衣霉素联合TRAIL促甲状腺癌细胞凋亡机制的研究

.TRAIL与衣霉素单用对甲状腺癌细胞均无明显杀伤作用,最高凋亡细胞比率分别为10.68%和10.14%;衣霉素与TRAIL联合组凋亡细胞比率最大达60.5%;但在存活素过表达甲状腺未分化癌ARO细胞中,凋亡细胞比率降低50%左右.结论:在体外单用TRAIL对甲状腺癌细胞增殖无明显抑制作用,衣霉素明显增加肿瘤细胞对TRAIL敏感性,其增敏机制至少部分与下调存活素水平有关.     

干扰素-α通过上调DR5和抑制Akt的活性增加胃癌细胞对TRAIL的敏感性

目的：观察IFN-α对TRAIL诱导胃癌细胞凋亡的影响,并对死亡受体DR5和PI3K/Akt通路在此过程中的作用机制进行探讨。方法：MTT法检测细胞增殖能力,流式细胞仪PI染色检测细胞凋亡,蛋白质印迹法检测蛋白的表达。结果：IFN-α预处理可协同TRAIL抑制胃癌MGC803细胞增殖。单独用IFN-α或TRAIL处理MGC803细胞,仅有少量的细胞发生凋亡,IFN-α和TRAIL联合用药细胞凋亡明显增加（P〈0.01）。IFN-α能上调DR5的表达,抑制Akt的磷酸化。结论：IFN-α能增强TRAIL诱导的胃癌细胞凋亡,其机制可能与其上调DR5的表达和抑制Akt的活性有关。     

顺铂增加人脑胶质瘤细胞U251对TRAIL敏感性的实验研究

目的 研究顺铂在增加人脑胶质瘤细胞U251对肿瘤坏死因子相关凋亡诱导配体(TRAIL)敏感性的作用,并探讨其分子机制.方法 通过倒置荧光显微镜下观察重组腺病毒载体携带的绿色荧光蛋白的表达确定最适感染复数MOI;运用MTT法检测细胞增殖活性;倒置荧光显微镜下观察Hoechst33342染色细胞凋亡形态;PI染色后通过流式细胞术检测诱导凋亡作用,RT-PCR法检测凋亡相关基因.结果 感染后TRAIL基因的表达明显上调.顺铂增敏TRAIL组与单独组相比,有显著抑制细胞增殖作用(P<0.05),Hoechst33342染色观察有明显的核固缩,核碎裂.流式细胞术检测细胞有凋亡峰的出现,增敏组与单独组有显著性差异(P<0.05).RT-PCR检测到TRAIL、DB5、caspase-3基因上调,survivin基因下调,DB4表达无明显变化.结论 顺铂可以增加人脑胶质瘤细胞U251对TRAIL的敏感性,其分子机制可能与DR5、caspase-3基因上调,survivin基因下调有关.     

TRAIL及其受体在非小细胞肺癌中的表达及其意义

目的 研究肿瘤坏死因子相关凋亡诱导配体(TRAIL)及其受体TRAIL-R2和TRAIL-R4在非小细胞肺癌(NSCLC)中的表达及其意义.方法 采用酶联免疫吸附试验(ELISA)检测79例NSCLC患者与80例正常人血清中TRAIL的表达水平,采用免疫组织化学法检测42例NSCLC组织及配对癌旁正常组织中TRAIL-R2和TRAIL-R4的表达水平.分析TRAIL、TRAIL-R2、TRAIL-R4与NSCLC患者临床病理特征的关系.结果 TRAIL在NSCLC患者血清中的表达明显低于正常人[(994.3±293.0) ng/ml∶(1 141.7 ±266.1) ng/ml],差异具有统计学意义(t=3.29,P=0.00).TRAIL的表达与NSCLC患者的临床分期(F=2.28,P=0.00)、分化程度(t=5.76,P=0.00)相关.TRAIL-R2在NSCLC组织中的阳性表达率为73.8％ (31/42),明显低于正常组织中的阳性表达率100.0％ (42/42)(x2=3.88,P=0.05),TRAIL-R2的表达与NSCLC患者的临床分期(x2=27.89,P=0.00)、分化程度(x2=9.50,P=0.00)相关.TRAIL-R4在NSCLC组织中的阳性表达率为81.0％ (34/42),明显高于正常组织中的阳性表达率50.0％(21/42)(x2=7.34,P=0.01),TRAIL-R4的表达与NSCLC患者的临床分期(x2=17.82,P=0.00)、分化程度(x2=4.47,P=0.03)相关.在NSCLC组织中,TRAIL-R4与TRAIL-R2的表达呈负相关(r=-0.67,P=0.01).结论 在NSCLC中,TRAIL、TRAIL-R2表达减少,TRAIL-R4表达增多,三者可能促进了NSCLC的发生发展,可为NSCLC临床治疗提供靶点.     

奥沙利铂通过上调死亡受体表达增强TRAIL诱导胃癌细胞凋亡的实验研究

目的:研究奥沙利铂对TRAIL诱导胃癌细胞凋亡的影响,探讨死亡受体5(DR5)在TRAIL诱导细胞凋亡中的作用.方法:采用MTT法测定细胞活力、采用流式细胞仪检测细胞凋亡,采用Western blot检测DR5蛋白表达.结果:100ng/ml的TRAIL导致轻度的增殖抑制,诱导不超过5%的细胞凋亡;TRAIL(100ng/ml)联合奥沙利铂(23.44μg/ml)引起明显的细胞增殖抑制和细胞凋亡(P＜0.05),TRAIL没有明显改变死亡受体5(DR5)的表达,而23.44 μg/ml奥沙利铂作用胃癌细胞24小时后,明显上调了DR5的表达.结论:奥沙利铂通过上调DR5的表达增强TRAIL诱导的胃癌细胞凋亡.     

肿瘤细胞TRAIL耐受与其受体的关系

肿瘤坏死因子相关凋亡诱导配体(TRAIL)能与含有死亡域的死亡受体DR4、DR5相结合,诱导多种肿瘤细胞发生凋亡而对正常细胞无明显毒性.但肿瘤细胞对TRAIL的耐受限制了其广泛应用于临床.受体与配体的结合是凋亡信号启动的起始及关键环节,因此肿瘤细胞发生TRAIL耐受的机制与受体的表达、定位、分布、功能等密切相关.联合其...     

TRAIL与卡铂联合诱导卵巢癌细胞凋亡的研究

目的探讨肿瘤坏死因子相关凋亡诱导配体（TRAIL）与卡铂（CBP）联合应用对体外培养的卵巢A2780细胞的生物学效应及其作用机制。方法采用MTT法、流式细胞术，检测体外培养的A2780细胞在卡铂和TRAIL共同作用下的细胞增殖抑制效应以及细胞凋亡程度，应用半定量逆转录多聚酶链反应（RT-PCR）法检测TRAIL受体的mRNA表达水平。结果A2780细胞对TRAIL敏感，TRAIL与卡铂联合用药对细胞的增殖抑制呈现高效协同作用，与单独用药组比较有显著性差异（P〈0．05）；流式细胞术分析协同性杀伤作用主要由于TRAIL和CBP联合诱导细胞凋亡引起；RT—PCR法检测结果显示A2780细胞在TRAIL与卡铂联合用药后均表现死亡受体DR4、DR5表达水平上调和诱骗受体DcR1、DcR2表达水平下调。结论在体外TRAIL与化疗药物联用能明显抑制肿瘤细胞增殖，诱导肿瘤细胞凋亡，卡铂能明显增强TRAIL对肿瘤细胞的敏感性，其诱导机制可能与死亡受体DR4、DR5表达水平上调和诱骗受体DcR1、DcR2表达水平下调相关。     

曲古抑菌素A增加人肝癌Bel7402细胞对TRAIL敏感度的实验

目的 探讨曲古抑菌素A(Trichostatin A,TSA)联合肿瘤坏死因子(tumor necrosis factor,TNF)相关凋亡诱导配体(TNF related apoptosis inducing ligand,TRAIL)对肝癌细胞Bel7402增殖凋亡的影响及其机制.方法 采用四甲基偶氮唑盐(MTT)染色法分别检测TSA、TRAIL及低浓度TSA联合TRAIL处理Bel7402细胞的生长抑制率;4,6-二脒基-2-苯基吲哚二盐酸盐(DAPI)染色法对药物联合处理后的细胞进行凋亡形态学观察;免疫细胞化学和Western blot技术观察药物联合作用后p65蛋白在细胞中表达和定位的变化.结果 不同浓度TSA作用6、12和24 h对人肝癌Bel7402细胞的增殖没有明显抑制作用,而作用48 h后的细胞增殖抑制率明显升高,和对照组比较差异有统计学意义(P＜0.05);不同浓度TRAIL处理Bel7402细胞存活率没有明显改变;低浓度TSA(20 ng/ml)预处理能够增加Bel7402细胞对TRAIL治疗的敏感度,TSA预处理联合TRAIL(100ng/ml)作用细胞24 h后,细胞生存率为(57.1±5.4)％,和单独药物处理组及对照组比较差异有统计学意义(P＜0.05);DAPI染色显示TSA和TRAIL联合作用后Bel7402细胞有核凋亡出现.荧光显微镜观察证明单独应用TSA(200 ng/ml)或TRAIL(100 ng/ml)处理的细胞p65蛋白有部分核内移位,而两种药物联合应用导致p65蛋白表达量降低,并且发生明显的核内转移和集聚.结论 低浓度TSA能够增加肝癌Bel7402细胞对TRAIL的敏感度;其机制可能是两种药物联合应用降低p65的表达和活性,从而诱导Bel7402细胞凋亡.     

新城疫病毒对人NK细胞表达TRAIL的促进作用

目的:研究新城疫病毒(Newcastle disease virus,NDV)刺激自然杀伤(natural killer,NK)细胞产生肿瘤坏死因子相关凋亡诱导配体(TNF-related apoptosis-inducing ligand,TRAIL)的作用机制。方法:采用免疫磁珠分选(magnetic activated cell sorting,MACS)法,分离得到高纯度的NK细胞;乳酸脱氢酶释放法检测NDV激活的NK细胞对人肝癌HepG2细胞的杀伤作用;实时荧光定量-PCR(real-time fluorogenic quantitative-PCR,RFQ-PCR)检测NDV刺激NK细胞4h后对TRAILmRNA表达水平的影响;FCM检测NDV刺激NK细胞16h时TRAIL蛋白的表达情况;ELISA法检测NDV刺激NK细胞后对其分泌干扰素γ(interferon-γ,IFN-γ)的影响;最后采用抗IFN-γ抗体中和IFN-γ后,再采用FCM检测NDV刺激的NK细胞中TRAIL蛋白的表达情况。结果:FCM检测结果显示,分离得到的NK细胞的纯度达(90.60±1.15)%;NDV刺激NK细胞16h后,明显增强NK细胞的杀伤肿瘤作用,NDV效价为204.8HU刺激组的杀伤率达到(22.28±0.84)%;RFQ-PCR及FCM检测结果显示,不同效价的NDV刺激NK细胞后,NK细胞中TRAILmRNA及蛋白表达水平均明显升高;ELISA检测结果显示,NDV刺激NK细胞后IFN-γ的分泌水平明显增加,在NDV效价为25.6HU刺激16h时达到峰值(796.47±37.87)pg/mL;采用抗IFN-γ抗体中和IFN-γ后,NK细胞中TRAIL蛋白表达水平明显下降。结论:NDV刺激NK细胞后可增强IFN-γ的分泌水平,进而通过IFN-γ上调TRAIL的表达,此途径为NDV刺激NK细胞表达TRAIL的机制之一。此外,NDV直接刺激NK细胞表达TRAIL是另一条重要的途径。     

TRAIL 联合喜树碱诱导胰腺癌细胞凋亡的研究

目的：探讨喜树碱（CPT）增强TRAIL 诱导胰腺癌细胞PANC-1 凋亡的机制。方法：通过MTT 检测TRAIL 组、喜树碱组、二者联合组对胰腺癌细胞PANC-1 生长抑制的影响;利用Hoechst33258 荧光染色检测各组细胞凋亡情况;应用Western-blot方法检测PANC-1 在喜树碱应用前后凋亡信号传导蛋白分子 DR4、DR5、Caspase- 8、Caspase- 3 以及c-flip 的表达情况。结果：PANC-1 细胞经不同浓度TRAIL（10、30、100、300、1 000ng/mL）作用24h 后,当浓度在100ng/mL 及以上时与对照组相比有统计学差异（P<0.01）,虽然存在差异,但当TRAIL 浓度为1 000ng/mL 时,其细胞抑制率仅为（10.60± 2.36）% ,TRAIL 与不同浓度的喜树碱（10、30、100、300、1 000ng/mL）联合作用后其细胞抑制率与单独应用喜树碱相比有显著性差异（P<0.05）;Hoechst33258 荧光染色结果显示：联合组有大量细胞出现细胞核聚集、边缘化、核碎裂等典型细胞凋亡形态,而其余各组未见明显凋亡征象;Western-blot结果显示：单独TRAIL 组及单独喜树碱组仅能看到Caspase- 8 和Caspase- 3 的前体,而二者联合后不仅看到了其前体,而且还看到其裂解带,并且发现经过喜树碱预处理后,PANC-1 细胞中c-Flip 蛋白的表达被明显下调了,CPT 组及联合组与对照组及单独TRAIL 相比均有显著性差异（P<0.05）,而喜树碱应用前后对DR4 和DR5 表达无显著效应。结论：TRAIL 联合喜树碱对胰腺癌细胞PANC-1 诱导的凋亡具有协同作用,其机制是喜树碱下调了c-Flip 蛋白的表达,解除了其对Caspase- 8 的抑制,激活了死亡受体通路,与DR4 和DR5 无关。     

表阿霉素提高胃癌BGC823细胞对TRAIL的敏感性

目的：研究表阿霉素对TRAIL诱导胃癌细胞BGC823细胞凋亡的影响,探讨脂筏和死亡受体4（DR4）在TRAIL诱导细胞凋亡中的作用。方法：采用MTT法测定细胞活力,流式细胞仪检测细胞凋亡,免疫荧光显微技术检测脂筏和DR4在细胞膜的分布。结果：（0.1-50）μg/ml表阿霉素处理BGC823细胞24h,抑制细胞增殖50%的药物浓度（IC50）为（4.61±0.62）μg/ml。在BGC823细胞中,100ng/ml的TRAIL导致轻度的增殖抑制和细胞凋亡,TRAIL（100ng/ml）联合表阿霉素（4.61μg/ml）引起明显的增殖抑制和细胞凋亡（P〈0.05）。与对照组相比,100ng/ml的TRAIL作用BGC823细胞24 h,没有引起明显的脂筏聚集或DR4聚集。表阿霉素（4.61μg/ml）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。表阿霉素和TRAIL联合作用24 h,同样观察到DR4定位在聚集的脂筏内。结论：表阿霉素通过促进DR4在脂筏聚集增强TRAIL诱导的胃癌BGC823细胞凋亡。     

2-Deoxy-D-glucose enhances TRAIL-induced apoptosis in human melanoma cells through XBP-1-mediated up-regulation of TRAIL-R2

Background

Osteosarcoma (OS) is the most common primary bone tumour in children and young adults. Despite improved prognosis, metastatic or relapsed OS remains largely incurable and no significant improvement has been observed in the last 20 years. Therefore, the search for alternative agents in OS is mandatory.

Results

We investigated phospho-ERK 1/2, MCL-1, and phospho-Ezrin/Radixin/Moesin (P-ERM) as potential therapeutic targets in OS. Activation of these pathways was shown by immunohistochemistry in about 70% of cases and in all OS cell lines analyzed. Mutational analysis revealed no activating mutations in KRAS whereas BRAF gene was found to be mutated in 4/30 OS samples from patients. Based on these results we tested the multi-kinase inhibitor sorafenib (BAY 43-9006) in preclinical models of OS. Sorafenib inhibited OS cell line proliferation, induced apoptosis and downregulated P-ERK1/2, MCL-1, and P-ERM in a dose-dependent manner. The dephosphorylation of ERM was not due to ERK inhibition. The downregulation of MCL-1 led to an increase in apoptosis in OS cell lines. In chick embryo chorioallantoic membranes, OS supernatants induced angiogenesis, which was blocked by sorafenib and it was also shown that sorafenib reduced VEGF and MMP2 production. In addition, sorafenib treatment dramatically reduced tumour volume of OS xenografts and lung metastasis in SCID mice.

Conclusion

In conclusion, ERK1/2, MCL-1 and ERM pathways are shown to be active in OS. Sorafenib is able to inhibit their signal transduction, both in vitro and in vivo, displaying anti-tumoural activity, anti-angiogenic effects, and reducing metastatic colony formation in lungs. These data support the testing of sorafenib as a potential therapeutic option in metastatic or relapsed OS patients unresponsive to standard treatments.     

2-Deoxy-D-glucose enhances TRAIL-induced apoptosis in human melanoma cells through XBP-1-mediated up-regulation of TRAIL-R2

Background  

Past studies have shown that sensitivity of melanoma cells to TRAIL-induced apoptosis is largely correlated with the expression levels of TRAIL death receptors on the cell surface. However, fresh melanoma isolates and melanoma tissue sections express generally low levels of death receptors for TRAIL. The clinical potential of TRAIL in the treatment of melanoma may therefore be limited unless given with agents that increase the cell surface expression of TRAIL death receptors. 2-Deoxy-D-glucose (2-DG) is a synthetic glucose analogue that inhibits glycolysis and glycosylation and blocks cell growth. It has been in clinical evaluation for its potential use as an anticancer agent. In this study, we have examined whether 2-DG and TRAIL interact to enhance their cytotoxicity towards melanoma cells.     

Chetomin induces degradation of XIAP and enhances TRAIL sensitivity in urogenital cancer cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is one of the most promising anti-cancer agents, but some tumor types develop resistance to TRAIL. Here, we report that chetomin, an inhibitor of hypoxia-inducible factors, is a potent enhancer of TRAIL-induced apoptosis. TRAIL or chetomin alone weakly induced apoptosis, but the combination of chetomin and TRAIL synergistically induced apoptosis in prostate cancer PC-3 cells. The combination of chetomin and TRAIL induces the activation of caspase-3, -8, -9 and -10. Among the apoptotic factors related to the TRAIL pathway, chetomin markedly decreased the X-linked inhibitor of apoptosis (XIAP) protein levels in a dose-dependent manner, but other IAP family members, TRAIL receptors and Bcl-2 family members were not altered by chetomin. Using XIAP siRNA instead of chetomin, down-regulation of XIAP sensitized PC-3 cells to TRAIL-induced apoptosis. Conversely, transient transfection of XIAP reduced the apoptotic response to combined treatment with chetomin and TRAIL. Treatment with chetomin induced a rapid decrease in XIAP protein levels but had no effect on XIAP mRNA levels. Since chetomin-mediated XIAP down-regulation was completely prevented by proteasome inhibitors, it was suggested that chetomin induces the degradation of the XIAP protein in a proteasome-dependent manner. Additionally, chetomin also sensitized renal cancer Caki-1 cells and bladder cancer UM-UC-3 cells to TRAIL-induced apoptosis via down-regulation of XIAP. Co-treatment of chetomin and TRAIL did not enhance apoptosis in normal peripheral blood mononuclear cells (PBMC). Taken together, these findings suggest that TRAIL and chetomin synergistically induce apoptosis in human urogenital cancer cells through a mechanism that involves XIAP down-regulation by chetomin.     

Survivin基因反义寡核苷酸增强胃癌细胞对顺铂的敏感性研究

目的探讨survivin基因的反义寡核苷酸(oligodeoxynucleotide,ODN)对胃癌细胞系(SGC7901)下调化疗药物的耐药性的研究.方法采用survivin基因反义ODN及其对照序列(空白对照和错配ODN)通过脂质体途径分别转染SGC7901细胞.经转染的各组SGC7901细胞分别采用蛋白印迹法观察survivin基因的表达、通过MTT法检测顺铂对癌细胞的毒性作用以及流式细胞仪(FCM)观察顺铂(CDDP)诱导各组SGC7901细胞的凋亡.结果经转染反义ODN之胃癌细胞出现survivin蛋白表达明显下降,反义ODN伴CDDP联合作用的细胞抑制率达49.9±2.1%,而单独应用CDDP或错配ODN联合CDDP的细胞抑制率分别为30.7±2.9%及34.8±3.4%,两者间呈明显差异(P＜0.01).细胞流式仪分析显示对照组、survivin反义ODN组、survivin错配ODN组的细胞凋亡率分别为8.1±0.9%、15.2±0.7%、8.5±0.8%,反义ODN组细胞凋亡率明显高于对照组、错配ODN组(P＜0.01).结论Survivin基因反义ODN能增强胃癌细胞株SGC7901对顺铂的敏感性,为临床提供一种增强化疗敏感性研究的途径.     

Functional expression of TRAIL receptors TRAIL-R1 and TRAIL-R2 in esophageal adenocarcinoma

The tumour necrosis factor (TNF) related apoptosis-inducing ligand (TRAIL/Apo2L) is a member of the TNF superfamily that preferentially induces apoptosis in cancer cells, while sparing normal cells. TRAIL induces apoptosis by interacting with its receptors TRAIL-R1 and TRAIL-R2. Recently, new humanized agonistic anti-TRAIL-R1 and anti-TRAIL-R2 antibodies have been developed, and are undergoing phase I/II clinical trails. Esophageal adenocarcinoma (EA) is associated with significantly poor outcome and is rapidly increasing in incidence in the United States and Western Europe, with virtually no effective non-surgical treatment. The aim of this study was to determine whether human EA tissue express TRAIL-R1 and/or TRAIL-R2, and whether EA cell lines Bic-1 and Seg-1 expresses functional TRAIL-R1 and/or TRAIL-R2. The expression of TRAIL-R1 and TRAIL-R2 was determined in sections from 18 human EA by immunohistochemistry (IHC). Sixteen (89%) of the EA expressed TRAIL-R1 and 17 (94%) expressed TRAIL-R2. Both cell lines were found to express TRAIL-R1 and TRAIL-R2 by western blot analysis, IHC, and flow cytometry. The fully human agonistic TRAIL-R1 (HGS-ETR1) and TRAIL-R2 (HGS-ETR2) antibodies induced apoptosis in Bic-1 and Seg-1 cells in a time and dose dependent manner. Our results show that the vast majority of primary human EA express TRAIL-R1 and TRAIL-R2 and that EA cells lines express functional TRAIL-R1 and TRAIL-R2. Targeting of these receptors by agonist monoclonal antibodies may be of therapeutic value in patients with EA.     

二十二碳六烯酸增加人胃癌细胞SGC-7901对多柔比星的敏感性

目的:本研究旨在探讨二十二碳六烯酸[docosahexaenoic acid (DHA),22:6 ω-3]逆转由多柔比星( adriamycin,ADR)引起的胃癌SGC-7901细胞的耐药性.方法:体外常规培养人低分化胃腺癌细胞SGC-7901,分别以DHA、ADR单药和DHA+ADR联合作用于细胞24 h后,采用MTT法检测各组细胞的增殖情况;倒置光学显微镜下观察细胞的形态;FCM法检测细胞的凋亡率;激光共聚焦显微镜以及高效液相色谱法检测细胞中ADR的蓄积情况;RT-PCR及蛋白质印迹法检测细胞中多药耐药蛋白(multidrug resistanceprotein,MDR) P-糖蛋白p-170的表达情况.结果:DHA与ADR联合作用于胃癌SGC-7901细胞后,DHA能够增加ADR对细胞的生长抑制作用;细胞形态观察发现死细胞明显增多;细胞凋亡率显著增加;细胞中的ADR蓄积量约增加2.1倍;细胞中p-170 mRNA以及蛋白表达降低(P均＜0.05).结论:DHA能够增加胃癌细胞SGC-7901对ADR的敏感性,放大ADR的肿瘤杀伤作用.