Oral recombinant methioninase increases TRAIL receptor-2 expression to regress pancreatic cancer in combination with agonist tigatuzumab in an orthotopic mouse model.

Methionine addiction is a fundamental and general hallmark of cancer. Gene expression analysis showed that methionine restriction (MR) of methionine-addicted cancer cells increases TNF-related apoptosis-induced ligand receptor-2 (TRAIL-R2) expression. Here, we determined the effects of MR on TRAIL-R2 targeted therapy in pancreatic cancer by the TRAIL-R2 agonist tigatuzumab. Human pancreatic cancer cell lines were cultured in control or methionine-free medium. The effects of MR on TRAIL-R2 expression and sensitivity to tigatuzumab were evaluated in vitro. An orthotopic pancreatic cancer mouse model was established to evaluate the efficacy of MR using oral recombinant methioninase (o-rMETase), and the efficacy of tigatuzumab and their combination. MR enabled tigatuzumab-induced apoptosis, by increasing TRAIL-R2 expression in pancreatic cancer cells in vitro. The protein expression level of the melanoma-associated antigen MAGED2, which reduces TRAIL-R2 expression, was decreased by MR. In the orthotopic pancreatic cancer mouse model, o-rMETase increased TRAIL-R2 expression level in the tumors and enabled the antitumor efficacy of tigatuzumab. MR, effected by o-rMETase, enabled the efficacy of the TRAIL-R2 agonist tigatuzumab by increasing TRAIL-R2 expression in pancreatic cancer. Our results suggest that o-rMETase has clinical potential for treating pancreatic cancer.

TRAIL receptors are differentially regulated and clinically significant in gallbladder cancer.

Deregulation of the receptors of TNF-related apoptosis inducing ligand (TRAIL) has been reported in various cancers. In an effort to define the role of these receptors we profiled their expression in gallbladder cancer (GBC) and explored their clinical significance. Expression of TRAIL receptors' mRNA in GBC was analysed through reverse transcriptase polymerase chain reaction (RT-PCR), and protein through western blotting, immunohistochemistry and enzyme-linked immunosorbent assay (ELISA). mRNA data show frequent higher expression of TRAIL receptors in GBC samples. Death receptors DR4 and DR5 showed significant negative correlation with tumour stage, T stage and tumour grade; DcR1 transcript showed positive correlation with tumour stage, N stage, M stage and tumour grade. Similarly, IHC showed frequent positive staining for DR4, DR5 and DcR1in GBC samples. Cytoplasmic and nuclear DR4 protein showed negative correlation with T stage and tumour grade, whereas cytoplasmic DcR1 protein showed positive correlation with tumour stage and N stage. Nuclear DcR1 showed positive correlation with N stage. ELISA results showed significantly higher expression of secretory DcR1 in GBC patients. Kaplan-Meier analysis demonstrated significantly decreased mean survival of patients with positive staining of cytoplasmic DcR1. High level of death receptors identified the patients with early gallbladder cancer, whereas high DcR1 expression served as a prognostic factor for poor outcome.

p-Hydroxylcinnamaldehyde from cochinchinamomordica seed reverses resistance to TRAIL in human oesophageal squamous cell carcinoma via the activation of the p38 mitogen-activated protein kinase signalling pathway.

BACKGROUND: Our previous studies have showed that p-Hydroxylcinnamaldehyde (CMSP) could induce the differentiation of ESCC cells via the cAMP-RhoA-MAPK signalling pathway, which suggests a new potential strategy for ESCC treatment. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in several tumour cells by binding to the death receptors DR4 and DR5. However, TRAIL has little effect on oesophageal squamous cell carcinoma (ESCC) cells due to the loss of the receptors. The present study determined the effect of CMSP, the firstly found chemical constituent of Cochinchinamomordica seed (CMS), on TRAIL-induced apoptosis and its mechanism in ESCC cells. METHODS: MTS assays were performed to examine the CMSP- and TRAIL-mediated inhibition of ESCC cell growth. Flow cytometry and Hoechst 33258 staining assays were used to detect apoptosis in ESCC cells treated with CMSP combined with TRAIL. Western blotting was used to determine the effect of CMSP on the expression of p38, p-p38, DR4, DR5, Bid and caspase-3/8 in ESCC cells treated with CMSP combined with TRAIL. Additionally, immunodeficient Balb-c/null mouse model was used to determine the chemotherapeutic efficacy of CMSP and TRAIL against ESCC tumour xenograft growth in vivo. RESULTS: We found that the combination of CMSP and TRAIL had a greater inhibitory effect on ESCC cell viability in vitro than CMSP or TRAIL alone. CMSP enhanced the TRAIL-induced apoptosis in ESCC cells by upregulating the expression of DR4 and DR5 via the p38 MAPK signalling pathway. Furthermore, the increased expression of DR4 and DR5 upon TRAIL-induced apoptosis in ESCC cells was mediated at least in part by subsequent caspase-3 and caspase-8 activation. Moreover, the in vivo model showed that tumour growth was significantly slower in CMSP and TRAIL combination-treated mice than in mice treated with CMSP or TRAIL alone. CONCLUSION: Taken together, our findings indicate that CMSP as an extract from TCM, might be as a potential sensitizer of TRAIL and thus provide a novel strategy for the clinical treatment of ESCC.

The expression of death receptor systems TRAIL-R1/-R2/-R4, CD95 and TNF-R1 and their cognate ligands in pancreatic ductal adenocarcinoma.

The expression of five members of the TNF receptor superfamily and two of their ligands in human pancreatic ductal adenocarcinoma were investigated in parallel by immunohistochemistry. 41 patients with histologically confirmed ductal carcinoma of the pancreas were enrolled in this study in order (i) to compare the individual TNFR-SF expression and their ligands in PDAC-cells and (ii) to investigate their correlation with survival data. All patients had undergone pancreaticoduodenectomy and were staged as pT3N1M0. Immunostaining was done on FFPE tissue sections of the tumor tissue, using antibodies directed against TRAIL-Receptor-1, -2 and -4, TRAIL, CD95, TNF-Receptor-1 and TNF-α. The intensity and quantity of immunostaining were evaluated separately for tumor cell cytoplasm and tumor cell nucleus. Immunostaining results were correlated with each other and with patient survival. All proteins were found to be expressed in the majority of the tumor cells. The expression (i) of the following members of TNFR-SF and their ligands correlated with each other: TNF-Receptor-1 and TNFα (cytoplasmatic scores, p=0.001), TNF-Receptor 1 and TRAIL (nuclear antigen expression p=0.005 and the main score p=0.001, which contains the overall intracellular antigen expression), TNF-Receptor 1 and CD95 (main score, p=0.001), TRAIL-Receptor-1 and TRAIL-Receptor-2 (nuclear parameters, p=0.023), TRAIL-Receptor-4 and TRAIL (main score p=0.041). In addition (ii), high cytoplasmatic expression of TNF-Receptor-1 and a strong cytoplasmatic and nuclear expression of CD95 correlated significantly with a better prognosis of the PDAC patients.

Cytoplasmic TRAIL-R1 is a positive prognostic marker in PDAC.

BACKGROUND: The death receptors TRAIL-R1 and TRAIL-R2 are frequently overexpressed in cancer and there is an emerging evidence for their important role in malignant progression, also in the case of pancreatic ductal adenocarcinoma (PDAC). In their canonical localization at the plasma membrane, TRAIL-R1/-R2 may induce cell death and/or pro-inflammatory signaling leading to cell migration, invasion and metastasis. Although, they have repeatedly been found intracellular, in the cytoplasm and in the nucleus, their functions in intracellular locations are still not well understood. Likewise, studies dealing with the prognostic relevance of TRAIL-Rs located in particular cellular compartments are very rare. For PDAC, the correlation of nuclear TRAIL-R2 with worse patients' prognosis has been shown recently. Corresponding data on TRAIL-R1 are not available so far. METHODS: In the present study we analyzed the expression of TRAIL-R1 in 106 PDACs and 28 adjacent, peritumoral non-malignant pancreatic ducts with special emphasis on its cytoplasmic and nuclear localization and correlated the immunohistochemical findings with clinico-pathological patient characteristics. RESULTS: TRAIL-R1 was found in 93.4% of all PDAC samples. Cytoplasmic staining was present with very similar intensity in tumor and normal tissue. In contrast, nuclear TRAIL-R1 staining was significantly stronger in tumor compared to normal tissue (p = 0.006). Interestingly, we found that the number of cells with cytoplasmic TRAIL-R1 staining negatively correlates with tumor grading (p = 0.043). No such correlation could be detected for nuclear TRAIL-R1. Neither, cytoplasmic nor nuclear TRAIL-R1 staining showed a correlation with other clinico-pathological parameter such as pTNM categories. However, Kaplan-Meier analyses revealed significantly prolonged median survival of patients with positive cytoplasmic TRAIL-R1 expression in more than 80% of tumor cells compared to patients with tumors containing a smaller quantity of cells positively stained for cytoplasmic TRAIL-R1 (20 vs. 8 months; p = 0.004). CONCLUSION: Cytoplasmic TRAIL-R1 is a positive prognostic marker for patients with PDAC. Our findings indicate that loss of cytoplasmic TRAIL-R1 results in recurrent disease with more malignant phenotype thus suggesting anti-tumor activities of cytoplasmic TRAIL-R1 in PDAC.

Comprehensive expression analysis of TNF-related apoptosis-inducing ligand and its receptors in colorectal cancer: Correlation with MAPK alterations and clinicopathological associations.

TNF-related, apoptosis-inducing ligand (TRAIL) apoptotic pathway constitutes a promising therapeutic target due to high selectivity and low toxicity of TRAIL targeting agents when administered in combination therapies. 106 colorectal cancers were examined for: relative mRNA expression of TRAIL pathway genes, decoy receptors TRAIL-R3 and TRAIL-R4 promoter methylation and the presence of KRAS, NRAS, BRAF mutations. Elevated mRNA levels were observed in 26%, 15%, 13%, 12% and 10% of the cases for TRAIL-R4, TRAIL-R3, TRAIL-R2, TRAIL-R1 and TRAIL genes respectively. Reduced mRNA levels were detected in 77%, 65%, 64%, 60% and 37% of the cases for TRAIL, TRAIL-R2, TRAIL-R3, TRAIL-R1 and TRAIL-R4 genes respectively. TRAIL-R3 and TRAIL-R4 promoter methylation was detected in 55% and 16% of the analysed samples respectively. TRAIL-R1, TRAIL-R2 elevated relative mRNA levels inversely correlated with tumor stage (p = .036, p = .048). Strong linear correlations of TRAIL receptors' mRNA levels were found: TRAIL-R1/TRAIL-R2 (R = 0.653, p < .001), TRAIL-R2/TRAIL-R3 (R = 0.573, p < .001). Finally, relative expression of TRAIL was correlated with KRAS, BRAF and NRAS mutation status, defining an inverse correlation between increased TRAIL expression and the absence of mutations in Mitogen-activated protein kinase (MAPK) pathway. In conclusion, simultaneous analysis of TRAIL pathway membrane components, pointed towards a significant deregulation of mRNA expression in colorectal tumours. Death receptor overexpression was an indicator of a less aggressive phenotype. The multiple expression patterns of TRAIL pathway components in colorectal tumours underscore the importance of patient selection in order to achieve maximum efficiency with TRAIL targeted therapy.

Alveolar epithelial cells in idiopathic pulmonary fibrosis display upregulation of TRAIL, DR4 and DR5 expression with simultaneous preferential over-expression of pro-apoptotic marker p53.

Idiopathic pulmonary fibrosis (IPF) is a progressive, debilitating, and fatal lung disease of unknown aetiology with no current cure. The pathogenesis of IPF remains unclear but repeated alveolar epithelial cell (AEC) injuries and subsequent apoptosis are believed to be among the initiating/ongoing triggers. However, the precise mechanism of apoptotic induction is hitherto elusive. In this study, we investigated expression of a panel of pro-apoptotic and cell cycle regulatory proteins in 21 IPF and 19 control lung tissue samples. We reveal significant upregulation of the apoptosis-inducing ligand TRAIL and its cognate receptors DR4 and DR5 in AEC within active lesions of IPF lungs. This upregulation was accompanied by pro-apoptotic protein p53 overexpression. In contrast, myofibroblasts within the fibroblastic foci of IPF lungs exhibited high TRAIL, DR4 and DR5 expression but negligible p53 expression. Similarly, p53 expression was absent or negligible in IPF and control alveolar macrophages and lymphocytes. No significant differences in TRAIL expression were noted in these cell types between IPF and control lungs. However, DR4 and DR5 upregulation was detected in IPF alveolar macrophages and lymphocytes. The marker of cellular senescence p21(WAF1) was upregulated within affected AEC in IPF lungs. Cell cycle regulatory proteins Cyclin D1 and SOCS3 were significantly enhanced in AEC within the remodelled fibrotic areas of IPF lungs but expression was negligible in myofibroblasts. Taken together these findings suggest that, within the remodelled fibrotic areas of IPF, AEC can display markers associated with proliferation, senescence, and apoptotosis, where TRAIL could drive the apoptotic response. Clear understanding of disease processes and identification of therapeutic targets will direct us to develop effective therapies for IPF.

Sensitization of hepatocellular carcinoma cells to Apo2L/TRAIL by a novel Akt/NF-κB signalling inhibitor.

Hepatocellular carcinoma (HCC) cells are intrinsically resistant to tumour necrosis factor-related apoptosis ligand (Apo2L/TRAIL), in part, due to the compensatory activation of nuclear factor-kappaB (NF-κB). To broaden the clinical utilization of Apo2L/TRAIL in HCC, OSU-A9, a potent indole-3-carbinol-derived Akt/NF-κB signalling inhibitor was used to overcome the intrinsic resistance. The antitumour effects of OSU-A9, Apo2L/TRAIL and the therapeutic combination were assessed by MTT assay, caspase activation and PARP cleavage, and the synergistic interactions were determined by Calcusyn analysis. NF-κB reporter gene and RT-PCR were tested for the activation of NF-κB and the expression of death receptors (DR)4 and 5. OSU-A9 could sensitize HCC cells to Apo2L/TRAIL with high potency through down-regulation of Akt/NF-κB signalling. OSU-A9 dose-dependently reduced Akt phosphorylation and the expression and nuclear localization of RelA/p65, accompanied by parallel decreases in the expression of NF-κB target products, including Bcl-xL, Mcl-1, cIAP1, cIAP2 and survivin. Moreover, OSU-A9 increased DR5 expression through a reactive oxygen species (ROS)-dependent mechanism. Concertedly, these mechanisms underlie the synergistic interaction between OSU-A9 and Apo2L/TRAIL in mediating apoptotic death in HCC cells. The ability of OSU-A9 to accentuate Apo2L/TRAIL-induced apoptosis by inactivating Akt/NF-κB signalling might foster a promising therapeutic strategy for HCC.

Expression of tumor necrosis factor-related apoptosis-inducing ligand death receptors DR4 and DR5 in human nonmelanoma skin cancer.

Death receptors 4 and 5 (DR4 and DR5) are cell surface receptors that when activated by their ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers apoptosis in most cancer cells but not in normal cells. Currently, it remains unclear whether DR4 and DR5 are involved in immune surveillance against nonmelanoma skin cancer (NMSC) progression. The aim of this study was to investigate the expression of DR4 and DR5 in NMSC and relate the results to the established clinicopathologic prognostic factors. This study was conducted on about 80 skin specimens from patients with NMSC (40 basal cell carcinoma and 40 squamous cell carcinoma) and diagnosed and confirmed by biopsy. Immunohistochemical analysis for DR4 and DR5 was carried out on formalin-fixed paraffin-embedded sections of skin tissues using avidin-biotin peroxidase method. Significant expression of both DR4 and DR5 was observed in NMSC cases. There was statistically significant association between DR4 and DR5 expression in squamous cell carcinoma and each of tumor site and lymph node metastasis. There was statistically significant association between DR4 expression in basal cell carcinoma and histopathologic subtypes (high expression in nodular type) and between DR5 expression and tumor site (high expression in sun-exposed area). In conclusion, expression of TRAIL receptors that mediate extrinsic apoptotic pathway in NMSC may be suggestive of a reassessment of the suitability of TRAIL-based strategy in future NMSC therapies.

Phosphorylated IκBα predicts poor prognosis in activated B-cell lymphoma and its inhibition with thymoquinone induces apoptosis via ROS release.

Activated B-cell lymphoma (ABC), one of the three subtypes of Diffuse Large B-cell Lymphoma (DLBCL) has the worst survival rate after upfront chemotherapy and is characterized by constitutively activated NFκB. We therefore studied the role of NFκB In a cohort of clinical DLBCL samples and ABC cell lines. In our clinical tissue microarray cohort of DLBCL samples, p-IκBα was detected in 38.3% of ABC DLBCL and was an independent prognostic marker for poor survival. In vitro, we found that Thymoquinone (TQ), a natural compound isolated from Nigella sativa caused release of ROS in ABC cells. TQ-mediated release of ROS in turn inhibited NFκB activity by dephosphorylating IκBα and decreased translocation of p65 subunit of NFκB in the nuclear compartment in ABC cell lines. This led to inhibition of cell viability and induction of mitochondrial dependent apoptosis in ABC-DLBCL cell lines. Additionally, TQ treatment also caused up-regulation of death receptor 5 (DR5), however, up-regulation of DR5 did not play a role in TQ-induced apoptosis. Finally, combination of sub-optimal doses of TQ and TRAIL induced efficient apoptosis in ABC-DLBCL cell lines. These data show that p-IκBα can be used as a prognostic marker and target for therapy in this aggressive sub-type of DLBCL and TQ may play an important role in the management of DLBCL in the future.

Possible role of apoptosis in the pathogenesis and clinical evolution of radicular cyst: an immunohistochemical study.

AIM: To investigate whether the apoptotic cascade is activated through the extrinsic pathway in epithelial lining and connective tissue of radicular cysts. METHODOLOGY: Fifteen radicular cysts were fixed in formalin, embedded in paraffin wax and processed for immunohistochemistry to evaluate the expression of polyclonal antibodies against Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), DR5 and caspase-3. Immunocomplexes were treated with the secondary antibodies and finally detected using the avidin-biotin-peroxidase complex. Immunoreactivity was visualized by development with 3,3'-diaminobenzidine. Data were analysed using the Mann-Whitney U-test; P < 0.05 was considered significant. RESULTS: The three antibodies were detected in connective tissue fibroblasts of all radicular cysts; TRAIL and DR5 immunoexpression was significantly greater (P < 0.05) compared with that of caspase-3. The three antibodies were also expressed in almost all epithelial layers and in endothelial cells of newly formed vessels. CONCLUSION: The involvement of apoptosis in the pathogenesis of radicular cysts, demonstrated by the immunoexpression patterns of TRAIL, DR5 and caspase-3 in lining epithelium and connective tissue, may explain their bland clinical aggressiveness and slow, benign evolution.

Multi-level disruption of the extrinsic apoptotic pathway mediates resistance of leukemia cells to TNF-related apoptosis-inducing ligand (TRAIL).

Disruption of apoptotic pathways belongs to commonly reported molecular mechanisms that underlie cancer drug resistance. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL, Apo2L) is a cytokine of the TNF family with selective anti-tumor activity and minimal toxicity toward healthy tissues. Primary leukemia cells are, however, largely intrinsically resistant to TRAIL-induced apoptosis. In this study we analyzed molecular differences between TRAIL-resistant K562 cell line and TRAIL-sensitive K562 clones. We demonstrate that TRAIL-sensitive K562 cells differ from the TRAIL-resistant cell line by cell surface downregulation of TRAIL decoy receptor 1, upregulation of both TRAIL death receptors, enhanced assembly and improved functioning of the death-inducing signaling complex, and increased cytoplasmic protein expression of CASP8 and key proapoptotic BCL2 members BID, BIM, BAD and BAK. The molecular basis of the intrinsic leukemia cell TRAIL resistance thus appears a consequence of the multi-level disruption of the extrinsic apoptotic pathway. The results of this study also suggest that the leukemia TRAIL-resistance is functional, leaving a possibility of overcoming the resistance by preexposure of the leukemia cells to potent TRAIL sensitizers, e.g. BH3-mimetics.

Real-time detection of cellular death receptor-4 activation by fluorescence resonance energy transfer.

Targeted therapy involving the activation of death receptors DR4 and/or DR5 by its ligand, TRAIL, can selectively induce apoptosis in certain tumor cells. In order to profile the dynamic activation or trimerization of TRAIL-DR4 in live cells in real-time, the development of an apoptosis reporter cell line is essential. Fluorescence resonance energy transfer (FRET) technology via a FRET pair, cyan fluorescence protein (CFP) and yellow fluorescence protein (YFP), was used in this study. DR4-CFP and DR4-YFP were stably expressed in human lung cancer PC9 cells. Flow cytometer sorting and limited dilution coupled with fluorescence microscopy were used to select a monoclonal reporter cell line with high and compatible expression levels of DR4-CFP and DR4-YFP. FRET experiments were conducted and FRET efficiencies were monitored according to the Siegel's YFP photobleaching FRET protocol. Upon TRAIL induction a significant increase in FRET efficiencies from 5% to 9% demonstrated the ability of the DR4-CFP/YFP reporter cell line in monitoring the dynamic activation of TRAIL pathways. 3D reconstructed confocal images of DR4-CFP/YFP reporter cells exhibited a colocalized expression of DR4-CFP and DR4-YFP mainly on cell membranes. FRET results obtained during this study complements the use of epi-fluorescence microscopy for FRET analysis. The real-time FRET analysis allows the dynamic profiling of the activation of TRAIL pathways by using the time-lapse fluorescence microscopy. Therefore, DR4-CFP/YFP PC9 reporter cells along with FRET technology can be used as a tool for anti-cancer drug screening to identify compounds that are capable of activating TRAIL pathways.

[Comparative study on tumor cell apoptosis in vitro induced by extracts of Stellera chamaejasme].

OBJECTIVE: To in vitro compare the induction of extracts of Stellera chamaejasme ESC, ESC-1 and ESC-2 on NCI-H157 cell apoptotic. METHOD: The apoptosis rate was inspected by flow cytometry; caspase-3, 8, 9 activities was measured by spectrophotometry. Fas, Fas-L, TNF-alpha, Trail-R, Cyto-C, Smac/diablo protein expressions of apoptosis pathway was observed by Elisa method. RESULT: Compared with the control group, ESC, ESC-1, ESC-2 can significantly improve the apoptosis rate of NCI-H157 cell. ESC significantly improved cells caspase-3, 8 activity, ESC-2 can significantly improve the activity of caspase-3, 8, 9. ESC, ESC-1, ESC-2 significantly increased Fas expression and ESC significantly increased Fas/Fas-L ratio. ESC, ESC-1, ESC-2 significantly increased TNF-alpha protein expression. ESC-1 significantly lowered TRAIL-R expression. ESC, ESC-1, ESC-2 had no significant effect on Cyto-C. ESC-1, ESC-2 significantly reduced Smac protein expression. CONCLUSION: The apoptotic effect induced by ESCs may be related to the regulation of death receptor pathway proteins. Induction mechanisms of ESCs were so complicated that it may have a two-way regulatory effect. Its induction in apoptosis is a result from comprehensive regulation and control.

An agonistic antibody to human death receptor 4 induces apoptotic cell death in head and neck cancer cells through mitochondrial ROS generation.

The proapoptotic death receptor 4 (DR4), along with DR5, is currently regarded as a promising target for development of agonistic anti-cancer agents due to its tumor-selective apoptosis-inducing ability with no significant cytotoxicity to normal cells. In this study, we examine susceptibility of various head and neck cancer (HNC) cells and mechanism of cell death to an anti-DR4 agonistic monoclonal antibody (mAb), AY4. AY4 as a single agent induced caspase-dependent apoptotic cell death of KB and HN9, but not in SNU899 and FaDu cell lines. AY4 treatment resulted in accumulation of intracellular reactive oxygen species (ROS) generated from mitochondria in AY4-sensitive cells. Blockade of ROS production by N-acetyl-l-cysteine (NAC) resulted in protection of AY4-sensitive cells against AY4-induced apoptosis. ROS generation induced by AY4 treatment triggered down-regulation of anti-apoptotic molecules of Bcl-xL and X-linked inhibitor of apoptosis (XIAP) without affecting the expression levels of DR4, Mcl-1, and survivin. AY4 also inhibited growth of pre-established HN9 tumors in a nude mouse xenograft model and did not show noticeable cytotoxicity in a zebrafish model. Our results provide further insight into the mechanism of DR4-mediated cell death and potential use of AY4 mAb as an anti-cancer therapeutic agent in AY4-sensitive HNC types.

DJ-1 inhibits TRAIL-induced apoptosis by blocking pro-caspase-8 recruitment to FADD.

DJ-1 was initially identified as an oncogene product involved in human tumorigenesis in cooperation with Ras. Increased DJ-1 expression is associated with tumorigenesis in many cancers, whereas the loss of DJ-1 function is linked to an autosomal recessive form of Parkinson's disease (PD). It has been reported that DJ-1 protects cells from TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)-induced apoptosis. However, the mechanism by which DJ-1 is involved is still largely unknown. Here we show that DJ-1 inhibits TRAIL-induced apoptosis by blocking Fas-associated protein death domain (FADD)-mediated pro-caspase-8 activation. Wild-type DJ-1, but not the PD-associated mutant L166P, binds to FADD to inhibit the formation of the death-inducing signaling complex (DISC). DJ-1 competes with pro-caspase-8 to bind to FADD at the death effector domain, thereby repressing the recruitment and activation of pro-caspase-8 to the active form of caspase-8. Thus, our study suggests that DJ-1 protects against TRAIL-induced apoptosis through the regulation of DISC formation.

Combined treatment with TRAIL and PPARγ ligands overcomes chemoresistance of ovarian cancer cell lines.

PURPOSE: Ovarian cancer accounts for the highest mortality among all gynecological cancers, mainly due to the fast developing chemoresistance. The death ligand TRAIL induces apoptosis and is able to sensitize tumor cells to cytostatic drugs without affecting physiological tissue. Combined treatment of TRAIL and the antidiabetic acting PPARγ ligands was shown to induce apoptosis synergistically in different ovarian cancer cell lines. METHODS: To investigate feasible TRAIL-dependent inhibition of proliferation and induction of apoptosis in chemoresistant ovarian cancer cell lines, the drug- and TRAIL-sensitive HEY cell line was utilized to develop subclones with selective resistance against cisplatin, etoposide, docetaxel, paclitaxel, gemcitabine and pemetrexed, as well as against TRAIL as control cell line. Expression of the key factors of the TRAIL signaling pathway, TRAIL receptors 1-4, caspase-8, FLIP and XIAP, was analyzed before and after TRAIL treatment by immunoblotting. RESULTS: Cell proliferation experiments showed TRAIL-dependent inhibition that was further increased by combination treatment with the PPARγ ligands. Simultaneous exposure of TRAIL and the PPARγ ligands also resulted in enhanced induction of apoptosis even in partial TRAIL-resistant HEY cell lines. In the parental HEY cell line, additional treatment with the PPARγ ligands led to an increased protein expression of DR5 and a further decline of XIAP expression. CONCLUSION: Therefore, the combinational treatment with TRAIL and PPARγ ligands might be a promising experimental therapy because the PPARγ ligands, especially d15-PGJ(2), sensitize drug-resistant ovarian cancer cells to TRAIL-induced apoptosis.

FSH inhibits ovarian cancer cell apoptosis by up-regulating survivin and down-regulating PDCD6 and DR5.

Ovarian epithelial cancer is the leading cause of death among gynecological malignancies. FSH may increase the risk of ovarian malignancy and play an important role in ovarian carcinogenesis. Our previous studies showed that FSH increases the expression of VEGF through survivin. In this study, the function and mechanism of FSH in ovarian cancer were further explored. We found that FSH promoted proliferation and prevented apoptosis of ovarian cancer cells by activating survivin through the SAPK/JNK and PI3K/AKT pathways. FSH also down-regulated the expression of programmed cell death gene 6 (PDCD6) and death receptor 5 (DR5), two molecules required for induction of apoptosis. RNA interference was applied to knock down survivin and PDCD6 expression, and we found that the blockage of survivin reversed the effects of FSH on apoptosis and proliferation, whereas knock down of PDCD6 enhanced these effects. The expression of DR5, cyclin D1, and cyclin E correlated with survivin expression, but PDCD6 did not. Using immunohistochemical staining, we further showed that ovarian serous cystadenocarcinoma samples had higher expression of survivin than did benign ovarian cystadenoma and borderline cystadenoma samples (P<0.01). Furthermore, survivin expression in the ovarian serous cystadenocarcinoma specimens was correlated with disease stage (P<0.05). Our results suggest that FSH promotes ovarian cancer development by regulating the expression of survivin, PDCD6, and DR5. Greater understanding of the molecular mechanisms of FSH in ovarian epithelial carcinogenesis and development will ultimately help in the development of a novel targeted therapy for ovarian cancer.

Enhancement of IL-2-induced cytotoxicity by interferon-alpha in renal cell carcinoma.

Metastatic renal cell carcinoma (mRCC) treatment consists of molecular targeted agents and cytokines that have fundamentally different mechanisms of action. Clinical responses also differ; complete response is rare with molecular targeted agents but is sometimes achieved with cytokine therapies. Because of the relatively high efficacy of combination therapy with low-dose interleukin-2 (IL-2) and interferon-alpha (IFN-alpha) against mRCC, it is important to reevaluate cytokine therapies in vitro. Here, we show that when IL-2 is administered in combination with IFN-alpha, a stronger cytotoxic effect of PBMCs on RCC cell lines is observed than when IL-2 is administered alone. The upregulation of TNF-related apoptosis-inducing ligand on NK cell by IL-2 and suppression of regulatory T cells (Tregs) by IFN-alpha were recognized at the same time when cytotoxicity of peripheral blood mononuclear cells (PBMCs) was enhanced. IL-2 is known to activate natural killer cell cytotoxicity; however, IL-2 also stimulates Treg expansion, which enhances immunosuppression. On the other hand, IFN-alpha negatively regulates Treg cells, thereby increasing the function of immune effector cells. Our in vitro results may explain, at least in part, the clinical efficacy of combination low-dose IL-2 and IFN-alpha therapy against mRCC.

A Phase 1b/2 trial of mapatumumab in patients with relapsed/refractory non-Hodgkin's lymphoma.

BACKGROUND: we conducted a multicentre Phase 1b/2 trial to evaluate the safety and efficacy of mapatumumab, a fully human agonistic monoclonal antibody to the tumour necrosis factor-related apoptosis-inducing ligand receptor 1 (TRAIL-R1) in patients with relapsed non-Hodgkin's lymphoma (NHL). METHODS: forty patients with relapsed or refractory NHL were treated with either 3 or 10 mg kg(-1) mapatumumab every 21 days. In the absence of disease progression or prohibitive toxicity, patients received a maximum of six doses. RESULTS: mapatumumab was well tolerated, with no patients experiencing drug-related hepatic or other dose-limiting toxicity. Three patients with follicular lymphoma (FL) experienced clinical responses, including two with a complete response and one with a partial response. Immunohistochemistry staining of the TRAIL-R1 suggested that strong staining in tumour specimens did not appear to be a requirement for mapatumumab activity in FL. CONCLUSIONS: mapatumumab is safe and has promising clinical activity in patients with FL.