Epigenetic Modification of Death Receptor Genes for TRAIL and TRAIL Resistance in Childhood B-Cell Precursor Acute Lymphoblastic Leukemia.

Immunotherapies specific for B-cell precursor acute lymphoblastic leukemia (BCP-ALL), such as anti-CD19 chimeric antigen receptor (CAR) T-cells and blinatumomab, have dramatically improved the therapeutic outcome in refractory cases. In the anti-leukemic activity of those immunotherapies, TNF-related apoptosis-inducing ligand (TRAIL) on cytotoxic T-cells plays an essential role by inducing apoptosis of the target leukemia cells through its death receptors (DR4 and DR5). Since there are CpG islands in the promoter regions, hypermethylation of the DR4 and DR5 genes may be involved in resistance of leukemia cells to immunotherapies due to TRAIL-resistance. We analyzed the DR4 and DR5 methylation status in 32 BCP-ALL cell lines by sequencing their bisulfite PCR products with a next-generation sequencer. The DR4 and DR5 methylation status was significantly associated with the gene and cell-surface expression levels and the TRAIL-sensitivities. In the clinical samples at diagnosis (459 cases in the NOPHO study), both DR4 and DR5 genes were unmethylated in the majority of cases, whereas methylated in several cases with dic(9;20), MLL-rearrangement, and hypodiploidy, suggesting that evaluation of methylation status of the DR4 and DR5 genes might be clinically informative to predict efficacy of immunotherapy in certain cases with such unfavorable karyotypes. These observations provide an epigenetic rational for clinical efficacy of immunotherapy in the vast majority of BCP-ALL cases.

Glucose deprivation-induced endoplasmic reticulum stress response plays a pivotal role in enhancement of TRAIL cytotoxicity.

Abnormalities of the tumor vasculature result in insufficient blood supply and development of a tumor microenvironment that is characterized by low glucose concentrations, low extracellular pH, and low oxygen tensions. We previously reported that glucose-deprived conditions induce metabolic stress and promote tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cytotoxicity. In this study, we examined whether the metabolic stress-associated endoplasmic reticulum (ER) stress response pathway plays a pivotal role in the enhancement of TRAIL cytotoxicity. We observed no significant cytotoxicity when human colorectal cancer SW48 cells were treated with various doses of TRAIL (2-100 ng/ml) for 4 h or glucose (0-25 mM) for 24 h. However, a combination of TRAIL and low glucose-induced dose-dependent apoptosis through activation of caspases (-8, -9, and -3). Studies with activating transcription factor 4 (ATF4), C/EBP-homologous protein (CHOP), p53 upregulated modulator of apoptosis (PUMA), or death receptor 5 (DR5)-deficient mouse embryonic fibroblasts or HCT116 cells suggest that the ATF4-CHOP-PUMA axis and the ATF4-CHOP-DR5 axis are involved in the combined treatment-induced apoptosis. Moreover, the combined treatment-induced apoptosis was completely suppressed in BH3 interacting-domain death agonist (Bid)- or Bcl-2-associated X protein (Bax)-deficient HCT116 cells, but not Bak-deficient HCT116 cells. Interestingly, the combined treatment-induced Bax oligomerization was suppressed in PUMA-deficient HCT116 cells. These results suggest that glucose deprivation enhances TRAIL-induced apoptosis by integrating the ATF4-CHOP-PUMA axis and the ATF4-CHOP-DR5 axis, consequently amplifying the Bid-Bax-associated mitochondria-dependent pathway.

TNF Family Cytokines Induce Distinct Cell Death Modalities in the A549 Human Lung Epithelial Cell Line when Administered in Combination with Ricin Toxin.

Ricin is a member of the ribosome-inactivating protein (RIP) family of toxins and is classified as a biothreat agent by the Centers for Disease Control and Prevention (CDC). Inhalation, the most potent route of toxicity, triggers an acute respiratory distress-like syndrome that coincides with near complete destruction of the lung epithelium. We previously demonstrated that the TNF-related apoptosis-inducing ligand (TRAIL; CD253) sensitizes human lung epithelial cells to ricin-induced death. Here, we report that ricin/TRAIL-mediated cell death occurs via apoptosis and involves caspases -3, -7, -8, and -9, but not caspase-6. In addition, we show that two other TNF family members, TNF-α and Fas ligand (FasL), also sensitize human lung epithelial cells to ricin-induced death. While ricin/TNF-α- and ricin/FasL-mediated killing of A549 cells was inhibited by the pan-caspase inhibitor, zVAD-fmk, evidence suggests that these pathways were not caspase-dependent apoptosis. We also ruled out necroptosis and pyroptosis. Rather, the combination of ricin plus TNF-α or FasL induced cathepsin-dependent cell death, as evidenced by the use of several pharmacologic inhibitors. We postulate that the effects of zVAD-fmk were due to the molecule's known off-target effects on cathepsin activity. This work demonstrates that ricin-induced lung epithelial cell killing occurs by distinct cell death pathways dependent on the presence of different sensitizing cytokines, TRAIL, TNF-α, or FasL.

Redundant modulatory effects of proinflammatory cytokines in human osteoblastic cells in vitro.

OBJECTIVES: The aim of our study was to investigate possible interaction of IL-17, TRAIL, and TNF-α in the modulation of osteoblast homeostasis in vitro, using human differentiated osteoblastic Saos-2 cells as in vitro model. METHODS: The effects of these cytokines on osteoblastic cell viability were assessed, by MTT assay, alone or in combination, at different times and concentrations. The effects of IL-17 and TNF-α on the regulatory system of osteoclast activity RANK/RANKL/ OPG were evaluated by Western blot and ELISA techniques in cell culture media. Quantitative expression of RANKL, OPG and pro-inflammatory factors were analysed at the mRNA level by quantitative real time RT-PCR. RESULTS: Effects of IL-17, TNF-α and TRAIL on osteoblastic cell viability indicated that IL-17 alone, or in combination with TNF-α did not alter Saos-2 cell viability. On the other hand, TRAIL, as expected, exhibited time- and concentration-dependent cytotoxicity. The expression both RANKL and OPG were increased at the mRNA level and protein release by IL-17 and TNF-α, either alone or in combination. The analysis of IL-17 and TNF-α on pro-inflammatory molecules mRNA expression, such as CXC family chemokines CXCL-1 and CXCL-5, COX-2 and IL-6 demonstrated an increase in these pro-inflammatory cytokines with cooperative effects of the combination. CONCLUSIONS: Overall, these results suggest that IL-17, TRAIL and TNF-α sustain bone tissue inflammation associated with decrease of calcified component. To do so, they act redundantly each other, to amplify the inflammatory response in the bone. In conclusion, unravelling novel molecular targets within the bone-cytokine network represents a platform for innovative treatment of bone diseases due to immunological diseases such as psoriatic arthritis.

N-Acetyl-Glucosamine Sensitizes Non-Small Cell Lung Cancer Cells to TRAIL-Induced Apoptosis by Activating Death Receptor 5.

BACKGROUND/AIMS: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potential anti-cancer agent due to its selective toxicity. However, many human non-small cell lung cancer (NSCLC) cells are partially resistant to TRAIL, thereby limiting its clinical application. Therefore, there is a need for the development of novel adjuvant therapeutic agents to be used in combination with TRAIL. METHODS: In this study, the effect of N-acetyl-glucosamine (GlcNAc), a type of monosaccharide derived from chitosan, combined with TRAIL was evaluated in vitro and in vivo. Thirty NSCLC clinical samples were used to detect the expression of death receptor (DR) 4 and 5. After GlcNAc and TRAIL co-treatment, DR expression was determined by real-time PCR and western blotting. Cycloheximide was used to detect the protein half-life to further understand the correlation between GlcNAc and the metabolic rate of DR. Non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to detect receptor clustering, and the localization of DR was visualized by immunofluorescence under a confocal microscope. Furthermore, a co-immunoprecipitation assay was performed to analyze the formation of death-inducing signaling complex (DISC). O-linked glycan expression levels were evaluated following DR5 overexpression and RNA interference mediated knockdown. RESULTS: We found that the clinical samples expressed higher levels of DR5 than DR4, and GlcNAc co-treatment improved the effect of TRAIL-induced apoptosis by activating DR5 accumulation and clustering, which in turn recruited the apoptosis-initiating protease caspase-8 to form DISC, and initiated apoptosis. Furthermore, GlcNAc promoted DR5 clustering by improving its O-glycosylation. CONCLUSION: These results uncovered the molecular mechanism by which GlcNAc sensitizes cancer cells to TRAIL-induced apoptosis, thereby highlighting a novel effective agent for TRAIL-mediated NSCLC-targeted therapy.

The Proinflammatory Cytokine GITRL Contributes to TRAIL-mediated Neurotoxicity in the HCN-2 Human Neuronal Cell Line.

BACKGROUND: Cytokines belonging to the TNF superfamily play a relevant role in neurodegenerative processes. Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL), released during neuronal injury, has proven to potently mediate and sustain neurotoxic processes leading to neuronal death. Similarly to TRAIL, the cytokine Glucocorticoid-induced TNF receptor ligand (GITRL) is able to transduce proapoptotic signals. In spite of the array of reports suggesting relationships between TRAIL and other cytokines, scanty data are, so far, available about a GITRL/TRAIL crosstalk. METHODS: Here, we investigated possible interactions between TRAIL and the GITRL system in an in vitro model of neurodegeneration, using the human cortical neuronal cell line HCN-2. Cultured HCN-2 neurons were incubated at different times with GITRL and/or TRAIL, and thereafter nucleic acid and protein expression were measured. Real-time PCR analysis showed that the human cortical neuronal cell line HCN-2 does not express GITRL mRNA, but the latter is induced after treatment with TRAIL. In addition, HCN-2 cells did not express the GITRL receptor GITR mRNA, neither in control cultures, nor after treatment with TRAIL. All mRNA data were confirmed by western blot analysis of proteins. Cell viability assay showed that TRAIL, when associated to GITRL, was able to exert additive toxic effects. A counterproof was provided in experiments performed blocking GITRL, in which TRAIL-mediated toxicity appeared significantly reduced. Results suggest that GITRL/TRAIL redundancy during neurodegenerative processes implies extended potentiation of detrimental effects of both cytokines on neurons, eventually leading to larger cell damage and death. CONCLUSION: Finally, characterization of novel molecular targets within the TRAIL/GITRL interplay may represent a platform for innovative therapy of neurodegenerative disorders.

N-glycosylation of mouse TRAIL-R and human TRAIL-R1 enhances TRAIL-induced death.

APO2L/TRAIL (TNF-related apoptosis-inducing ligand) induces death of tumor cells through two agonist receptors, TRAIL-R1 and TRAIL-R2. We demonstrate here that N-linked glycosylation (N-glyc) plays also an important regulatory role for TRAIL-R1-mediated and mouse TRAIL receptor (mTRAIL-R)-mediated apoptosis, but not for TRAIL-R2, which is devoid of N-glycans. Cells expressing N-glyc-defective mutants of TRAIL-R1 and mouse TRAIL-R were less sensitive to TRAIL than their wild-type counterparts. Defective apoptotic signaling by N-glyc-deficient TRAIL receptors was associated with lower TRAIL receptor aggregation and reduced DISC formation, but not with reduced TRAIL-binding affinity. Our results also indicate that TRAIL receptor N-glyc impacts immune evasion strategies. The cytomegalovirus (CMV) UL141 protein, which restricts cell-surface expression of human TRAIL death receptors, binds with significant higher affinity TRAIL-R1 lacking N-glyc, suggesting that this sugar modification may have evolved as a counterstrategy to prevent receptor inhibition by UL141. Altogether our findings demonstrate that N-glyc of TRAIL-R1 promotes TRAIL signaling and restricts virus-mediated inhibition.

Antitherapeutic antibody-mediated hepatotoxicity of recombinant human Apo2L/TRAIL in the cynomolgus monkey.

Apo2L/TRAIL is a member of the tumor necrosis factor superfamily and an important inducer of apoptosis. Recombinant human (rhu) Apo2L/TRAIL has been attractive as a potential cancer therapeutic because many types of tumor cells are sensitive to its apoptosis-inducing effects. Nonclinical toxicology studies were conducted to evaluate the safety of rhuApo2L/TRAIL for possible use in humans. The cynomolgus monkey was chosen for this safety assessment based on high protein sequence homology between human and cynomolgus Apo2L/TRAIL and comparable expression of their receptors. Although hepatotoxicity was observed in repeat-dose monkey studies with rhuApo2L/TRAIL, all animals that displayed hepatotoxicity had developed antitherapeutic antibodies (ATAs). The cynomolgus ATAs augmented the cytotoxicity of rhuApo2L/TRAIL but not of its cynomolgus counterpart. Of note, human and cynomolgus Apo2L/TRAIL differ by four amino acids, three of which are surface-exposed. In vivo studies comparing human and cynomolgus Apo2L/TRAIL supported the conclusion that these distinct amino acids served as epitopes for cross-species ATAs, capable of crosslinking rhuApo2L/TRAIL and thus triggering hepatocyte apoptosis. We describe a hapten-independent mechanism of immune-mediated, drug-related hepatotoxicity - in this case - associated with the administration of a human recombinant protein in monkeys. The elucidation of this mechanism enabled successful transition of rhuApo2L/TRAIL into human clinical trials.

TRAIL causes deletions at the HPRT and TK1 loci of clonogenically competent cells.

When chemotherapy and radiotherapy are effective, they function by inducing DNA damage in cancerous cells, which respond by undergoing apoptosis. Some adverse effects can result from collateral destruction of non-cancerous cells, via the same mechanism. Therapy-related cancers, a particularly serious adverse effect of anti-cancer treatments, develop due to oncogenic mutations created in non-cancerous cells by the DNA damaging therapies used to eliminate the original cancer. Physiologically achievable concentrations of direct apoptosis inducing anti-cancer drugs that target Bcl-2 and IAP proteins possess negligible mutagenic activity, however death receptor agonists like TRAIL/Apo2L can provoke mutations in surviving cells, probably via caspase-mediated activation of the nuclease CAD. In this study we compared the types of mutations sustained in the HPRT and TK1 loci of clonogenically competent cells following treatment with TRAIL or the alkylating agent ethyl methanesulfonate (EMS). As expected, the loss-of-function mutations in the HPRT or TK1 loci triggered by exposure to EMS were almost all transitions. In contrast, only a minority of the mutations identified in TRAIL-treated clones lacking HPRT or TK1 activity were substitutions. Almost three quarters of the TRAIL-induced mutations were partial or complete deletions of the HPRT or TK1 genes, consistent with sub-lethal TRAIL treatment provoking double strand breaks, which may be mis-repaired by non-homologous end joining (NHEJ). Mis-repair of double-strand breaks following exposure to chemotherapy drugs has been implicated in the pathogenesis of therapy-related cancers. These data suggest that TRAIL too may provoke oncogenic damage to the genomes of surviving cells.

Dexamethasone protects normal human liver cells from apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand by upregulating the expression of P-glycoproteins.

Glucocorticoids are effective for the treatment of acute-on-chronic pre-liver failure, severe chronic hepatitis B and acute liver failure; however, the mechanism underlying the effects of treatment by glucocorticoids remains to be fully elucidated. The role and detailed mechanism of how glucocorticoids prevent liver disease progression can be elucidated by investigating the apoptosis of hepatocytes following glucocorticoid treatment. P­glycoproteins (P­gps) also confer resistance to apoptosis induced by a diverse range of stimuli. Glucocorticoids, particularly dexamethasone (DEX), upregulate the expression of P­gp in several tissues. In the present study, the normal human L­02 liver cell line was used, and techniques, including immunocytochemistry, western blot analysis, flow cytometry and reverse transcription­quantitative polymerase chain reaction analysis were used for determining the expression levels of P­gps, and for evaluating the effect of DEX pretreatment on the expression of P­gps. DEX (1­10 µM) was added to the cell culture media and incubated for 24­72 h. The results revealed that DEX upregulated the mRNA and protein levels of P­gp in a dose­ and time­dependent manner. Subsequently, tumor necrosis factor­related apoptosis­inducing ligand (TRAIL) was used for the induction of apoptosis in the cells, followed by a terminal deoxynucleotidyl transferase dUTP nick end labeling assay to assess the apoptotic stages. The results demonstrated that apoptosis in the group of cells, which were pre­treated with DEX was significantly lower than that in the control group. Treatment with tariquidar, a P­gp inhibitor, reduced the anti­apoptotic effects of DEX. These results established that DEX protects normal human liver cells from TRAIL­induced apoptosis by upregulating the expression of P-gp. These observations may be useful for elucidating the mechanism of DEX for preventing the progression of liver disease.

Impact of Procyanidins from Different Berries on Caspase 8 Activation in Colon Cancer.

SCOPE: The aim of this work is to identify which proapoptotic pathway is induced in human colon cancer cell lines, in contact with proanthocyanidins extracted from various berries. METHODS AND RESULTS: Proanthocyanidins (Pcys) extracted from 11 berry species are monitored for proapoptotic activities on two related human colon cancer cell lines: SW480-TRAIL-sensitive and SW620-TRAIL-resistant. Apoptosis induction is monitored by cell surface phosphatidylserine (PS) detection. Lowbush blueberry extract triggers the strongest activity. When tested on the human monocytic cell line THP-1, blueberry Pcys are less effective for PS externalisation and DNA fragmentation is absent, highlighting a specificity of apoptosis induction in gut cells. In Pcys-treated gut cell lines, caspase 8 (apoptosis extrinsic pathway) but not caspase 9 (apoptosis intrinsic pathway) is activated after 3 hours through P38 phosphorylation (90 min), emphasizing the potency of lowbush blueberry Pcys to eradicate gut TRAIL-resistant cancer cells. CONCLUSION: We highlight here that berries Pcys, especially lowbush blueberry Pcys, are of putative interest for nutritional chemoprevention of colorectal cancer in view of their apoptosis induction in a human colorectal cancer cell lines.

Metformin Causes G1-Phase Arrest via Down-Regulation of MiR-221 and Enhances TRAIL Sensitivity through DR5 Up-Regulation in Pancreatic Cancer Cells.

Although many chemotherapeutic strategies against cancer have been developed, pancreatic cancer is one of the most aggressive and intractable types of malignancies. Therefore, new strategies and anti-cancer agents are necessary to treat this disease. Metformin is a widely used drug for type-2 diabetes, and is also known as a promising candidate anti-cancer agent from recent studies in vitro and in vivo. However, the mechanisms of metformin's anti-cancer effects have not been elucidated. We demonstrated that metformin suppressed the expression of miR-221, one of the most well-known oncogenic microRNAs, in human pancreatic cancer PANC-1 cells. Moreover, we showed that the down-regulation of miR-221 by metformin caused G1-phase arrest via the up-regulation of p27, one of the direct targets of miR-221. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is also a promising agent for cancer treatment. While recent studies showed that treatment with only TRAIL was not effective against pancreatic cancer cells, the present data showed that metformin sensitized p53-mutated pancreatic cancer cells to TRAIL. Metformin induced the expressions of death receptor 5 (DR5), a receptor for TRAIL, and Bim with a pro-apoptotic function in the downstream of TRAIL-DR5 pathway. We suggest that the up-regulation of these proteins may contribute to sensitization of TRAIL-induced apoptosis. The combination therapy of metformin and TRAIL could therefore be effective in the treatment of pancreatic cancer.

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.

Bortezomib sensitizes primary meningioma cells to TRAIL-induced apoptosis by enhancing formation of the death-inducing signaling complex.

A meningioma is the most common primary intracranial tumor in adults. Here, we investigated the therapeutic potential of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in 37 meningiomas. Freshly isolated primary meningioma cells were treated with TRAIL with or without different sensitizing protocols, and apoptotic cell death was then quantified. Mechanisms of TRAIL sensitization were determined by a combination of Western blotting, flow cytometry, receptor complex immunoprecipitation, and siRNA-mediated knockdown experiments. Tumor necrosis factor-related apoptosis-inducing ligand receptor expression was analyzed using immunohistochemistry and quantified by an automated software-based algorithm. Primary tumor cells from 11 (29.7%) tumor samples were sensitive to TRAIL-induced apoptosis, 12 (32.4%) were intermediate TRAIL resistant, and 14 (37.8%) were completely TRAIL resistant. We tested synergistic apoptosis-inducing cotreatment strategies and determined that only the proteasome inhibitor bortezomib potently enhanced expression of the TRAIL receptors TRAIL-R1 and/or TRAIL-R2, the formation of the TRAIL death-inducing signaling complex, and activation of caspases; this treatment resulted in sensitization of all TRAIL-resistant meningioma samples to TRAIL-induced apoptosis. Bortezomib pretreatment induced NOXA expression and downregulated c-FLIP, neither of which caused the TRAIL-sensitizing effect. Native TRAIL receptor expression could not predict primary TRAIL sensitivity. This first report on TRAIL sensitivity of primary meningioma cells demonstrates that TRAIL/bortezomib cotreatment may represent a novel therapeutic option for meningiomas.

Gingerol sensitizes TRAIL-induced apoptotic cell death of glioblastoma cells.

Glioblastoma multiforme (GBM) is the most lethal and aggressive astrocytoma of primary brain tumors in adults. Although there are many clinical trials to induce the cell death of glioblastoma cells, most glioblastoma cells have been reported to be resistant to TRAIL-induced apoptosis. Here, we showed that gingerol as a major component of ginger can induce TRAIL-mediated apoptosis of glioblastoma. Gingerol increased death receptor (DR) 5 levels in a p53-dependent manner. Furthermore, gingerol decreased the expression level of anti-apoptotic proteins (survivin, c-FLIP, Bcl-2, and XIAP) and increased pro-apoptotic protein, Bax and truncate Bid, by generating reactive oxygen species (ROS). We also found that the sensitizing effects of gingerol in TRAIL-induced cell death were blocked by scavenging ROS or overexpressing anti-apoptotic protein (Bcl-2). Therefore, we showed the functions of gingerol as a sensitizing agent to induce cell death of TRAIL-resistant glioblastoma cells. This study gives rise to the possibility of applying gingerol as an anti-tumor agent that can be used for the purpose of combination treatment with TRAIL in TRAIL-resistant glioblastoma tumor therapy.

Synergistic effect of celecoxib in tumor necrosis factor­related apoptosis­inducing ligand treatment in osteosarcoma cells.

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

CHF5074 protects SH-SY5Y human neuronal-like cells from amyloidbeta 25-35 and tumor necrosis factor related apoptosis inducing ligand toxicity in vitro.

Alzheimer's disease (AD) is contributed by multiple pathogenic causes. The anomalous protein amyloid-ß (Aß) is regarded as a pivotal factor in AD, and originates from enzymatic cleavage of a precursor protein by the secretase family. 1-(3',4'-Dichloro-2-fluoro[1,1'-biphenyl]-4-yl)-cyclopropanecarboxylic acid (CHF5074) is a non-steroidal antiinflammatory derivative able to inhibit Aß deposition in the brain of transgenic mouse models of AD. The proapoptotic cytokine TRAIL has been reported to mediate Aß-dependent neurotoxicity. Here, the effects of CHF5074 on Aß25-35- triggered TRAIL toxicity were evaluated in the differentiated human neuroblastoma cell line SH-SY5Y in vitro. Cells were pre-treated 1h with CHF5074 at graded concentrations (range: 1 nM-1 uM) and then challenged for 72 h with either Aß25-35 or TRAIL. Results show that CHF5074 treatment prevented apoptotic death in SH-SY5Y cell line in a concentration- dependent fashion. Its maximally active concentration was 10 nM. Then, investigation of related molecular mechanisms underlying such protective effect of CHF5074 suggested that the levels of caspases, as well as of various kinases, including stress and MAP kinases, are modulated by CHF5074. Finally, treatment of injured human neuroblastoma cell line SH-SY5Y with CHF5074 resulted in prominent protection from apoptotic death. The bulk of these data suggest that CHF5074 represents a potential candidate for pharmacological neuroprotective treatment in neurodegenerative disorders.

Epigenetic silencing of apoptosis-inducing gene expression can be efficiently overcome by combined SAHA and TRAIL treatment in uterine sarcoma cells.

The lack of knowledge about molecular pathology of uterine sarcomas with a representation of 3-7% of all malignant uterine tumors prevents the establishment of effective therapy protocols. Here, we explored advanced therapeutic options to the previously discovered antitumorigenic effects of the histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) by combined treatment with the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo-2L). In addition, we investigated the uterine sarcoma cell lines, MES-SA and ESS-1, regarding the underlying molecular mechanisms of SAHA and TRAIL-induced apoptosis and their resistance towards TRAIL. Compared to single SAHA or TRAIL treatment, the combination of SAHA with TRAIL led to complete cell death of both tumor cell lines after 24 to 48 hours. In contrast to single SAHA treatment, apoptosis occured faster and was more pronounced in ESS-1 cells than in MES-SA cells. Induction of SAHA- and TRAIL-induced apoptosis was accompanied by upregulation of the intrinsic apoptotic pathway via reduction of mitochondrial membrane potential, caspase-3, -6, and -7 activation, and PARP cleavage, but was also found to be partially caspase-independent. Apoptosis resistance was caused by reduced expression of caspase-8 and DR 4/TRAIL-R1 in ESS-1 and MES-SA cells, respectively, due to epigenetic silencing by DNA hypermethylation of gene promoter sequences. Treatment with the demethylating agent 5-Aza-2'-deoxycytidine or gene transfer therefore restored gene expression and increased the sensitivity of both cell lines against TRAIL-induced apoptosis. Our data provide evidence that deregulation of epigenetic silencing by histone acetylation and DNA hypermethylation might play a fundamental role in the origin of uterine sarcomas. Therefore, tumor growth might be efficiently overcome by a cytotoxic combinatorial treatment of HDAC inhibitors with TRAIL.

Cellular inhibitor of apoptosis (cIAP)-mediated ubiquitination of phosphofurin acidic cluster sorting protein 2 (PACS-2) negatively regulates tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) cytotoxicity.

Lysosomal membrane permeabilization is an essential step in TRAIL-induced apoptosis of liver cancer cell lines. TRAIL-induced lysosomal membrane permeabilization is mediated by the multifunctional sorting protein PACS-2 and repressed by the E3 ligases cIAP-1 and cIAP-2. Despite the opposing roles for PACS-2 and cIAPs in TRAIL-induced apoptosis, an interaction between these proteins has yet to be examined. Herein, we report that cIAP-1 and cIAP-2 confer TRAIL resistance to hepatobiliary cancer cell lines by reducing PACS-2 levels. Under basal conditions, PACS-2 underwent K48-linked poly-ubiquitination, resulting in PACS-2 proteasomal degradation. Biochemical assays showed cIAP-1 and cIAP-2 interacted with PACS-2 in vitro and co-immunoprecipitation studies demonstrated that the two cIAPs bound PACS-2 in vivo. More importantly, both cIAP-1 and cIAP-2 directly mediated PACS-2 ubiquitination in a cell-free assay. Single c-Iap-1 or c-Iap-2 gene knock-outs in mouse hepatocytes did not lead to PACS-2 accumulation. However, deletion of both cIAP-1 and cIAP-2 reduced PACS-2 ubiquitination, which increased PACS-2 levels and sensitized HuH-7 cells to TRAIL-induced lysosomal membrane permeabilization and apoptosis. Correspondingly, deletion of cIAPs sensitized wild-type, but not PACS-2-deficient hepatocarcinoma cells or Pacs-2-/- mouse hepatocytes to TRAIL-induced apoptosis. Together, these data suggest cIAPs constitutively downregulate PACS-2 by polyubiquitination and proteasomal degradation, thereby restraining TRAIL-induced killing of liver cancer cells.

Site-specific PEGylation of a mutated-cysteine residue and its effect on tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL).

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a promising antitumor agent that specifically induces apoptosis in broad-spectrum tumor cell lines, meanwhile leaving normal cells unaffected. Unfortunately, the clinical development of TRAIL was hampered, and could be attributed to its instability, bioavailability or poor delivery. Although N-terminal specific PEGylation provides a means to improve the pharmacokinetic and stability of TRAIL, it took a bit longer time to accomplish the PEGylation process than expected. We therefore designed another PEGylation approach, mutated Cys-SH site-specific PEGylation, to conjugate methoxypoly(ethylene glycol) maleimide (mPEG-MAL) with TRAIL (95-281) mutant N109C. Asn-109 was chosen as the PEGylated site for it is a potential N-linked glycosylation site. It was shown that ~90% TRAIL mutant N109C could be PEGylated by mPEG-MAL within 40 min. And mPEG(MAL)-N109C was revealed to possess superior in vitro stability and antitumor activity than N-terminal specifically PEGylated TRAIL (114-281) (mPEG(ALD)-TRAIL(114-281)). What's more, mPEG(MAL)-N109C exhibited more therapeutic potentials than mPEG(ALD)-TRAIL(114-281) in tumor xenograft model, benefitting from better drug delivery and bioavailability. These results have demonstrated mutated Cys-SH specific PEGylation is an alternative to site-specifically PEGylate TRAIL efficiently and effectively other than N-terminal specific PEGylation.