Antitumor effect of tumor necrosis factor-related apoptosis inducing ligand combined with mevastatin on a human glioma cell line SWO-38

BACKGROUND： Previous studies have reported that statins are less toxic to the human body and have greater antitumor activity; however, few studies have addressed the antitumor effect of statins combined with tumor necrosis factor-related apoptosis inducing ligand （TRAIL）. OBJECTIVE： To explore the effect of TRAIL combined with mevastatin on the proliferation and apoptotic cell death of a human glioma cell line SWO-38, and to study its mechanism of action. DESIGN, TIME AND SETTING： An in vitro control experiment was performed at the Central Laboratory of the Third Hospital Affiliated to Sun Yat-sen University, between January and April 2009. MATERIALS： The human SWO-38 cell line was provided by Cell Research, Department of Animal Experimental Center of Sun Yat-sen University; human recombinant soluble TRAIL by R＆D, USA; and mevastatin by Sigma, USA. METHODS： SWO-38 cells were separately incubated in TRAIL （100, 200, 300, 400, and 500 tJg/L） and mevastatin （5, 10, 20, 30, and 40 pmol/L） for 72 hours. In addition, SWO-38 cells were incubated in TRAIL （300 μg/L）, mevastatin （30 μmol/L）, and a solution containing both TRAIL and mevastatin for 12, 24, 48 and 72 hours. MAIN OUTCOME MEASURES： Cell proliferation was detected using methyl thiazolyl tetrazolium assay; cell apoptosis was observed using Hoechst 33258 staining and fluorescence microscopy and was measured using Annexin V/propidium iodide flow cytometry; TRAIL R1/DR4 and TRAIL R2/DR5 protein expressions levels were measured using indirect immunofluorescence staining combined with flow cytometry in the recombinant soluble TRAIL （rsTRAIL, 300 tJg/L）, mevastatin （30 IJmol/L） and combination groups; TRAIL R1/DR4 and TRAIL R2/DR5 mRNA expression was detected using real-time polymerase chain reaction. RESULTS： rsTRAIL, mevastatin and their combination inhibited tumor proliferation in a time- and dose-dependent manner. The proliferation inhibitory rate and apoptosis rate of human SWO-38 cells in the combined group were significantly greater than the rsTRAIL or mevastatin alone group （P 〈 0.01）. TRAIL R1/DR4 and TRAIL R2/DR5 protein and mRNA expressions were increased in the combination group compared with mevastatin or rsTRAIL alone after 72 hours （P 〈 0.01）. CONCLUSION： Both rsTRAIL and mevastatin inhibit the proliferation and apoptosis of the human glioma cell line SWO-38, while their combination enhances the anti-tumor effect. The mechanism of action possibly correlates to the upregulation of TRAIL R1/DR4 and TRAIL R2/DR5 mRNA expression by mevastatin, thereby enhancing the cell sensitivity to rsTRAIL.

Expression of tumor necrosis factor related apoptosis inducing ligand receptor in glioblastoma

BACKGROUND： Receptors for tumor necrosis factor related apoptosis inducing ligand （TRAIL） include death receptor 4, death receptor 5, decoy receptor 1, and decoy receptor 2. Activation of death receptor 4 and 5 selectively kills tumor cells. OBJECTIVE： To detect TRAIL receptor expression in glioblastoma by immunohistochemistry and RT-PCR, and to compare this expression to that in normal brain tissue. DESIGN： Observational analysis. SETTING： Department of Pathology, the First Affiliated Hospital of Zhengzhou University; Henan Tumor Pathology Key Laboratory. PARTICIPANTS： Twenty-five patients （17 males and 8 females） who received glioblastoma resection were selected from the Fifth Affiliated Hospital of Zhengzhou University, between September 2003 to June 2004. All glioblastoma samples were diagnosed pathologically. Twenty patients （12 males and 8 females） with craniocerebral injury who received normal brain tissue resection were selected in the same time period. There were no significant differences in sex and age between glioblastoma patients or between craniocerebral injury patients （P 〉 0.05）. All patients and appropriate relatives provided informed consent, and this study was approved by the local research ethics committee. METHODS： Polyclonal antibody against TRAIL receptors and an immunohistochemical kit （batch number： 200502） were purchased from Boster Company, Wuhan. Immunohistochemistry： Expression of death receptor 4, death receptor 5, decoy receptor l, and decoy receptor 2 were observed in both glioblastoma and normal brain tissue. The experiment was performed according to the kit instructions, and positive staining was brown-yellow. Assessment： There were no positive signals （-）; weakly positive signals, positive cells 〈 25% （＋）; weakly positive signals, positive cells 25%-50% （＋＋）; strongly positive signals, positive cells 50%-75% （＋＋＋）; strongly positive signals, positive cells 〉 75% （＋＋＋＋）. Evaluation： Expression levels of TRAIL receptors were estimated in both normal brain tissue and glioblastoma. Expression of decoy receptor 1 and decoy receptor 2 mRNA in glioblastoma were detected by reverse transcription polymerase chain reaction, and expression of decoy receptor in glioblastoma was estimated. MAIN OUTCOME MEASURES： Comparison of death receptor and decoy receptor protein expression between glioblastoma and normal brain tissue; decoy receptor mRNA expression in glioblastoma. RESULTS： Death receptor protein expression was strongly positive （＋＋＋） in glioblastoma, while it was weakly positive （＋, ＋＋） in normal brain tissue. Therefore, expression rate of death receptor protein in the glioblastoma was significantly higher than that in the normal brain tissue （.~ 2 = 18.48, 23.03, P 〈 0.01）. Decoy receptor protein expression in the glioblastoma was significantly lower than that in the normal brain tissue （ x2 = 6.65, 18.76, P 〈 0.01）. The level of decoy receptor mRNA expression in glioblastoma was significantly higher than those of protein expression （ x 2 = 9.82, 10.09, P〈 0.01）. CONCLUSION： High expression of death receptor and low expression of decoy receptor are frequently observed in glioblastoma, suggesting that TRAIL receptor genes show an anti-tumor and expressive response during the initiation and development of the tumor. There are significant differences in decoy receptor expression between normal brain tissue and glioblastoma, suggesting that the restricted expression of decoy receptor in glioblastoma is regulated at the post-transcriptional level.

Role of TNF-related apoptosis-inducing ligand （TRAIL） in the pathogenesis of varicocele-induced testicular dysfunction

The higher frequency of varicocele in men with infertility has drawn attention and resulted in increased research at the molecular level towards treatments. The aim of this study was to investigate the role of tumor necrosis factor （TNF）-related apoptosis-inducing ligand （TRAIL） and its receptors in varicocele-induced testicular dysfunction in an experimental rat model. The rats were divided into three groups： control, sham and varicocele. Varicoceles in rats were induced by partial ligation of the left renal vein and left testes. The rats were analyzed 13 weeks after surgery. The degree of DNA fragmentation within cells in the testis was determined using terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling （TUNEL） assay. Tubule degeneration was evaluated using the Johnsen score. The expression of TRAIL and its receptors was detected by immunohistochemical and Western blotting techniques. The apoptotic index, Johnsen score and the expression of TRAIL and TRAIL receptors were examined. The data are presented as the mean-.＋s.d, and were analyzed using computer software. The KruskaI-Wallis and Dunn＇s multiple comparison tests were used in the statistical analyses. The germ cell apoptotic index was increased in rats with varicoceles when compared with the sham and control groups （P=0.0031）. The Johnsen score was significantly decreased in the varicocele group when compared with the sham and control groups （P〈O.O001）. Immunohistochemical and Western blotting analyses showed that after varicocele induction, the expression of TRAIL-R1 and TRAIL-R4 in germ cells was increased and the expression of TRAIL-R2 was decreased. There are no significant differences among the groups in terms of TRAIL and TRAIL-R3 receptor expression. The results of this study indicate that TRAIL and its receptors may have a potential role in the pathogenesis of varicocele-induced testicular dysfunction.

Induction of apoptosis in osteogenic sarcoma cells by combination of tumor necrosis factor-related apoptosis inducing ligand and chemotherapeutic agents

Background Osteosarcoma is one of the most common primary malignant tumors of bone with poor prognosis. TNF-related apoptosis inducing ligand （TRAIL） is a member of the tumor necrosis factor （TNF） cytokine family. TRAIL induces apoptosis in various tumor cell lines but is not found to be cytotoxic to many normal cell types in vitro. We investigated the cytotoxic activity of TRAIL and chemotherapeutic agents, including methotrexate （MTX）, doxorubicin （DOX） and cisplatin （CDDP）, on established osteosarcoma cell line-OS-732. Methods OS-732 cells were incubated with chemotherapeutic agents MTX,DOX and CDDP at various peak plasma concentrations（PPC）, 0.1PPC,1PPC and 10PPC, alone or with 100 ng/ml of TRAIL for 24 hours or 48 hours. MTT was used to evaluate the cytotoxic activity of different agents on OS-732. The apoptosis proportion was assayed by flow cytometry. Cellular morphologic changes were observed by phase contrast microscope, scan electron microscope, and transmission electron microscope. Results The inhibitory rate was （24.438±3.414）% with TRAIL of 100 ng/ml for 24 hours. The cells were responsive to DOX and CDDP with a dose-effect relationship （P〈0.05）. In OS-732 cells, DOX and CDDP cooperated synergistically with TRAIL when incubated the cells with them for 24 hours （the combined inhibitory rate is （58.360±2.146）% and （54.101±-2.721）%, respectively）. TRAIL alone or drugs alone induced the apoptosis rate was less than 25% （P〈0.05）. However, the combination of TRAIL and MTX did not present synergistic effects on OS-732 cells （P〉0.05, compared with TRAIL alone）. Conclusions Osteosarcoma OS-732 cells were not responsive to TRAIL-induced apoptosis. DOX and CDDP sensitize osteosarcoma OS-732 cells to TRAIL-induced apoptosis. The combination of TRAIL and MTX presented no synergistic effects on killing OS-732 cells.

TRAIL-induced apoptosis of hepatocellular carcinoma cells is augmented by targeted therapies

AIM:To analyze the effect of chemotherapeutic drugs and specific kinase inhibitors,in combination with the death receptor ligand tumor necrosis factor-related apoptosis inducing ligand(TRAIL),on overcoming TRAIL resistance in hepatocellular carcinoma(HCC)and to study the efficacy of agonistic TRAIL antibodies,as well as the commitment of antiapoptotic BCL-2 proteins, in TRAIL-induced apoptosis. METHODS:Surface expression of TRAIL receptors (TRAIL-R1-4)and expression levels of the antiapoptotic BCL-2 proteins MCL-1 and BCL-xL were analyzed by flow cytometry and Western blotting,respectively. Knock-down of MCL-1 and BCL-xL was performed by transfecting specific small interfering RNAs.HCC cellswere treated with kinase inhibitors and chemotherapeutic drugs.Apoptosis induction and cell viability were analyzed via flow cytometry and 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. RESULTS:TRAIL-R1 and-R2 were profoundly expressed on the HCC cell lines Huh7 and Hep-G2. However,treatment of Huh7 and Hep-G2 with TRAIL and agonistic antibodies only induced minor apoptosis rates.Apoptosis resistance towards TRAIL could be considerably reduced by adding the chemotherapeutic drugs 5-fluorouracil and doxorubicin as well as the kinase inhibitors LY294002[inhibition of phosphoinositol- 3-kinase(PI3K)],AG1478(epidermal growth factor receptor kinase),PD98059(MEK1),rapamycin(mam- malian target of rapamycin)and the multi-kinase inhibitor Sorafenib.Furthermore,the antiapoptotic BCL-2 proteins MCL-1 and BCL-xL play a major role in TRAIL resistance:knock-down by RNA interference increased TRAIL-induced apoptosis of HCC cells.Additionally, knock-down of MCL-1 and BCL-xL led to a significant sensitization of HCC cells towards inhibition of both c-Jun N-terminal kinase and PI3K.CONCLUSION:Our data identify the blockage of survival kinases,combination with chemotherapeutic drugs and targeting of antiapoptotic BCL-2 proteins as promising ways to overcome TRAIL resistance in HCC.

COMBINATION OF γ-INTERFERON WITH TRAIL AND CISPLATIN OR ETOPOSIDE INDUCES APOPTOSIS IN HUMAN NEUROBLASTOMA CELL LINE SH-SY5Y

Objective To study the effect of γ-interferon （IFNγ）, tumor necrosis factor related apoptosis inducing ligand （TRAIL）, and cisplatin or etoposide induced apoptosis in human neuroblastoma cell line SH-SY5Y and its possible molecular mechanisms. Methods The expressions of Caspase 8 mRNA and protein were detected with RT-PCR and Western blot analysis. The effects of IFNγ, TRAIL, IFNγ ＋ TRAIL, IFNγ ＋ Caspase 8 inhibitor ＋ TRAIL, IFNγ ＋ cisplatin ＋ TRAIL, and IFNγ ＋ etoposide ＋ TRAIL on the growth and apoptosis of SH-SY5Y cells were detected with the methods of MTT and flow cytometry. The relative Caspase 8 activity was measured with colorimetric assay. Results Caspase 8 was undetectable in SH-SY5Y cells but an increased expression of Caspase 8 mRNA and protein was found after treatment with IFNγ. SH-SY5Y ceils themselves were not sensitive to TRAIL, but those expressing Caspase 8 after treatment with IFNγ were. The killing effect of TRAIL on SH-SY5Y cells expressing Caspase 8 was depressed by Caspase 8 inhibitor. Cisplatin and etoposide could enhance the sensitivity of TRAIL on SH-SY5Y cells. The relative Caspase 8 activity of SH-SY5Y cells in IFNγ ＋ TRAIL group was significantly higher than those of control group, IFNγ group, TRAIL group, and inhibitor group （ P 〈 0. 01 ）. There was no significant difference among IFNγ ＋ TRAIL group, IFNγ ＋ cisplatin ＋ TRAIL group, and IFNγ ＋ etoposide ＋ TRAIL group. Conclusions IFNγ could sensitize SH-SY5Y cells to TRAIL-induced apoptosis and this may be realized by the up-regulation of Caspase 8. Cisplatin and etoposide could enhance the killing effect of TRAIL on SH-SY5Y cells.

Apoptosis and Expression of Protein TRAIL in Granulosa Cells of Rats with Polycystic Ovarian Syndrome

The relationship between apoptosis of granulosa cells and follicle development arrest in polycystic ovarian syndrome (PCOS) rats, and the contribution of tumor necrosis factor related apoptosis inducing ligand (TRAIL) in apoptosis of granulosa cells were explored. By using sodium prasterone sulfate rat PCOS model was induced. The apoptosis of granulosa cells in ovaries of rats was observed by TdT-mediated dUTP-biotin nick end-labeling (TUNEL), and the expression of TRAIL protein and mRNA in granulosa cells was detected by using immunhistochemical staining and reverse transcription polymerase chain reaction (RT-PCR) respectively. The apoptotic rate and the expression of protein TRAIL in granulosa cells were significantly higher in antral follicles from the PCOS rats than in those from the control rats (P<0.01, P<0.05). There was no significant difference in apoptotic rate and the expression of TRAIL protein in granulosa cells of preantral follicles between the PCOS rats and the control rats (P>0.05). No apoptosis and the expression of TRAIL protein in granulosa cells of primordial follicles were found in the two groups. The expression of TRAIL mRNA was significantly stronger in granulosa cells from the PCOS rats than in those from the con- trol rats (P<0.01). It was suggested that the apoptotic rate in granulosa cells was significantly higher in antral follicle from the PCOS rats than in those from the control rats. TRAIL played a role in regu- lating the apoptosis of granulosa cells in PCOS rats.