistance in Brain Tumors

Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) induced apoptosis specifically in tumor cells. However, with approximately half of all known tumor lines being resistant to TRAIL, the identification of TRAIL sensitizers and their mechanism of action become critical to broadly use TRAIL as a therapeutic agent. In this study, we explored whether c-Met protein contributes to TRAIL sensitivity. We found a direct correlation between the c-Met expression level and TRAIL resistance. We show that the knock down c-Met protein, but not inhibition, sensitized brain tumor cells to TRAIL-mediated apoptosis by interrupting the interaction between c-Met and TRAIL cognate death receptor (DR) 5. This interruption greatly induces the formation of death-inducing signaling complex (DISC) and subsequent downstream apoptosis signaling. Using intracranially implanted brain tumor cells and stem cell (SC) lines engineered with different combinations of fluorescent and bioluminescent proteins, we show that SC expressing a potent and secretable TRAIL (S-TRAIL) have a significant anti-tumor effect in mice bearing c-Met knock down of TRAIL-resistant brain tumors. To our best knowledge, this is the first study that demonstrates c-Met contributes to TRAIL sensitivity of brain tumor cells and has implications for developing effective therapies for brain tumor patients.     

Alpha-tocopheryl succinate induces DR4 and DR5 expression by a p53-dependent route: implication for sensitisation of resistant cancer cells to TRAIL apoptosis

We evaluated the ability of alpha-tocopheryl succinate (alpha-TOS) to sensitise TRAIL-resistant malignant mesothelioma (MM) cells to TRAIL-induced apoptosis. We show that alpha-TOS activates expression of DR4/DR5 in a p53-dependent manner and re-establishes sensitivity of resistant MM cells to TRAIL-mediated apoptosis, as documented in p53wt MM cells but not in their p53null counterparts. MM cells selected for TRAIL resistance expressed low cell surface levels of DR4 and DR5. Treatment with sub-lethal doses of alpha-TOS restored expression of DR4 and DR5. The ability of alpha-TOS to modulate expression of pro-apoptotic genes may play a role in sensitisation of tumour cells to immunological stimuli.     

Phenethyl isothiocyanate sensitizes glioma cells to TRAIL-induced apoptosis

Tumor necrosis factor-related apoptosis-induced ligand (TRAIL) is a promising antitumor therapy. However, many cancer cells, including malignant glioma cells, tend to be resistant to TRAIL, highlighting the need for strategies to overcome TRAIL resistance. Here we show that in combination with phenethyl isothiocyanate (PEITC), exposure to TRAIL induced apoptosis in TRAIL-resistant glioma cells. Subtoxic concentrations of PEITC significantly potentiated TRAIL-induced cytotoxicity and apoptosis in glioma cells. PEITC dramatically upregulated DR5 receptor expression but had no effects on DR4 receptor. PEITC enhances TRAIL-induced apoptosis through the downregulation of cell survival proteins and the upregulation of DR5 receptors through actions on the ROS-induced-p53.     

Lupulone, a hop bitter acid, activates different death pathways involving apoptotic TRAIL-receptors, in human colon tumor cells and in their derived metastatic cells

Our study aimed to compare death signalling pathways triggered by lupulone in TRAIL-sensitive human colon cancer cells (SW480) and in their derived TRAIL-resistant metastatic cells (SW620). Lupulone (40 μg/ml) up-regulated expression of TRAIL DR4/DR5 death receptors at the cell surface of both cell lines, even in the absence of exogenous TRAIL ligand. Cell death induced by lupulone was inhibited in SW480 and SW620 cells exposed to blocking anti-DR4/DR5 antibodies. In SW480 cells, lupulone triggered cell death through a cross-talk between TRAIL-DR4/DR5 and the mitochondrial (intrinsic) pathways involving caspase-8 activation and Bid protein cleavage. As a consequence mitochondrial cytochrome c was released into the cytosol and activation of caspases-9 and -3 was observed. In the metastatic SW620 cells, lupulone restored the sensibility of these cells to TRAIL ligand and activated the extrinsic apoptotic pathway via DR4/DR5 death receptors and the involvement of the caspase-8/caspase-3 cascade. The demonstration that lupulone is able to activate TRAIL-death signalling pathways even in TRAIL resistant cancer cells highlights the potential of this natural compound for cancer prevention and therapy.     

Sensitization of human bladder tumor cells to TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis with a small molecule IAP antagonist

Urothelial carcinoma of the bladder accounts for approximately 5% of all cancer deaths in humans. The large majority of bladder tumors are non-muscle invasive at diagnosis, but even after local surgical therapy there is a high rate of local tumor recurrence and progression. Current treatments extend time to recurrence but do not significantly alter disease survival. The objective of the present study was to investigate the tumoricidal potential of combining the apoptosis-inducing protein TNF-related apoptosis-inducing ligand (TRAIL) with a small molecule inhibitor of apoptosis proteins (IAP) antagonist to interfere with intracellular regulators of apoptosis in human bladder tumor cells. Our results demonstrate that the IAP antagonist Compound A exhibits high binding affinity to the XIAP BIR3 domain. When Compound A was used at nontoxic concentrations in combination with TRAIL, there was a significant increase in the sensitivity of TRAIL-sensitive and TRAIL-resistant bladder tumor lines to TRAIL-mediated apoptosis. In addition, modulation of TRAIL sensitivity in the TRAIL-resistant bladder tumor cell line T24 with Compound A was reciprocated by XIAP small interfering RNA-mediated suppression of XIAP expression, suggesting the importance of XIAP-mediated resistance to TRAIL in these cells. These results suggest the potential of combining Compound A with TRAIL as an alternative therapy for bladder cancer.     

Gefitinib enhances human colon cancer cells to TRAIL-induced apoptosis of via autophagy- and JNK-mediated death receptors upregulation

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a potent cancer cell-specific apoptosis-inducing cytokine with little toxicity to most normal cells. Here, we report that gefitinib and TRAIL in combination produce a potent synergistic effect on TRAIL-sensitive human colon cancer HCT116 cells and an additive effect on TRAIL-resistant HT-29 cells. Interestingly, gefitinib increases the expression of cell surface receptors DR4 and DR5, possibly explaining the synergistic effect. Knockdown of DR4 and DR5 by siRNA significantly decreases gefitinib- and TRAIL-mediated cell apoptosis, supporting this idea. Because the inhibition of gefitinib-induced autophagy by 3-MA significantly decreases DR4 and DR5 upregulation, as well as reduces gefitinib- and TRAIL-induced apoptosis, we conclude that death receptor upregulation is autophagy mediated. Furthermore, our results indicate that death receptor expression may also be regulated by JNK activation, because pre-treatment of cells with JNK inhibitor SP600125 significantly decreases gefitinib-induced death receptor upregulation. Interestingly, SP600125 also inhibits the expression CHOP, yet CHOP has no impact on death receptor expressions. We also find here that phosphorylation of Akt and ERK might also be required for TRAIL sensitization. In summary, our results indicate that gefitinib effectively enhances TRAIL-induced apoptosis, likely via autophagy and JNK- mediated death receptor expression and phosphorylation of Akt and ERK.     

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.     

Enhanced caspase-8 recruitment to and activation at the DISC is critical for sensitisation of human hepatocellular carcinoma cells to TRAIL-induced apoptosis by chemotherapeutic drugs

Tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) exhibits potent antitumour activity upon systemic administration in mice without showing the deleterious side effects observed with other apoptosis-inducing members of the TNF family such as TNF and CD95L. TRAIL may, thus, have great potential in the treatment of human cancer. However, about 60% of tumour cell lines are not sensitive to TRAIL. To evaluate the mechanisms of tumour resistance to TRAIL, we investigated hepatocellular carcinoma (HCC) cell lines that exhibit differential sensitivity to TRAIL. Pretreatment with chemotherapeutic drugs, for example, 5-fluorouracil (5-FU), rendered the TRAIL-resistant HCC cell lines sensitive to TRAIL-induced apoptosis. Analysis of the TRAIL death-inducing signalling complex (DISC) revealed upregulation of TRAIL-R2. Caspase-8 recruitment to and its activation at the DISC were substantially increased after 5-FU sensitisation, while FADD recruitment remained essentially unchanged. 5-FU pretreatment downregulated cellular FLICE-inhibitory protein (cFLIP) and specific cFLIP downregulation by small interfering RNA was sufficient to sensitise TRAIL-resistant HCC cell lines for TRAIL-induced apoptosis. Thus, a potential mechanism for TRAIL sensitisation by 5-FU is the increased effectiveness of caspase-8 recruitment to and activation at the DISC facilitated by the downregulation of cFLIP and the consequent shift in the ratio of caspase-8 to cFLIP at the DISC.     

Cetuximab enhances TRAIL-induced gastric cancer cell apoptosis by promoting DISC formation in lipid rafts

TRAIL is a member of the tumor necrosis factor family that selectively induces cancer cell apoptosis. However, gastric cancer cells are insensitive to TRAIL. Our and others studies showed that the inhibition of EGFR pathway activation could increase the sensitivity of TRAIL in cancer cells. But the detailed mechanism is not fully understood. In the present study, compared with TRAIL or cetuximab (an anti-EGFR monoclonal antibody) alone, treatment with the TRAIL/cetuximab combination significantly promoted death receptor 4 (DR4) clustering as well as the translocation of both DR4 and Fas-associated death domain-containing protein (FADD) into lipid rafts. This in turn resulted in caspase-8 cleavage and the formation of the death-inducing signaling complex (DISC) in these lipid rafts. Cholesterol-depletion with methyl-β-cyclodextrin partially prevented DR4 clustering and DISC formation, and thus partially reversed apoptosis induced by the TRAIL/cetuximab dual treatment. These results indicate that cetuximab increases TRAIL-induced gastric cancer cell apoptosis at least partially through the promotion of DISC formation in lipid rafts.