BAP1 and YY1 regulate expression of death receptors in malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is a rare, aggressive, and incurable cancer arising from the mesothelial lining of the pleura, with few available treatment options. We recently reported that loss of function of the nuclear deubiquitinase BRCA1-associated protein 1 (BAP1), a frequent event in MPM, is associated with sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. As a potential underlying mechanism, here we report that BAP1 negatively regulates the expression of TRAIL receptors: death receptor 4 (DR4) and death receptor 5 (DR5). Using tissue microarrays of tumor samples from MPM patients, we found a strong inverse correlation between BAP1 and TRAIL receptor expression. BAP1 knockdown increased DR4 and DR5 expression, whereas overexpression of BAP1 had the opposite effect. Reporter assays confirmed wt-BAP1, but not catalytically inactive BAP1 mutant, reduced promoter activities of DR4 and DR5, suggesting deubiquitinase activity is required for the regulation of gene expression. Co-immunoprecipitation studies demonstrated direct binding of BAP1 to the transcription factor Ying Yang 1 (YY1), and chromatin immunoprecipitation assays revealed BAP1 and YY1 to be enriched in the promoter regions of DR4 and DR5. Knockdown of YY1 also increased DR4 and DR5 expression and sensitivity to TRAIL. These results suggest that BAP1 and YY1 cooperatively repress transcription of TRAIL receptors. Our finding that BAP1 directly regulates the extrinsic apoptotic pathway will provide new insights into the role of BAP1 in the development of MPM and other cancers with frequent BAP1 mutations.

TRAIL produced by SAM-1-activated CD4+ and CD8+ subgroup T cells induces apoptosis in human tumor cells through upregulation of death receptors.

Bacterial superantigens potently activate conventional T-cells to induce massive cytokine production and mediate tumor cell death. To engineer superantigens for immunotherapy against tumors in clinic, we previously generated SAM-1, a staphylococcal enterotoxins C2 (SEC2) mutant, that exhibited significantly reduced toxicity but maintained the superantigen activity in animal models. This present study aimed to investigate whether SAM-1 activates T cells and induces apoptosis in human tumor cells. We found that SAM-1 induced the maturation of dendritic cells (DCs) with upregulating expression of the surface markers CD80, CD86 and HLA-DR, which secreted high levels of IL-12p70 by activating TLR2-NF-κB signaling pathways. SAM-1 could activate human CD4+ subgroup T cells and CD8+ subgroup T cells in the presence of mature dendritic cells (DCs), leading to the productions of cytokines TRAIL, IL-2, IFN-γ and TNF-α. We observed that TRAIL mediated the apoptosis and S-phase and G2/M-phase arrest in HGC-27 tumor cells via binding to upregulated death receptors DR4 and DR5. Using shRNA knockdown in HGC-27 cells or constitutive overexpression in ES2 cells for DR4 and DR5, we demonstrated the vital requirement of DR4 and DR5 in apoptosis of tumor cells in response to TRAIL secreted from SAM-1-activated T cells. Collectively, our results will facilitate better understanding of SAM-1-based immunotherapies for cancer.

Luteolin enhances TRAIL sensitivity in non-small cell lung cancer cells through increasing DR5 expression and Drp1-mediated mitochondrial fission.

Cancer cells exhibit extreme sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) over normal cells, highlighting TRAIL's potential as a novel and effective cancer drug. However, the therapeutic effect of TRAIL is limited due to drug resistance. In the present study, we sought to investigate the potential effects of luteolin as a TRAIL sensitizer in non-small cell lung cancer (NSCLC) cells. A549 and H1975 cells had low sensitivity or were resistant to TRAIL. Luteolin alone or in combination with TRAIL decreased cell viability and increased apoptosis. Furthermore, luteolin alone or in combination with TRAIL enhanced death receptor 5 (DR5) expression and dynamin-related protein 1 (Drp1)-dependent mitochondrial fission. However, the synergistic effect of luteolin on cell viability and apoptosis was reversed by DR5 and Drp1 inhibition, suggesting that DR5 upregulation and mitochondrial dynamics may be essential for luteolin as a sensitizer of TRAIL-based therapy in NSCLC. Moreover, luteolin treatment alone or in combination with TRAIL increased the phosphorylation of c-Jun N-terminal kinase (JNK), while SP600125 (the JNK inhibitor) significantly abolished the synergistic effect on DR5 expression and Drp1 translocation, indicating that JNK signaling activation was greatly associated with the synergistic effect exerted by luteolin in NSCLC cells. Therefore, TRAIL combined with luteolin could be as an effective chemotherapeutic strategy for NSCLC.

Non-Thermal Plasma Couples Oxidative Stress to TRAIL Sensitization through DR5 Upregulation.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in various tumor cells without affecting most normal cells. Despite being in clinical testing, novel strategies to induce TRAIL-mediated apoptosis are in need to overcome cancer cell unresponsiveness and resistance. Plasma-activated medium (PAM) markedly stimulates reactive oxygen/nitrogen species (ROS/RNS)-dependent apoptosis in cancer cells. We investigate the capability of PAM and TRAIL (PAM/TRAIL) combination therapy to overcome TRAIL resistance and improve the anticancer efficacy of TRAIL. The combinatorial treatment of PAM and TRAIL shows synergistic effects on growth inhibition in TRAIL-resistant cancer cells via augmented apoptosis by two attributes. DR5 (TRAIL-R2) transcription by CHOP is upregulated in a PAM-generated ROS/RNS-dependent manner, and PAM itself upregulates PTEN expression mediated by suppression of miR-425 which is involved in Akt inactivation, leading to increased apoptosis induction. Treatment of cancer cell lines with the antioxidant N-acetylcysteine reduces the extent of membrane dysfunction and the expression of both CHOP-DR5 and miR-425-PTEN axes, attenuating PAM/TRAIL-induced cancer cell apoptosis. These data suggest that PAM/TRAIL treatment is a novel approach to sensitizing cancer cells to TRAIL-induced apoptosis and overcoming TRAIL resistance. PAM is a promising candidate for further investigations as a chemotherapeutic sensitizer in the treatment of cancer.

A New Efficient Method for Production of Recombinant Antitumor Cytokine TRAIL and Its Receptor-Selective Variant DR5-B.

The cytokine TRAIL induces apoptosis in tumor cells of various origin without affecting normal cells. Clinical trials of TRAIL-receptor (DR4 and DR5) agonists (recombinant TRAIL or death receptors antibodies) have largely failed because most human tumors were resistant to them. Currently, a second generation of agents targeted at TRAIL-R with increased efficiency has been developed. To this end, we have developed DR5-B, a variant of TRAIL selectively interacting with DR5. We have developed a new efficient method for production of TRAIL and DR5-B using expression of these proteins in Escherichia coli strain SHuffle B. The proteins were isolated from the cytoplasmic fraction of cells and purified to a high degree of homogeneity using metal-affinity and ion-exchange chromatography. The protein yield was 211 and 173 mg from one liter of cell culture for DR5-B and TRAIL, respectively, which significantly exceeded the results obtained by other methods. DR5-B killed tumor cells of different origin more efficiently and rapidly compared with TRAIL. The resulting preparations can be used for the study of TRAIL signaling pathways and in preclinical and clinical trials as antitumor agents.

The long non-coding RNA HOTAIR enhances pancreatic cancer resistance to TNF-related apoptosis-inducing ligand.

Pancreatic cancer is a malignant neoplasm with a high mortality rate. Therapeutic agents that activate TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis have shown promising efficacy, but many pancreatic cancers are resistant to TRAIL therapy. Epigenetic regulation plays important roles in tumor pathogenesis and resistance, and a recent study indicated that the long non-coding RNA HOX transcript antisense RNA (HOTAIR) is overexpressed in pancreatic cancer. However, the role of HOTAIR in pancreatic cancer resistance to anticancer agents is unknown. The present study determined the role of HOTAIR in pancreatic cancer TRAIL resistance and investigated the underlying molecular mechanisms. We observed that TRAIL-resistant pancreatic cancer cells had higher levels of HOTAIR expression, whereas TRAIL-sensitive pancreatic cancer cells had lower HOTAIR levels. Overexpressing HOTAIR in TRAIL-sensitive cells attenuated TRAIL-induced apoptosis, and shRNA-mediated HOTAIR knockdown in TRAIL-resistant PANC-1 cells sensitized them to TRAIL-induced apoptosis. These results support a causative effect of HOTAIR on TRAIL sensitivity. Mechanistically, we found that increased HOTAIR expression inhibited the expression of the TRAIL receptor death receptor 5 (DR5), whereas HOTAIR knockdown increased DR5 expression. We further demonstrated that HOTAIR regulates DR5 expression via the epigenetic regulator enhancer of zeste homolog 2 (EZH2) and that EZH2 controls histone H3 lysine 27 trimethylation on the DR5 gene. Taken together, these results demonstrate that high HOTAIR levels increase the resistance of pancreatic cancer cells to TRAIL-induced apoptosis via epigenetic regulation of DR5 expression. Our study therefore supports the notion that targeting HOTAIR function may represent a strategy to overcome TRAIL resistance in pancreatic cancer.

Expression of Death Receptor 4 Is Positively Regulated by MEK/ERK/AP-1 Signaling and Suppressed upon MEK Inhibition.

Death receptor 4 (DR4) is a cell surface receptor for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and triggers apoptosis upon ligation with TRAIL or aggregation. MEK/ERK signaling is a well known and the best-studied effector pathway downstream of Ras and Raf. This study focuses on determining the impact of pharmacological MEK inhibition on DR4 expression and elucidating the underlying mechanism. We found that several MEK inhibitors including MEK162, AZD6244, and PD0325901 effectively decreased DR4 protein levels including cell surface DR4 in different cancer cell lines. Accordingly, pre-treatment of TRAIL-sensitive cancer cell lines with a MEK inhibitor desensitized them to TRAIL-induced apoptosis. These results indicate that MEK inhibition negatively regulates DR4 expression and cell response to TRAIL-induced apoptosis. MEK inhibitors did not alter DR4 protein stability, rather decreased its mRNA levels, suggesting a transcriptional regulation. In contrast, enforced activation of MEK/ERK signaling by expressing ectopic B-Raf (V600E) or constitutively activated MEK1 (MEK1-CA) or MEK2 (MEK2-CA) activated ERK and increased DR4 expression; these effects were inhibited when a MEK inhibitor was present. Promoter analysis through deletion and mutation identified the AP-1 binding site as an essential response element for enhancing DR4 transactivation by MEK1-CA. Furthermore, inhibition of AP-1 by c-Jun knockdown abrogated the ability of MEK1-CA to increase DR4 promoter activity and DR4 expression. These results suggest an essential role of AP-1 in mediating MEK/ERK activation-induced DR4 expression. Our findings together highlight a previously undiscovered mechanism that positively regulates DR4 expression through activation of the MEK/ERK/AP-1 signaling pathway.

Remote Limb Preconditioning Generates a Neuroprotective Effect by Modulating the Extrinsic Apoptotic Pathway and TRAIL-Receptors Expression.

As remote limb preconditioning (RPC) ameliorates brain damage after ischemic cerebral stroke (ICS), the purpose of the present study was to explore the molecular mechanisms in the course of RPC. Results of TUNEL staining and cleaved caspase-3 expression showed that ischemia-induced neuronal apoptosis was inhibited by RPC. The expression changes in cleaved caspase-8, cFLIP, Bid itself, and its truncated form represented that RPC suppressed the activation of extrinsic apoptotic pathway during ICS. Then, the level of cytoplasmic cytochrome c was also decreased by RPC. In addition, RPC might partially suppress TNF-related apoptosis-inducing ligand (TRAIL)-induced extrinsic apoptosis through downregulation of TRAIL death receptors and upregulation of TRAIL decoy receptors. As a counterproof, immunoneutralization of TRAIL in dMCAO rats resulted in significant restraint of tissue damage and in a marked functional recovery. Our data complemented the knowledge of RPC neuroprotective mechanism and provided new evidence for its clinical application.

3-Bromopyruvate enhances TRAIL-induced apoptosis in human nasopharyngeal carcinoma cells through CHOP-dependent upregulation of TRAIL-R2.

Past reports have shown that the sensitivity of cancer cells to TRAIL-induced apoptosis is related to their expression of TRAIL-death receptors on the cell surface. However, the level of TRAIL-death receptors expression on cancer cells is always low. Our previous research showed that nasopharyngeal carcinoma (NPC) cells have a poor sensitivity to low doses of TRAIL. Here, we evaluated combined treatment with the energy inhibitor 3-bromopyruvate (3BP) and TRAIL as a method to produce an increased apoptotic response in NPC cells. The results showed that 3BP and TRAIL together produced higher cytotoxicity and increased TRAIL-R2 expression in NPC cells compared with the effects of either 3BP or TRAIL alone. These findings led us to hypothesize that 3BP may sensitize NPC cells to TRAIL. 3BP is a metabolic blocker that inhibits hexokinase II activity, suppresses ATP production, and induces endoplasmic reticulum (ER) stress. Our results showed that 3BP also activated AMP-activated protein kinase, which we found to play an important role in the induction of ER stress by 3BP. Furthermore, the induction of TRAIL-R2 expression and the sensitization of the NPC cells to TRAIL by 3BP were reduced when we inhibited the expression of CHOP. Taken together, our results showed that a low dose of 3BP sensitized NPC cells to TRAIL-induced apoptosis by the upregulation of CHOP, which was mediated by the activation of AMP-activated protein kinase and ER stress. The results showed that 3BP is a promising candidate agent for enhancing the therapeutic response to TRAIL in NPC.

DDIT3 and KAT2A Proteins Regulate TNFRSF10A and TNFRSF10B Expression in Endoplasmic Reticulum Stress-mediated Apoptosis in Human Lung Cancer Cells.

TNFRSF10A and TNFRSF10B are cell surface receptors that bind to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and mediate the extrinsic pathway of apoptosis. However, the mechanisms of transcriptional regulation of TNFRSF10A and TNFRSF10B remain largely uncharacterized. In this study, two putative DDIT3 binding sites (-1636/-1625; -374/-364) and a putative AP-1 binding site (-304/-298) were identified in the TNFRSF10A promoter region. We found that DDIT3 interacts with phospho-JUN, and the DDIT3·phospho-JUN complex binds to the AP-1 binding site (-304/-298) within the TNFRSF10A promoter region. In addition, we confirmed that KAT2A physically interacts with the N-terminal region (amino acids 1-26) of DDIT3. Importantly, knockdown of KAT2A down-regulated TNFRSF10A and TNFRSF10B and dramatically decreased promoter activity of cells transfected with luciferase reporter plasmid containing the AP-1 binding site (-304/-298) of the TNFRSF10A promoter, as well as cells transfected with luciferase reporter plasmid containing DDIT3 binding site (-276/-264) of the TNFRSF10B promoter. ChIP results suggest that KAT2A may participate in a KAT2A·DDIT3·phospho-JUN complex, or may participate in a KAT2A·DDIT3 complex and acetylate H3K9/K14, respectively. Moreover, we verified that TNFRSF10A mediates apoptosis triggered by endoplasmic reticulum stress in human lung cancer cells. Collectively, we demonstrate that DDIT3 and KAT2A cooperatively up-regulate TNFRSF10A and TNFRSF10B. Our findings highlight novel mechanisms underlying endoplasmic reticulum stress-induced TNFRSF10A and TNFRSF10B expressions and apoptosis. These findings will be helpful for elucidating mechanisms related to anticancer drugs in mediating apoptosis.

The alkyllysophospholipid edelfosine enhances TRAIL-mediated apoptosis in gastric cancer cells through death receptor 5 and the mitochondrial pathway.

The ether phospholipid edelfosine is the prototype of a group of synthetic antitumor alkyllysophospholipid (ALP) compounds that exert pro-apoptotic effects in various types of cancer cells through cell type-dependent mechanisms. In this study, we examined the antitumor effect of edelfosine in human gastric cancer cells. Edelfosine decreased cell viability and induced autophagic death at a moderate concentration (~30 µM), whereas it induced apoptotic cell death at concentrations over 30 µM. Interestingly, low concentrations of edelfosine (5-10 µM) effectively enhanced recombinant human tumor necrosis factor (TNF)-related apoptosis-inducing ligand (rhTRAIL/TNFSF10)-induced apoptosis and clonogenicity in gastric cancer cells, including TRAIL-resistant AGS cells. Edelfosine upregulated the protein level of death receptor 5 (DR5/TNFRSF10B) and/or increased DR5 upregulation in lipid rafts. In addition, edelfosine-mediated rhTRAIL sensitization was regulated by the DR5 pathway. Edelfosine also activated p38MAPK (MAPK14), and edelfosine-mediated rhTRAIL sensitization was partially regulated by a p38-mediated decrease in mitochondrial membrane potential. This study suggests a novel therapeutic strategy targeting gastric cancer cells by using the combination of edelfosine and TRAIL.

Caffeic acid phenethyl ester enhances TRAIL-mediated apoptosis via CHOP-induced death receptor 5 upregulation in hepatocarcinoma Hep3B cells.

Caffeic acid phenethyl ester (CAPE) exhibits various pharmaceutical properties, including anti-bacterial, anti-inflammatory, anti-viral, anti-cancer, and anti-oxidative activity. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been a promising anti-cancer agent that preferentially induces cancer cell apoptosis with negligible cytotoxicity toward normal cells. Therefore, the present study investigated whether CAPE promotes TRAIL-mediated cytotoxicity in hepatocarcinoma Hep3B cells. The present study demonstrated that CAPE sensitized TRAIL-mediated cell death in Hep3B carcinoma cells. The percentages of the apoptotic cells and annexin-V(+) cells significantly increased in combined treatment with CAPE and TRAIL (CAPE/TRAIL). Treatment with pancaspase inhibitor, z-VAD-fmk, attenuated CAPE/TRAIL-induced apoptosis, suggesting that the combined treatment triggers caspase-dependent apoptosis. Additionally, we found that CAPE stimulated the expression of death receptor 5 (DR5) and treatment with DR5/Fc chimera protein significantly blocked CAPE/TRAIL-induced apoptosis, which indicates that CAPE/TRAIL stimulated apoptosis through the binding of TRAIL to DR5. Moreover, expression of transcription factor C/EBP homologous protein (CHOP) markedly increased in response to CAPE and transient knockdown of CHOP abolished CAPE/TRAIL-mediated apoptosis. These results suggest that CHOP is a key regulator in CAPE/TRAIL-mediated apoptosis. Taken together, the present study found that CAPE significantly enhanced TRAIL-mediated apoptosis in Hep3B carcinoma cells and suggested that CAPE has promising potential in chemoprevention of hepatocellular carcinomas.

IL-32α has differential effects on proliferation and apoptosis of human melanoma cell lines.

BACKGROUND: Interleukin-32 (IL-32) is a recently recognized intracellular, proinflammatory cytokine which may play a role in cancer metastasis and patient survival. The role of IL-32 in cancer, especially its direct effect on cancer cells, is not well understood. MATERIAL AND METHODS: Clonogenic assay, PCNA staining, Quick Cell Proliferation assay, TUNEL staining, and caspase-3 activity assay were used to investigate the in vitro role for IL-32α in human melanoma growth. We further investigated the possible molecular mechanisms using RT-PCR and immunohistochemical staining. RESULTS: Exogenous administration of IL-32α inhibited proliferation of the HTB-72 human melanoma cell line, but had little effect on other melanoma cell lines. Inhibition of proliferation in HTB-72 correlated with increased expression of p21 and p53. IL-32α administration also increased apoptosis in HTB-72. This finding correlated with increased expression of TRAILR1. CONCLUSIONS: The data presented suggest a direct effect of IL-32α on the growth of human melanoma and give some insight into the mechanisms which may in part govern this effect. J. Surg. Oncol. 2016;113:364-369. © 2016 Wiley Periodicals, Inc.

COX-2- and endoplasmic reticulum stress-independent induction of ULBP-1 and enhancement of sensitivity to NK cell-mediated cytotoxicity by celecoxib in colon cancer cells.

In the present study, we investigated whether celecoxib could induce the expression of NKG2D ligands in clonogenic colon cancer cells, and increase their susceptibility to NK cell-mediated cell death. Celecoxib and its non-coxib analog, 2,5-dimethyl celecoxib, induced ULBP-1 and DR5 in both COX-2 negative HCT-15 cells and COX-2 positive HT-29 cells. Celecoxib increased their susceptibility to NK92 cells in both DELFIA assay and soft agar colony forming assay. The inducibility of ULBP-1 and DR5 by celecoxib was not different between CD44- and CD44+ HCT-15 cells, and CD133- and CD133+ HT-29 cells. Celecoxib increased the susceptibility of highly clonogenic CD44+ HCT-15 and CD133+ HT-29 cells to NK92 cells, at least comparable to less clonogenic CD44- HCT-15 and CD133- HT-29 cells, respectively. In addition, celecoxib induced CHOP, and thapsigargin, an inducer of ER (endoplasmic reticulum) stress, induced DR5 but not ULBP1 in HCT-15. Taken together, these findings suggest that celecoxib induces the expression of ULBP-1 as well as DR5 in clonogenic colon cancer cells via COX-2 and ER stress-independent pathways, and increases their susceptibility to NK cells.

Poly(ADP-ribose) polymerase inhibitors sensitize cancer cells to death receptor-mediated apoptosis by enhancing death receptor expression.

Recombinant human tumor necrosis factor-α-related apoptosis inducing ligand (TRAIL), agonistic monoclonal antibodies to TRAIL receptors, and small molecule TRAIL receptor agonists are in various stages of preclinical and early phase clinical testing as potential anticancer drugs. Accordingly, there is substantial interest in understanding factors that affect sensitivity to these agents. In the present study we observed that the poly(ADP-ribose) polymerase (PARP) inhibitors olaparib and veliparib sensitize the myeloid leukemia cell lines ML-1 and K562, the ovarian cancer line PEO1, non-small cell lung cancer line A549, and a majority of clinical AML isolates, but not normal marrow, to TRAIL. Further analysis demonstrated that PARP inhibitor treatment results in activation of the FAS and TNFRSF10B (death receptor 5 (DR5)) promoters, increased Fas and DR5 mRNA, and elevated cell surface expression of these receptors in sensitized cells. Chromatin immunoprecipitation demonstrated enhanced binding of the transcription factor Sp1 to the TNFRSF10B promoter in the presence of PARP inhibitor. Knockdown of PARP1 or PARP2 (but not PARP3 and PARP4) not only increased expression of Fas and DR5 at the mRNA and protein level, but also recapitulated the sensitizing effects of the PARP inhibition. Conversely, Sp1 knockdown diminished the PARP inhibitor effects. In view of the fact that TRAIL is part of the armamentarium of natural killer cells, these observations identify a new facet of PARP inhibitor action while simultaneously providing the mechanistic underpinnings of a novel therapeutic combination that warrants further investigation.

α-Hispanolol sensitizes hepatocellular carcinoma cells to TRAIL-induced apoptosis via death receptor up-regulation.

Hispanolone derivatives have been previously described as anti-inflammatory and antitumoral agents. However, their effects on overcoming Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance remain to be elucidated. In this study, we analyzed the cytotoxic effects of the synthetic hispanolone derivative α-hispanolol (α-H) in several tumor cell lines, and we evaluated the induction of apoptosis, as well as the TRAIL-sensitizing potential of α-H in the hepatocellular carcinoma cell line HepG2. Our data show that α-H decreased cell viability in a dose-dependent manner in HeLa, MDA-MB231, U87 and HepG2 cell lines, with a more prominent effect in HepG2 cells. Interestingly, α-H had no effect on non-tumoral cells. α-H induced activation of caspase-8 and caspase-9 and also increased levels of the proapoptotic protein Bax, decreasing antiapoptotic proteins (Bcl-2, X-IAP and IAP-1) in HepG2 cells. Specific inhibition of caspase-8 abrogated the cascade of caspase activation, suggesting that the extrinsic pathway has a critical role in the apoptotic events induced by α-H. Furthermore, combined treatment of α-H with TRAIL enhanced apoptosis in HepG2 cells, activating caspase-8 and caspase-9. This correlated with up-regulation of both the TRAIL death receptor DR4 and DR5. DR4 or DR5 neutralizing antibodies abolished the effect of α-H on TRAIL-induced apoptosis, suggesting that sensitization was mediated through the death receptor pathway. Our results demonstrate that α-H induced apoptosis in the human hepatocellular carcinoma cell line HepG2 through activation of caspases and induction of the death receptor pathway. In addition, we describe a novel function of α-H as a sensitizer on TRAIL-induced apoptotic cell death in HepG2 cells.

The XIAP inhibitor Embelin enhances TRAIL-induced apoptosis in human leukemia cells by DR4 and DR5 upregulation.

The present study was designed to explore the effects of low-toxicity Embelin on TRAIL-induced apoptosis and its possible mechanism in human leukemia cells. Our study showed that low-toxicity Embelin enhanced TRAIL-induced apoptosis through DR4 and DR5 upregulation and caspase activation in HL-60 cells. Pan-caspase inhibitor Z-VAD-FMK inhibited cell apoptosis induced by TRAIL alone or combined with low-toxicity Embelin, which indicated the cytotoxic effect is mediated by caspase-dependent apoptosis. Although Embelin is an X chromosome-linked inhibitor-of-apoptosis protein (XIAP) inhibitor, an XIAP independent effect on cell death was detected in HL-60 cells exposed to low-toxicity Embelin and TRAIL. Low-toxicity Embelin upregulated DR4 and DR5 expression to enhance TRAIL-induced apoptosis. The sensitizing effects of Embelin on TRAIL-induced apoptosis were markedly attenuated when DR4/DR5 was knocked down. These data suggested that low-toxicity Embelin enhanced TRAIL-induced cell apoptosis through DR4 and DR5 upregulation, indicating that combination of low-toxicity Embelin and TRAIL may become as a potential antileukemia strategy.

Shogaol overcomes TRAIL resistance in colon cancer cells via inhibiting of survivin.

In this study, we showed the ability of representative shogaol, which as a major component of ginger, to overcome TRAIL resistance by increasing apoptosis in colon cancer cells. Shogaol increased death receptor 5 (DR5) levels. Furthermore, shogaol decreased the expression level of antiapoptotic proteins (survivin and Bcl-2) and increased pro-apoptotic protein, Bax. Shogaol treatment induced apoptosis and a robust reduction in the levels of the antiapoptotic protein survivin but did not affect the levels of many other apoptosis regulators. Moreover, knockdown of survivin sensitized colon cancer cells to resistant of TRAIL-induced apoptosis. Therefore, we showed the functions of shogaol as a sensitizing agent to induce cell death of TRAIL-resistant colon cancer cells. This study gives rise to the possibility of applying shogaol as an antitumor agent that can be used for the purpose of combination treatment with TRAIL in TRAIL-resistant colon tumor therapy.

The enhanced expression of death receptor 5 (DR5) mediated by HBV X protein through NF-kappaB pathway is associated with cell apoptosis induced by (TNF-α related apoptosis inducing ligand) TRAIL in hepatoma cells.

BACKGROUND: HBV X protein (HBX) is associated with cell apoptosis mediated by TNF-α related apoptosis inducing ligand (TRAIL), while the role of HBX on the expressions of TRAIL receptors death receptor 4 (DR4) and DR5 are unclear. In this study, we detected the cell apoptosis induced by TRAIL as well as gene and protein expressions of DR4 and DR5 in Huh-7 cells steadily transfected with HBX (Huh-7-HBX cells). In addition, we investigated the activation of different pathways associated with the expressions of TRAIL receptors in Huh-7-HBX cells. METHODS: The apoptosis of Huh-7-HBX cells induced by TRAIL was evaluated by flow cytometry analysis. The levels of DR4 and DR5 expression in cells were determined by real-time PCR and western blotting analysis. The activities of JNK pathway and NF-kappaB (NF-κB) pathway were demonstrated by western blotting assay. RESULTS: Compared to control cells, the percentage of cell apoptosis was increased in Huh-7-HBX cells. The increased expressions of DR4 and DR5 on gene and protein levels were observed in Huh-7-HBX cells. Further researches suggested that activation of JNK pathway was increased but not involved in the expression of TRAIL receptors in HBX positive cells. The activation of NF-κB pathway increased and was responsible for DR5 expression and cell apoptosis in HBX positive cells. CONCLUSIONS: These results demonstrate that increased apoptosis induced by TRAIL is associated with increased expression of DR5 that mediated by HBX through NF-κB pathway. This finding provides a critical insight into the mechanism of hepatocyte apoptosis mediated by HBX in HBV infection.

Ubiquitination by the membrane-associated RING-CH-8 (MARCH-8) ligase controls steady-state cell surface expression of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) receptor 1.

The eleven members of the membrane-associated RING-CH (MARCH) ubiquitin ligase family are relatively unexplored. Upon exogenous (over)expression, a number of these ligases can affect the trafficking of membrane molecules. However, only for MARCH-1 endogenous functions have been demonstrated. For the other endogenous MARCH proteins, no functions or substrates are known. We report here that TRAIL-R1 is a physiological substrate of the endogenous MARCH-8 ligase. Human TRAIL-R1 and R2 play a role in immunosurveillance and are targets for cancer therapy, because they selectively induce apoptosis in tumor cells. We demonstrate that TRAIL-R1 is down-regulated from the cell surface, with great preference over TRAIL-R2, by exogenous expression of MARCH ligases that are implicated in endosomal trafficking, such as MARCH-1 and -8. MARCH-8 attenuated TRAIL-R1 cell surface expression and apoptosis signaling by virtue of its ligase activity. This suggested that ubiquitination of TRAIL-R1 was instrumental in its down-regulation by MARCH-8. Indeed, in cells with endogenous MARCH expression, TRAIL-R1 was ubiquitinated at steady-state, with the conserved membrane-proximal lysine 273 as one of the potential acceptor sites. This residue was also essential for the interaction of TRAIL-R1 with MARCH-1 and MARCH-8 and its down-regulation by these ligases. Gene silencing identified MARCH-8 as the endogenous ligase that ubiquitinates TRAIL-R1 and attenuates its cell surface expression. These findings reveal that endogenous MARCH-8 regulates the steady-state cell surface expression of TRAIL-R1.