Tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in glioma U87 cells

Studies have shown that tumor necrosis factor-related apoptosis-inducing ligand（TRAIL）exhibits strong induction of apoptosis in human glioma cells.It remains unclear whether the mitochondrion pathway,an important apoptosis signaling pathway,is involved in TRAIL-induced glioma cell apoptosis.In the present study,in vitro cultured human glioma U87 cells were treated with human recombinant soluble TRAIL.Apoptosis of glioma U87 cells,mitochondrial transmembrane potential（Δψm）,cytoplasmic cytochrome c concentration and changes in caspase-3,-8 and-9 activity following human recombinant soluble TRAIL treatment were investigated to determine the mechanism of glioma U87 cell apoptosis induced by TRAIL.Additionally,blocking caspase-8resulted in TRAIL-induced mitochondrion pathway activation,suggesting that TRAIL,through activating caspase-8,initiated a series of mitochondrial events and resulted in apoptosis of glioma U87 cells.

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.

Polyphyllin Ⅰ enhances tumor necrosis factor-related apoptosis-inducing ligand-induced inhibition of human osteosarcoma cell growth via downregulating the Wnt/β-catenin pathway

OBJECTIVE:To investigate the synergistic effects of polyphyllin Ⅰ(PPⅠ)combined with tumor necrosis factorrelated apoptosis-inducing ligand(TRAIL)on the growth of osteosarcoma cells through downregulating the Wnt/β-catenin signaling pathway.METHODS:Cell viability,apoptosis and cell cycle distribution were examined using cell counting kit-8 and flow cytometry assays.The morphology of cancer cells was observed with inverted phase contrast microscope.The migration and invasion abilities were examined by xCELLigence real time cell analysis DP system and transwell assays.The expressions of poly(adenosine diphosphate-ribose)polymerase,C-Myc,Cyclin B1,cyclin-dependent kinases 1,N-cadherin,Vimentin,Active-β-catenin,β-catenin,p-glycogen synthase kinase 3β(GSK-3β)and GSK-3βwere determined by Western blotting assay.RESULTS:PPⅠ sensitized TRAIL-induced decrease of viability,migration and invasion,as well as increase of apoptosis and cell cycle arrest of MG-63 and U-2 OS osteosarcoma cells.The synergistic effect of PPⅠwith TRAIL in inhibiting the growth of osteosarcoma cells was at least partially realized through the inactivation of Wnt/β-catenin signaling pathway.CONCLUSION:The combination of PPⅠ and TRAIL is potentially a novel treatment strategy of osteosarcoma.

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.

重组变构人TNF相关促凋亡配体联合三氧化二砷诱导白血病耐药细胞株凋亡

本研究探讨重组变构人TNF相关促凋亡配体(recombinant mutant human TNF-related apoptosis-inducing ligand,rmhTRAIL)联合三氧化二砷(As2O3)对阿霉素诱导的人慢性髓系白血病红白血病变耐药细胞株K562/AO2(mdr-1+)的促凋亡作用。以亲本阿霉素敏感细胞株K562(mdr-1-)为对照,观察rmhTRAIL作用后细胞形态变化,采用MTT法测定rmhTRAIL、As2O3单药及联合作用后细胞增殖抑制率;碘化丙锭染色的流式细胞术(FACSCalibur)定量检测rmhTRAILL、As2O3单药及联合作用后sub-G1峰占检测细胞的百分比(sub-G1%)。结果表明:rmhTRAIL联合As2O3对K562/AO2和K562增殖的抑制作用优于单药,采用国内金(正均)氏公式判断rmhTRAIL和As2O3联合作用具有协同作用;流式细胞术定量检测sub-G1%显示,rmhTRAIL2 000ng/ml与As2O32μmol/L单独作用下K562/AO2组与K562组凋亡率分别为(34.93±0.10)%、(10.53±0.16)%(p<0.01);(5.95±0.07)%、(3.50±0.01)%(p<0.05),联合作用下细胞凋亡率分别为(50.95±0.91)%、(20.75±0.95)%(p<0.05),K562/AO2组凋亡效应强于K562组。结论:rmhTRAIL可诱导K562、K562/AO2细胞凋亡;rmhTRAIL联合凋亡诱导剂As2O3对白血病耐药细胞株K562/AO2和K562细胞株的凋亡具有协同作用;rmhTRAIL、As2O3对K562/AO2的促凋亡作用优于其亲本K562细胞。

亚细胞毒性剂量As_2O_3增加胃癌细胞对rhTRAIL的敏感性及其机制

目的:研究亚细胞毒性剂量As2O3对rhTRAIL诱导胃癌细胞凋亡的影响及其机制．方法:人胃腺癌细胞株SGC 7901以As2O3(1μmol／L),rhTRAIL(500μg／L)及两者联用处理:采用AnnexinV-FITC和PI双染色流式细胞仪检测细胞凋亡:用间接免疫荧光染色结合流式细胞技术检测细胞表面TRAIL R1／DR4和TRAIL R2／DR5分子表达;RT-PCR方法检测TRAIL R1／DR4和TRAIL R2／DR5 mRNA表达．结果:rhTRAIL在单用或与As2O3联用时均可以诱导胃癌SGC7901细胞凋亡,并且随着作用时间延长(12-72 h),细胞凋亡率逐渐增高．As2O3与rhTRAIL联用24,48,72 h后,SGC7901细胞凋亡率分别显著高于单用rhTRAIL处理相同时间细胞(36．49％±7．12％,47．13％±3．44％,55．63％±7．16％vs 29．78％±3.18％、38．56％±1．89％,43．12％±6．23％,P<0．05); As2O3单用或联用rhTRAIL处理SGC7901细胞24 h后,细胞表面TRAIL R1／DR4和TRAIL R2／DR5分子的平均荧光密度(MFI)较对照组细胞显著增加(R1／DR4:29．44±4．29,26．14±3．40 vs13．45±3．81,P<0．05或P<0．01;R2／DR5:28．04±0．79．3 1．47±4．56vs16．45±5．07,P<0．05或P<0．01);与此同时,TRAIL R1／DR4和TRAIL R2／DR5mRNA表达水平增高．结论:亚细胞毒性剂量As2O3可能通过增加TRAIL R1／DR4和TRAIL R2／DR5基因表达、上调细胞表面TRAIL死亡受体从而增加胃癌细胞SGC7901对rhTRAIL的敏感性,两药可能联合用于治疗胃癌．

葡萄糖、雌二醇对MG63细胞株TRAIL,OPG,OPGL mRNA表达的影响

目的观察在不同浓度葡萄糖、雌二醇环境下人成骨肉瘤MG63细胞株肿瘤坏死因子相关凋亡诱导配体(TRAIL)及其护骨素(OPG)、护骨素配体(OPGL)的表达,探讨绝经后糖尿病女性骨质疏松症的发病机制。方法用不同浓度葡萄糖(5.5、16.7、33.3 mmol/L)和17β-E2分别刺激培养的MG63细胞24 h,RT-PCR法检测TRAIL、OPG、OPGL mRNA的表达。结果葡萄糖对MG63细胞中TRAIL、OPGL mRNA的表达,均按照对照组、5.5 mmol/L组、16.7 mmol/L组、33.3 mmol/L组顺序递增(P<0.05),OPG mRNA的表达按照此顺序递减(P<0.05)。33.3 mmol/L组TRAIL mRNA的水平明显高于对照组[(1.004±0.070)vs(0.740±0.023),P<0.05],而17β-E2可增加OPG mRNA的表达,减少TRAIL、OPGL mRNA的表达。结论高糖环境可能导致成骨细胞中TRAIL和OPGL表达增多,OPG的表达减少,从而导致骨质疏松。而雌二醇对葡萄糖环境下的MG63细胞中上述因子的表达可产生一定的对抗效应。这可能是绝经后糖尿病女性骨质疏松症患者雌激素替代治疗的依据之一。

奥沙利铂对TRAIL诱导胃癌BGC823细胞凋亡的增强作用

目的:探讨奥沙利铂和肿瘤坏死因子相关凋亡诱导配体(TRAIL)对胃癌BGC823细胞联合作用的机制.方法:不同浓度的TRAIL和/或奥沙利铂作用于人胃癌细胞系BGC823细胞,MTT检测细胞增殖能力.流式细胞术PI染色检测细胞凋亡.TRAIL和/或奥沙利铂作用BGC823细胞后,用抗CTX-B,抗死亡受体4(DR4)和罗丹明标记的荧光二抗染色.免疫荧光显微技术检测脂筏和DR4在细胞膜的分布.结果:1-1000μg/L的TRAIL作用BGC823细胞24h,细胞增殖抑制率不超过20%.100μg/L的TRAIL对细胞的增殖抑制率在10%左右.100μg/L的TRAIL作用BGC823细胞24h,诱导4.12%±1.26%的细胞凋亡.1-50mg/L奥沙利铂处理BGC823细胞24h,抑制细胞增殖50%的药物浓度(IC50)为37.36mg/L±8.12mg/L.与单药奥沙利铂相比,奥沙利铂(38mg/L,24h的IC50)联合TRAIL(100μg/L)作用24h,细胞凋亡比率明显提高(19.83%±4.21%vs40.42%±5.78%,P<0.05).与对照组相比,100μg/L的TRAIL作用BGC823细胞24h,没有引起明显的脂筏聚集或DR4聚集.奥沙利铂(38mg/L)明显促进脂筏聚集和DR4聚集,同时观察到DR4和脂筏的共定位.奥沙利铂和TRAIL联合作用24h,同样观察到DR4定位在聚集的脂筏内.结论:奥沙利铂通过促进DR4在脂筏聚集增强TRAIL诱导的胃癌BGC823细胞凋亡.

Combination of oridonin and TRAIL induces apoptosis in uveal melanoma cells by upregulating DR5

AIM:To investigate the inhibitory effect of the combined use of tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)and oridonin on choroidal melanoma cell lines,and to explore its underlying mechanism.METHODS:MUM-2B and C918 cells were treated with different concentrations of TRAIL and oridonin,and MTT assay used to evaluate the inhibition rate of the two compounds on cells.Then,the cell cycle distribution and apoptosis were detected by flow cytometry,and changes in apoptosis-related proteins such as death receptor 5(DR5),a-caspase-3,and x-linked inhibitor of apoptosis protein(XIAP)were detected by Western blot.MUM-2B cells were transfected with si-DR5,which interfered with the expression of the DR5 gene.MTT and Western blot assay were used to detect cell activity and apoptosis-related proteins.RESULTS:When TRAIL and oridonin were simultaneously administered to the MUM-2B cells,the apoptosis rate was significantly higher than that by the two drugs individually.However,the effect of combined use of TRAIL and oridonin on C918 cells was not significantly different from that used alone.Cell cycle analysis showed that TRAIL and oridonin could induce G2/M arrest in MUM-2B cells.The Western blot results showed that the protein expression levels of the DR5,a-caspase-3,and BAX increased,while the expression levels of the anti-apoptosis-related proteins XIAP and BCL-2 were suppressed when TRAIL and oridonin simultaneously administered to MUM-2B cells.Interfering the expression of DR5 gene in MUM-2B cells could reverse the inhibitory effect of oridonin and TRAIL on the proliferation and apoptosis induction of MUM-2B cells.CONCLUSION:The inhibitory effects of oridonin and TRAIL on MUM-2B cells are significantly enhanced when they were administered as a combined treatment,which may ascribe to up-regulation of DR5.

肿瘤坏死因子相关凋亡诱导配体与肝细胞凋亡的相关性

肿瘤坏死因子相关的调亡诱导配体是TNF家族成员之一,通过其死亡受体诱导凋亡.研究显示TRAIL/死亡受体途径参与多种肝脏病理过程,本文就TRAIL/死亡受体途径的特性、致凋亡的机制及与肝细胞凋亡关系的最新进展作一综述.

Downregulation of cyclooxygenase-1 is involved in gastric mucosal apoptosis via death signaling in portal hypertensive rats

Portal hypertension （PHT） gastropathy is a frequent complication of fiver cirrhosis and one of the leading causes of death from cirrhosis. Apoptosis is widely considered to be an active energy-dependent mode of cell death and a distinct entity from necrotic cell death. It is unclear whether gastric mucosal apoptosis is involved in PHT gastropa- thy. Prostaglandins （PGs） produced through cyclooxygenase （COX） are thought to play a key role in protection of the gastrointestinal mucosa from injury and apoptosis. However, the role of COX in PHT gastropathy is still not clearly understood. The aims of this study were to investigate whether （1） gastric mucosal apoptosis is involved in PHT gas- tropathy and （2） downregulation of COX contributes to this apoptosis. In this study, we show that gastric mucosal apoptosis was remarkably increased while mucosal proliferation was inhibited in PHT rats. Gastric mucosal COX- 1 was significantly suppressed at both the mRNA and protein levels, and PGE2 was reduced in PHT rats. Further, PGE2 treatment suppressed gastric mucosal apoptosis in PHT rats. However, gastric mucosal COX-2 levels did not differ between sham-operated rats and PHT rats. Gastric mucosal levels of tumor necrosis factor-α （TNF-α） and Fas ligand, but not TNF-related apoptosis-inducing ligand, were increased, and activated caspase-8 and caspase-3 levels were upregulated in PHT rats. The release of cytochrome c from the mitochondria to the cytosol was not observed in PHT rats. Our data indicate that downregulation of COX-1 is involved in gastric mucosal apoptosis via death signal- ing-mediated type-I cell death in PHT rats.

Bcl-2 degradation is an additional pro-apoptotic effect of polo-like kinase inhibition in cholangiocarcinoma cells

To examine the influence on apoptotic mechanisms following inhibition of polo-like kinases as therapeutically approach for cholangiocellular cancer treatment.METHODSAs most cholangiocarcinomas are chemotherapy-resistant due to mechanisms preventing tumor cell death, we investigated the effect of Cisplatin on cholangiocellular carcinoma (CCA) cell lines KMCH-1 and Mz-Ch-1. Polo-like kinases (PLK) are important regulators of the cell cycle and their inhibition is discussed as a potential therapy while PLK inhibition can regulate apoptotic mediators. Here, cells were treated with PLK inhibitor BI6727 (Volasertib), Cisplatin, and in combination of both compounds. Cell viability was assessed by MTT; apoptosis was measured by DAPI staining and caspase-3/-7 assay. Western blot and qRT-PCR were used to measure expression levels of apoptosis-related molecules Bax and Bcl-2.RESULTSThe cell viability in the CCA cell lines KMCH-1 and Mz-Ch-1 was reduced in all treatment conditions compared to vehicle-treated cells. Co-treatment with BI6727 and cisplatin could even enhance the cytotoxic effect of cisplatin single treatment. Thus, co-treatment of cisplatin with BI6727 could slightly enhance the cytotoxic effect of the cisplatin in both cell lines whereas there was evidence of increased apoptosis induction solely in Mz-Ch-1 as compared to KMCH-1. Moreover, PLK inhibition decreases protein levels of Bcl-2; an effect that can be reversed by the proteasomal degradation inhibitor MG-132. In contrast, protein levels of Bax were not found to be altered by PLK inhibition. These findings indicate that cytotoxic effects of Cisplatin in Mz-Ch-1 cells can be enhanced by cotreatment with BI6727.CONCLUSIONIn conclusion, BI6727 treatment can sensitize CCA cells to cisplatin-induced apoptosis with proteasomal Bcl-2 degradation as an additional pro-apoptotic effect.

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.

EGCG Enhances TRAIL-mediated Apoptosis in Human Melanoma A375 Cell Line

Tumor necrosis factor-related apoptosis-inducing ligand （TRAIL） is a promising anti-cancer agent. Epigallocatechin-3-gallate （EGCG） is a polyphenolic constituent of green tea. In this study, inhibitory effect of combined use of EGCG and TRAIL on human melanoma A375 cells was examined and the possible mechanism investigated. The cells were divided into 4 groups： control group, EGCG group （EGCG： 10, 20 μg/mL）, TRAIL group （TRAIL： 25 ng/mL） and EGCG＋TRAIL group （combined group）. The growth inhibition was measured in the A375 cells treated with different concentrations of TRAIL （（25, 50, 75, 100, 125, 150 ng/mL） by MTT assay. The apoptosis was assessed by flow cytometry. The expressions of DR4 and DR5 were detected by flow cytometry and western blotting. The activities of caspase-8 and caspase-3 were determined by colorimetric assay. The results showed that TRAIL could dose-dependently inhibit the growth of A375 cells and the IC50 of TRAIL was 150 ng/mL. The apoptosis rate was 11.8% in the TRAIL group, 5%–7% in the EGCG group and 48.9%–59.1% in the combined group. Significant difference was found in the apoptosis rate between the combined group and the EGCG or TRAIL group （P〈0.05 for each）. The expression of DR4 instead of DR5 was significantly increased in the EGCG group. The activity of caspase-3 rather than caspase-8 was substantially enhanced in the EGCG group. These results suggest that EGCG is useful for the TRAIL-based treatment for melanoma.

Expression of caspase-3 and TRAIL receptors in CD4^+ and CD8^+ T cells of SLE patients

Objective: To study the expression of caspase-3 and tumor necrosis factor-related apoptosisinducing ligand (TRAIL) receptors in the CD4+ and CD8+ T cells of systemic lupus enythematosus (SLE) patients. Methods: RT-PCR was used to analyze the expression of caspase-3 and TRAIL receptors in CD4+ and CD8+ T cells of SLE patients and normal subjects. Results: The death domain-containing TRAIL-R1/R2 as well as 'decoy' TRAIL-R3/R4 were co-expressed in majority of CD4+ and CD8+ T cells in both SLE patients and normal subjects. The CD8+ T cells from SLE patients showed significantly higher expression of caspase-3 and TRAIL-R2 than those from normal subjects,and the expression was correlated with the activity of the disease. Conclusion: The TRAIL-R2 signal pathway might contribute to the apoptosis of T cells in SLE.

Piperlongumine inhibits cell growth and enhances TRAIL-induced apoptosis in prostate cancer cells

Objective: To investigate whether piperlongumine can sensitize prostate cancer cells to tumor necrosis factor-related apoptosisinducing ligand(TRAIL) and trigger apoptosis in prostate cells. Methods: Human prostate cancer cell lines PC3, LNCa P, and VCa P were cultured with piperlongumine and TRAIL. Then, cell proliferation, migration, caspase activation, apoptotic protein expressions, and death receptor expressions were measured.Results: Piperlongumine inhibited cell proliferation at low doses(<10 μM) alone and in combination with TRAIL(25 ng/m L), induced apoptosis, and suppressed cyclooxygenase activation. Additionally, piperlongumine induced expression of death receptors which potentiated TRAIL-induced apoptosis in cancer cells but did not affect decoy receptors. Piperlongumine also downregulated tumor cell-survival pathways, inhibited colony formation and migration of cancer cells alone or in combination with TRAIL. The combination of piperlongumine with TRAIL was found to be synergistic. Conclusions: Our findings indicate that piperlongumine can sensitize cancer cells to TRAIL through the upregulation of death receptors and can trigger apoptosis with the downregulation of antiapoptotic proteins.

The flavonoid casticin enhances TRAIL-induced apoptosis of colon cancer cells through endoplasmic reticulum stress-mediated up-regulation of DR5

Objective： The aim of this study was to explore the mechanisms by which the flavonoid casticin enhances tumor necrosis factor-related apoptosis-inducing ligand （TRAIL）-induced apoptosis in colon cancer cells. Methods： Human colon cancer HT-29 cells were treated with TRAIL or casticin. Cytotoxicity was examined by MTT assay, and apoptosis determined by morphological observation and flow cytometric analysis. Death receptor 5 （DRS）, DR4, and endoplasmic reticulum （ER） stress response markers, including glucose regulating protein 78 （GRP78）, activating transcription factor 4 （ATF4） and CHOP （CCAAT/enhancer binding protein homologous protein）, were examined with western blot. Small interfering RNA （siRNA） transfection was employed to knock down CHOP. Results： HT-29 cells were resistance to TRAIL-induced apoptosis, but casticin, at subtoxic concentrations, potentiated HT-29 cells to TRAIL-induced apoptosis. Casticin up-regulated the expression of DR5 time-and dose-dependent manners, but had no effect on the expression of DR4. Also, casticin increased the levels of ER stress response markers （GRP78, ATF4 and CHOP） in a similar way to DR5. Knockdown of CHOP by specific siRNA, or salubrinal, an ER stress inhibitor, abolished the up-regulation of DR5 and enhancement of TRAIL-induced apoptosis by casticin. Conclusion： Casticin enhances TRAIL-induced apoptosis of colon cancer cells by ER stress-mediated up-regulation of DR5.

TRAIL and Celastrol Combinational Treatment Suppresses Proliferation, Migration, and Invasion of Human Glioblastoma Cells via Targeting Wnt/β-catenin Signaling Pathway

Objective To investigate the mechanistic basis for the anti-proliferation and anti-invasion effect of tumor necrosis factor-related apoptosis-induced ligand(TRAIL)and celastrol combination treatment(TCCT)in glioblastoma cells.Methods Cell counting kit-8 was used to detect the effects of different concentrations of celastrol(0-16µmol/L)and TRAIL(0-500 ng/mL)on the cell viability of glioblastoma cells.U87 cells were randomly divided into 4 groups,namely control,TRAIL(TRAIL 100 ng/mL),Cel(celastrol 0.5µmol/L)and TCCT(TRAIL 100 ng/mL+celastrol 0.5µmol/L).Cell proliferation,migration,and invasion were detected by colony formation,wound healing,and Transwell assays,respectively.Quantitative reverse transcription polymerase chain reaction and Western blotting were performed to assess the levels of epithelial-mesenchymal transition(EMT)markers(zona occludens,N-cadherin,vimentin,zinc finger E-box-binding homeobox,Slug,and β-catenin).Wnt pathway was activated by lithium chloride(LiCl,20 mol/L)and the mechanism for action of TCCT was explored.Results Celastrol and TRAIL synergistically inhibited the proliferation,migration,invasion,and EMT of U87 cells(P<0.01).TCCT up-regulated the expression of GSK-3β and down-regulated the expression of β-catenin and its associated proteins(P<0.05 or P<0.01),including c-Myc,Cyclin-D1,and matrix metalloproteinase(MMP)-2.In addition,LiCl,an activator of the Wnt signaling pathway,restored the inhibitory effects of TCCT on the expression of β-catenin and its downstream genes,as well as the migration and invasion of glioblastoma cells(P<0.05 or P<0.01).Conclusions Celastrol and TRAIL can synergistically suppress glioblastoma cell migration,invasion,and EMT,potentially through inhibition of Wnt/β-catenin pathway.This underlies a novel mechanism of action for TCCT as an effective therapy for glioblastoma.

矽肺病患者肺泡巨噬细胞死亡受体表达及意义

目的探讨矽肺病患者肺泡巨噬细胞(AM)死亡受体(DR)的表达水平及其与肺纤维化程度的关系。方法以61例进行肺灌洗的汉族男性观察对象及矽肺病患者为观察组,以13例肺部X射线表现完全正常的汉族男性为对照组,分离、纯化及培养各研究对象的AM,检测3种DR[凋亡蛋白-1(Fas)、肿瘤坏死因子受体-1(TNFR1)和肿瘤坏死因子相关凋亡诱导配体受体-2]的表达水平,采用酶联免疫吸附试验检测AM培养上清中的可溶性DR(sDR)水平,采用免疫印迹法检测AM裂解液中3种膜结合型DR(mDR)水平,分析DR表达水平与粉尘接触各因素的关系。结果矽肺病患者膜结合型Fas(mFas)及膜结合型TNFR1(mTNFR1)的相对水平均高于对照组及观察对象(P<0.05),且随着肺部病变的加重而升高;矽肺病患者可溶性Fas(sFas)及可溶性TNFR1(sTNFR1)水平均低于观察对象(P<0.05),且sFas随着肺部病变的加重而下降。结论矽尘诱导的mFas和mTNFR1表达上调、sFas表达下调在矽肺病发病及进展中起重要作用,DR信号通路活化可能是矽肺病发病的重要机制之一。

Bcl-XL小分子干扰RNA逆转人结肠癌获得性肿瘤坏死因子相关凋亡诱导配体的耐药

目的探讨Bcl-XL小分子干扰RNA（siRNA）在逆转人结肠癌细胞获得性肿瘤坏死因子相关的凋亡诱导配体（TRAIL）耐药中的作用。方法以Bcl-XL siRNA转染获得性TRAIL耐药的人结肠癌DLD1-TRAIL／R细胞24h，并用TRAIL蛋白处理，通过细胞计数和流式细胞仪检测细胞的存活率，并通过Western blot法检测各种凋亡相关蛋白的活化情况。结果Bcl-XL siRNA能有效下调DLD1-TRAIL／R细胞中Bcl-XL蛋白的表达水平，并且能够有效地逆转其对TRAIL蛋白的耐药。DLDl-TRAIL／R细胞经过Bcl-XLsiRNA和TRAIL蛋白共同处理2h后，其细胞凋亡率〉50％；共同处理4h后，其细胞存活率〈40％；而对照组和TRAIL蛋白处理组的细胞凋亡率和生存率无明显变化（P〈0．05）。Western blot法检测结果表明，经Bcl-XL siRNA与TRAIL蛋白联合处理后，DLDl-TRAIL／R细胞中caspase-8、caspase-9、Bid、caspase-3以及多聚腺苷酸二磷酸核糖聚合酶（PARP）均明显活化，细胞色素C的释放显著增加。结论Bcl-XL siRNA能够有效逆转结肠癌细胞获得性TRAIL耐药，这将为治疗肿瘤耐药提供一种新的思路。