Tumor Necrosis Factor-related Apoptosis-inducing Ligand Resistance Research Articles

Abstract 251: Role of ß-catenin in prostate cancer cell apoptosis

Abstract Introduction: Evasion of apoptosis is a hallmark of cancer cells, and apoptotic signaling pathways are attractive targets for cancer therapy. TNF-related apoptosis inducing ligand (TRAIL) has gained much importance recently due to its ability to preferentially induce apoptosis in transformed cells and not in normal cells. Since many tumor cells develop TRAIL resistance, developing combinatorial treatment strategies to enhance TRAIL sensitivity is a major challenge of current cancer research. The reasons behind TRAIL resistance are multiple, and might be linked with aberrant activation of various oncogenic pathways. ß-catenin is a well established oncogene being overexpressed in various cancers and might be involved in TRAIL resistance. Normally, it functions as a component of both the adherens junction complex and the Wnt/wingless signaling pathway. Our earlier studies have shown that treatment of cancer cells with Troglitazone (TZD), a synthetic ligand for Peroxisome Proliferator-activator receptor gamma can reduce cell proliferation and ß-catenin expression. When combined with TZD, TRAIL was capable of inducing potent apoptosis in various TRAIL-resistant cancer cells. Aims: The present study was designed to determine the involvement of ß-catenin during TRAIL-TZD-induced apoptosis of prostate cancer cells. Experimental procedures: Different prostate cancer cell lines (LNCaP, 22RV1, PC-3, DU145) were used to determine the effect of TRAIL-TZD on apoptosis and ß-catenin pathway, utilizing Western Blots, Immunoprecipitation, and luciferase assays. Results: Treatment with a combination of TRAIL and TZD resulted in significant apoptosis (PARP, caspase-3 cleavage) and ß-catenin cleavage in LNCaP but not in PC-3 and DU145 cells, and 22RV1 showed partial PARP cleavage. TRAIL-TZD-induced ß-catenin cleavage was antagonized by caspase inhibitors, suggesting this to be downstream of caspase activation. Luciferase assays with ß-catenin/TCF-responsive reporter indicated a significant reduction of luciferase activity with this treatment. Interestingly the cleaved ß-catenin fragment retained strong interaction with E-Cadherin, as indicated by immunoprecipitation studies. Detergent fractionation of the cell extracts treated with the drug combination revealed that ß-catenin cleavage was enriched in the cytosolic fraction. To understand any participation of HDACs, TRAIL treatment was performed in combination with various HDAC inhibitors. These results showed a strong increase in apoptosis and ß-catenin cleavage with TRAIL and valproic acid treatment, suggesting an involvement of HDACs in this apoptotic resistance. Conclusion: These results show that targeting ß-catenin signaling pathway might be effective means of increasing TRAIL sensitivity in prostate cancer, and combining TRAIL with TZD or valproic acid might be a promising treatment regimen for drug-resistant cells, especially those with aberrant ß-catenin expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 251. doi:1538-7445.AM2012-251

Abstract 3724: Wogonin enhances tumor necrosis factor-related apoptosis-inducing ligand's antitumor activity in vivo through H2O2-mediated downregulation of X-linked inhibitor of apoptosis protein and cellular inhibitor of apoptosis protein 1 and 2

Abstract Numerous malignant cells are resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cytotoxicity, limiting value of TRAIL as an antitumor therapeutics. Combination of TRAIL with other agents is a promising strategy to overcome TRAIL resistance. Wogonin (5, 7-dihydroxy-8-methoxyflavone), a flavonoid originated from the root of the medicinal herb Scutellaria baicalensis Georgi, has been shown to enhance TRAIL-induced apoptosis in malignant cells in in vitro studies. However, whether wogonin enhances TRAIL's antitumor activity in vivo has never been studied. In this study, we first found that wogonin sensitized a variety of cancer cells including A549, HeLa and SKOV3 to TRAIL-induced apoptosis, as indicated by increased annexin V staining and enhanced activation of caspase-8 and -3 in TRAIL and wogonin cotreated cells, whereas having marginal effect on non-transformed bronchial epithelial cells and cervical epithelial cells. Then the effect of combination of TRAIL and wogonin was tested in a xenografted tumor model with A549 cells in nude mice. The results show that wogonin greatly enhanced TRAIL-induced suppression of tumor growth, which was accompanied with increased apoptosis in tumor tissues determined by a terminal deoxynucleotidyl-transferase -mediated dUTP nick-end labeling (TUNEL) assay. The protein levels of X-linked inhibitor of apoptosis protein (XIAP) and cellular inhibitor of apoptosis protein 1 and 2 (cIAP-1 and cIAP-2) were markedly reduced in both cultured cells and xenografted tumor tissues with co-treatment of wogonin and TRAIL. The down-regulation of these antiapoptotic proteins was likely mediated through proteasomal degradation and involved intracellular H2O2, as wogonin robustly induced H2O2 accumulation in A549 cells and the reactive oxygen species scavengers butylated hydroxyanisole (BHA) and N-acetyl-L-cysteine (NAC) and the proteasome inhibitor MG132 restored the expression of these antiapoptotic proteins and suppressed cytotoxicity in cells co-treated with wogonin and TRAIL. These results provide important in vivo evidence supporting the application of wogonin as TRAIL sensitizer for cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3724. doi:1538-7445.AM2012-3724

Abstract LB-72: A20 ubiquitin ligase-mediated RIP1 polyubiquitin chains inhibits TRAIL-induced apoptosis in glioblastoma

Abstract The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) apoptotic pathway has emerged as a cancer therapeutic target; however, clinical trials have proven that the vast majority of human cancers are resistant to TRAIL apoptotic pathway-targeted therapies. We show here that A20-mediated ubiquitination inhibits caspase-8 cleavage and TRAIL-induced apoptosis in glioblastoma through two signaling complexes. A20 is highly expressed in glioblastomas and, together with the death receptor 5 (DR5) and receptor-interacting protein 1 (RIP1), forms a DR5-associated plasma membrane bound signaling complex that we named the preligand assembly complex (PLAC) under physiologic conditions. TRAIL treatment leads to the recruitment of caspase-8 to the PLAC for the assembly of a death-inducing signaling complex (DISC). In the DISC, the C-terminal Zinc finger domain of the A20 ubiquitin ligase mediates RIP1 ubiquitination through lysine (K)-63-linked polyubiquitin chains, which bind to the protease domain of caspase-8 and inhibits its dimerization, cleavage and the initiation of TRAIL-induced apoptosis in glioblastoma-derived cell lines and tumor-initiating cells. These results identify A20 ubiquitin ligase as a therapeutic target whose inhibition can overcome TRAIL resistance in glioblastoma and thus have an impact on ongoing clinical trials of TRAIL apoptotic pathway-targeted combination cancer therapies (the manuscript was published online first in Cancer Discovery). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-72. doi:1538-7445.AM2012-LB-72

Inostamycin enhanced TRAIL-induced apoptosis through DR5 upregulation on the cell surface

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been considered as a possible therapeutic agent for cancer treatment. This is because of its selective cytotoxicity against various cancer cells without a detrimental effect on normal cells. However, recent studies have reported that the potential application of TRAIL in cancer therapy is limited, as many cancer cells have been found to be resistant to TRAIL. Therefore, small molecule compounds that potentiate the cytotoxicity of TRAIL would be strategic candidates for therapeutic applications in combination with TRAIL. Here we found that a combined treatment of inostamycin and TRAIL synergistically induced caspase-dependent apoptosis in HCT116 cells. Inostamycin upregulated DR5, and a knockdown of DR5 suppressed the apoptosis that was synergistically induced by co-treatment with inostamycin and TRAIL. Moreover, inostamycin increased the expression of DR5 on the cell surface. Therefore, inostamycin-increased cell surface expression of DR5 may have contributed to the enhancement of TRAIL-induced apoptosis. Our study suggests that combined treatment with inostamycin and TRAIL may offer a strategy to overcome TRAIL resistance in tumor cells.

The effect of lentivirus-mediated expression of tumor necrosis factor related apoptosis-inducing ligand and shRNA against Bcl-2 on the growth of lymphoma cells

It has been well established that tumor necrosis factor related apoptosis-inducing ligand (TRAIL) effectively induces apoptosis in tumor cells. However, tumor resistance to TRAIL, especially of hematological tumor cells, has become a major problem in the potential use of TRAIL in clinical practice. Among many factors that contribute to TRAIL resistance, overexpression of Bcl-2 is commonly seen in many kinds of tumors, particularly in lymphoma. In this study, we developed a lentivirus system that encodes recombinant human TRAIL cDNA for overexpression and Bcl-2 shRNA for down-regulation of Bcl-2 (lenti-TRAIL-shBcl-2) simultaneously. The efficiency of recombinant lentiviruses infecting different lymphoma cell lines was assessed by flow cytometric analysis and fluorescence microscopy. Reverse transcription polymerase chain reaction and Western blot assay were carried out to evaluate the expression of TRAIL and Bcl-2 in lymphoma cells after infection. We also examined the growth inhibition effect of recombinant lentivirus on lymphoma cell proliferation by CCK-8 (Cell Counting Kit-8) assay and its effect on bystander cells by flow cytometric analysis. The results showed that lymphoma cells were effectively infected by recombinant lentivirus and that TRAIL was exogenously expressed and Bcl-2 expression was down-regulated in lymphoma cells simultaneously. Results of this study demonstrated that lenti-TRAIL-shBcl-2 induced apoptosis in bystander cells as well as infected lymphoma cells and inhibited the growth of lymphoma cells.

Apigenin Sensitizes Huh-7 Human Hepatocellular Carcinoma Cells to TRAIL-induced Apoptosis

TNF-related apoptosis-inducing ligand (TRAIL) is a promising agent for management of cancer because of its selective cytotoxicity to cancer cells. However, some cancer cells have resistance to TRAIL. Accordingly, novel treatment strategies are required to overcome TRAIL resistance. Here, we examined the synergistic apoptotic effect of apigenin in combination with TRAIL in Huh-7 cells. We found that combined treatment of TRAIL and apigenin markedly inhibited Huh-7 cell growth compared to either agent alone by inducing apoptosis. Combined treatment with apigenin and TRAIL induced chromatin condensation and the cleavage of poly (ADP-ribose) polymerase (PARP). In addition, enhanced apoptosis by TRAIL/apigenin combination was quantified by annexin V/PI flow cytometry analysis. Western blot analysis suggested that apigenin sensitizes cells to TRAIL-induced apoptosis by activating both intrinsic and extrinsic apoptotic pathway-related caspases. The augmented apoptotic effect by TRAIL/apigenin combination was accompanied by triggering mitochondria-dependent signaling pathway, as indicated by Bax/Bcl-2 ratio up-regulation. Our results demonstrate that combination of TRAIL and apigenin facilitates apoptosis in Huh-7 cells.

Proteasome inhibition overcomes TRAIL resistance in human hepatoblastoma cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is responsible for cell death in many cancer cells while being non-toxic for most normal cells. In this study, we investigated the role of TRAIL in human hepatoblastoma (HB) cells and analyzed different approaches to reverse TRAIL resistance in these tumors. Death receptors DR4 and DR5 expression was found on all analyzed primary HB samples and on the cell lines HuH6 and HepT1 by immunofluorescence staining. Recombinant TRAIL alone did not induce in vitro cytotoxicity. Decoy receptor blocking by antibodies led to moderate effects in HepT1 but not in HUH6 cells, whereas FLIP knock-down using siRNA rendered HUH6 cells but not HepT1 cells sensible to TRAIL. Bcl-2 inhibition with ABT-737 enhanced TRAIL-mediated apoptosis in all HB cells. Strongest cytotoxic TRAIL effects were seen in HB cell lines with synchronous proteasome inhibition using bortezomib. FLIP and Bcl-2 contributed to the TRAIL resistance in HB. Overcoming TRAIL resistance in HB by proteasome inhibitors has been identified a possible additive to improve treatment results in HB patients with drug resistant tumors.

Proteasome inhibition overcomes TRAIL resistance in human hepatoblastoma cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is responsible for cell death in many cancer cells while being non-toxic for most normal cells. In this study, we investigated the role of TRAIL in human hepatoblastoma (HB) cells and analyzed different approaches to reverse TRAIL resistance in these tumors. Death receptors DR4 and DR5 expression was found on all analyzed primary HB samples and on the cell lines HuH6 and HepT1 by immunofluorescence staining. Recombinant TRAIL alone did not induce in vitro cytotoxicity. Decoy receptor blocking by antibodies led to moderate effects in HepT1 but not in HUH6 cells, whereas FLIP knock-down using siRNA rendered HUH6 cells but not HepT1 cells sensible to TRAIL. Bcl-2 inhibition with ABT-737 enhanced TRAIL-mediated apoptosis in all HB cells. Strongest cytotoxic TRAIL effects were seen in HB cell lines with synchronous proteasome inhibition using bortezomib. FLIP and Bcl-2 contributed to the TRAIL resistance in HB. Overcoming TRAIL resistance in HB by proteasome inhibitors has been identified a possible additive to improve treatment results in HB patients with drug resistant tumors.

Kinome profiling of non-canonical TRAIL signaling reveals RIP1-Src-STAT3 dependent invasion in resistant non-small cell lung cancer cells

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) triggers apoptosis selectively in tumor cells through interaction with TRAIL-R1/DR4 or TRAIL-R2/DR5 and this process is considered a promising avenue for cancer treatment. TRAIL resistance, however, is frequently encountered and hampers anti-cancer activity. Here we show that whereas H460 non-small cell lung cancer (NSCLC) cells display canonical TRAIL-dependent apoptosis, A549 and SW1573 NSCLC cells are TRAIL resistant and display pro-tumorigenic activity, in particular invasion, following TRAIL treatment. We exploit this situation to contrast TRAIL effects on the kinome of apoptosis-sensitive cells to that of NSCLC cells in which non-canonical effects predominate, employing peptide arrays displaying 1024 different kinase pseudosubstrates more or less comprehensively covering the human kinome. We observed that failure of a therapeutic response to TRAIL coincides with the activation of a non-canonical TRAIL-induced signaling pathway involving, amongst others, Src, STAT3, FAK, ERK and Akt. The use of selective TRAIL variants against TRAIL-R1 or TRAIL-R2 subsequently showed that this non-canonical migration and invasion is mediated through TRAIL-R2. Short-hairpin-mediated silencing of RIP1 kinase prevented TRAIL-induced Src and STAT3 phosphorylation and reduced TRAIL-induced migration and invasion of A549 cells. Inhibition of Src or STAT3 by shRNA or chemical inhibitors including dasatinib and 5,15-diphenylporphyrin blocked TRAIL-induced invasion. FAK, AKT and ERK were activated in a RIP1-independent way and inhibition of AKT sensitized A549 cells to TRAIL-induced apoptosis. We thus identified RIP1-dependent and -independent non-canonical TRAIL kinase cascades in which Src and AKT are instrumental and could be exploited as co-targets in TRAIL therapy for NSCLC.

Wogonin and Related Natural Flavones Overcome Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL) Protein Resistance of Tumors by Down-regulation of c-FLIP Protein and Up-regulation of TRAIL Receptor 2 Expression

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent that kills various tumor cells without damaging normal tissues. However, many cancers remain resistant to TRAIL. To overcome TRAIL resistance, combination therapies using sensitizers of the TRAIL pathway would be an efficacious approach. To investigate potential sensitizers of TRAIL-induced apoptosis, we used TRAIL-resistant human T cell leukemia virus type 1 (HTLV-1)-associated adult T cell leukemia/lymphoma (ATL) cells as a model system. So far, HTLV-1-associated ATL is incurable by presently known therapies. Here, we show that wogonin and the structurally related natural flavones apigenin and chrysin break TRAIL resistance in HTLV-1-associated ATL by transcriptional down-regulation of c-FLIP, a key inhibitor of death receptor signaling, and by up-regulation of TRAIL receptor 2 (TRAIL-R2). This effect is mediated through transcriptional inhibition of the p53 antagonist murine double minute 2 (Mdm2), leading to an increase in p53 levels and, consequently, to up-regulation of the p53 target gene TRAIL-R2. We also show that these flavones can sensitize to TNFα- and CD95-mediated cell death. Furthermore, we show that wogonin, apigenin, and chrysin also enhance TRAIL-mediated apoptosis in other human cancer cell lines including breast cancer cell line MDA-MB-231, colon cancer cell line HT-29, hepatocellular carcinoma cell line HepG2, melanoma cell line SK-MEL-37, and pancreatic carcinoma cell line Capan-1 by the same mechanism. Thus, our study suggests the potential use of these flavones as an adjuvant for TRAIL-mediated anticancer therapy.

Wilm's Tumor 1 Protein Confers TRAIL Resistance in Myeloid Leukemia Cells Through Bcl-Xl

Abstract 2557 INTRODUCTION:Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is an attractive therapeutic agent because of its selective potential to induce apoptosis in variety of tumors but not in normal cells. However, many cancers including myeloid leukemias exhibit resistance to TRAIL and the underlying mechanisms are not fully understood. Wilms' tumor 1 (WT1) protein is an aberrantly overexpressed protein that is associated with leukemia blast survival, chemoresistance and shortened disease-free survival in acute myeloid leukemia (AML). Here, we report a previously unknown protective role of WT1 against TRAIL-induced apoptosis in leukemia cells. METHODS:Various myeloid leukemia cell lines with variable WT1 expression levels were tested for TRAIL sensitivity. The effect of pharmacological or short hairpin RNA (shRNA)-mediated inhibition of WT1 on TRAIL-induced apoptosis was evaluated by AnnexinV/PI staining method. The mRNA levels of WT1 and Bcl-xL were measured by reverse transcription-PCR (RT-PCR) and the expression of apoptosis regulators were analyzed by western blotting. RESULTS:We observed a strong correlation between higher levels of WT1 and increased TRAIL resistance in myeloid leukemia cell lines. WT1 downregulation by shRNA significantly sensitized leukemia cells to TRAIL-induced apoptosis and importantly, ectopic expression of shRNA resistant WT1 (WT1SR) in WT1-knockdown cells restored the TRAIL resistance. WT1 influences apoptosis through transcriptional regulation of Bcl-2 family members such as Bcl-2 and Bak. We found for the first time, a positive correlation between WT1 and Bcl-xL expression in leukemia cell lines and primary AML samples. Furthermore, using chromatin immunoprecipitation (ChIP) analysis, we show that Bcl-xL is a bonafide WT1-target gene as WT1 transctivates Bcl-xL by binding to its promoter. We have (Bansal et al., Blood 2010) recently shown that the expression and oncogenic functions of WT1 could be abrogated by heat shock protein 90 (Hsp90) inhibitors in myeloid leukemia cells. In order to establish whether Hsp90- mediated regulation of WT1 has any functional significance in TRAIL-resistance we treated leukemia cell with the Hsp90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG). The results confirm that Hsp90 inhibition was able to overcome TRAIL resistance, by reducing levels of WT1 and Bcl-xL and by increasing the levels of the proapoptotic protein Bak. Taken together, our results reveal that increased expression of Bcl-xL, along with low Bak, confer WT1-mediated TRAIL resistance. CONCLUSION:Our data indicate that TRAIL resistance in myeloid leukemia cells is mediated by overexpression of the panleukemic marker WT1 and this can be overcome by either silencing WT1 or by pharmacologic inhibitors such as 17-AAG. of Hsp90. Our study highlights the potential therapeutic benefit of the combining TRAIL and Hsp90 inhibitors for the treatment of myeloid leukemias. Disclosures:No relevant conflicts of interest to declare.

Cell-surface galectin-3 confers resistance to TRAIL by impeding trafficking of death receptors in metastatic colon adenocarcinoma cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis and preferentially kills tumor cells by engaging specific glycosylated death receptors, resulting in the internalization of ligand/receptor complexes and recruitment of the initiator caspase-8 to an activation platform known as the death-inducing signaling complex (DISC). However, emergence of TRAIL-resistant sub-populations may contribute to therapeutic failure. To investigate resistance mechanisms, we isolated a stable TRAIL-resistant sub-population of the metastatic colon cancer cell line LS-LIM6, designated LIM6-TR. LIM6-TR cells are impaired in endocytosis of TRAIL/death receptors complexes and failed to recruit/activate caspase-8 to the DISC upon TRAIL stimulation. Differential activation of Wnt and JNK pathways is not responsible for acquisition of TRAIL resistance. LIM6-TR cells display a marked increase in cell-surface expression of galectin-3, an endogenous lectin, which co-localizes with and binds death receptors. Silencing of galectin-3 restores TRAIL sensitivity and promotes TRAIL-mediated endocytosis of TRAIL/death receptors complexes. Inhibitors of galectin-3 and glycosylation also re-sensitize LIM6-TR to TRAIL and restore internalization of ligand/receptors complexes. These studies identify a novel TRAIL-resistance mechanism in which galectin-3 impedes trafficking of death receptor by anchoring them in glycan nano-clusters, blocking the execution of the apoptosis signal.

Simultaneously Targeting Bcl-2 and Akt Pathways Sensitizes Nasopharyngeal Carcinoma to Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand

The 5-year survival rate of nasopharyngeal carcinoma (NPC) is disappointing despite the much improved technologies in its treatment. Thus, finding more effective treatment for NPC has become an urgent priority. The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can selectively induce apoptosis in most tumor cells while sparing normal cells. However, its potential in the treatment of NPC has been limited by the eventual emergence of drug resistance. Bcl-2 and Akt contribute to TRAIL resistance in some cancer cells. In this study, CNE-2 was found to be the most resistant NPC cell line to TRAIL, and whether Bcl-2 small-interfering RNA (siRNA) and phosphatidylinositol 3-kinase (PI3-K) inhibitors (LY294002 and Wortmannin) could prevent TRAIL resistance in CNE-2 was also investigated. Results showed that both Bcl-2 siRNA and PI3-K inhibitors could prevent TRAIL resistance in CNE-2. Bcl-2 siRNA sensitized CNE-2 by activating the intrinsic apoptotic pathway and PI3-K inhibitors sensitized CNE-2 by activating both intrinsic and extrinsic pathways. Further, simultaneously targeting Bcl-2 and Akt was found to be a more efficient approach to prevent TRAIL resistance in CNE-2 and this synergistic effect happened at the level of Bid downstream. At last, the combinative treatments did not enhance toxicity of TRAIL in MRC5, a human benign fibroblast cell line. This study suggests that simultaneously targeting Bcl-2 and Akt pathway might be effective in preventing TRAIL resistance of NPC cells.

Latent membrane protein 1 mediates the resistance of nasopharyngeal carcinoma cells to TRAIL-induced apoptosis by activation of the PI3K/Akt signaling pathway

The 5-year survival rate of nasopharyngeal carcinoma (NPC) is still disappointing despite the much improved technologies in its treatment. Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) can selectively induce apoptosis in most tumor cells while sparing normal cells. However, its potential in the treatment of NPC has been limited by the eventual emergence of drug resistance. Latent membrane protein 1 (LMP1) is a major oncogene of the human DNA tumor virus Epstein-Barr virus (EBV) and is associated with the development of NPC and the emergence of chemo-resistance in NPC. In this study, we investigated the potential role of LMP1 in TRAIL resistance in CNE-1 NPC cells. Results show that overexpression of LMP1 could induce TRAIL resistance in NPC cells without influencing death receptors. The LMP1-induced TRAIL resistance is associated with increased expression of FLIP and elevated cleavage of caspase-8 without altering the TRAIL-mediated mitochondrial events and Bid cleavage. Knockdown of the FLIP gene with siRNA prevented the LMP1-induced TRAIL resistance. Furthermore, we found that overexpression of LMP1 activated Akt. Inhibition of Akt with LY294002 completely prevented the LMP1-induced FLIP expression and TRAIL resistance. Together, these results show that LMP1 can inhibit the TRAIL-mediated apoptosis through activation of PI3K/Akt and expression of FLIP in CNE-1 NPC cells, and may provide new methods to prevent and reverse drug resistance in NPC.

MiR-130a targets MET and induces TRAIL-sensitivity in NSCLC by downregulating miR-221 and 222

Non-small cell lung cancer (NSCLC) accounts for ∼80% of all lung cancers. Although some advances in lung cancer therapy have been made, patient survival is still quite poor. Two microRNAs, miR-221 and miR-222, upregulated by the MET proto-oncogene, have been already described to enhance cell survival and to induce TNF-related apoptosis-inducing ligand (TRAIL) resistance in NSCLC cell lines, through the downregulation of p27(kip1), PTEN and TIMP3. Here, we further investigated this pathway and showed that miR-130a, expressed at low level in lung cancer cell lines, by targeting MET was able to reduce TRAIL resistance in NSCLC cells through the c-Jun-mediated downregulation of miR-221 and miR-222. Moreover, we found that miR-130a reduced migratory capacity of NSCLC. A better understanding of MET-miR-221 and 222 axis regulation in drug resistance is the key in developing new strategies in NSCLC therapy.

TRAIL-mediated signaling in prostate, bladder and renal cancer

Tumor necrosis factor related apoptosis inducing ligand (TRAIL) is a death receptor ligand that has the ability to preferentially initiate apoptosis in malignant cells with minimal toxicity to normal cells. TRAIL-based therapeutics, including recombinant TRAIL, TRAIL-receptor agonistic antibodies and TRAIL gene therapy, have now entered clinical trials. Although these therapeutics are promising, concerns regarding TRAIL resistance are causing research efforts to shift towards the identification of effective combination therapies. Small-molecule inhibitors, natural compounds, and drugs approved for treatment of diseases other than cancer have been shown to affect TRAIL receptors, antiapoptotic proteins and survival pathways in prostate, bladder and renal cell lines and in preclinical models. Changes in endogenous TRAIL and TRAIL receptor expression during the development of genitourinary malignancies and the way in which the expression pattern is affected by treatment are of great interest, and understanding the biological consequences of such changes will be important to maximize the potential of TRAIL-based therapeutics.

Epidermal Growth Factor Receptor-mediated Tissue Transglutaminase Overexpression Couples Acquired Tumor Necrosis Factor-related Apoptosis-inducing Ligand Resistance and Migration through c-FLIP and MMP-9 Proteins in Lung Cancer Cells

Acquired chemoresistance not only blunts anticancer therapy but may also promote cancer cell migration and metastasis. Our previous studies have revealed that acquired tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance in lung cancer cells is associated with Akt-mediated stabilization of cellular caspase 8 and Fas-associated death domain (FADD)-like apoptosis regulator-like inhibitory protein (c-FLIP) and myeloid cell leukemia 1 (Mcl-1). In this report, we show that cells with acquired TRAIL resistance have significantly increased capacities in migration and invasion. By gene expression screening, tissue transglutaminase (TGM2) was identified as one of the genes with the highest expression increase in TRAIL-resistant cells. Suppressing TGM2 dramatically alleviated TRAIL resistance and cell migration, suggesting that TGM2 contributes to these two phenotypes in TRAIL-resistant cells. TGM2-mediated TRAIL resistance is likely through c-FLIP because TGM2 suppression significantly reduced c-FLIP but not Mcl-1 expression. The expression of matrix metalloproteinase 9 (MMP-9) was suppressed when TGM2 was inhibited, suggesting that TGM2 potentiates cell migration through up-regulating MMP-9 expression. We found that EGF receptor (EGFR) was highly active in the TRAIL-resistant cells, and suppression of EGFR dramatically reduced TGM2 expression. We further determined JNK and ERK, but not Akt and NF-κB, are responsible for EGFR-mediated TGM2 expression. These results identify a novel pathway that involves EGFR, MAPK (JNK and ERK), and TGM2 for acquired TRAIL resistance and cell migration in lung cancer cells. Because TGM2 couples TRAIL resistance and cell migration, it could be a molecular target for circumventing acquired chemoresistance and metastasis in lung cancer.

Up-regulation of the DR5 Expression by Proteasome Inhibitor MG132 Augments TRAIL-Induced Apoptosis in Soft Tissue Sarcoma Cell Lines

PurposeCurrent chemotherapeutics for treating locally advanced or metastatic soft tissue sarcomas (STS) are limited. Accordingly, the present in vitro study was conducted to evaluate the effects of treatment of STS cells with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) applied as a single agent or in combination with a proteasome inhibitor, MG132.Materials and MethodsSensitivity to TRAIL and activity of TRAIL-induced apoptotic pathways were analyzed in four STS cell lines: HTB-82 (rhabdomyosarcoma), HT-1080 (fibrosarcoma), HTB-93 (synovial sarcoma), and HTB-94 (chondrosarcoma). Reduction of the dye dimethylthiazolyl 2,5 diphenyltetrazolium bromide (MTT) was used to evaluate cytotoxic activity; western blots were used to evaluate TRAIL-induced apoptosis.ResultsTRAIL induced apoptosis in HTB-93 cells, but had little effect in HTB-82, HT-1080, or HTB-94 cells. Expression of TRAIL receptor-1 and -2 did not correlate with sensitivity to TRAIL. Co-incubation of cells with TRAIL and a proteasome inhibitor, MG132, augmented the apoptotic effect of TRAIL in both TRAIL-sensitive and TRAIL-resistant cells. This effect was due to up-regulation of TRAIL receptors and members of the pro-apoptotic BCL-2 family by MG132.ConclusionThese data show that combining TRAIL with MG132 enhances apoptosis and overcomes TRAIL resistance. This restoration of TRAIL sensitivity occurs through an increase in the expression of death receptor 5 and of pro-apoptotic BCL-2 family members such as BAX.

Knockdown of MADD and c-FLIP overcomes resistance to TRAIL-induced apoptosis in ovarian cancer cells

The clinical utility of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in the treatment of established human malignancies is limited by the development of resistance to TRAIL. We hypothesized that knockdown of map-kinase activating death domain containing protein (MADD), a TRAIL-resistance factor, may overcome TRAIL resistance in ovarian cancer cells. MADD expression in resected ovarian cancer specimens and cell lines was quantified with the use of polymerase chain reaction. Sensitivity of ovarian cancer cell lines to TRAIL, with or without MADD knockdown, was assessed. MADD is expressed at relatively higher levels in human malignant ovarian cancer tissues and cell lines, compared with normal ovarian tissues. The cell lines OVCA429 and OVCAR3 were susceptible, and cell lines CAOV-3 and SKOV-3 were resistant to TRAIL. MADD knockdown in CAOV-3 cells, but not in SKOV-3 cells, conferred TRAIL sensitivity. Knockdown of cellular Fas-associated death domain-like interleukin-1 beta-converting enzyme-inhibitory protein (c-FLIP) in SKOV-3 cells increased spontaneous and TRAIL-induced apoptosis, which was further increased on MADD knockdown. MADD/c-FLIP(L) knockdown can render TRAIL-resistant ovarian cancer cells susceptible to TRAIL.

Artesunate enhances TRAIL-induced apoptosis in human cervical carcinoma cells through inhibition of the NF-κB and PI3K/Akt signaling pathways

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis and kills cancer cells with little or no adverse effects on normal cells. TRAIL is relatively safe for clinical applications. However, TRAIL resistance is widely found in cancer cells leading to limitations in utilizing TRAIL as a therapeutic agent for cancer treatment. Recently, artesunate, an effective and safe anti-malarial drug, was also described as a promising candidate for cancer therapy. It would be of importance to determine whether combination treatment of TRAIL together with artesunate could overcome drug-resistance of tumors. Here, we demonstrate the first evidence that artesunate effectively enhances TRAIL-mediated cytotoxicity by suppressing pro-survival proteins, such as survivin, XIAP and Bcl-XL. Upon treatment with artesunate, the levels of survival proteins were strongly suppressed in HeLa cells. The down-regulation of these survival proteins could be regulated by repressing activation of NF-κB and Akt. Artesunate also inhibited TRAIL-induced transcriptional activity of NF-κB. In addition, this substance significantly enhanced both extrinsic and intrinsic apoptosis, which were induced by TRAIL. Taken together, the results of the present study suggest that artesunate exhibits an ability to overcome TRAIL resistance and combination treatment of TRAIL together with artesunate may be an effective strategy for cancer therapy.