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

Trailing TRAIL Resistance: Novel Targets for TRAIL Sensitization in Cancer Cells.

Resistance to chemotherapeutic drugs is the major hindrance in the successful cancer therapy. The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor (TNF) family of ligands, which initiates apoptosis in cancer cells through interaction with the death receptors DR4 and DR5. TRAIL is perceived as an attractive chemotherapeutic agent as it specifically targets cancer cells while sparing the normal cells. However, TRAIL therapy has a major limitation as a large number of the cancer develop resistance toward TRAIL and escape from the destruction by the immune system. Therefore, elucidation of the molecular targets and signaling pathways responsible for TRAIL resistance is imperative for devising effective therapeutic strategies for TRAIL resistant cancers. Although, various molecular targets leading to TRAIL resistance are well-studied, recent studies have implicated that the contribution of some key cellular processes toward TRAIL resistance need to be fully elucidated. These processes primarily include aberrant protein synthesis, protein misfolding, ubiquitin regulated death receptor expression, metabolic pathways, epigenetic deregulation, and metastasis. Novel synthetic/natural compounds that could inhibit these defective cellular processes may restore the TRAIL sensitivity and combination therapies with such compounds may resensitize TRAIL resistant cancer cells toward TRAIL-induced apoptosis. In this review, we have summarized the key cellular processes associated with TRAIL resistance and their status as therapeutic targets for novel TRAIL-sensitizing agents.

Novel TRAIL sensitizer Taraxacum officinale F.H. Wigg enhances TRAIL-induced apoptosis in Huh7 cells.

TRAIL (TNF-related apoptosis inducing ligand) is a promising anti-cancer drug target that selectively induces apoptosis in cancer cells. However, many cancer cells are resistant to TRAIL-induced apoptosis. Therefore, reversing TRAIL resistance is an important step for the development of effective TRAIL-based anti-cancer therapies. We previously reported that knockdown of the TOR signaling pathway regulator-like (TIPRL) protein caused TRAIL-induced apoptosis by activation of the MKK7-c-Jun N-terminal Kinase (JNK) pathway through disruption of the MKK7-TIPRL interaction. Here, we identified Taraxacum officinale F.H. Wigg (TO) as a novel TRAIL sensitizer from a set of 500 natural products using an ELISA system and validated its activity by GST pull-down analysis. Furthermore, combination treatment of Huh7 cells with TRAIL and TO resulted in TRAIL-induced apoptosis mediated through inhibition of the MKK7-TIPRL interaction and subsequent activation of MKK7-JNK phosphorylation. Interestingly, HPLC analysis identified chicoric acid as a major component of the TO extract, and combination treatment with chicoric acid and TRAIL induced TRAIL-induced cell apoptosis via JNK activation due to inhibition of the MKK7-TIPRL interaction. Our results suggest that TO plays an important role in TRAIL-induced apoptosis, and further functional studies are warranted to confirm the importance of TO as a novel TRAIL sensitizer for cancer therapy. © 2015 Wiley Periodicals, Inc.

Withanolide E sensitizes renal carcinoma cells to TRAIL-induced apoptosis by increasing cFLIP degradation.

Withanolide E, a steroidal lactone from Physalis peruviana, was found to be highly active for sensitizing renal carcinoma cells and a number of other human cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. Withanolide E, the most potent and least toxic of five TRAIL-sensitizing withanolides identified, enhanced death receptor-mediated apoptotic signaling by a rapid decline in the levels of cFLIP proteins. Other mechanisms by which TRAIL sensitizers have been reported to work: generation of reactive oxygen species (ROS), changes in pro-and antiapoptotic protein expression, death receptor upregulation, activation of intrinsic (mitochondrial) apoptotic pathways, ER stress, and proteasomal inhibition proved to be irrelevant to withanolide E activity. Loss of cFLIP proteins was not due to changes in expression, but rather destabilization and/or aggregation, suggesting impairment of chaperone proteins leading to degradation. Indeed, withanolide E treatment altered the stability of a number of HSP90 client proteins, but with greater apparent specificity than the well-known HSP90 inhibitor geldanamycin. As cFLIP has been reported to be an HSP90 client, this provides a potentially novel mechanism for sensitizing cells to TRAIL. Sensitization of human renal carcinoma cells to TRAIL-induced apoptosis by withanolide E and its lack of toxicity were confirmed in animal studies. Owing to its novel activity, withanolide E is a promising reagent for the analysis of mechanisms of TRAIL resistance, for understanding HSP90 function, and for further therapeutic development. In marked contrast to bortezomib, among the best currently available TRAIL sensitizers, withanolide E's more specific mechanism of action suggests minimal toxic side effects.

Microwave-Assisted Claisen Rearrangement: Synthesis of Naturally Occurring TRAIL-Resistance-Overcoming Tyrosine Derivative

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a tumor necrosis factor (TNF) family ligand that binds on the death receptors, DR4 and DR5, activating apoptotic pathways selectively in cancer cells and thus has become a promising cancer therapeutic agent. Compound 1, isolated from Streptomyces sp. IFM 10937, has shown activity in overcoming TRAIL resistance in AGS cells. Synthesis of 1 has been accomplished from L-tyrosine in an overall high-yielding reaction sequence.

2'-Hydroxy-4-methylsulfonylchalcone enhances TRAIL-induced apoptosis in prostate cancer cells.

Prostate cancer is the most common malignant cancer in men and the second leading cause of cancer deaths. Previously, we have shown that 2'-hydroxy-4-methylsulfonylchalcone (RG003) induced apoptosis in prostate cancer cell lines PC-3 and DU145. Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent, some cancer cells are resistant to TRAIL treatment. PC-3 and LNCaP prostatic cancer cell lines have been reported to be resistant to TRAIL-induced apoptosis. Here, we show for the first time that RG003 overcomes TRAIL resistance in prostate cancer cells. RG003 can enhance TRAIL-induced apoptosis through DR5 upregulation and downregulation of Bcl-2, PI3K/Akt, NF-κB, and cyclooxygenase-2 (COX-2) survival pathways. When used in combined treatment, RG003 and TRAIL amplified TRAIL-induced activation of apoptosis effectors and particularly activation of caspase-8 and the executioner caspase-3, leading to increased poly-ADP-ribose polymerase cleavage and DNA fragmentation in prostate cancer cells. Furthermore, we showed that RG003 reduced COX-2 expression in cells. Previously, we showed that COX-2 was involved in resistance to an apoptosis mechanism; then, its inhibition by RG003 could render cells more sensitive to TRAIL treatment. We showed that nuclear factor-κB activation was inhibited after RG003 treatment. This inhibition was correlated with reduction in COX-2 expression and induction of apoptosis. Overall, we conclude, for the first time, that RG003 can enhance TRAIL-induced apoptosis in human prostate cancer cells. The significance of our in-vitro study with RG003 and TRAIL combined is very encouraging, suggesting the relevance of testing this combined treatment in xenograft animal models.

Prenylated flavonoids and resveratrol derivatives isolated from Artocarpus communis with the ability to overcome TRAIL resistance.

In a screening program on natural products that can abrogate tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) resistance, four new prenylated flavonoid and resveratrol derivatives (1-4) were isolated from Artocarpus communis, together with eight known prenylflavonoids (5-12). The structures of 1-4 were elucidated spectroscopically. Pannokin D [corrected] (1) (2 μM) and artonin E (5) (3 μM) potently exhibited the ability to overcome TRAIL resistance. Artonin E (5) induced caspase-dependent apoptosis in combination with TRAIL, increased caspase 3/7 activity, and enhanced the protein levels of p53 and DR5. Moreover, this substance decreased cell viability in combination with TRAIL and enhanced the protein levels of DR5, and these effects were mediated by increases in the production of ROS (reactive oxygen species). Thus, artonin E (5) was found to induce extrinsic apoptotic cell death by the ROS- and p53-mediated up-regulation of DR5 expression in AGS cells.

Parthenolide enhances sensitivity of colorectal cancer cells to TRAIL by inducing death receptor 5 and promotes TRAIL-induced apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic agent. Recombinant human TRAIL has been evaluated in clinical trials, however, various malignant tumors are resistant to TRAIL. Parthenolide (PT) has recently been demonstrated as a highly effective anticancer agent and has been suggested to be used for combination therapy with other anticancer agents. In this study, we investigate the molecular mechanisms by which PT sensitizes colorectal cancer (CRC) cells to TRAIL-induced apoptosis. HT-29 (TRAIL-resistant) and HCT116 (TRAIL-sensitive) cells were treated with PT and/or TRAIL. The results demonstrated that combined treatment induced apoptosis which was determined using MTT, cell cycle analysis, Annexin V assay and Hoechst 33258 staining. Interestingly, we confirmed that HCT116 cells have much higher death receptor (DR) 5 than HT-29 cells and PT upregulates DR5 protein level and surface expression in both cell lines. Apoptosis through the mitochondrial pathway was confirmed by detecting regulation of Bcl-2 family members, p53 cytochrome C release, and caspase cascades. These results suggest that PT sensitizes TRAIL-induced apoptosis via upregulation of DR5 and mitochondria-dependent pathway. Combination treatment using PT and TRAIL may offer an effective strategy to overcome TRAIL resistance of certain CRC cells.

XIAP-targeting drugs re-sensitize PIK3CA-mutated colorectal cancer cells for death receptor-induced apoptosis.

Mutations in the oncogenic PIK3CA gene are found in 10–20% of colorectal cancers (CRCs) and are associated with poor prognosis. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and agonistic TRAIL death receptor antibodies emerged as promising anti-neoplastic therapeutics, but to date failed to prove their capability in the clinical setting as especially primary tumors exhibit high rates of TRAIL resistance. In our study, we investigated the molecular mechanisms underlying TRAIL resistance in CRC cells with a mutant PIK3CA (PIK3CA-mut) gene. We show that inhibition of the constitutively active phosphatidylinositol-3 kinase (PI3K)/Akt signaling pathway only partially overcame TRAIL resistance in PIK3CA-mut-protected HCT116 cells, although synergistic effects of TRAIL plus PI3K, Akt or cyclin-dependent kinase (CDK) inhibitors could be noted. In sharp contrast, TRAIL triggered full-blown cell death induction in HCT116 PIK3CA-mut cells treated with proteasome inhibitors such as bortezomib and MG132. At the molecular level, resistance of HCT116 PIK3CA-mut cells against TRAIL was reflected by impaired caspase-3 activation and we provide evidence for a crucial involvement of the E3-ligase X-linked inhibitor of apoptosis protein (XIAP) therein. Drugs interfering with the activity and/or the expression of XIAP, such as the second mitochondria-derived activator of caspase mimetic BV6 and mithramycin-A, completely restored TRAIL sensitivity in PIK3CA-mut-protected HCT116 cells independent of a functional mitochondrial cell death pathway. Importantly, proteasome inhibitors and XIAP-targeting agents also sensitized other CRC cell lines with mutated PIK3CA for TRAIL-induced cell death. Together, our data suggest that proteasome- or XIAP-targeting drugs offer a novel therapeutic approach to overcome TRAIL resistance in PIK3CA-mutated CRC.

The role of Cullin3-mediated ubiquitination of the catalytic subunit of PP2A in TRAIL signaling

Protein phosphatase 2A (PP2A) is the major serine-threonine phosphatase that regulates a number of cell signaling pathways. PP2A activity is controlled partially through protein degradation; however, the underlying mechanism is not fully understood. Here we show that PP2A/C, a catalytic subunit of PP2A, is degraded by the Cullin3 (Cul3) ligase-mediated ubiquitin-proteasome pathway. In response to death receptor signaling by tumor-necrosis factor-related apoptosis-inducing ligand (TRAIL), PP2A/C, caspase-8 and Cul3, a subunit of the cullin family of E3 ligases, are recruited into the death-inducing signaling complex (DISC) where the Cul3 ligase targets PP2A/C for ubiquitination and subsequent degradation. Functionally, knockdown of PP2A/C expression by siRNA or pharmacological inhibition of PP2A activity increases TRAIL-induced apoptosis. In cancer cells that have developed acquired TRAIL resistance, PP2A phosphatase activity is increased, and PP2A/C protein is resistant to TRAIL-induced degradation. Thus, this work identifies a new mechanism by which PP2A/C is regulated by Cul3 ligase-mediated degradation in response to death receptor signaling and suggests that inhibition of PP2A/C degradation may contribute to resistance of cancer cells to death receptor-induced apoptosis.

The molecular mechanisms of TRAIL resistance in cancer cells: help in designing new drugs.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which is capable of selectively inducing apoptosis of cancer cells, is a potential targeted drug for cancer therapy. Many clinical trials have verified the safety, tolerability, and therapeutic efficacy of TRAIL or TRAIL agonists in patients. However, the resistance to TRAIL in multiple cancer cells resulted in limited treatment response and poor prognosis. In this review, the molecular mechanisms of TRAIL resistance in cancer cells are summarized. How TRAIL receptors, structure of the cellular membrane, the Protein Kinase B (Akt) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways involve in regulating TRAIL resistance is described. A full understanding of the exact molecular mechanisms of TRAIL resistance in cancer cells could help to design more suitable strategies and new drugs to overcome TRAIL resistance and obtain better therapeutic outcomes.

C-Rel is a critical mediator of NF-κB-dependent TRAIL resistance of pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) represents one of the deadliest malignancies with an overall life expectancy of 6 months despite current therapies. NF-κB signalling has been shown to be critical for this profound cell-autonomous resistance against chemotherapeutic drugs and death receptor-induced apoptosis, but little is known about the role of the c-Rel subunit in solid cancer and PDAC apoptosis control. In the present study, by analysis of genome-wide patterns of c-Rel-dependent gene expression, we were able to establish c-Rel as a critical regulator of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in PDAC. TRAIL-resistant cells exhibited a strong TRAIL-inducible NF-κB activity, whereas TRAIL-sensitive cells displayed only a small increase in NF-κB-binding activity. Transfection with siRNA against c-Rel sensitized the TRAIL-resistant cells in a manner comparable to siRNA targeting the p65/RelA subunit. Gel-shift analysis revealed that c-Rel is part of the TRAIL-inducible NF-κB complex in PDAC. Array analysis identified NFATc2 as a c-Rel target gene among the 12 strongest TRAIL-inducible genes in apoptosis-resistant cells. In line, siRNA targeting c-Rel strongly reduced TRAIL-induced NFATc2 activity in TRAIL-resistant PDAC cells. Furthermore, siRNA targeting NFATc2 sensitized these PDAC cells against TRAIL-induced apoptosis. Finally, TRAIL-induced expression of COX-2 was diminished through siRNA targeting c-Rel or NFATc2 and pharmacologic inhibition of COX-2 with celecoxib or siRNA targeting COX-2, enhanced TRAIL apoptosis. In conclusion, we were able to delineate a novel c-Rel-, NFATc2- and COX-2-dependent antiapoptotic signalling pathway in PDAC with broad clinical implications for pharmaceutical intervention strategies.

Abstract 2286: Regulation of GSK3β axis by combination treatment with TRAIL and Troglitazone in cancer cells

Abstract Prostate cancer is estimated to account for 29% of all new cancers and is the second leading cause of cancer-related death in men in the United States. Hormonal therapy is the only treatment for advanced forms, but androgen-independence eventually develops in most patients. Developing new treatment strategies are urgently needed, which needs a deeper molecular understanding of the events that lead to tumor progression. TNF-related apoptosis inducing ligand (TRAIL) has gained much importance recently due to its ability to preferentially induce cell death in malignant and transformed cells. However, since many tumor cells develop resistance to TRAIL, recent approaches are focused on developing combinatorial therapeutic regimens that can enhance TRAIL sensitivity. There are multiple reasons behind TRAIL resistance, and recent studies (including ours) indicate that, Glycogen Synthase Kinase 3β (GSK3β) might be a key player in mediating this. Thus pathways that can antagonize GSK3β axis are important targets for cancer drug development. GSK3β is a serine/threonine kinase, initially identified as a critical mediator of glycogen metabolism and insulin signaling and is now well accepted to regulate various cellular processes including cell survival. In our earlier studies, combinatorial treatment with Troglitazone (TZD), a synthetic ligand for peroxisome proliferator-activator receptor gamma (PPARγ) and TRAIL induced significant apoptosis in TRAIL-resistant cancer cells. Utilizing this combination, we also observed an increase in GSK3βSer9 phosphorylation that preceded the onset of apoptosis. At a later time, however, TRAIL-TZD combination produced a dramatic reduction of total GSK3β levels, suggesting that GSK3β is being targeted by multiple pathways following this treatment. Similar results were observed in various prostate as well as pancreatic cancer cells, indicating this to be a generalized event. Interestingly, pretreatment of Bx-PC3 pancreatic cancer cells (with a shorter half-life of GSK3β) with protein synthesis inhibitor Cycloheximide (CHX) significantly reduced TRAIL-TZD-induced inhibition of total GSK3β expression, suggesting mostly a transcriptional regulation. Luciferase assays carried out in both pancreatic and prostate cancer cells indicated dramatic reduction of GSK3β promoter activity with TRAIL-TZD. Knockdown of endogenous PPARγ expression by PPARγ- siRNA resulted in a reduction of PPARγ levels with siRNA transfection, which was unable to antagonize TRAIL-TZD-induced reduction of GSK3β transcription or protein expression. These suggested a potential PPARγ-independent regulation of GSK3β in this pathway. Since the mechanisms that regulate GSK3β expression in cancer cells are largely unknown, these studies indicate a novel strategy of targeting GSK3β, elucidation of which might provide newer insights to improve our understanding of TRAIL resistance. Citation Format: Sunipa Majumdar, Sreevidya Santha, Ajay Rana, Basabi Rana. Regulation of GSK3β axis by combination treatment with TRAIL and Troglitazone in cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2286. doi:10.1158/1538-7445.AM2014-2286

Abstract 789: Parthenolide sensitizes colorectal cancer cells to TRAIL-induced apoptosis by regulating mitochonrial pathway

Abstract Combination therapy utilizing of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in conjunction with other anticancer agents, is a promising strategy to overcome TRAIL resistance in malignant cells. Recently, parthenolide (PT) has proved to be a promising anticancer agent, and several studies have explored its use in combination therapy. Here, we investigated the molecular mechanisms by which PT sensitizes colorectal cancer (CRC) cells to TRAIL-induced apoptosis. TRAIL inhibited HCT116 cell growth in a dose-dependent manner; however, this reduction did not occur in TRAIL resistant HT-29 cells with an even higher dose of TRAIL. A combination of PT with TRAIL significantly inhibited cell growth of TRAIL resistant HT-29 cells. Consistent with cell growth inhibition, apoptotic cell death was significantly increased by a combination of PT with TRAIL in both of HCT116 and HT-29 cells. Results of flow cytometry analysis demonstrated that TRAIL-sensitive HCT116 cells had much higher death receptor (DR) 5 than TRAIL-resistant HT-29 cells. Interestingly, treatment of PT and/or TRAIL did not affect DR4/DR5, these results indicate that the apoptotic effect of combination is death receptor-independent apoptosis. We observed that the synergistic effect was associated with Bcl-2 family members, p53 and cytochrome C. Moreover, activation of caspase -3, -8 and -9 was increased by combination treatment in both of TRAIL-resistant and -sensitive cells. These results suggest that PT sensitizes TRAIL-induced apoptosis via death receptor-independent and mitochondrial-dependent pathway. Combination treatment using PT and TRAIL might offer an effective strategy to overcome TRAIL resistance in certain CRC cells. Citation Format: Se Lim Kim, Sang-Wook Kim, Soo-Teik Lee, Seong Hun Kim, In Hee Kim, Seung Ok Lee, Dae Ghon Kim. Parthenolide sensitizes colorectal cancer cells to TRAIL-induced apoptosis by regulating mitochonrial pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 789. doi:10.1158/1538-7445.AM2014-789

Abstract 5118: Decursin synergistically enhances TRAIL-induced apoptosis through CHOP dependent DR5 signaling in TRAIL resistant lung cancer cells

Abstract Targeting the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which has been clinically established for anticancer therapy, is the attractive strategy to specifically kill cancer cells through the death receptors. Nonetheless, its therapeutic potential is limited due to the resistance in cancer cells. Thus, in the present study, we evaluated the therapeutic potential of Decursin, a pyranocoumarin isolated from the Korean Angelica gigas root as a TRAIL sensitizer in a variety of resistant lung cancer cells. Co-treatment of Decursin with TRAIL synergistically induced apoptosis in TRAIL resistant lung cancer cells, A545, H1299, H596 and Calu-1. This synergistic effect through the induction of DR5 in Decursin treated cells was also confirmed by immunoblotting and immunoanalysis of endogeneous and surface expression of DR5, respectively. In contrast, in DR knockdown cells, combination treatment Decursin and TRAIL did not induce apoptosis at all. Furthermore, we found that DR5 induction by Decursin was mediated by ER-stress and its byproduct CHOP. Depletion of CHOP via siRNA transfection completely blocked Decusin induced apoptosis cell death in TRAIL resistant lung cancer cells. Taken together, induction of the DR5 via upregulation of CHOP and its inducer, ER stress, by Decursin leads to synergistically increase the TRAIL mediated cell death in TRAIL resistant lung cancer cells. Our results indicate that Decursin is a useful therapeutic strategy for overcoming TRAIL resistance in lung tumor treatment. Key words: Decursin, DR-5, ER stress, CHOP, Trail resistant, Lung cancer Conflict of Interests: The authors disclose no potential conflicts of interest. Acknowledgment: This work was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korea Government (MEST) (no. 2012-0005755). Citation Format: Kim Jae Kwang. Decursin synergistically enhances TRAIL-induced apoptosis through CHOP dependent DR5 signaling in TRAIL resistant lung cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5118. doi:10.1158/1538-7445.AM2014-5118

Aplysin sensitizes cancer cells to TRAIL by suppressing P38 MAPK/survivin pathway.

TNF-related apoptosis-inducing ligand (TRAIL) is a tumor-selective apoptosis inducer and has been shown to be promising for treating various types of cancers. However, the application of TRAIL is greatly impeded by the resistance of cancer cells to its action. Studies show that overexpression of some critical pro-survival proteins, such as survivin, is responsible for TRAIL resistance. In this study, we found that Aplysin, a brominated compound from marine organisms, was able to restore the sensitivity of cancer cells to TRAIL both in vitro and in vivo. Aplysin was found to enhance the tumor-suppressing capacity of TRAIL on several TRAIL-resistant cancer cell lines. TRAIL-induced apoptosis was also potentiated in A549 and MCF7 cells treated with Aplysin. Survivin downregulation was identified as a mechanism by which Aplysin-mediated TRAIL sensitization of cancer cells. Furthermore, the activation of p38 MAPK was revealed in Aplysin-treated cancer cells, and its inhibitor SB203580 was able to abrogate the promoting effect of Aplysin on the response of cancer cells to TRAIL action, as evidenced by restored survivin expression, elevated cell survival and reduced apoptotic rates. In conclusion, we provided evidence that Aplysin acts as a sensitizer for TRAIL and its effect on p38 MAPK/survivin pathway may partially account for this activity. Considering its low cytotoxicity to normal cells, Aplysin may be a promising agent for cancer treatment in combination with TRAIL.

Synergistic effect of celecoxib in tumor necrosis factor‑related apoptosis‑inducing ligand treatment in osteosarcoma cells.

Tumor necrosis factor-related apoptosis‑inducing ligand (TRAIL) is under clinical development as a cancer therapeutic as it has been shown to induce apoptosis in numerous types of cancer cells without significant toxicity towards normal cells. However, the majority of osteosarcoma (OS) tumors are resistant to TRAIL. Thus, the development of cancer therapeutics that overcome TRAIL resistance is required. In the present study, celecoxib (CXB), a non-steroidal anti‑inflammatory drug, was administered in combination with TRAIL to induce cell apoptosis and the doses of the two drugs were simultaneously reduced. The effects of this combination treatment were examined in MG-63 human OS cancer cell lines in culture. Assays of proliferation, apoptosis and tumor growth were performed, along with analysis of the proteins involved. The results revealed that CXB sensitized TRAIL-resistant MG-63 OS cells to TRAIL‑induced apoptosis through downregulation of cellular B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein, caspase-8 and caspase-3. Furthermore, combination treatment reduced tumor growth in a nude rat model. In conclusion, the experimental results provided evidence that the combined administration of CXB and TRAIL is potentially a novel treatment method of OS tumors.

Overcoming hypoxic-resistance of tumor cells to TRAIL-induced apoptosis through melatonin.

A solid tumor is often exposed to hypoxic or anoxic conditions; thus, tumor cell responses to hypoxia are important for tumor progression as well as tumor therapy. Our previous studies indicated that tumor cells are resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cell apoptosis under hypoxic conditions. Melatonin inhibits cell proliferation in many cancer types and induces apoptosis in some particular cancer types. Here, we examined the effects of melatonin on hypoxic resistant cells against TRAIL-induced apoptosis and the possible mechanisms of melatonin in the hypoxic response. Melatonin treatment increased TRAIL-induced A549 cell death under hypoxic conditions, although hypoxia inhibited TRAIL-mediated cell apoptosis. In a mechanistic study, hypoxia inducible factor-1α and prolyl-hydroxylase 2 proteins, which increase following exposure to hypoxia, were dose-dependently down-regulated by melatonin treatment. Melatonin also blocked the hypoxic responses that reduced pro-apoptotic proteins and increased anti-apoptotic proteins including Bcl-2 and Bcl-xL. Furthermore, melatonin treatment reduced TRAIL resistance by regulating the mitochondrial transmembrane potential and Bax translocation. Our results first demonstrated that melatonin treatment induces apoptosis in TRAIL-resistant hypoxic tumor cells by diminishing the anti-apoptotic signals mediated by hypoxia and also suggest that melatonin could be a tumor therapeutic tool by combining with other apoptotic ligands including TRAIL, particularly in solid tumor cells exposed to hypoxia.

Effect of Snake Venom Toxin on Inhibition of Colorectal Cancer HT29 Cells Growth via Death Receptors Mediated Apoptosis

Objectives : We investigated whether snake venom toxin(SVT) from Vipera lebetina turanica sensitizes HT29 human epithelial colorectal cancer cells to tumor necrosis factor(TNF)-related apoptosis-inducing ligand(TRAIL) induced apoptosis in cancer cells. Methods : Cell viability assay was used to assess the inhibitory effect of TRAIL on cell growth of HT29 human colorectal cancer cells. And 6-diamidino-2-phenylindole(DAPI), terminal deoxynucleotidyl transferase mediated dUTP nick end labeling assay(TUNEL) staining assay were used to evaluate cell-apoptosis. Western blot analysis were conducted to observe apoptosis related proteins and death receptor. To assess whether the synergized inhibitory effect of SVT and TRAIL on reactive oxygen species(ROS) generation was reversed by strong anti-oxidative agent. Results : SVT with TRAIL inhibited HT29 cell growth different from TRAIL alone. Consistent with cell growth inhibition, the expression of TRAIL receptors; Expression of death receptor(DR)4 and DR5 was significantly increased and intrinsic pro-apoptotic cleaved caspase-3, -9 was subsequently increased together with increase of Bax/Bcl-2 ratio and extrinsic pro-apototic caspase-8 was also activated. In addition, the expression of anti-apoptotic survival proteins, a marker of TRAIL resistance(eg, cFLIP, survivin, X-linked inhibitor of apoptosis protein(XIAP) and Bcl-2) was suppressed by the combination treatment of SVT and TRAIL. Pretreatment with the ROS scavenger N-acetylcysteine abolished the SVT and TRAIL-induced upregulation of DR4 and DR5 expression and expression of the intrinsic pro-apoptotic caspase-3 and-9. Conclusion : The collective results suggest that SVT facilitates TRAIL-induced apoptosis in HT29 human epithelial colorectal cancer cells through up-regulation of the TRAIL receptors; DR4 and DR5 and consecutive induction of bilateral apoptosis via regulating apoptosis related proteins.

TRAIL-R2-specific antibodies and recombinant TRAIL can synergise to kill cancer cells.

TRAIL induces apoptosis in cancer cells whilst sparing normal tissues. Despite promising pre-clinical results, few patients responded to treatment with recombinant TRAIL (Apo2L/Dulanermin) or TRAIL-R2-specific antibodies, such as conatumumab (AMG655). It is unknown whether this was due to intrinsic TRAIL resistance within primary human cancers or insufficient agonistic activity of the TRAIL-R-targeting drugs. FcγR-mediated crosslinking increases the cancer-cell-killing activity of TRAIL-R2-specific antibodies in vivo. We tested this phenomenon using FcγR-expressing immune cells from patients with ovarian cancer. However, even in the presence of high numbers of FcγR-expressing immune cells, as found in ovarian cancer ascites, AMG655-induced apoptosis was not enabled to any significant degree, indicating that this concept may not translate into clinical use. On the basis of these results we next set out to determine whether AMG655 possibly interferes with apoptosis induction by endogenous TRAIL which could be expressed by immune cells. To do so, we tested how AMG655 affected apoptosis induction by recombinant TRAIL. This, however, resulted in the surprising discovery of a striking synergy between AMG655 and non-tagged TRAIL (Apo2L/TRAIL) in killing cancer cells. This combination was as effective in killing cancer cells as highly active recombinant isoleucine-zipper-tagged TRAIL (iz-TRAIL). The increased killing efficiency was due to enhanced formation of the TRAIL death-inducing signalling complex (DISC), enabled by concomitant binding of Apo2L/TRAIL and AMG655 to TRAIL-R2. The synergy of AMG655 with Apo2L/TRAIL extended to primary ovarian cancer cells and was further enhanced by combination with the proteasome inhibitor bortezomib or a SMAC mimetic. Importantly, primary human hepatocytes were not killed by the AMG655-Apo2L/TRAIL combination, also not when further combined with bortezomib or a SMAC mimetic. We therefore propose that clinical-grade non-tagged recombinant forms of TRAIL, such as dulanermin, could be combined with antibodies such as AMG655 to introduce a highly active TRAIL-R2-agonistic therapy into the cancer clinic.

MiR-942 decreases TRAIL-induced apoptosis through ISG12a downregulation and is regulated by AKT.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an attractive death ligand in targeted cancer therapy. Many cancer cells are refractory to TRAIL-induced cell death and the mechanisms underlying resistance are unclear. The molecular mechanisms of HCC and gastric cancer cells resistant to TRAIL-induced apoptosis were explored using molecular biological and immunological methods. In vivo experiments were conducted to study the effect of interferon stimulated gene 12a (ISG12a) on human liver cancer xenografts in mice. ISG12a decreases in TRAIL-resistant cancer cells. ISG12a regulates the sensitivity of cancer cells to TRAIL in vitro and in vivo. MicroRNA-942 (miR-942) is inversely correlated with ISG12a expression in cancer cells and tissues. Forced expression of miR-942 in TRAIL-sensitive cells significantly reduces endogenous ISG12a level and changes the TRAIL sensitive phenotype to a resistant one. Knockdown of miR-942 expression in TRAIL-resistant cells restores the expression of ISG12a and sensitizes the cells to TRAIL treatment. AKT control TRAIL resistance of cancer cells through downregulation of ISG12a by miR-942. Downregulation of ISG12a by miR-942 is needed to maintain the TRAIL-resistant phenotype of cancer cells and favors cancer cell survival. MiR-942 may offer a novel drug response marker with important implications in designing new therapeutics for TRAIL resistant tumors.