Tumor Necrosis Factor-related Apoptosis-inducing Ligand Resistant Cells Research Articles

CDH1 overexpression sensitizes TRAIL resistant breast cancer cells towards rhTRAIL induced apoptosis.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is well known for its unique ability to induce apoptosis in cancer cells but not normal cells. However, a subpopulation of cancer cells exist that does not respond to toxic doses of TRAIL. In this study, we aimed to identify key factors regulating TRAIL resistance in breast cancer. rhTRAIL (recombinant human TRAIL) resistant cells (TR) isolated from TRAIL sensitive MDA-MB-231 parental cells (TS) were confirmed using trypan blue assay, cell viability assay and AO/EtBr (acridine orange/ethidium bromide) staining. Microarray was performed followed by analysis using DAVID and Cytoscape bioinformatics software to identify the candidate hub gene. Gene expression of the candidate gene was confirmed using real-time PCR and western blot. Candidate gene was overexpressed via transient transfection to identify its significance in the context of rhTRAIL. Breast cancer patient data was obtained from The Cancer Genome Atlas (TCGA) database. Whole transcriptome analysis identified 4907 differentially expressed genes (DEGs) between TS and TR cells. CDH1 was identified as the candidate hub gene, with 18-degree centrality. We further observed CDH1 protein to be downregulated, overexpression of which increased apoptosis in TR cells after rhTRAIL treatment. TCGA patient data analysis also showed CDH1 mRNA to be low in TRAIL resistant patient group compared to TRAIL sensitive group. CDH1 overexpression sensitizes TR cells towards rhTRAIL induced apoptosis. Therefore, we can hypothesize that CDH1 expression should be taken into account while performing TRAIL therapy in breast cancer.

Abstract 4377: The role of Src in TRAIL signaling in non-small cell lung cancer cells

Abstract Introduction: Non-small cell lung cancer (NSCLC) is the most prevalent form of lung cancer and accounts for most cancer-related deaths worldwide. Despite progress in the treatment of subgroups of patients with targeted therapies, better treatments are required to improve overall prognosis. Agents that target the tumor-necrosis factor (TNF) related apoptosis-inducing ligand (TRAIL) receptors (TRAIL-R1 and -R2) are able to trigger selective apoptosis in tumor cells, however showed discouraging activity in clinical studies. Resistance to TRAIL-induced apoptosis and non-canonical pro-tumorigenic signaling hampers therapeutic efficacy. Previously, we found that TRAIL induces migration and invasion via RIPK1/Src/STAT3 pathway in apoptosis resistant NSCLC cells. Here we aim to analyze in more detail the role of Src in TRAIL non-canonical signaling and apoptosis resistance. Methods: Cytotoxicity to rhTRAIL was assessed by MTT assays. Src was chemically inhibited or genetically ablated by short hairpin(sh)RNA or CRISPR/CAS9. Src phosphorylation was studied by western blotting. Protein interactions were examined by co-immunoprecipitation (IP) and subsequent western blotting. Src interacting proteins were examined by co-IP experiments in conjunction with LC-mass spectrometric (MS) analyses. Results: The function of Src in TRAIL signaling was examined in TRAIL resistant A549 and sensitive H460 NSCLC cells. rhTRAIL treatment revealed distinct Src phosphorylation patterns, indicating that Src is differentially activated by TRAIL. Subsequently, co-IP experiments were performed showing that in A549 cells Src interacts with RIPK1 and Caspase-8 upon TRAIL treatment, but not in H460 cells. Next, we explored the possible role of Src in regulating TRAIL-induced apoptosis by chemical modulation of Src or by gene silencing or genetic knockout of Src. We found no role for Src in regulating sensitivity or resistance to TRAIL-induced apoptosis. To further investigate possible biological consequences of TRAIL-dependent Src activation we performed co-IP coupled with LC-MS analysis. Abundant differences were found in the Src interactome of A549 and H460 cells and in absence and presence of TRAIL. Various proteins known to be involved in tumor signaling were identified to be in complex with Src, including components of the RAF/MEK/ERK, Wnt and SMAD3 signaling pathways. Currently, mechanistic and validation studies are in progress to elucidate the role of these proteins. Conclusions: Src has no role in controlling sensitivity or resistance to TRAIL-induced apoptosis in the examined NSCLC cells. On the contrary, the Src interactome showed the activation of various pro-tumorigenic pathways by TRAIL. We anticipate that a deeper knowledge of TRAIL signaling will lead to novel therapeutic strategies to improve TRAIL-receptor targeted therapy. Supported by a grant from the Dutch Cancer Society (KWF 2011-5211). Citation Format: Margot de Looff, Steven de Jong, Frank A.E. Kruyt. The role of Src in TRAIL signaling in non-small cell lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4377.

1176 Mechanistic analysis of B16F10 melanoma cell death induced by treatment with recombinant TRAIL and bortezomib

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces caspase-dependent apoptosis in cancer cells after binding to its corresponding receptors. Previously, we demonstrated that a recombinant human TRAIL, ILz:rhTRAIL, potentiated TRAIL killing activity by multimerization through its isoleucine zipper hexamerization motif in human colon carcinoma and leukemic T lymphocytes. Additionally, treatment of tumor cells with the proteasome inhibitor bortezomib results in multiple biological effects including inhibition of the cell cycle and increased apoptosis. In this study, we examined the role of ILz:rhTRAIL and bortezomib in inducing cell death in mouse TRAIL resistant B16F10 melanoma cells. Treatment with 100 ng/ml ILz:rhTRAIL and 100 nM bortezomib efficiently eliminated B16F10 melanoma cell growth in vitro in a manner dependent on caspase 3 and 8 activation suggesting a role for apoptosis in the mechanism of action of these two agents. The B16F10 cell death induced by treatment with ILz:rhTRAIL and bortezomib was not carried out through autophagy or in response to endoplasmic reticulum stress. In addition, in vivo tumor progression and metastasis induced by B16F10 injection in the syngeneic mouse melanoma model was also inhibited by treatment with 200 ng/kg ILz:rhTRAIL and 200 pmol/kg bortezomib. Therefore, our results suggest that the combination treatment of ILz:rhTRAIL and bortezomib may represent a new potential therapeutic approach to melanoma.

Irigenin sensitizes TRAIL-induced apoptosis via enhancing pro-apoptotic molecules in gastric cancer cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) holds promising value for cancer therapy due to its capacity to induce apoptosis in cancer cells. Nevertheless, TRAIL therapy is greatly hampered by its resistance. Irigenin (Iri), isoflavonoids, can be isolated from the rhizome of Belamcanda chinensis, and has been shown anti-cancer properties. In this study, we explored if Iri could enhance TRAIL-regulated apoptosis in TRAIL resistant gastric cancer cells. Iri significantly potentiated TRAIL-triggered cytotoxicity. Iri alone and TRAIL alone showed no effective role in apoptosis induction, whereas combined treatment with Iri and TRAIL markedly induced apoptosis in cancer cells, as evidenced by the up-regulation of cleaved Caspase-8/-9/-3 and PARP. Additionally, the sensitization to TRAIL was along with the enhancement of pro-apoptotic proteins, including FAS-associated protein with death domain (FADD), death receptor 5 (DR5) and Bax. And suppressing FADD, DR5 and Bax by si RNA significantly reduced the apoptosis and enhanced the cell viability induced by the co-application of Iri and TRAIL. Moreover, the sensitization to TRAIL was accompanied by the decrease of Cellular-FLICE inhibitory protein (c-FLIP), Bcl-2 and Survivin. Additionally, Iri could sensitize TRAIL to produce reactive oxygen species (ROS). Pre-treatment of N-acetyl-cysteine (NAC), ROS scavenger, attenuated Iri plus TRAIL-induced apoptosis and improved cell viability. Finally, combination of Iri and TRAIL inhibited tumor growth in the xenograft model. Collectively, our present study gave new insights into the effects of Iri on potentiating TRAIL-sensitivity, and suggested that Iri could be a potential candidate for sensitizer of TRAIL-resistant cancer cell treatment.

Novel targets for sensitizing breast cancer cells to TRAIL-induced apoptosis with siRNA delivery.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in variety of cancer cells without affecting most normal cells, which makes it a promising agent for cancer therapy. However, TRAIL therapy is clinically not effective due to resistance induction. To identify novel regulators of TRAIL that can aid in therapy, protein targets whose silencing sensitized breast cancer cells against TRAIL were screened with an siRNA library against 446 human apoptosis-related proteins in MDA-231 cells. Using a cationic lipopolymer (PEI-αLA) for delivery of library members, 16 siRNAs were identified that sensitized the TRAIL-induced death in MDA-231 cells. The siRNAs targeting BCL2L12 and SOD1 were further evaluated based on the novelty and their ability to sensitize TRAIL induced cell death. Silencing both targets sensitized TRAIL-mediated cell death in MDA-231 cells as well as TRAIL resistant breast cancer cells, MCF-7. Combination of TRAIL and siRNA silencing BCL2L12 had no effect in normal human umbilical vein cells and human bone marrow stromal cell. The silencing of BCL2L12 and SOD1 enhanced TRAIL-mediated apoptosis in MDA-231 cells via synergistically activating capsase-3 activity. Hence, here we report siRNAs targeting BCL2L12 and SOD1 as a novel regulator of TRAIL-induced cell death in breast cancer cells, providing a new approach for enhancing TRAIL therapy for breast cancer. The combination of siRNA targeting BCL2L12 and TRAIL can be a highly effective synergistic pair in breast cancer cells with minimal effect on the non-transformed cells.

Bakuchiol sensitizes cancer cells to TRAIL through ROS- and JNK-mediated upregulation of death receptors and downregulation of survival proteins

We investigated whether bakuchiol, an analog of resveratrol enhances the apoptosis ability of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) in cancer cells. Bakuchiol enhanced expression of cell death receptor (DR) in TRAIL-sensitive and -resistant colon cancer cells in a dose-dependent manner. A combination of bakuchiol with TRAIL significantly inhibited cell growth of TRAIL sensitive HCT116 and TRAIL resistant HT-29 cells. The expression of TRAIL receptors; DR4 and DR5 was significantly increased by treatment of bakuchiol, however, the expression of survival proteins (e.g., cFLIP, survivin, XIAP and Bcl2) was suppressed. Moreover, the expression of apoptosis related proteins such as cleaved caspase-3, -8, -9 and PARP was increased by combination treatment of bakuchiol and TRAIL. Depletion of DR4 or DR5 by small interfering RNA significantly reversed the cell growth inhibitory effects of bakuchiol in HCT116 and HT-29 cells. Pretreatment with the c-Jun N-terminal kinase (JNK) inhibitor SP600125 and the reactive oxygen species (ROS) scavenger N-acetylcysteine reduced the bakuchiol induced cell growth inhibitory effects. The collective results suggest that bakuchiol facilitates TRAIL-induced apoptosis in colon cancer cells through up-regulation of the TRAIL receptors; DR4 and DR5 via ROS/JNK pathway signals.

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.

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

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

ALS2CR7 (CDK15) attenuates TRAIL induced apoptosis by inducing phosphorylation of survivin Thr34

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered a promising agent for medical applications because it induces apoptosis selectively in a variety of cancer cells without toxicity to normal human cells. However, its therapeutic potential has been limited by the existence of several cancer cells with TRAIL resistance. TRAIL resistance results from a variety of mechanisms, which occur at various points in the cellular signaling pathways. In this study, we demonstrate that ALS2CR7 (CDK15) can mediate resistance to TRAIL. We also demonstrate that cell viability of TRAIL sensitive HCT116 and MDA-MB-231 cells increased after TRAIL treatment in ALS2CR7 transfected cancer cells compared with vector transfected cancer cells. Furthermore, cell viability was decreased by TRAIL treatment after knockdown with ALS2CR7 siRNA in TRAIL resistant HT29 and MCF-7 cells. We also show that the activated form of apoptotic proteins such as caspase-3, -8 and -9 and PARP increased after TRAIL treatment in the control group, but decreased in the ALS2CR7 transfected group. The expression of survival proteins such as bcl2 and survivin in TRAIL sensitive cancer cells increased in the ALS2CR7 transfected group, but decreased in TRAIL resistant cancer cells treated with ALS2CR7 siRNA. Other survival proteins such as FLIP and XIAP were not affected. ALS2CR7 appears to bind with only survivin, and not bcl2. The phospho-survivin (Thr34) critical in drug resistance was increased by transfection with ALS2CR7, but the expression of death receptors such as DR4 and DR5 was not affected. ALS2CR7 did not bind with any of the death receptors in our study. In summary, our results suggest that ALS2CR7 confers TRAIL resistance to cancer cells via phosphorylation of survivin.

Triptolide sensitizes pancreatic cancer cells to TRAIL-induced activation of the Death Receptor pathway

The tumor necrosis factor related apoptosis-inducing ligand (TRAIL) causes cancer cell death, but many cancers, including pancreatic cancer, are resistant to TRAIL therapy. A combination of TRAIL and the diterpene triepoxide, triptolide, is effective in inducing pancreatic cancer cell death. Triptolide increases levels of death receptor DR5 and decreases the pro-survival FLICE-like inhibitory protein (c-FLIP), which contribute to the activation of caspase-8. This combination further causes both lysosomal and mitochondrial membrane permeabilization, resulting in cell death. Our study provides a mechanism by which triptolide sensitizes TRAIL resistant cells, which may become a novel therapeutic strategy against pancreatic cancer.

MK-2206 induces cell cycle arrest and apoptosis in HepG2 cells and sensitizes TRAIL-mediated cell death

It has become evident that AKT inhibitors have great potential in cancer treatment. In this study, we investigate the anticancer activity of MK-2206, a novel AKT inhibitor, on HepG2 hepatocellular carcinoma cell, and to show whether MK-2206 enhances the apoptosis-inducing potential of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). The cell growth inhibition was evaluated by MTT assay and colony formation assay. Cell cycle distribution was assessed by propidium iodide flow cytometry. Apoptosis was determined by AnnexinV-FITC/PI double staining assay and caspase-9, casapse-7, caspase-3, and PARP cleavage. The results of present study showed that MK-2206-induced G1-phase arrest was associated with a marked decrease in the protein expression of cyclin D1 with concomitant induction of p21 and p27. MK-2206-induced apoptosis was characterized by cleavage of a pro-caspase in a concentration-dependent manner. Moreover, the MAP family kinases p38 kinase and JNK were activated by exposure to MK-2206. SB203580, an p38-specific inhibitor, partially blocked MK-2206-induced death of HepG2 cells and caspase activation. A combination of MK-2206 with TRAIL significantly inhibited growth of TRAIL resistant HepG2 cells. Taken together, our findings provide a new insight to better understand anticancer mechanisms of MK-2206, at least in HepG2 cell. Using of MK-2206 as a potent sensitizer to TRAIL-induced apoptotic cell death offers a promising means of enhancing the efficacy of TRAIL-based HCC treatments.

Novel Insights into the Synergistic Interaction of a Thioredoxin Reductase Inhibitor and TRAIL: The Activation of the ASK1-ERK-Sp1 Pathway

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces cell death in various types of cancer cells but has little or no effects on normal cells. Unfortunately, not all cancer cells respond to TRAIL; therefore, TRAIL sensitizing agents are currently being explored. Here, we reported that 6-(4-N,N-Dimethylaminophenyltelluro)-6-deoxy-β-cyclodextrin (DTCD), a cyclodextrin-derived diorganyl telluride which has been identified as an excellent inhibitor of thioredoxin reductase (TrxR), could sensitize TRAIL resistant human ovarian cancer cells to undergo apoptosis. In vitro, DTCD enhanced TRAIL-induced cytotoxicity in human ovarian cancer cells through up-regulation of DR5. Luciferase analysis and CHIP assays showed that DTCD increased DR5 promoter activity via Sp1 activation. Additionally, DTCD stimulated extracellular signal-regulated kinase (ERK) activation, while the ERK inhibitor PD98059 blocked DTCD-induced DR5 expression and suppressed binding of Sp1 to the DR5 promoter. We further demonstrated that DTCD could induce the release of ASK1 from its complex with Trx-1, and recovered its kinase activity. Meanwhile, suppression of ASK1 by RNA interference led to decreased ERK phosphorylation induced by DTCD. The underlying mechanisms reveal that Trx-1 is heavily oxidized in response to DTCD treatment, in accordance with the fact that DTCD could inhibit the activity of TrxR that reduces oxidized Trx-1. Moreover, using an A2780 xenograft model, DTCD plus TRAIL significantly inhibited the growth of tumor in vivo. Our results suggest that Trx/TrxR system inhibition may play a critical role in apoptosis by combined treatment with DTCD and TRAIL, and raise the possibility that their combination may be a promising strategy for ovarian carcinoma treatment.

Down-Regulation of Survivin by Nemadipine-A Sensitizes Cancer Cells to TRAIL-Induced Apoptosis

The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor family of cytokines. TRAIL selectively induces apoptotic cell death in various tumors and cancer cells, but it has little or no toxicity in normal cells. Agonism of TRAIL receptors has been considered to be a valuable cancer-therapeutic strategy. However, more than 85% of primary tumors are resistant to TRAIL, emphasizing the importance of investigating how to overcome TRAIL resistance. In this report, we have found that nemadipine-A, a cell-permeable L-type calcium channel inhibitor, sensitizes TRAIL-resistant cancer cells to this ligand. Combination treatments using TRAIL with nemadipine-A synergistically induced both the caspase cascade and apoptotic cell death, which were blocked by a pan caspase inhibitor (zVAD) but not by autophagy or a necrosis inhibitor. We further found that nemadipine-A, either alone or in combination with TRAIL, notably reduced the expression of survivin, an inhibitor of the apoptosis protein (IAP) family of proteins. Depletion of survivin by small RNA interference (siRNA) resulted in increased cell death and caspase activation by TRAIL treatment. These results suggest that nemadipine-A potentiates TRAIL-induced apoptosis by down-regulation of survivin expression in TRAIL resistant cells. Thus, combination of TRAIL with nemadipine-A may serve a new therapeutic scheme for the treatment of TRAIL resistant cancer cells, suggesting that a detailed study of this combination would be useful.

Galangin and TRAIL cooperate to suppress A549 lung cancer proliferation via apoptosis and p38 MAPK activation

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) is a promising antitumor therapy against lung cancer which is currently undergoing a phase III clinical trial in China. Unfortunately some cancer patients in the clinical trial displayed resistance to TRAIL treatment. In investigating ways to overcome this resistance, we evaluated the inhibitory effect of galangin on TRAIL resistant A549 human lung adenocarcinoma cells. Here we report that, in comparison with the single agents, the combination of galangin and TRAIL markedly suppressed proliferation of A549 cells and induced apoptosis as shown by DAPI and JC-1 staining. The combination of galangin and TRAIL induced PARP cleavage, activation of caspase-8 and p38 MAPK (mitogen-activated protein kinases). These findings indicate that the combination of galangin and TRAIL may constitute a promising strategy for the treatment of lung cancer.

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

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

Abstract 255: Identification of a novel TRAIL sensitizer in TRAIL-resistant cancer cells

Abstract The Tumor Necrosis Factor Related Apoptosis Inducing Ligand (TRAIL), a TNF-family member is a highly promising agent for cancer therapy as it selectively induces apoptosis in various cancer cells but not in normal cells. However, many primary tumors are resistant to TRAIL-induced apoptosis and the resistant mechanisms are not fully understood. To identify novel TRAIL signaling regulatory molecules, we screened a siRNA library that target 719 whole human protein kinases in TRAIL-resistant lung cancer cell line (A549 cell). As a screening result, we identified a member of NIMA-related kinases as a new regulator of TRAIL-induced cell death. NEK family play key role in controlling mitosis but the role in TRAIL signaling was not elucidated. Down-regulation of NEK by siRNA dramatically increased TRAIL-induced apoptosis in TRAIL resistant cancer cells including A549 cells. However it was not effective on both etoposide and TNF/CHX-mediated apoptosis. In addition, inhibition of NEK highly activated caspase-3 in response to TRAIL. On the other hand, over-expression of NEK reduced TRAIL-induced apoptosis in TRAIL sensitive cells. Our findings suggest that NEK is a novel negative modulator of TRAIL-induced apoptosis in cancer cells. 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 255. doi:1538-7445.AM2012-255

Prediction of proapoptotic anticancer therapeutic response in vivo based on cell death visualization and TRAIL death ligand-receptor interaction

Tumor growth is often associated with insufficient apoptosis. The Tumor Necrosis Factor (TNF)-Related Apoptosis-Inducing Ligand (TRAIL) and its proapoptotic receptors death receptor 4 (DR4) and DR5 agonistic monoclonal antibodies are being developed as targeted therapeutics because they kill cancer cells while sparing normal cells. A challenge to targeted therapeutics is the selection of patients who are most likely to benefit from targeted drugs because of the heterogeneity of cancer. Molecular imaging may be useful in targeted drug development by assessing the target expression and drug-target interaction, and for predicting therapeutic response. We hypothesized that the cell surface expression level of DR4/5 may predict the proapoptotic targeted therapeutic response if the signaling pathway downstream is intact. The goal of this proof-of-concept study was to develop a molecular imaging strategy to predict proapoptotic anti-cancer therapy response at an early stage of treatment. TRAIL and the DR5 agonistic monoclonal antibody HGS-ETR2 (Lexatumumab, TRM-2) were labeled with a near-infrared dye and these were used to image the TRAIL receptors on cultured TRAIL sensitive and TRAIL resistant human tumor cells as well as tumor xenografts. Imaging of cells and tumor-bearing animals was conducted with near infrared fluorescence imagers and apoptosis in cells was assessed by western blots of PARP-cleavage and flow cytometry of sub-G1 content. Apoptosis in tumors was evaluated by imaging near-infrared dye-labeled Annexin V and tumor tissue activated caspase-3 staining. Both in vitro and in vivo studies showed that imaging of death inducing ligand-receptor interaction was consistent with the apoptosis readout. Thus TRAIL sensitive tumors that express TRAIL receptors underwent cell death following treatment whereas tumors lacking TRAIL receptor expression were shown to be TRAIL resistant. In vivo molecular imaging of TRAIL receptor expression correlated with response to TRAIL therapy and an apoptotic response in vivo.

Abstract 4099: F-box protein 10, an NF-κB-dependent anti-apoptotic protein, regulates TRAIL-induced apoptosis through modulating c-Fos/c-FLIP pathway

Abstract Background: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) holds great promise as a potential anti-cancer agent, but some tumors develop resistance to TRAIL. AP-1 family member, c-Fos, is an important modulator of apoptosis. Although F-box protein 10 (FBXL10) has been implicated to regulate an AP-1 family protein, significance and mechanism of FBXL10 in apoptosis has not been investigated. Methods: In this study, different types of TRAIL-sensitive cells (A498, PC3, MBA-MD-231) and TRAIL-resistant cells (LNCaP, PC3TR) were used, and a quantitative analysis of apoptosis was performed by using MTS and caspase-3, PARP activity assay and flow cytometry assay of annexin V- and propidium iodide-stained cells. RNAi, Chromatin immunoprecipitation (ChIP), EMSA, hydrodynamic-based transfection, oligonucleotide decoy technologies were performed to analyze the molecular interaction between FBXL10, c-Fos and NF-κB. The anti-apoptotic role of FBXL10 in vivo was analyzed by Lexatumumab (an agonistic human monoclonal antibody against TRAIL-R2) intravenous injection into mice carrying xenograft prostate cancers. All statistical tests were two-sided. Results: We found that FBXL10 was suppressed and c-Fos was upregulated in TRAIL sensitive cancer cells and xenografts after treatment with TRAIL. However, in TRAIL resistant cancer cells and xenografts, FBXL10 and c-Fos were not affected. Expression of FBXL11, a FBXL10 homologous protein and c-Jun, was not affected by TRAIL. Silencing of FBXL10 sensitized resistant cells to TRAIL. Conversely, over-expression of FBXL10 repressed TRAIL-induced apoptosis. To behave as an anti-apoptotic molecule, we found that FBXL10 directly binds and represses c-Fos promoter in vitro and in vivo. In addition, FBXL10 regulates c-FLIP, another anti−apoptotic molecule, by a c-Fos dependent pathway. We also found that expression of FBXL10 is NF-κB – dependent, and TRAIL or proteasome inhibitors down-regulate FBXL10 via inhibiting NF-κB signaling. Using ChIP, EMSA assays and oligonucleotide decoy technologies, we found that NF-κB -p65 directly binds the FBXL10 promoter in vivo and in vitro, and promotes expression of FBXL10. Conclusions: Differentiating molecular mechanisms between TRAIL sensitive and TRAIL resistant cancer cells will improve the efficacy of apoptotic therapies for cancer patients. In this study, we demonstrate a novel functional role for FBXL10 as an anti-apoptotic molecule, and describe a new apoptotic mediated pathway that involves NF-κB/FBXL10/c-Fos/c-FLIP. Therefore, silencing FBXL10 can help overcome resistant cancer cells for pro-apoptotic therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4099. doi:10.1158/1538-7445.AM2011-4099

Activation of the Akt Survival Pathway Contributes to TRAIL Resistance in Cancer Cells

The mechanism of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance in cancer cells is not fully understood. Here, we show that the Akt survival pathway plays an important role in TRAIL resistance in human cancer cells. Specifically, we found that TRAIL treatment activates the Akt survival pathway and that inhibition of this pathway by the PI3K inhibitor LY294002 or knockdown of Akt sensitizes resistant cancer cells to TRAIL. Since Akt is negatively regulated by the tumor suppressor PTEN, we examined the TRAIL sensitivity in PTEN knockdown mouse prostate epithelial cells and found that PTEN−/− cells are more resistant than PTEN+/+ cells while the sensitivity of PTEN+/− cells fell in between. Further, we showed that overexpression of a mutant PTEN confers TRAIL resistance in PTEN+/+ cells, supporting a role of PTEN in TRAIL sensitivity. In TRAIL resistant breast T47D cells, overexpression of the mutant PTEN further increased their resistance to TRAIL. Taken together, our data indicate that inactivation of functional PTEN and the consequent activation of the Akt pathway prevents TRAIL-induced apoptosis, leading to TRAIL resistance. Therefore, our results suggest that TRAIL resistance can be overcome by targeting PTEN or the Akt survival pathway in cancer cells.