TNF related apoptosis-inducing ligand and its receptors in ocular tumors

Most of the ocular tumors have poor prognosis, and they remain a difficult problem in the area of ophthalmology. With the rapid development of molecular biology and immunologic techniques and the deep research on ocular tumor related genes, it becomes possible to diagnose and treat malignant tumors from the molecular level. The tumor necrosis factor related apoptosis-inducing ligand (TRAIL), a member of the tumor necrosis factor (TNF) super family, is a promising candidate, either alone or in combination with established cancer therapies, since it can initiate apoptosis through the activation of their death receptors. The ability of TRAIL to selectively induce apoptosis of transformed, virus-infected or tumor cells but not normal cells promotes the development of TRAIL-based cancer therapy. Here, we will review TRAIL and its receptors' structure, function, mechanism of action and application in ocular tumors therapy.

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.

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.

肺源性心脏病并发肺动脉高压患者血清β-NGF和TRAIL水平检测在临床诊断及预后评估中的意义

目的探讨肺源性心脏病(pulmonary heart disease,PHD)并发肺动脉高压(pulmonary heart disease,PAH)患者血清β-神经生长因子(β-nerve growth factor,β-NGF)、肿瘤坏死因子相关凋亡诱导配体(tumor necrosis factor-related apoptosis-inducing ligand,TRAIL)表达水平及其在临床诊断及预后评估中的意义。方法采用1∶1病例-对照研究设计选取2019年1月~2022年6月北京市大兴区人民医院86例并发PAH的PHD患者为病例组,86例单纯PHD患者为对照组,进行回顾性分析。将病例组根据肺动脉收缩压(pulmonary arterial systolic pressure,PASP)分为轻度PAH组(n=39)、中度PAH组(n=25)和重度PAH组(n=22),根据出院后一年的结果分为预后良好组(n=75)和预后不良组(n=11)。收集研究对象人口学资料和实验室检查指标,采用酶联免疫吸附(ELISA)法检测血清β-NGF,TRAIL水平。Pearson积矩相关分析β-NGF,TRAIL与PASP的关系,Logistic回归分析PHD患者PAH影响因素,ROC曲线评估β-NGF,TRAIL对PAH的诊断价值,COX比例风险回归分析β-NGF,TRAIL与PHD并发PAH患者预后不良的关系,ROC曲线评估其对预后不良的预测价值。结果与对照组比较,病例组PHD病程长(8.63±1.27年vs 5.49±1.15年),血清β-NGF(26.97±8.25 ng/ml vs 22.14±7.32 ng/ml)和TRAIL(2.83±0.76 ng/ml vs 1.71±0.68 ng/ml)水平升高,差异具有统计学意义(t=17.006,4.064,10.183,均P<0.05)。血清β-NGF,TRAIL对PHD患者PAH有诊断价值,AUC分别为0.842,0.838,二者联合诊断的AUC为0.920,诊断价值高于单一指标(Z=3.416,3.508,均P<0.05)。轻度PAH组、中度PAH组和重度PAH组血清β-NGF(23.26±5.13 ng/ml,27.83±5.57 ng/ml,32.57±6.02 ng/ml),TRAIL(2.24±0.65 ng/ml,2.89±0.71 ng/ml,3.81±0.90 ng/ml)水平依次升高,差异具有统计学意义(F=20.624,31.972,均P<0.05)。病例组血清β-NGF,TRAIL与PASP呈正相关(r=0.673,0.659,均P<0.05)。预后不良组血清β-NGF(36.34±8.05 ng/ml),TRAIL(3.49±1.01 ng/ml)水平高于预后良好组(25.59±7.28 ng/ml,2.73±0.89 ng/ml),差异具有统计学意义(t=4.516,2.604,均P<0.05)。Logistic回归分析结果显示,PHD病程[OR(95%CI):1.784(1.135~2.806)]、β-NGF[OR(95%CI):1.976(1.108~3.523)],TRAIL[OR(95%CI):1.866(1.123~3.101)]是PHD患者发生PAH的独立危险因素(均P<0.05)。多因素COX比例风险回归结果显示,PHD病程[OR(95%CI):1.167(1.082~1.364]、β-NGF[OR(95%CI):1.322(1.134~1.649)],TRAIL[OR(95%CI):1.259(1.087~1.590)]是PHD并发PAH患者预后不良的独立危险因素(均P<0.05)。血清β-NGF,TRAIL可预测PHD并发PAH患者预后不良发生风险,AUC分别为0.863,0.881,二者联合检测的AUC为0.907,诊断价值高于单一指标检测(Z=2.905,3.128,均P<0.05)。结论血清β-NGF和TRAIL升高是PHD患者PAH独立危险因素,并与PAH严重程度有关,早期联合β-NGF和TRAIL检测可提高对PAH的诊断价值及对患者预后不良的预测效果。

血清TRAIL水平与晚期非小细胞肺癌患者免疫治疗反应的关联性研究

目的探讨血清肿瘤坏死因子相关凋亡诱导配体(TRAIL)水平与晚期非小细胞肺癌(NSCLC)患者免疫治疗反应的关联性。方法回顾性分析2019年1月至2020年5月唐山市人民医院收治的70例晚期NSCLC患者的临床资料。在免疫治疗前24 h内采用酶联免疫吸附试验法(ELISA)测定患者血清TRAIL水平。免疫治疗反应通过客观缓解率(ORR)和临床获益率(CBR)进行评估。分析患者血清TRAIL水平与免疫治疗反应、肺功能及肺气肿、临床预后的关联性。结果经免疫治疗后,NSCLC患者获得客观缓解23例,临床获益46例。获得客观缓解患者的血清TRAIL水平显著高于未获得客观缓解者[27.77(23.13,39.13)pg/mL vs 12.36(8.76,18.15)pg/mL;Z=4.508,P<0.001]。临床获益患者的血清TRAIL水平显著高于临床未获益者[23.13(16.99,30.63)pg/mL vs 11.75(8.76,15.56)pg/mL;Z=4.887,P<0.001]。Spearman秩相关性分析结果显示,血清TRAIL水平与1秒用力呼气容积/用力肺活量(FEV1/FVC)呈正相关(rs=0.288,P=0.016),与肺气肿总比值(rs=-0.257,P=0.032)、肺叶肺气肿比率(LER)(rs=-0.324,P=0.006)呈负相关。多因素logistic回归分析结果显示,以血清TRAIL>27.46 pg/mL为参考,血清TRAIL<18.15 pg/mL患者经免疫治疗不能获得客观缓解和临床获益的风险显著增加(P<0.05)。ROC曲线分析结果显示,血清TRAIL水平可有效预测NSCLC患者对免疫治疗的反应(P<0.05)。TRAIL高水平组(血清TRAIL≥18.15 pg/mL)的总体生存(OS)、无进展生存(PFS)预后显著优于TRAIL低水平组(血清TRAIL<18.15 pg/mL)(P<0.05)。结论血清TRAIL低水平与晚期NSCLC患者免疫治疗无反应以及临床预后不良有关,该指标监测有助于筛选能从免疫治疗中获益的NSCLC患者。

血清骨硬化蛋白、OPG、OPG/TRAIL比值对高血压伴慢性心力衰竭患者发生主要不良心血管事件的预测价值

目的探讨血清骨硬化蛋白、骨保护素(OPG)/肿瘤坏死因子相关凋亡诱导配体(TRAIL)比值对高血压伴慢性心力衰竭(CHF)患者发生主要不良心血管事件(MACE)的预测价值。方法选取2019年10月至2022年10月于该院就诊的134例高血压伴CHF患者作为研究对象,根据患者治疗1年内是否发生MACE将其分为MACE组(46例)和无MACE组(88例)。另选取同期于该院进行体检的74例健康者作为对照组。采用酶联免疫吸附实验检测3组血清骨硬化蛋白、OPG和TRAIL水平。收集所有患者的临床资料(包括冠心病史、糖尿病史、收缩压、舒张压)。检测3组血清胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)、肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-6和IL-10水平。采用Pearson相关分析高血压伴CHF患者血清骨硬化蛋白、OPG和TRAIL水平与相关实验室指标水平的关系。采用多因素Logistic回归分析高血压伴CHF患者发生MACE的危险因素。绘制受试者工作特征(ROC)曲线评估血清骨硬化蛋白、OPG/TRAIL比值单独及2项指标联合检测对MACE的预测价值。结果无MACE组和MACE组血清骨硬化蛋白、OPG水平及OPG/TRAIL比值均明显高于对照组,TRAIL水平明显低于对照组,差异均有统计学意义(P<0.05)。MACE组血清骨硬化蛋白、OPG水平及OPG/TRAIL比值均明显高于无MACE组,TRAIL水平明显低于无MACE组,差异均有统计学意义(P<0.05)。MACE组TNF-α、IL-10、IL-6水平均高于无MACE组,差异均有统计学意义(P<0.05)。MACE组和无MACE组冠心病史和糖尿病史患者比例、收缩压、舒张压以及TC、TG、HDL-C、LDL-C水平比较,差异均无统计学意义(P>0.05)。高血压伴CHF患者血清骨硬化蛋白、OPG水平与TNF-α、IL-10、IL-6水平均呈正相关(P<0.05),TRAIL水平与TNF-α、IL-10、IL-6水平均呈负相关(P<0.05)。多因素Logistic回归分析结果显示,骨硬化蛋白水平升高、OPG水平升高均为高血压伴CHF患者发生MACE的危险因素(P<0.05),而TRAIL水平升高为高血压伴CHF患者发生MACE的保护因素(P<0.05)。血清骨硬化蛋白、OPG/TRAIL比值单独及2项指标联合检测预测高血压伴CHF患者发生MACE的曲线下面积(AUC)分别为0.847、0.803、0.927,且2项指标联合检测的AUC优于血清骨硬化蛋白、OPG/TRAIL比值单独检测的AUC(Z=2.350、2.824,P<0.05)。结论高血压伴CHF患者血清骨硬化蛋白、OPG/TRAIL比值联合检测患者预后发生MACE的预测价值较高。

血清SIGIRR、sTRAIL与传染性单核细胞增多症患儿外周血T细胞亚群和肝脏损伤的关系研究

目的 探讨传染性单核细胞增多症(infectious mononucleosis, IM)患儿血清单免疫球蛋白白细胞介素1相关受体(single immunoglobulin interleukin-1 related receptor, SIGIRR)、可溶性肿瘤坏死因子相关的凋亡诱导配体(soluble tumor necrosis factor-related apoptosis-inducing ligand, sTRAIL)与外周血T细胞亚群和肝脏损伤的关系。方法 选取2020-01/2023-01月作者医院收治的95例IM患儿为IM组,另选取同期100例体检健康儿童为对照组。根据IM患儿是否发生肝脏损伤分为肝脏损伤组(n=49)和无肝脏损伤组(n=46)。采用酶联免疫吸附试验法检测血清SIGIRR、sTRAIL水平,流式细胞术检测外周血T细胞亚群(CD3^(+)、CD16^(+)、CD4^(+)、CD8^(+)、CD4^(+)/CD8^(+))。通过多因素Logistic回归分析探讨IM患儿肝脏损伤的影响因素,受试者工作特征(receiver operating characteristic, ROC)曲线分析血清SIGIRR、sTRAIL对IM患儿肝脏损伤的预测价值。结果 与对照组健康儿童比较,IM组患儿血清SIGIRR水平和外周血CD16^(+)、CD4^(+)、CD4^(+)/CD8^(+)降低,血清sTRAIL水平和外周血CD3^(+)、CD8^(+)升高(P均<0.05)。Pearson相关性分析显示,IM患儿血清SIGIRR水平与外周血CD16^(+)、CD4^(+)、CD4^(+)/CD8^(+)呈正相关关系,与CD3^(+)、CD8^(+)呈负相关关系(P<0.05),血清sTRAIL水平与外周血CD16^(+)、CD4^(+)、CD4^(+)/CD8^(+)呈负相关关系,与CD3^(+)、CD8^(+)呈正相关关系(P<0.05)。单因素分析结果显示,病程、白细胞计数、CD3^(+)、CD16^(+)、CD4^(+)、CD8^(+)、CD4^(+)/CD8^(+)、SIGIRR水平、sTRAIL水平为IM患儿肝脏损伤的影响因素(P均<0.05)。多因素Logistic回归分析结果显示,CD3^(+)、CD16^(+)、sTRAIL升高为IM患儿肝脏损伤的独立危险因素,CD4^(+)/CD8^(+)、SIGIRR升高为其保护因素(P<0.05)。ROC曲线分析结果显示,血清SIGIRR、sTRAIL单独和二者联合预测IM患儿肝脏损伤的曲线下面积(area under the curve, AUC)分别为0.786、0.737、0.836,二者联合对IM患儿肝脏损伤预测效能的AUC大于SIGIRR、sTRAIL单独的预测效能。结论 IM患儿血清SIGIRR水平降低和sTRAIL水平升高与外周血T细胞亚群异常、肝脏损伤密切相关,血清SIGIRR、sTRAIL水平联合检测对IM患儿肝脏损伤的预测价值更高。

Human Soluble TRAIL Protein Inducing Apoptosis in Osteosarcoma Cell

This study is to examine the effect of human recombinant soluble TRAIL （TNF-related apoptosis-inducing ligand） protein inducing apoptosis in MG-63 human osteosarcoma cells. The inhibitive rates of TRAIL to MG-63 cells were detected by MTT assay. The apoptosis induced by TRAIL in MG-63 human osteosarcoma cells was analyzed with FACS and TUNEL and the apoptotic bodies were observed by transmission electron microscope. MTT assay showed that the inhibitive rates of 500, 1 000, 2 000 and 4 000 ng/mL TRAIL for 24 h were 10.1%, 24.3%, 50.6% and 97.7% respectively. Flow cytometric analysis showed that after MG-63 cells were treated with 2 gg/mL TRAIL for 6 h, obvious apoptotic peak would immediately appear before diploid peak. Human soluble TRAIL protein can quickly kill MG-63 osteosarcoma cells selectively, and may have potential value for clinical treatment of osteosarcoma.

强直性脊柱炎患者Toll样受体4表达和sTRAIL、TNF-α、IL-12水平的研究

目的探讨Toll样受体(toll-like receptor,TLR)4、可溶性肿瘤坏死因子相关凋亡诱导配体(soluble TNF-related apoptosis-inducing ligand,sTRAIL)、白介素(interleukin,IL)-12、肿瘤坏死因子(tumornecrosis factor,TNF)-α在强直性脊柱炎(ankylosing spondylitis,AS)发病中的作用。方法采用ELISA双抗体夹心法检测110例AS患者和60例正常对照的血清IL-12、TNF-α及sTRAIL水平,并采用逆转录聚合酶链反应(RT-PCR)法检测两组对象外周血单个核细胞TLR4基因水平,同时采用两点终点法测定C反应蛋白(C-reactive protein,CRP)、Wintnobe法检测红细胞沉降率(erythrocyte sedimentation rate,ESR),分析各指标与AS的关联。结果 AS患者TLR4 mRNA表达、血清sTRAIL含量均明显高于正常对照组,差异有统计学意义(P<0.05)。但TNF-α、IL-12与AS发病的相关性无统计学意义(P>0.05)。AS患者组中,血清sTRAIL与CRP(r=0.445,P<0.05)、TNF-α与ESR(r=0.622,P<0.01)均呈正相关。结论 TLR4通路异常及sTRAIL水平增高可能在AS的发病及炎症反应中发挥一定的作用。

LPS、rh-TNFα刺激PBL诱导TRAIL表达的初步研究

选择人ＰＢＬ作为人ＴＲＡＩＬ基因的来源，抽提细胞在ＬＰＳ和ｒｈ－ＴＮＦα刺激２、１０、１８ｈ的总ＲＮＡ，通过ＲＴ－ＰＣＲ观察ＴＲＡＩＬ基因转录ｍＲＮＡ的水平，然后用ＰＣＲ扩增ＴＲＡＩＬ基因，并用免疫印迹法分析ＴＲＡＩＬｍＲＮＡ翻译蛋白的水平。结果发现：在ＬＰＳ和ｒｈ－ＴＮＦα刺激细胞并进一步诱导细胞凋亡可能与诱导表达ＴＲＡＩＬ有关。本实验得到了ＴＲＡＩＬ基因，为重组表达ＴＲＡＩＬ打下了基础。

LPS、TNFα刺激PBL诱导TRAIL表达的初步研究

近来的实验显示从 EST来源的 TRAIL在体外能特异性地诱导肿瘤细胞凋亡。我们选择人PBL作为人 TRAIL基因的来源 ,抽提细胞在 LPS和 rh- TNFα刺激 2、1 0、1 8h的总 RNA,通过RT- PCR观察 TRAIL基因转录 m RNA的水平 ,然后用 PCR扩增 TRAIL基因 ,并用免疫印迹法分析 TRAIL m RNA翻译蛋白的水平。结果发现 :在 LPS和 rh- TNFα的刺激下可以从 1 0 6 ～ 1 0 7个人 PBL中扩增出 TRAIL基因。提示 LPS和 rh- TNFα刺激细胞并进一步诱导细胞凋亡可能与诱导表达 TRAIL有关。本实验得到了 TRAIL基因 ,为重组表达 TRAIL打下了基础。

肝癌组织中TRAIL表达及其与癌细胞增殖、凋亡、耐药的相关性分析

目的:研究肿瘤坏死因子相关凋亡诱导配体(tumor-necrosis-factor-related apoptosis-inducing ligand, TRAIL蛋白在肝细胞癌(hepatocellular carcinoma, HCC)组织中的表达水平,分析其与临床病理特征及预后的相关性,并探讨TRAIL对HCC细胞凋亡、增殖及耐药的影响。方法 :利用免疫组织化学法检测186例HCC患者中HCC组织及癌旁肝组织TRAIL、Caspase-3、NF-κB、P-糖蛋白(P-glycoprotein, P-gP)及Ki-67的表达,分析TRAIL与临床病理特征及预后的关系,并探讨TRAIL与HCC生物学行为(增殖、凋亡、耐药)的相关性。结果:HCC组织中TRAIL表达明显低于癌旁肝组织(P<0.01)。HCC中Caspase-3、NF-κB、P-gP的阳性表达率分别为38.2%、84.4%、66.1%,Ki-67在HCC中的高表达率为75.8%,低表达率为24.2%。Spearman相关性分析显示TRAIL与Caspase-3的表达呈正相关(P<0.01),与NF-κB、P-gP、Ki-67的表达均呈负相关(均P<0.05)。Kaplan-Meier生存分析显示,TRAIL阳性表达患者的平均生存时间显著长于阴性表达者(P<0.01)。单因素和Cox模型多因素分析结果显示TRAIL低表达与术后复发是HCC预后的独立影响因素(P<0.05)。结论:TRAIL在HCC中低表达,并与脉管癌栓、肿瘤病理分级、术后复发有关,提示TRAIL低表达是HCC恶性进展中的关键性分子事件。检测TRAIL表达在评价HCC分级、浸润、复发及预后中有一定的价值。

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.

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 cells 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 upregulation of Caspase 8. Cisplatin and etoposide could enhance the killing effect of TRAIL on SH-SY5Y cells.

抗肿瘤坏死因子单抗对脂多糖诱导的人牙周膜成纤维细胞TRAIL和TNF-α的影响

目的:观察脂多糖对人牙周膜成纤维细胞(HPLFs)肿瘤坏死因子(TNF)受体相关凋亡诱导配体(TRAIL)和TNF-α的诱导作用及抗肿瘤坏死因子单抗(抗TNF单抗)对其的影响,探讨TNF超家族参与牙周病的可能作用机制。方法:HPLFs培养至第6代,加入不同浓度的脂多糖(0、0.1、1、10、100μg/m L)培养24 h,分为命名为Z0组、Z0.1组、Z1组、Z10组、Z100组,并取Z1组浓度的脂多糖,在加药的同时加抗TNF单抗75μg/m L(Z1+75组)。分别采用实时荧光定量PCR法或Western blot法测定TRAIL和TNF-αm RNA或蛋白的表达。结果:Z1组、Z10组HPLFs TRAIL m RNA的表达水平显著高于Z0组或Z0.1组(均P<0.05),Z1组与Z10组之间差异无统计学意义(P>0.05);Z100组显著高于Z0组(P<0.05)且与Z0.1组、Z1组和Z10组比较差异无统计学意义(均P>0.05)。Z1组或Z10组TNF-αm RNA的表达水平显著高于Z0组或Z100组(均P<0.05);Z1组与Z10组之间,Z0组、Z0.1组和Z100组之间差异无统计学意义(均P>0.05)。HPLFs TRAIL蛋白的表达水平在Z100组<Z0组<Z0.1组<Z1组(均P<0.05或<0.01);Z10组显著高于Z0组或Z100组(均P<0.01),但与Z0.1组或Z1组之间差异无统计学意义(均P>0.05)。抗TNF单抗治疗后TRAIL m RNA及蛋白和TNF-αm RNA的表达水平显著低于Z1组(P<0.05和P<0.01)。TRAIL和TNF-αm RNA的表达水平呈显著直线正相关(r=0.819,n=30,P<0.01)。结论:HPLFs经脂多糖诱导后,其TRAIL和TNF-α的表达呈现先升高后下降,且随脂多糖的浓度变化而相应地变化,这种诱导作用能被抗TNF单抗所抑制。

TRAIL联合顺铂诱导三阴乳腺癌MDA-MB-231细胞过度性自噬的分子机制

流行病学报道,乳腺癌成为目前女性发病率和死亡率最高的癌症[1]。而铂类药物也是临床上最常用的周期非特异性抗肿瘤药物,在临床治疗实体肿瘤中显示了良好的疗效[2]。其中顺铂作为一线临床的传统用药,其靶点选择范围窄、疗效差、易耐药等问题,尚未得到解决。肿瘤坏死因子相关凋亡诱导配体(tumor necrosis factor-related apoptosis-inducing ligand,TRAIL)属于肿瘤坏死因子(tumor necrosis factor,TNF)超家族。

EGFR inhibitors sensitize non-small cell lung cancer cells to TRAIL-induced apoptosis

Apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand(TRAIL) can be regulated by the epidermal growth factor(EGF) signaling pathway.In this study,recombinant adenoviral vectors that encode TRAIL gene from the hTERT/RGD promoter(AdTRAIL) was combined with drugs including gefitinib,elotinib,and cetuximab that inhibit EGFR and the EGF signaling pathway in non-small cell lung cancer(NSCLC) cell lines to investigate their antitumor activity.In vitro,compared to single reagent,AdTRAIL combined with EGFR inhibitors reduced proliferation and enhanced apoptosis in H460,A549,and SW1573 cell lines.Western blot results suggested that these effects were relative to up-regulation of pro-apoptosis protein BAX and down-regulation of p-AKT.In vivo,AdTRAIL combined with cetuximab resulted in a significant growth reduction in H460 xenografts without damage to the main organs of nude mice.Histological examination and TUNEL analyses of xenografts showed that cetuximab enhanced cell apoptosis induced by AdTRAIL.These results indicate that EGFR inhibitors enhanced AdTRAIL anti-tumor activity in NSCLC cell lines and that inhibiting the AKT pathway played an important role in this enhancement.

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.