TRAIL-mediated Apoptosis Research Articles

The role of apoptosis defects in malignant mesothelioma pathogenesis with an impact on prognosis and treatment.

Malignant mesothelioma (MM) is a highly aggressive tumor that is strongly related to asbestos fiber exposure. The tumorigenesis procedure in MM is complex, and many pathogenetic mechanisms including chronic inflammation, deregulation of cell death, and the genomic copy-number losses and gains may contribute to carcinogenesis. MM cells are resistant to TRAIL-mediated apoptosis due to defects in extrinsic apoptotic pathway. CAPS, a regulator of cell cycle and death, may contribute to the MM development as well. BAP1 is the most frequently inactivated gene in MPM; BAP1 deficiency triggers malignant transformation via disruption of DNA repair, transcription regulation, cell metabolism, apoptosis, and ferroptosis. In addition, bcl-2 family proteins as well as abnormal activation of PI3K/Akt/mTOR pathway and deregulation of the Wnt signaling pathway may result in MM tumorigenesis. Finally, the Hippo pathway plays a critical role in MPM development. Mutations of NF2 and LATS lead to YAP activation in MPM. Thus, inhibition of YAP activity by YAP inhibitors could be a potentially promising treatment option for MM. In conclusion, extensive genetic alterations exist in mesotheliomas associated with the signaling of apoptotic HM cells death. The comprehension of these pathways may contribute to enhancing survival via developing new effective therapeutic strategies.

Histone Deacetylase Inhibitors Sensitize TRAIL-Induced Apoptosis in Colon Cancer Cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered as a promising anti-cancer therapeutic. However, many cancers have been found to be or to become inherently resistant to TRAIL. A combination of epigenetic modifiers, such as histone deacetylase inhibitors (HDACi’s), with TRAIL was effective to overcome TRAIL resistance in some cancers. Broad spectrum HDACi’s, however, show considerable toxicity constraining clinical use. Since overexpression of class I histone deacetylase (HDAC) has been found in colon tumors relative to normal mucosa, we have focused on small spectrum HDACi’s. We have now tested agonistic receptor-specific TRAIL variants rhTRAIL 4C7 and DHER in combination with several class I specific HDACi’s on TRAIL-resistant colon cancer cells DLD-1 and WiDr. Our data show that TRAIL-mediated apoptosis is largely improved in WiDr cells by pre-incubation with Entinostat-a HDAC1, 2, and 3 inhibitor- and in DLD-1 cells by RGFP966-a HDAC3-specific inhibitor- or PCI34051-a HDAC8-specific inhibitor. We are the first to report that using RGFP966 or PCI34051 in combination with rhTRAIL 4C7 or DHER represents an effective cancer therapy. The intricate relation of HDACs and TRAIL-induced apoptosis was confirmed in cells by knockdown of HDAC1, 2, or 3 gene expression, which showed more early apoptotic cells upon adding rhTRAIL 4C7 or DHER. We observed that RGFP966 and PCI34051 increased DR4 expression after incubation on DLD-1 cells, while RGFP966 induced more DR5 expression on WiDr cells, indicating a different role for DR4 or DR5 in these combinations. At last, we show that combined treatment of RGFP966 with TRAIL variants (rhTRAIL 4C7/DHER) increases apoptosis on 3D tumor spheroid models.

Natural Product Mediated Regulation of Death Receptors and Intracellular Machinery: Fresh from the Pipeline about TRAIL-Mediated Signaling and Natural TRAIL Sensitizers.

Rapidly developing resistance against different therapeutics is a major stumbling block in the standardization of therapy. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-mediated signaling has emerged as one of the most highly and extensively studied signal transduction cascade that induces apoptosis in cancer cells. Rapidly emerging cutting-edge research has helped us to develop a better understanding of the signaling machinery involved in inducing apoptotic cell death. However, excitingly, cancer cells develop resistance against TRAIL-induced apoptosis through different modes. Loss of cell surface expression of TRAIL receptors and imbalance of stoichiometric ratios of pro- and anti-apoptotic proteins play instrumental roles in rewiring the machinery of cancer cells to develop resistance against TRAIL-based therapeutics. Natural products have shown excellent potential to restore apoptosis in TRAIL-resistant cancer cell lines and in mice xenografted with TRAIL-resistant cancer cells. Significantly refined information has previously been added and continues to enrich the existing pool of knowledge related to the natural-product-mediated upregulation of death receptors, rebalancing of pro- and anti-apoptotic proteins in different cancers. In this mini review, we will set spotlight on the most recently published high-impact research related to underlying mechanisms of TRAIL resistance and how these deregulations can be targeted by natural products to restore TRAIL-mediated apoptosis in different cancers.

Hsp90 Inhibitor SNX-2112 Enhances TRAIL-Induced Apoptosis of Human Cervical Cancer Cells via the ROS-Mediated JNK-p53-Autophagy-DR5 Pathway.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent cancer cell apoptosis-inducing factor that can induce apoptosis in a variety of cancer cells. However, resistance to TRAIL in cancer cells is a huge obstacle in creating effective TRAIL-targeted clinical therapies. Thus, agents that can either enhance the effect of TRAIL or overcome its resistance are needed. In this study, we combined TRAIL with SNX-2112, an Hsp90 inhibitor we previously developed, to explore the effect and mechanism that SNX-2112 enhanced TRAIL-induced apoptosis in cervical cancer cells. Our results showed that SNX-2112 markedly enhanced TRAIL-induced cytotoxicity in HeLa cells, and this combination was found to be synergistic. Additionally, we found that SNX-2112 sensitized TRAIL-mediated apoptosis caspase-dependently in TRAIL-resistant HeLa cells. Mechanismly, SNX-2112 downregulated antiapoptosis proteins, including Bcl-2, Bcl-XL, and FLIP, promoted the accumulation of reactive oxygen species (ROS), and increased the expression levels of p-JNK and p53. ROS scavenger NAC rescued SNX-2112/TRAIL-induced apoptosis and suppressed SNX-2112-induced p-JNK and p53. Moreover, SNX-2112 induced the upregulation of death-receptor DR5 in HeLa cells. The silencing of DR5 by siRNA significantly decreased cell apoptosis by the combined effect of SNX-2112 and TRAIL. In addition, SNX-2112 inhibited the Akt/mTOR signaling pathway and induced autophagy in HeLa cells. The blockage of autophagy by bafilomycin A1 or Atg7 siRNA abolished SNX-2112-induced upregulation of DR5. Meanwhile, ROS scavenger NAC, JNK inhibitor SP600125, and p53 inhibitor PFTα were used to verify that autophagy-mediated upregulation of DR5 was regulated by the SNX-2112-stimulated activation of the ROS-JNK-p53 signaling pathway. Thus, the combination of SNX-2112 and TRAIL may provide a novel strategy for the treatment of human cervical cancer by overcoming cellular mechanisms of apoptosis resistance.

Xanthohumol increases death receptor 5 expression and enhances apoptosis with the TNF-related apoptosis-inducing ligand in neuroblastoma cell lines.

High-risk neuroblastoma (NB) is lethal childhood cancer. Published data including ours have reported the anti-proliferative effect of Xanthohumol (XN), a prenylated chalcone, in various cancer types suggesting that XN could be a useful small molecule compound against cancer. The TNF-Related Apoptosis-Inducing Ligand (TRAIL) is an endogenous ligand that is expressed in various immune cells. TRAIL mediates apoptosis through binding of transmembrane receptors, death receptor 4 (DR4) and/or death receptor 5 (DR5). Cancer cells are frequently resistant to TRAIL-mediated apoptosis, and the cause of this may be decreased expression of death receptors. This study aimed to identify combination therapies that exploit XN for NB. First, the effect of XN on cellular proliferation in human NB cell lines NGP, SH-SY-5Y, and SK-N-AS were determined via MTT assay, colony forming assay, and real-time live cell imaging confluency. XN treatment causes a statistically significant decrease in the viability of NB cells with IC50 values of approximately 12 μM for all three cell lines. Inhibition of cell proliferation via apoptosis was evidenced by an increase in pro-apoptotic markers (cleaved PARP, cleaved caspase-3/-7, and Bax) and a decrease in an anti-apoptotic marker, Bcl-2. Importantly, XN treatment inhibited PI3K/Akt pathway and associated with increased expression of DR5 by both mRNA and protein levels. Furthermore, a statistically significant synergistic reduction was observed following combination treatment (50%) compared to either TRAIL (5%) or XN (15%) alone in SK-N-AS cells. Therefore, this study shows XN treatment reduces NB cell growth via apoptosis in a dose-dependent manner, and enhanced growth reduction was observed in combination with TRAIL. This is the first study to demonstrate that XN alters the expression of DR5 as well as the synergistic effect of XN on TRAIL in NB and provides a strong rationale for further preclinical analysis.

CRISPR-mediated upregulation of DR5 and downregulation of cFLIP synergistically sensitize HeLa cells to TRAIL-mediated apoptosis

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has received attention as an anticancer therapy because it mediates apoptosis of several cancer cell types but not normal human cell types. In this study, we implemented genome editing techniques to upregulate DR5 and downregulate cFLIP in HeLa cells to stimulate TRAIL-induced apoptosis. We designed and validated sgRNAs to enrich the endogenous level of DR5 by dead Cas9 (dCas9). Similarly, we designed two sgRNAs to disrupt the cFLIP gene by CRISPR/Cas9. We analyzed the effect of TRAIL on tumor cells by co-transfecting HeLa cells with the best combinations of sgRNAs regulating DR5 and cFLIP genes. TRAIL-induced apoptosis in HeLa cells was evaluated by the γH2AX foci formation assay to check for double-strand break and propidium iodide and Annexin V staining to quantify apoptotic cells. Viable cells were identified by CCK-8 assay, and cleaved-PARP level was evaluated by Western blot. This is the first study to demonstrate that genome editing techniques can be used as an effective combinatorial treatment strategy to induce apoptosis of cancer cells. In particular, enhancement of DR5 expression and inhibition of cFLIP expression by genome editing had a synergistic effect of inhibiting proliferation and inducing apoptosis in TRAIL-resistant HeLa cells. These results suggest that combinatorial treatment strategies mediated by the CRISPR/Cas9 system may be effective for design of other human TRAIL-resistant cell types.

E3 ubiquitin ligases and deubiquitinases as modulators of TRAIL-mediated extrinsic apoptotic signaling pathway

The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) initiates the extrinsic apoptotic pathway through formation of the death-inducing signaling complex (DISC), followed by activation of effector caspases. TRAIL receptors are composed of death receptors (DR4 and DR5), decoy receptors (DcR1 and DcR2), and osteoprotegerin. Among them, only DRs activate apoptotic signaling by TRAIL. Since the levels of DR expressions are higher in cancer cells than in normal cells, TRAIL selectively activates apoptotic signaling pathway in cancer cells. However, multiple mechanisms, including down-regulation of DR expression and pro-apoptotic proteins, and up-regulation of anti-apoptotic proteins, make cancer cells TRAIL-resistant. Therefore, many researchers have investigated strategies to overcome TRAIL resistance. In this review, we focus on protein regulation in relation to extrinsic apoptotic signaling pathways via ubiquitination. The ubiquitin proteasome system (UPS) is an important process in control of protein degradation and stabilization, and regulates proliferation and apoptosis in cancer cells. The level of ubiquitination of proteins is determined by the balance of E3 ubiquitin ligases and deubiquitinases (DUBs), which determine protein stability. Regulation of the UPS may be an attractive target for enhancement of TRAIL-induced apoptosis. Our review provides insight to increasing sensitivity to TRAIL-mediated apoptosis through control of post-translational protein expression.

Auriculasin sensitizes primary prostate cancer cells to TRAIL-mediated apoptosis through up-regulation of the DR5-dependent pathway

Primary prostate cancer cells frequently develop resistance toward chemotherapy as well as most chemotherapeutics have been reported to induce undesirable cytotoxicity in normal cells. In this study, we performed sensitizing activity analysis of auriculasin (AC) to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in RC-58T/h/SA#4 primary prostate cancer cells without significant cytotoxicity in RWPE-1 prostate epithelial cells. Combined treatment with AC and TRAIL at optimal concentrations resulted in tumor-specific apoptotic cell death in RC-58T/h/SA#4 cells, characterized by DNA fragmentation, accumulation of apoptotic cell population, and nuclear condensation. Compared to single treatment with AC or TRAIL, co-treatment with AC and TRAIL significantly increased expression of Bax, cleaved PARP, AIF, endo G, and cytochrome c but decreased expression of phosphorylation of AKT and mammalian target of rapamycin (mTOR), phosphoinositide 3-kinase (PI3K), Bcl-2 and caspases-9, -8, -3, and -10. The sensitizing effect of AC to TRAIL was well correlated with inhibition of death receptor 5 (DR5) CHOP, and p53 expression. Moreover, pre-treatment with a chimeric blocking antibody for DR5 effectively reduced AC-TRAIL-induced cell death and apoptosis-related protein expression. These results suggest that non-toxic concentrations of AC sensitize TRAIL-resistant primary prostate cancer cells to TRAIL-mediated apoptosis via up-regulation of DR5 and downstream signaling pathways.

Hydroxychavicol sensitizes imatinib-resistant chronic myelogenous leukemia cells to TRAIL-induced apoptosis by ROS-mediated IAP downregulation.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of cytokine superfamily, induces apoptosis in a number of tumor cells through the activation of extrinsic apoptotic pathway but shows little or no cytotoxicity toward normal cells. However some tumor cells are inherently resistant to TRAIL-mediated apoptosis, which needs to be addressed to establish TRAIL as a potential chemotherapeutic drug. In this study, our aim was to manipulate TRAIL-apoptosis pathway by hydroxychavicol (HCH), a polyphenol from Piper betel leaf, for the induction of apoptosis in TRAIL resistant chronic myeloid leukemia cell. When imatinib-resistant K562 cells were treated with HCH, it made these K562 cells sensitive to TRAIL. It was observed that HCH downregulated antiapoptotic proteins XIAP and FLIP, whereas the expression of TRAIL receptors, DR4 and DR5, remains unchanged. Moreover, we observed that reactive oxygen species or ROS played a crucial role in the downregulation of FLIP and XIAP because ROS scavenger significantly reversed the decrease of XIAP, and FLIP. Ubiquitin-proteasome pathway was observed to play a crucial role in the downregulation of XIAP and FLIP, as proteasomal inhibitor MG132 significantly reversed the downregulation of XIAP and FLIP. In conclusion, this study demonstrates the combinatorial treatment of TRAIL and HCH as promising alternative therapeutic approach to treat the imatinib-resistant leukemia, which are also resistant to TRAIL.

Diallyl disulfide (DADS) boosts TRAIL-Mediated apoptosis in colorectal cancer cells by inhibiting Bcl-2

Ever since several targeted agents were introduced a decade ago, progress in new therapeutic strategies for colorectal cancer (CRC) has been much slower than that for other cancers. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is widely known to induce cellular apoptosis in numerous cancer cell types. However, many cancer cells are resistant to the effects of TRAIL, and thus, approaches are needed to overcome TRAIL resistance. We demonstrated that non-cytotoxic doses of diallyl disulfide (DADS) increased TRAIL-associated cell death in CRC cell lines. Additionally, synergistic effects between DADS and TRAIL were validated in vivo in nude mice. One process involved in these effects includes down-regulation of the anti-apoptotic protein Bcl-2, and the synergistic effect of DADS with TRAIL was attenuated in Bcl-2-over-expressing cells. Taken together, the results of this study give new insights into the role of DADS in TRAIL-related repression of CRC progression by inhibition of Bcl-2.

PIM kinases mediate resistance of glioblastoma cells to TRAIL by a p62/SQSTM1-dependent mechanism

Glioblastoma (GBM) is the most common and aggressive brain tumor and is associated with poor prognosis. GBM cells are frequently resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and finding new combinatorial therapies to sensitize glioma cells to TRAIL remains an important challenge. PIM kinases are serine/threonine kinases that promote cell survival and proliferation and are highly expressed in different tumors. In this work, we studied the role of PIM kinases as regulators of TRAIL sensitivity in GBM cells. Remarkably, PIM inhibition or knockdown facilitated activation by TRAIL of a TRAIL-R2/DR5-mediated and mitochondria-operated apoptotic pathway in TRAIL-resistant GBM cells. The sensitizing effect of PIM knockdown on TRAIL-induced apoptosis was mediated by enhanced caspase-8 recruitment to and activation at the death-inducing signaling complex (DISC). Interestingly, TRAIL-induced internalization of TRAIL-R2/DR5 was significantly reduced in PIM knockdown cells. Phospho-proteome profiling revealed a decreased phosphorylation of p62/SQSTM1 after PIM knockdown. Our results also showed an interaction between p62/SQSTM1 and the DISC that was reverted after PIM knockdown. In line with this, p62/SQSTM1 ablation increased TRAIL-R2/DR5 levels and facilitated TRAIL-induced caspase-8 activation, revealing an inhibitory role of p62/SQSTM1 in TRAIL-mediated apoptosis in GBM. Conversely, upregulation of TRAIL-R2/DR5 upon PIM inhibition and apoptosis induced by the combination of PIM inhibitor and TRAIL were abrogated by a constitutively phosphorylated p62/SQSTM1S332E mutant. Globally, our data represent the first evidence that PIM kinases regulate TRAIL-induced apoptosis in GBM and identify a specific role of p62/SQSTM1Ser332 phosphorylation in the regulation of the extrinsic apoptosis pathway activated by TRAIL.

Kaempferol Improves TRAIL-Mediated Apoptosis in Leukemia MOLT-4 Cells by the Inhibition of Anti-apoptotic Proteins and Promotion of Death Receptors Expression.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL or Apo2L) is a member of the Tumor Necrosis Factor (TNF) superfamily, which stimulates apoptosis in a wide range of cancer cells through binding to Death Receptors 4 and 5 (DR4/5). Nevertheless, TRAIL has noticeable anti-cancer abilities; some cancer cells acquire resistance to TRAIL, and consequently, its potential for inducing apoptosis in target cells is strongly diminished. Acute lymphoblastic leukemia MOLT-4 cell line is one of the most resistant cells to TRAIL that developed resistance to TRAIL through different pathways. TRAIL plus kaempferol was used to eliminate the resistance of the MOLT-4 cells to TRAIL. Firstly, IC50 for kaempferol (95μM) was determined by using the MTT assay. Secondly, the viability of the MOLT-4 cells was assayed by FACS after Annexin V/PI staining, following treatment with TRAIL (50 and 100nM) and kaempferol (95μM) alone and in combination. Finally, the expression levels of the candidate genes involved in resistance to TRAIL were assayed by real-time PCR technique. Kaempferol plus TRAIL induced apoptosis robustly in MOLT-4 cells at 12, 24 and 48 hours after treatment. Additionally, it was found that kaempferol could inhibit the expression of c-FLIP, X-IAP, cIAP1/2, FGF-8 and VEGF-beta, and conversely augment the expression of DR4/5 in MOLT-4 cells. It is suggested that co-treatment of MOLT-4 cells with TRAIL plus kaempferol is a practical and attractive approach to eliminate cancers' resistance to TRAIL by inhibition of the intracellular anti-apoptotic proteins, upregulation of DR4/5 and also by suppression of the VEGF-beta (VEGFB) and FGF-8 expressions.

Suppression of miR-21-3p enhances TRAIL-mediated apoptosis in liver cancer stem cells by suppressing the PI3K/Akt/Bad cascade via regulating PTEN.

BackgroundTNF-related apoptosis-inducing ligand (TRAIL) functions as a selective apoptosis-inducing ligand in cancer cells with normal cells remaining unaffected; however, resistance limits its anticancer properties. Cancer stem cells (CSCs) are involved in the treatment of resistant cancer cases including liver cancer (LC). The aim of this study was to look into the approaches for increasing the sensitivity of liver cancer stem cells (LCSCs) toward TRAIL.MethodologyPLC, HepG2 and Huh7 LC cell lines were used in this study. Quantitative reverse transcription PCR (qRT-PCR) analysis was done for evaluating the expression of miR-21-3b. Fluorescent-activated cell-sorting equipment was used for separation and identification of LCSCs and non-LCSCs. The cells were transfected with RNA along with miR-21-3p mimics, anti- miR-21-3p, miR-NC and the phosphatase and tensin homologue (PTEN) siRNA. MTT assay for cell viability, Luciferase assay for luciferase activity, Western blots for the expression of proteins and flow cytometry for the measurement of ROS and apoptosis, respectively, were carried out. Tumor xenografts nude mice were used for tumor growth in vivo.ResultsWe found that miR-21-3p was overexpressed in LCSCs compared to non-LCSCs and that the suppression of miR-21-3p along with anti-miR-21-3p enhanced the sensitivity of LCSCs to TRAIL-mediated apoptosis. We further found that miR-21-3p regulated the expression of PTEN in Huh7-LCSCs directly and that the suppression of miR-21-3p enhanced the levels of PTEN. The study confirmed that inhibition of the PI3K/Akt/Bad signaling pathway was involved in enhancing TRAIL-mediated apoptosis of LC cells.ConclusionThe study suggested that overexpression of miR-21-3p suppresses the sensitivity to TRAIL in LCSCs. This study concludes that the suppression of miR-21-3p is a potential approach for enhancing the sensitivity of LC cells toward TRAIL by PI3K/Akt/Bad cascade via the miR-21-3p/PTEN axis.

Delivery of TRAIL-expressing plasmid DNA to cancer cells in vitro and in vivo using aminoglycoside-derived polymers.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a death ligand that can preferentially induce apoptosis in cancer cells over normal cells. The transmembrane form of TRAIL has been shown to elicit much stronger activity than its soluble counterpart but delivery is a potential challenge. Here, we investigated the potential of aminoglycoside-derived polymers to enhance delivery of a plasmid (pEF-TRAIL) that expresses the transmembrane form of TRAIL in order to determine the effect on cell death in vitro and tumor growth in vivo. Transgene delivery efficacy and toxicity of aminoglycoside-derived polymers was first evaluated using a GFP-expressing plasmid (pEF-GFP) at different plasmid amounts and plasmid : polymer ratios in UMUC3 bladder cancer and HeLa cervical cancer cells. Delivery of the TRAIL plasmid using aminoglycoside-derived polymers resulted in up to 60% cell death in UMUC3 and HeLa cells; TRAIL protein expression was confirmed using Western blots. TRAIL plasmid delivery resulted in a decrease in cellular procaspase-8 and an increase in TRAIL receptor DR5 levels, suggesting a role for the death receptor and caspase cascade in TRAIL-mediated apoptosis. The TRAIL plasmid did not cause cell death in normal human or mouse fibroblasts. The in vivo delivery of the TRAIL plasmid using a paromomycin-derived polymer resulted in significant reduction in tumor burden and increased survival in tumor-bearing live mice.

Andrographolide Enhances TRAIL-Induced Apoptosis via p53-Mediated Death Receptors Up-Regulation and Suppression of the NF-кB Pathway in Bladder Cancer Cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an effective chemotherapeutic agent that specifically impairs cancer cells while sparing normal cells; however, some cancer cells develop resistance to TRAIL. Here, we identified Andrographolide, a diterpenoid lactone derived from a traditional herbal medicine Andrographis paniculata, as an ideal sensitizer for TRAIL to overcome bladder cancer. Our results showed that combination treatment of Andro and TRAIL retarded growth, attenuated proliferation, decreased colony formation, inhibited migration and promoted caspases-mediated apoptosis in T24 cells. Additionally, the sensitization by Andro is achieved through up-regulation of death receptors (DR4 and DR5) of TRAIL in a p53-dependent manner. Crucially, Andro is also capable of inactivating NF-κB signaling pathway via transcriptional down-regulation p65/RelA, which is further contributed to enhancement of TRAIL-mediated cytotoxicity. These results indicated that non-toxic doses of Andrographolide sensitized bladder cancer cells to TRAIL-mediated apoptosis, suggesting it as an effective therapeutic agent for TRAIL resistant human bladder cancers.

GSK-3 inhibitors enhance TRAIL-mediated apoptosis in human gastric adenocarcinoma cells.

Resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis has been reported in some cancer cells, including AGS human gastric adenocarcinoma cells. Reducing this resistance might shed light on the treatment of human gastric adenocarcinoma. In this study, we examined whether glycogen synthase kinase-3 (GSK-3) inhibitors can restore TRAIL responsiveness in gastric adenocarcinoma cells. The effect of two GSK-3 inhibitors, SB-415286, and LiCl, on apoptosis signaling of TRAIL in human gastric adenocarcinoma cell lines and primary gastric epithelial cells was analyzed. Both inhibitors can sensitize gastric adenocarcinoma cells, but not primary gastric epithelial cells, to TRAIL-induced apoptosis by increasing caspase-8 activity and its downstream signal transmission. Adding p53 siRNA can downregulate GSK-3 inhibitor-related sensitization to TRAIL-induced apoptosis and caspase-3 activity. GSK-3 inhibitors strongly activate the phosphorylation of JNK. Inhibition of JNK leads to earlier and more intense apoptosis, showing that the activation of JNK may provide anti-apoptotic equilibrium of pro-apoptotic cells. Our observations indicate that GSK-3 inhibitors can sentize AGS gastric adenocarcinoma cells to TRAIL-induced apoptosis. Therefore, in certain types of gastric adenocarcinoma, GSK-3 inhibitor might enhance the antitumor activity of TRAIL and mightbe a promising candidate for the treatment of certain types of gastric adenocarcinoma.

Tumor necrosis factor‑related apoptosis‑inducing ligand inhibits the growth and aggressiveness of colon carcinoma via the exogenous apoptosis signaling pathway

Colon cancer is one of the most common types of gastrointestinal tumor. Previous studies have demonstrated that tumor necrosis factor-(TNF)-related apoptosis-inducing ligand (TRAIL) reduces the aggressiveness of colon cancer tumors and promotes the apoptosis of colon carcinoma cells. In the present study, the inhibitory effects of TRAIL were investigated and the potential mechanism of TRAIL-mediated apoptosis was explored in colon cancer cells. Reverse transcription-quantitative polymerase chain reaction, western blotting, immunofluorescence, immunohistochemistry, TUNEL and flow cytometry assays were used to analyze the effects of TRAIL on the growth, migration, invasion and apoptosis of colon tumor cells. In vivo experiments were performed in mice to analyze the therapeutic effects of TRAIL. The results demonstrated that TRAIL significantly suppressed the growth of colorectal tumor cells in a dose-dependent manner (0.5–2.5 mg/ml) and also promoted colon tumor cell death. The migration and invasion of colon tumor cells were inhibited by the downregulation of fibronectin, Vimentin and E-cadherin. The apoptotic rate revealed that TRAIL (2.0 mg/ml) significantly promoted the apoptosis of colon tumor cells by regulating apoptosis-related gene expression. TRAIL administration promoted the apoptosis of colon tumor cells via the exogenous apoptosis signaling pathway due to the upregulation of caspase-3, caspase-8 and nuclear factor-κB protein expression. In vivo assays revealed that TRAIL administration significantly inhibited tumor growth and promoted apoptotic body and lymphocyte infiltration, which led to increased survival in tumor-bearing mice compared with the control group. Immunohistochemistry revealed that P53 and B-cell lymphoma-2 were downregulated in TRAIL-treated tumors. In conclusion, TRAIL treatment significantly inhibited the growth and aggressiveness of colon tumors by inducing apoptosis via the exogenous apoptosis pathway, which suggests that TRAIL may be a potential anticancer agent for colon carcinoma therapy.

Cepharanthine Enhances TRAIL-Mediated Apoptosis Through STAMBPL1-Mediated Downregulation of Survivin Expression in Renal Carcinoma Cells.

Cepharanthine (CEP) is a natural plant alkaloid, and has anti-inflammatory, antineoplastic, antioxidative and anticancer properties. In this study, we investigated whether CEP could sensitize renal carcinoma Caki cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. CEP alone and TRAIL alone had no effect on apoptosis. However, combined CEP and TRAIL treatment markedly enhanced apoptotic cell death in cancer cells, but not in normal cells. CEP induced downregulation of survivin and cellular-FLICE inhibitory protein (c-FLIP) expression at post-translational levels. Ectopic expression of survivin blocked apoptosis by combined treatment with CEP plus TRAIL, but not in c-FLIP overexpression. Interestingly, CEP induced survivin downregulation through downregulation of deubiquitin protein of STAM-binding protein-like 1 (STAMBPL1). Overexpression of STAMBPL1 markedly recovered CEP-mediated survivin downregulation. Taken together, our study suggests that CEP sensitizes TRAIL-mediated apoptosis through downregulation of survivin expression at the post-translational levels in renal carcinoma cells.

Lipocalin 2 inversely regulates TRAIL sensitivity through p38 MAPK-mediated DR5 regulation in colorectal cancer.

TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis through death receptors (DRs)4 and/or 5expressed on the cell surface. Multiple clinical trials are underway to evaluate the antitumor activity of recombinant human TRAIL and agonistic antibodies to DR4 or DR5. However, their therapeutic potential is limited by the high frequency of cancer resistance. In this study, we provide evidence demonstrating the role of lipocalin2 (LCN2) in the TRAIL-mediated apoptosis of human colorectal cancer(CRC). By analyzing the mRNA expression data of 71CRC tissues from patients, we found that DR5 was preferentially expressed in CRC tissues with a low LCN2 expression level compared to tissues with a high LCN2 expression level. Moreover, we analyzed the association between DR5 and LCN2 expression and this analysis revealed that DR5 expression in CRC tended to be inversely associated with LCN2 expression. By contrast, no association was found between the DR4 and LCN2 expression levels. The expression patterns of LCN2 in human CRC cell lines also exhibited an inverse association with DR5 expression. The knockdown of LCN2 by siRNA in the TRAIL‑resistant CRC cells expressing high levels of LCN2 led to a significant increase in TRAIL-induced apoptosis through the upregulation of DR5 protein and mRNA expression. The mechanism through which LCN2 silencing sensitized the CRC cells to TRAIL was dependent on the extrinsic pathway of apoptosis. In addition, we identified that the knockdown of LCN2 enhanced the sensitivity of the cells to TRAIL through the p38 MAPK/CHOP-dependent upregulation of DR5. Taken together, the findings of this study suggest that LCN2 is responsible for TRAIL sensitivity and LCN2 may thus prove to be a promising target protein in DR-targeted CRC therapy.

MG-132 treatment promotes TRAIL-mediated apoptosis in SEB-1 sebocytes

AimsThis study aimed to identify the mechanism of how MG-132 stimulates cell death in SEB-1 sebocytes. Materials and methodsTUNEL staining and annexin-FITC/PI flow cytometry were utilized to examine the apoptotic cell number of SEB-1 sebocytes and HaCaT keratinocytes upon MG-132 and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) treatment. MTT assay and CCK-8 assay monitored the proliferative rate and viability of both cell lines with different treatment. Western blotting (WB) and qPCR were performed to detect the expression of TRAIL and members of Bcl-2 family at protein and gene level. Additionally, RNA interfering was used to knockdown the mRNA transcription of TRAIL and BIK gene. Key findingsMG-132 treatment enhanced cell death in SEB-1 sebocytes but not in HaCaT keratinocytes. Meanwhile, TRAIL concentrations in SEB-1 sebocytes treated with MG-132 were markedly elevated. Furthermore, treatment with TRAIL or the TRAIL receptor-specific monoclonal antibody AY4 at various doses stimulated cell death in SEB-1 sebocytes in a time- and dose-dependent manner. Silencing of TRAIL restored the cell viability of SEB-1 cells to a normal level after MG-132 treatment. Combined treatment of SEB-1 sebocytes with TRAIL and MG-132 synergistically triggered cell death, suppressed cell proliferation and survival, and promoted BIK expression. Furthermore, BCL2 Interacting Killer (BIK) knockdown via RNA interference participated in the recovery of cell survival reduced by treatment with TRAIL and MG-132. SignificanceThese findings suggest that treatment with the selective proteasome suppressor MG-132 and TRAIL induces cell death in sebocytes through upregulation of BIK, a member of the Bcl-2 family.