Tumor Necrosis Factor-related Apoptosis-inducing Ligand Protein Research Articles

Conditionally replicating adenovirus combined with gene-targeted radiotherapy induces apoptosis via TRAIL death receptors in MDA-MB-231 cells

Malignant tumors are usually treated using monotherapies, which are not always effective. Therefore, combination therapies have gained increasing attention. The aims of this study were to investigate the effects of conditionally replicating adenovirus (CRAd) in combination with X-ray irradiation on the proliferation and apoptosis of MDA-MB-231 cells, as well as to determine the molecular mechanisms involved. MDA-MB-231 cells were treated simultaneously with CRAd and X-ray irradiation. Then, cell viability was measured using Cell Counting Kit-8. Cell apoptosis was assessed by flow cytometry with Annexin V and propidium iodide (PI) double-staining. The expression of tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL), death receptor 5 (DR5), caspase-3 and caspase-8 mRNA was detected by quantitative polymerase chain reaction. The expression of TRAIL, DR5, caspase-3 and caspase-8 protein was measured by enzyme-linked immunosorbent assay (ELISA) and western blotting, respectively. The results showed that CRAd, in combination with irradiation, inhibited cell proliferation, promoted cell apoptosis and significantly increased the expression of TRAIL, DR5, caspase-3 and caspase-8 mRNA and proteins in MDA-MB-231 cells. Therefore, three aspects, including the targeted killing effect of CRAd, the apoptosis-promoting role of TRAIL and the direct killing effect of ionizing radiation to MDA-MB-231 cells, contribute to the mechanisms of CRAd in combination with irradiation to inhibit the proliferation of MDA-MB-231 cells. The pro-apoptotic effect may involve the interaction between TRAIL, DR5, caspase-3 and caspase-8.

Up-regulation of miR-146b and Down-regulation of miR-200b Contribute to the Cytotoxic Effect of Histone Deacetylase Inhibitors onras-Transformed Thyroid Cells

Histone deacetylase inhibitors (HDACis) are anticancer agents that inhibit tumor cell growth and/or survival. However, their mechanism of action remains largely undefined. Recently, we have demonstrated that HDACis induce apoptosis in a model of rat thyroid cells transformed by the v-ras-Ki oncogene (FRTL-5 v-ras-Ki). The stabilization of TNF-related apoptosis-inducing ligand (TRAIL) protein, due to its reduced ubiquitination and proteasome degradation, accounts for the apoptotic effect induced specifically by suberoylanilide hydroxamic acid (SAHA, Vorinostat) in the v-ras-Ki thyroid transformed cells. The aim of this work was to investigate whether SAHA may induce its cytotoxic effects also deregulating microRNA (miRNA) expression levels. We analyzed the miRNA expression profile of the thyroid transformed cells, FRTL-5 v-ras-Ki, upon SAHA treatment. Here we report that SAHA induces the down-regulation of 18 and the up-regulation of 11 miRNAs with a fold change higher than 2 in the transformed cells. Then, we focus on the miR-146b and miR-200b, respectively up-regulated and down-regulated by SAHA. We show that both these miRNAs target genes coding for proteins with a critical role in proteasome composition and ubiquitin degradation. These results suggest a role of miRNA deregulation in TRAIL protein stabilization responsible for SAHA-induced apoptotic effect in thyroid transformed cells.

Induction of non-small cell lung carcinoma apoptosis using soluble RGD-TRAIL by targeting the integrin receptor of tumor cells

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising candidate for cancer therapeutics that exhibits the ability to preferentially induce apoptosis in malignant cells. RGD peptides bind to the integrins, ανβ3 and ανβ5, which are selectively expressed in tumor neovasculature and at the surface of certain tumor cells. To enhance the antitumor effect, an RGD-TRAIL protein, in which TRAIL was fused with the RGD motif-containing cell adhesive sequence, GRGDNP (gly-arg-gly-asp-asn-pro), was constructed and evaluated for bioactivity. The soluble TRAIL and RGD-TRAIL proteins were expressed in Escherichia coli BL21 (DE3) and purified using a non-denaturing method. The antitumor effect of the purified RGD-TRAIL on cell proliferation was evaluated invitro using MTT and wound healing assays and cell apoptosis was assessed by Hoechst 33342 staining and PARP expression analysis. The results revealed that RGD-TRAIL inhibited the proliferation of multiple tumor cell lines (A549, NCI-H1299 and HCC827). Western blot analysis demonstrated that the treatment of tumor cells with RGD-TRAIL activated the apoptotic pathway by the cleavage of PARP, in the same way as wild-type TRAIL (wtTRAIL). These results demonstrate that RGD-TRAIL possesses more potent antitumor effects than wtTRAIL and, therefore, merits further investigation in preclinical and clinical studies.

Sp1, acetylated histone‐3 and p300 regulate TRAIL transcription: Mechanisms of PDGF‐BB‐mediated VSMC proliferation and migration

We recently reported that TNF-related apoptosis-inducing ligand (TRAIL) is important in atherogenesis, since it can induce vascular smooth muscle cell (VSMC) proliferation and arterial thickening following injury. Here we show the first demonstrate that TRAIL siRNA reduces platelet-derived growth factor-BB (PDGF-BB)-stimulated VSMC proliferation and migration. PDGF-BB-inducible VSMC proliferation was completely inhibited in VSMCs isolated from aortas of TRAIL(-/-) mice; whereas inducible migration was blocked compared to control VSMCs. TRAIL transcriptional control mediating this response is not established. TRAIL mRNA, protein and promoter activity was increased by PDGF-BB and subsequently inhibited by dominant-negative Sp1, suggesting that the transcription factor Sp1 plays a role. Sp1 bound multiple Sp1 sites on the TRAIL promoter, including two established (Sp1-1 and -2) and two novel Sp1-5/6 and -7 sites. PDGF-BB-inducible TRAIL promoter activity by Sp1 was mediated through these sites, since transverse mutations to each abolished inducible activity. PDGF-BB stimulation increased acetylation of histone-3 (ac-H3) and expression of the transcriptional co-activator p300, implicating chromatin remodelling. p300 overexpression increased TRAIL promoter activity, which was blocked by dominant-negative Sp1. Furthermore, PDGF-BB treatment increased the physical interaction of Sp1, p300 and ac-H3, while chromatin immunoprecipitation studies revealed Sp1, p300 and ac-H3 enrichment on the TRAIL promoter. Taken together, our studies demonstrate for the first time that PDGF-BB-induced TRAIL transcriptional activity requires the cooperation of Sp1, ac-H3 and p300, mediating increased expression of TRAIL which is important for VSMC proliferation and migration. Our findings have the promising potential for targeting TRAIL as a new therapeutic for vascular proliferative disorders.

Abstract 4886: PTHrP sensitizes breast cancer cells to TRAIL-induced apoptosis

Abstract Parathyroid hormone-related protein (PTHrP) is a multifunctional protein that has been implicated in breast tumor biology. Breast cancers commonly express PTHrP, and when such tumors are grown in bone in murine models, PTHrP results in enhanced osteolysis. Whilst clinical studies have determined that PTHrP expression by primary breast cancers was an independent predictor of improved prognosis (Henderson et al., 2006). Thus, the action of PTHrP upon breast cancers directly remains to be elucidated. The aim of this study was to assess the interaction of PTHrP with molecules associated with cell survival and cell death pathways, such as Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines that induces apoptosis in a wide range of transformed cells but not in normal cells. To understand the role of PTHrP in relation to TRAIL in breast cancer cells, PTHrP was overexpressed in two breast cancer cell lines; MDA-MB-231 (TRAIL-sensitive) and MCF-7 (TRAIL-resistant), and functional assays were performed. Over-expression of PTHrP in both MCF-7 and MDA-MB-231 cells enhanced cell cycle progression and cell growth rate in comparison to parental and vector controls. However, treatment of PTHrP overexpressing breast cancer cells with TRAIL resulted in an enhancement of TRAIL-induced apoptosis in the TRAIL sensitive cell line, MDA-MB-231, and a sensitisation to TRAIL-induced apoptosis in the TRAIL-resistant cell line, MCF-7. Western blot showed decreased levels of Caspase-10, Caspase-9, Caspase-6, Capase-7 precursor proteins and increased PARP cleavage levels in PTHrP overexpressing cells treated with TRAIL when compared to controls. There was no change observed in levels of Caspase-8 percursor protein, suggesting that the apoptosis signal is acting via the intrinsic pathway in PTHrP overexpressing cells. To determine TRAIL sensitivity in MCF-7 cells overexpressing PTHrP, TRAIL receptor mRNA and protein expression was assessed by qRT-PCR and FACS analysis. TRAIL receptors; DR4, DR5, DcR1 and DcR2, mRNA expression was not altered in PTHrP overexpressing cells. However, flow cytometric analysis demonstrated an increase in cell surface expression of both death receptors (DR4 and DR5) in cells overexpressing PTHrP. To determine whether TRAIL-induced apoptosis in PTHrP overexpressing cells was signaling either through DR4, DR5 or a combination of both death receptors, antagonistic antibodies against DR4 or DR5 were used. Pre-incubation of PTHrP overexpressing cells with DR5 antagonistic antibody attenuated TRAIL-induced apoptosis. This demonstrated that TRAIL preferentially signals through DR5 in the PTHrP overexpressing cells to activate the intrinsic apoptotic pathway. In conclusion, these results demonstrate a new function for PTHrP in breast cancer and may provide utility when TRAIL is used as a treatment for breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4886. doi:1538-7445.AM2012-4886

Hypoxia-Ischemia Upregulates TRAIL and TRAIL Receptors in the Immature Rat Brain

The immature brain is susceptible to inflammatory injury induced by hypoxia-ischemia (HI) or infection, which causes serious neurodevelopmental disabilities in the survivors of preterm births. Recently, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors (death receptor DR4/5 and decoy receptor DcR1/2) were reported to mediate various neuroinflammatory responses. However, little information is available regarding the role of TRAIL and its receptors in the immature brain after HI. The purpose of this study was to evaluate the expression of TRAIL and its receptors in the immature brain after HI and relate this expression to neurological function. We performed right common carotid artery ligation followed by hypoxia (6% O₂, 37°C) for 2.5 h to induce HI in postnatal day 3 rats. The distribution of TRAIL and its receptors, caspase-3 and CD68-labeled microglia/macrophages was evaluated 24 h after HI by immunostaining. The protein and mRNA expression of TRAIL and DR5 was measured by Western blot and real-time PCR, respectively. Delayed neuronal loss was evaluated by NeuN and Nissl staining 7 days after HI. Furthermore, neurological deficits were evaluated by a righting reflex test, time of eye opening and T-maze test. The expression of TRAIL, DR5 and DcR1/2 receptors and caspase-3 was more pronounced in the ipsilateral hemisphere compared with the contralateral part and the control group 24 h after HI. DR5/active caspase-3 double-positive cells were observed at 24 h after HI in the ipsilateral hemisphere but not in the contralateral hemisphere. The TRAIL and CD68 double-labeled cells were more pronounced in the ipsilateral cortical regions compared with the corresponding regions of the contralateral part. HI also resulted in a significant increase in TRAIL and DR5 protein and mRNA expression at 24 h, which corresponded to neuronal cell loss 7 days after HI. Furthermore, the HI group displayed impaired neurobehavioral development compared with the control group (p < 0.05). Altogether our results show that the TNF-α superfamily ligand TRAIL is induced on CD68+ microglia/macrophages after perinatal HI and that one of its receptors, DR5, is induced on neocortical neurons and glial cells. That many DR5+ cells were also caspase-3+ strongly supports the conclusion that these signaling molecules are involved in the delayed loss of neurons in the neocortex and in the neurobehavioral deficits that are often seen after perinatal HI.

BID is a Critical Factor Controlling Cell Viability Regulated by IFN-α

Clinical applications of human interferon (IFN)-α have met with varying degrees of success. Nevertheless, key molecules in cell viability regulated by IFN-α have not been clearly identified. Our previous study indicated that IFN (α, β, and ω) receptor (IFNAR) 1/2- and IFN regulatory factor 9-RNA interference (RNAi) completely restored cell viability after IFN-α treatment in human ovarian adenocarcinoma OVCAR3 cells sensitive to IFN-α. In this study, IFNAR1/2- and IFN regulatory factor 9-RNAi inhibited the gene expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), but not of Fas ligand, after IFN-α treatment. In fact, TRAIL but not Fas ligand inhibited the viability of OVCAR3 cells. IFN-α notably upregulated the levels of TRAIL protein in the supernatant and on the membrane of OVCAR3 cells. After TRAIL signaling, caspase 8 inhibitor and BH3 interacting domain death agonist (BID)-RNAi significantly restored cell viability in response to IFN-α and TRAIL in OVCAR3 cells. Furthermore, BID-RNAi prevented both IFN-α and TRAIL from collapsing the mitochondrial membrane potential (ΔΨm). Finally, we provided important evidence that BID overexpression led to significant inhibition of cell viability after IFN-α or TRAIL treatments in human lung carcinoma A549 cells resistant to IFN-α. Thus, this study suggests that BID is crucial for cell viability regulated by IFN-α which can induce mitochondria-mediated apoptosis, indicating a notable potential to be a targeted therapy for IFN-α resistant tumors.

In vitro and in vivo growth inhibition of drug-resistant ovarian carcinoma cells using a combination of cisplatin and a TRAIL-encoding retrovirus

Retroviruses encoding the TNF-related apoptosis-inducing ligand (TRAIL) gene were generated by transient transfection of the retrovirus packing cell line BOSC 23 using TRAIL-encoding plasmid. The retrovirus was able to transduce drug-resistant A2780/DDP ovarian carcinoma cells in vitro and induce TRAIL expression in the cells, as detected by western blot assay. Furthermore, the TRAIL protein led to the growth inhibition of the cells via a caspase-activated apoptotic mechanism. It was confirmed that exposure of such cells to cisplatin in combination with the TRAIL-encoding retrovirus resulted in higher anticancer activity in vitro and in the xenograft A2780/DDP tumor in a nude mouse model. This study suggests that chemotherapy in combination with TRAIL gene therapy may be an efficient approach to treat drug-resistant ovarian cancer.

Salmonella‐mediated tumor‐targeting TRAIL gene therapy significantly suppresses melanoma growth in mouse model

Attenuated Salmonella typhimurium (S. typhimurium) strains can selectively grow and express exogenous genes in tumors for targeted therapy. We engineered S. typhimurium strain VNP20009 to secrete tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) under the control of a hypoxia-induced nirB promoter and examined the efficacy of Salmonella-mediated targeted expression of TRAIL in mice bearing melanoma tumor and in TRAIL-resistant RM-1 tumor. We found that VNP preferentially accumulated in tumor tissues and the nirB promoter effectively drove targeted expression of TRAIL. Compared with recombinant TRAIL protein and VNP20009 combination therapy, VNP20009 expressing TRAIL significantly suppressed melanoma growth but failed to suppress RM-1 tumor growth. Furthermore, we confirmed that VNP20009 expressing TRAIL yielded its antitumor effect by inducing melanoma apoptosis. Our findings indicate that Salmonella-mediated tumor-targeted therapy with TRAIL could reduce tumor growth and extend host survival.

Novel transcript variants of TRAIL show different activities in activation of NF-κB and apoptosis

Aims Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has many transcript variants, but whether they possess distinct function is not completely known. In the present study, we compared the function of these TRAIL variants. Main methods A bioinformatics analysis was performed to examine potential TRAIL variants. For the functional study, over-expression of TRAIL isoforms was used to examine their NF-κB inducing and apoptotic activities in both cancer and normal cells. Moreover, soluble TRAIL E4 variant protein was expressed and purified in prokaryotic cells, and was used for apoptotic assay. Key findings We cloned seven truncated TRAIL variants, designated as AK, E2, E3, E4, DA, BX424, and BX439. In comparison with the wild type TRAIL protein expressed from full-length RefSeq, over-expression of all these TRAIL variants activated NF-κB and its targeting genes in human cells at varying degrees. Some isoforms including BX424, DA and E4 even showed NF-κB, IL8, CCL4 and CCL20 promoter activating activity stronger than the wild type protein. All truncated variant proteins had no toxicity to normal human cells, similar to the wild type protein; however, they all failed to induce apoptosis in cancer cells that are sensitive to TRAIL. Recombinant soluble TRAIL E4 protein also failed to antagonize TRAIL-induced apoptosis in cancer cells. Significance Truncated TRAIL variant proteins lost apoptotic activity but retained or even enhanced the NF-κB activating potentials, these results suggest that TRAIL variants may play roles in non-apoptotic cellular processes that are more important than we previously thought.

TRAIL contributes to the apoptotic effect of 13-cis retinoic acid in human sebaceous gland cells

The full mechanism of action of isotretinoin [13-cis retinoic acid (13-cis RA)] in treating acne is unknown. 13-cis RA induces key genes in sebocytes that are involved in apoptosis, including Tumor necrosis factor Related Apoptosis Inducing Ligand (TRAIL). In this study, we investigated the role of 13-cis RA-induced TRAIL within SEB-1 sebocytes. Using 13-cis RA and recombinant human TRAIL (rhTRAIL) protein, we assessed induction of TRAIL and apoptosis in SEB-1 sebocytes, normal keratinocytes and patient skin biopsies. Treatment with rhTRAIL protein increased TUNEL-positive staining in SEB-1 sebocytes. TRAIL siRNA significantly decreased the percentage of TUNEL-positive SEB-1 sebocytes in response to 13-cis RA treatment. Furthermore, TRAIL expression increased in the skin of patients with acne after 1 week of isotretinoin therapy compared with baseline. TRAIL expression localized within sebaceous glands. Unlike sebocytes, TRAIL protein expression was not increased in normal human epidermal keratinocytes in response to 13-cis RA, nor did rhTRAIL induce apoptosis in keratinocytes, suggesting that TRAIL is key in the sebocyte-specific apoptotic effects of 13-cis RA. Taken together, our data suggest that TRAIL, like the neutrophil gelatinase-associated lipocalin, is involved in mediating 13-cis RA apoptosis of sebocytes.

Inhibition of the ubiquitin-proteasome system sensitizes TRAIL-resistant prostate cancer cells by up-regulation of death receptor 5

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a type II transmembrane cytokine and a potent inducer of apoptosis in cancer cells. However, some cancer cells, particularly prostate cancer cells, such as LNCaP cells, were found to be resistant to TRAIL. In the present study, we demonstrate that the proteasome inhibitor ALLN significantly enhanced TRAIL-induced apoptosis by up-regulating TRAIL/Apo2L death receptor 5 expression in LNCaP cells. LNCaP cells were exposed to ALLN for 3 h and treated with recombinant TRAIL protein. ALLN alone induced a 20% cell death after a 3‑h treatment; however, pretreatment with ALLN induced death to more than 80% of cells after 3 h of TRAIL treatment. ALLN also enhanced the cell death of TRAIL-sensitive/resistant prostate cancer and other cancer cell lines. Western blotting results showed that the combination of ALLN and TRAIL increased the levels of activated caspase-8, -3 and DR-5 in LNCaP cells. Furthermore, we observed an increase in DR-5 expression following 3 h of treatment of ALLN alone. Taken together, our findings indicate that ALLN enhances TRAIL-induced apoptosis in LNCaP cells by up-regulating DR-5 expression. Thus, our results suggest that the combination of ALLN and TRAIL is a novel therapeutic strategy in TRAIL-resistant tumors.

Functional Identification of a Non-Fusion TRAIL Extracellular Protein and Preparation of Its Polyclonal Antibody

Human tumor necrosis factor related apoptosis inducing ligand (TRAIL) can selectively induce apoptosis in a variety of transformed cells and is currently being developed as a cancer therapeutic drug. Here we expressed the TRAIL protein including extracellular (114-281aa) without any tag protein named TRAIL-NT, and prepared anti-TRAIL polyclonal antibodies (Poly-Ab). The human TRAIL extracellular gene was amplified from PBMC and cloned into pGEM-T-Easy vector for sequence analysis. The expression vector pET-28a/TRAIL was constructed using the DNA recombinant method, and the recombinant protein without any tag protein was expressed in Escherichia coli BL21(DE3). The TRAIL-NT protein was purified by cation ion-exchange column and identified by SDS-PAGE and Western blot analysis. The proliferation inhibition activity of TRAIL-NT was detected by the MTT method, Wright-Giemsa staining assay, and FACS. The polyclonal antibody of TRAIL-NT was obtained after the BALB/C mice were immunized with purificated TRAIL-NT protein. Results showed that the target protein expressed in E. coli BL21(DE3) has the same molecular weight as that expected and could be recognized by anti-TRIAL Poly-Ab. The TRAIL-NT protein could also inhibit proliferation and induced apoptosis of Jurkat cells but no cytotoxicity to human liver cells and PBMC was observed. This preliminary research laid a solid foundation for further research on its biological activity and application in anti-tumor therapy.

Cancer targeting Gene-Viro-Therapy of liver carcinoma by dual-regulated oncolytic adenovirus armed with TRAIL gene

Liver cancer is a common and aggressive malignancy, but available treatment approaches remain suboptimal. Cancer targeting Gene-Viro-Therapy (CTGVT) has shown excellent anti-tumor effects in a preclinical study. CTGVT takes advantage of both gene therapy and virotherapy by incorporating an anti-tumor gene into an oncolytic virus vector. Potent anti-tumor activity is achieved by virus replication and exogenous expression of the anti-tumor gene. A dual-regulated oncolytic adenoviral vector designated Ad·AFP·E1A·E1B (Δ55) (Ad·AFP·D55 for short thereafter) was constructed by replacing the native viral E1A promoter with the simian virus 40 enhancer/α-fetoprotein (AFP) composite promoter (AFPep) based on an E1B-55K-deleted construct, ZD55. Ad·AFP·D55 showed specific replication and cytotoxicity in AFP-positive hepatoma cells. It also showed enhanced safety in normal cells when compared with the mono-regulated vector ZD55. To improve the anti-hepatoma activities of the virus, the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene was introduced into Ad·AFP·D55. Ad·AFP·D55-TRAIL exhibited remarkable anti-tumor activities in vitro and in vivo. Treatment with Ad·AFP·D55-TRAIL can induce both autophagy owing to the Ad·AFP·D55 vector and caspase-dependent apoptosis owing to the TRAIL protein. Therefore, Ad·AFP·D55-TRAIL could be a potential anti-hepatoma agent with anti-tumor activities due to AFP-specific replication and TRAIL-induced apoptosis.

Tumor Necrosis Factor‐Related Apoptosis‐Inducing Ligand and Vascular Calcification

Vascular calcification is frequent in patients with chronic kidney disease. Osteoprotegerin (OPG, a soluble factor which blocks osteoclast differentiation) has recently been implicated in the genesis of vascular calcification. Given that OPG can bind the pro-apoptotic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), we hypothesized that the TRAIL protein is involved in the formation of vascular calcification both in vitro and in vivo. Using an immunohistochemical approach, we evaluated TRAIL and OPG expression on aortic valves slides from non-uremic and uremic wild type and apolipoprotein knockout (Apo E(-/-) ) mice. We also tested the in vitro effects of TRAIL on cultured primary human vascular smooth muscle cells (hVSMC). We further assayed serum soluble TRAIL (sTRAIL) levels in hemodialysis patients and correlated them with vascular calcification scores. Our results demonstrated that: (i) TRAIL and OPG were expressed inside the atheroma plaque in non-uremic ApoE(-/-) mice, but not in wild type mice; and (ii) uremia enhanced the expression levels. TRAIL enhanced the phosphate-induced mineralization of hVSMCs in a dose-dependent manner. In clinical terms, we demonstrated that sTRAIL is depressed in the sera of hemodialysis patients, but was not correlated with vascular calcification. Our results suggest that TRAIL may be involved in the formation of vascular calcification in certain experimental settings; however, its role in chronic kidney disease patients requires further evaluation.

Bisphosphonate enhances TRAIL sensitivity to human osteosarcoma cellsviadeath receptor 5 upregulation

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily of cytokines, is one of the most promising candidates for cancer therapeutics. However, many osteosarcomas are resistant to TRAIL. Bisphosphonates are very effective in the treatment of bone problems associated with malignancies; the antitumor effects are due to the inhibition of protein prenylation that is essential for cell function and survival. The purpose of this study was to determine the effects of bisphosphonates on TRAIL-resistant MG 63 human osteosarcoma cells. The cells showed no response to TRAIL alone; however, pre-treatment with bisphosphonates significantly increased TRAIL-mediated apoptosis and cellular activation of caspase-3. Bisphosphonates significantly induced mRNA and protein expression of the TRAIL receptor, DR5. Bisphosphonates induced protein unprenylation in MG 63 cells; in addition, co-treatment with TRAIL also significantly increased protein unprenylation. Blocking of protein unprenylation using geranylgeraniol attenuated the cellular responses, including cell apoptosis and protein unprenylation induced by bisphosphonates and TRAIL. This is the first study to demonstrate that bisphosphonates markedly enhanced TRAIL-induced apoptosis in human osteosarcoma cells. These findings suggest that bisphosphonates may be a new and effective anticancer treatment with TRAIL proteins for TRAIL-resistant cancer cells.

Evaluation of TNF superfamily molecules release by neutrophils and B leukemic cells of patients with chronic B – cell lymphocytic leukemia

It was demonstrated that TNF superfamily proteins may affect significantly the time of leukemic cells' survival in the course of B-cell chronic lymphocytic leukemia (B-CLL). The aim of our study was to evaluate the expression and release of BAFF (B-cell activating factor), APRIL (a proliferation-inducing ligand) and TRAIL (TNF-related apoptosis inducing ligand) molecules belonging to the cytokines of the superfamily of the tumor necrosis factor (TNF) by neutrophils (PMNs) and, for comparison, B cells isolated from the blood of patients with B-CLL vs. their concentration in the blood serum. 40 patients suffering from B-CLL and a control group of 15 healthy subjects were included in the study. Cytoplasmic fractions of PMNs and B cells were analyzed with the use of western blotting for the presence of TRAIL, BAFF and APRIL. Soluble TRAIL, BAFF and APRIL in the culture supernatants and the serum were assessed using ELISA kits. PMNs and B cells of patients with B-CLL before treatment demonstrated the statistically significantly higher expression of APRIL and BAFF proteins when compared with the control group of healthy subjects. In contrast, the expression of TRAIL protein in both types of cells of patients was statistically significantly lower than its expression in the control cells. In the supernatants of PMN and B lymphocytes of patients the decreased concentrations of sBAFF, unchanged of APRIL and increased of sTRAIL molecules were demonstrated. The results of studies carried out in patients with B-CLL before treatment indicate that the relations demonstrated between APRIL, BAFF and TRAIL molecules, released by neutrophils and B cells and relations between their concentrations in the serum can significantly influence the development of B-CLL.

Evaluation of TNF Superfamily Molecules Release by Neutrophils and B Leukemic Cells of Patients with Chronic B – Cell Lymphocytic Leukemia

AbstractAbstract 4855It was demonstrated that TNF superfamily proteins may affect significantly the time of leukemic cells' survival in the course of B-cell chronic lymphocytic leukemia (B-CLL).The aim of our study was to evaluate the expression and release of BAFF (B-cell activating factor), APRIL (a proliferation-inducing ligand) and TRAIL (TNF-related apoptosis inducing ligand) molecules belonging to the cytokines of the superfamily of the tumor necrosis factor (TNF) by neutrophils (PMNs) and, for comparison, B cells isolated from the blood of patients with B-CLL vs. their concentration in the blood serum.40 patients suffering from B-CLL and a control group of 15 healthy subjects were included in the study. Cytoplasmic fractions of PMNs and B cells were analyzed with the use of western blotting for the presence of TRAIL, BAFF and APRIL. Soluble TRAIL, BAFF and APRIL in the culture supernatants and the serum were assessed using ELISA kits.PMNs and B cells of patients with B-CLL before treatment demonstrated the statistically significantly higher expression of APRIL and BAFF proteins in comparison in relation to the control group of healthy subjects. In contrast, the expression of TRAIL protein in both types of cells of patients was statistically significantly lower than its expression in the control cells.In the supernatants of PMN and B lymphocytes of patients the decreased concentrations of sBAFF, unchanged of APRIL and increased of sTRAIL molecules were demonstrated.The results of studies carried out in patients with B-CLL before treatment indicate that the relations demonstrated between APRIL, BAFF and TRAIL molecules, released by neutrophils and B cells and relations between their concentrations in the serum can significantly influence the development of B-CLL. Disclosures:No relevant conflicts of interest to declare.

Expression and significance of TRAIL and its receptor DR5 in murine myocardium with acute viral myocarditis

Objective To deterine the expression of TNF-related apoptosis inducing ligand (TRAIL) and its receptor DR5 in murine myocardium with acute viral myocarditis(VM).Methods We builted the model of VM.Eight mice of the VM group and the normal control group were sacrificed on the 7th,10th,14th,21st,28th day after inoculation CVB3 virus.The myocardial histopathological scores were counted.The terminal reansferase-mediated dUPT-biotin nick end-labeling (TUNEL) assays was used to quantified apoptosis rate.The expression of TRAIL and DR5 protein and mRNA were determined by the method of immunohistochemistry and reverse-transcription polymerase chain reaction (RT-PCR).Results The expression of TRAIL and DR5 protein and mRNA were found in myocardium of both the normal control group and the VM group.The expression of TRAIL protein of the VM group(14th) were significantly higher than which in the normal group at the same time and the VM group(7th)(F=9.17,P<0.01).The DR5 protein of the VM group(10th,14th,21st)were significantly higher than which in the normal group at the same time and the VM group(7th)(F=13.32,P<0.01).The expression of TRAIL and DR5 protein were positive correlated with the myocardial histopathological scores and the apoptosis rate.The expression of TRAIL mRNA of the VM group(10th) were significantly higher than which in the normal group at the same time and the VM group(7th)(F=10.86,P<0.01).The DR5 mRNA of the VM group(10th,14th)were significantly higher than which in the normal group at the same time and the VM group(7th)(F=22.75,P<0.01).Conclusion High characteristic expressions of protein/mRNA TRAIL and DR5 were observed in the myocardium of mice with VM.The level was positive correlationed with the account of pathology and the rate of apoptosis.The apoptosis induced by TRAIL and DR5 may participate in the pathophysiological processes of VM. Key words: Coxsackie virus; Myocarditis; Tunor necrosis factor related apoptosis inducing ligand; Death receptor 5; Apoptosis

Hypoxia inducing factor-1α regulates tumor necrosis factor-related apoptosis-inducing ligand sensitivity in tumor cells exposed to hypoxia

Hypoxia is a common environmental stress. Particularly, the center of rapidly-growing solid tumors is easily exposed to hypoxic conditions. Hypoxia is well known to attenuate the therapeutic response to radio and chemotherapies including tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) protein. HIF-1α is a critical mediator of the hypoxic response. However, little is known about the function of hypoxia-inducible factor-1α (HIF-1α) on hypoxic inhibition of TRAIL-mediated apoptosis. In this study, we investigated whether hypoxic inhibition of TRAIL-mediated apoptosis can be regulated by modulating HIF-1α protein. Hypoxia- and DEF-induced HIF-1α activation inhibited the TRAIL-mediated apoptosis in SK-N-SH, HeLa, A549 and SNU-638 cells. And also, HIF-1α inactivating reagents including DOX increased the sensitivity to TRAIL protein in tumor cells exposed to hypoxia. Furthermore, knock-down of HIF-1α using lentiviral RNA interference sensitized tumor cells to TRAIL-mediated cell death under hypoxic condition. Taken together, these results indicate that HIF-1α inactivation increased TRAIL sensitivity in hypoxia-induced TRAIL-resistant tumor cells and also suggest that HIF-1α inhibitors may have benefits in combination therapy with TRAIL against hypoxic tumor cells.