不同分子亚型乳腺癌组织中TRAIL蛋白表达及意义

[目的]探讨不同分子亚型乳腺癌组织中TRAIL蛋白(肿瘤坏死因子相关凋亡诱导配体,TRAIL)的表达差异及TRAIL与乳腺癌各临床病理因素之间的关系.[方法]采用streptavidin-perosidase (SP)法检测110例乳腺癌及12例乳腺良性病变组织中TRAIL蛋白的表达水平,分析不同分子亚型乳腺癌组织中TRAIL蛋白表达差异,以及TRAIL表达与乳腺癌临床分期、淋巴结转移以及Ki-67表达等临床因素的关系.[结果]与其他分子亚型相比较,TRAIL蛋白在Luminal B HER-2+型乳腺癌组织中的表达水平明显下调(P=0.035),在Ki-67高表达的乳腺癌组织中也发现了TRAIL表达水平下调(P=0.045),并且TRAIL在乳腺癌组织中的表达水平还受到肿瘤大小、淋巴结状态和TNM分期等因素影响(P=0.004、P=0.006、P=0.001),而不受乳腺癌的病理类型、癌细胞分级、肿瘤有无坏死和神经浸润及患者年龄等因素的影响.[结论]TRAIL蛋白在不同分子亚型乳腺癌组织中的表达有显著差异,并且其与乳腺癌的临床分期、肿瘤大小、淋巴结有无转移等临床特征密切相关,可能有助于判断乳腺癌的预后.     

Treatment with gemcitabine and TRA-8 anti-death receptor-5 mAb reduces pancreatic adenocarcinoma cell viability in vitro and growth in vivo

Gemcitabine is a first line agent for pancreatic cancer, but yields minimal survival benefit. This study evaluated in vitro and in vivo effects of a monoclonal antibody (TRA-8) to human death receptor 5, combined with gemcitabine, using two human pancreatic cancer cell lines, S2VP10 and MIA PaCa-2. A subcutaneous model of pancreatic cancer was employed to test in vivo efficacy. S2VP10 and MIA PaCa-2 cells were treated with varying doses of gemcitabine and TRA-8. Cell viability and apoptosis were determined with an adenosine triphosphate assay and annexin V staining, respectively. Mitochondrial membrane destabilization was evaluated with fluorescence-activated cell sorting analysis of JC-1 stained cells. Caspase activation was evaluated by Western blot analysis. MIA PaCa-2 subcutaneous xenografts in athymic nude mice were evaluated for response to treatment with 200 μg of TRA-8 (intraperitoneal on days 9, 13, 16, 20, 23, and 27 postimplant) and 120 mg/kg gemcitabine (I.P. on days 10, 17, and 24). Tumor growth was measured with calipers. MIA PaCa-2 and S2VP10 cells receiving combination treatment with TRA-8 and gemcitabine demonstrated enhanced cytotoxicity, annexin V staining, and mitochondrial destabilization compared to either agent alone. Combination treatment produced enhanced caspase-3 and -8 activation in both cell lines compared with either agent alone. In vivo studies demonstrated mean subcutaneous tumor surface area (produce of two largest diameters) doubling times of 38 days untreated, 32 days gemcitabine, 49 days TRA-8, and 64 days combination treatment. TRA-8 is an apoptosis-inducing agonistic monoclonal antibody that produced synergistic cytotoxicity in combination with gemcitabine in vitro through enhanced caspase activation. These findings, with substantial inhibition of tumor growth in a mouse pancreatic cancer xenograft model receiving combination therapy, are encouraging for anti-death receptor therapy in the treatment of pancreatic cancer. Presented at the Forty-Seventh Annual Meeting of The Society for Surgery of the Alimentary Tract, Los Angeles, California, May 20–24, 2006 (poster presentation). Supported by the National Institutes of Health/NRSA T32 CA91078 Research Training Program in Surgical Oncology Training Grant (Dr. Kirby Bland P.I.) and NIH SPORE in Pancreatic Cancer 1 P20 CA10195-01.     

非增殖型和靶向增殖型腺病毒携带TRAIL基因治疗肝癌的实验研究

目的比较携带人TRAIL基因的增殖型腺病毒（CNHK500-hTRAIL）和非增殖型腺病毒（Ad-hTRAIL）对TRAIL基因的表达以及对SMMC-7721肝癌细胞的杀伤能力。方法通过病毒增殖实验评估增殖型腺病毒CNHK500-hTRAIL的选择性增殖能力。通过MTT实验,评估增殖型腺病毒CNHK500-hTRAIL以及非增殖型腺病毒Ad-hTRAIL对人正常肝细胞株L02、人肝癌细胞株SMMC-7721的杀伤能力。采用ELISA法检测CNHK500-hTRAIL和Ad-hTRAIL感染SMMC-7721肝癌细胞后TRAIL基因的表达情况。以及通过流式细胞术（FCM）检测其对细胞早期凋亡的影响。结果CNHK500-hTRAIL能选择性地在SMMC-7721细胞内大量增殖,感染96 h后增殖达225137倍,在极低的MOI值（MOI=0.1）即可大量杀伤SMMC-7721细胞,明显强于Ad-hTRAIL,而对L02细胞无明显杀伤。CNHK500-hTRAIL和Ad-hTRAIL感染SMMC-7721细胞后,其TRAIL基因表达量,前者是后者的近10倍;CNHK500-hTRAIL可选择性地诱导SMMC-7721细胞早期凋亡,其能力显著高于Ad-hTRAIL。结论靶向增殖型腺病毒载体携带TRAIL基因对肿瘤细胞的杀伤能力和目的基因的表达,明显优于传统的非增殖型腺病毒载体,应用前景广阔。     

脂质体介导的cFLIP反义寡核苷酸对肾细胞癌的凋亡作用

目的探讨cFLIP反义寡核苷酸（ASODN）对肾细胞癌的抑制作用。方法设计合成cFLIP的ASODN，按不同浓度在脂质体介导下转染786—0肾癌细胞株，设无义（NSODN）和空白对照组进行比较。观察细胞的生长状态，Western blot检测cFLIP蛋白的表达，MTT法检测细胞生长抑制率，应用流式细胞仪检测其凋亡率。结果与NSODN、空白对照组比较，ASODN组cFLIP蛋白表达显著降低（P〈0．05），细胞抑制率（分别为10μmol／L组34．20％，20μmol／L组39．50％）显著增高，上述效应呈浓度依赖性；镜下观察ASODN转染细胞代谢衰退，当转染浓度至20μmol／L时，其凋亡率（56．11％）显著高于空白对照组（7．29％）和NSODN（4．69％）组。结论AS0DN能特异性封闭肾癌细胞cFLIP基因的表达，并可抑制肾癌细胞的增殖，诱导其凋亡。     

肿瘤坏死因子相关凋亡诱导配体治疗肝细胞癌的研究

目的探讨肿瘤坏死因子相关凋亡诱导配体(TRAIL)与其受体在肝细胞肝癌(hepatocellularcarcinoma,HCC)中的表达及意义,及利用TRAIL对HCC的治疗作用。方法采用免疫组化技术及原位杂交方法分别检测了100例肝癌组织,100例癌旁组织,40例正常肝组织中TRAIL及TRAILR的表达,并结合临床资料进行分析;采用不同浓度TRAIL处理肝癌细胞株HepG2、SMMC7721,观察经药物处理前后肿瘤细胞的凋亡发生率。结果TRAIL在正常肝组织中无表达,癌旁组织中的表达明显高于癌组织。86例肝癌组织不表达诱捕受体DcR1(86%),55例肝癌组织不表达诱捕受体DcR2(55%),40例正常肝组织两种诱捕受体均有表达。肝癌组织中死亡受体为高表达,诱捕受体为低表达,正常肝组织则相反,两者间有显著差异性(P<0.05)。肝癌组织中死亡受体的表达与肿瘤的分化呈正相关(P<0.01),与肿瘤分级呈负相关(P<0.05),与病人的性别、年龄、AFP水平、肿瘤的大小以及是否转移无关。经TRAIL(100ng/ml)处理24h,肝癌细胞凋亡发生率约10%,而Jurkat细胞凋亡率达70%以上,胆管癌细胞QBC939凋亡发生率约50%。结论HCC时,TRAILR普遍表达,但存在受体类型的表达差异,其中DcR1大多缺失,这为利用TRAIL治疗HCC提供了理论依据,然而,单一的TRAIL治疗只能有限的诱导肝癌细胞HepG2、SMMC7721发生凋亡,HCC对TRAIL诱导的凋亡存在耐药现象。     

TRAIL受体在胆管癌组织中的表达及意义

目的 :探讨肿瘤坏死因子相关凋亡诱导配体 (TRAIL)的受体在人胆管癌组织、癌旁胆管组织和胆管癌细胞株 (QBC939)的表达及其临床意义。 方法 :应用原位杂交组织化学法检测TRAIL受体mRNA在人胆管癌组织、癌旁胆管组织和胆管癌细胞株 (QBC939)中的表达。 结果 :DR4mRNA、DR5mRNA在人胆管癌组织、癌旁胆管组织和胆管癌细胞株 (QBC939)均呈阳性表达。只有 3例DcR1(3/5 2 )和 7例DcR2 (7/5 2 )在人胆管癌组织呈弱阳性表达。DcR1、DcR2 mRNA在癌旁胆管组织呈阳性表达 ,而在胆管癌细胞株 (QBC939)均不表达。 结论 :TRAIL死亡受体和诱捕受体在人胆管癌、癌旁胆管组织和胆管癌细胞株 (QBC939)的表达不同 ,这些受体表达的变化可能在调控人胆管癌凋亡中发挥重要作用。     

TRAIL受体在子宫内膜癌细胞株的表达

目的:研究肿瘤坏死因子相关凋亡诱导配体(TNF-related apoptosis inducing ligand,TRAIL)受体在正常子宫内膜组织、子宫内膜癌组织和癌旁组织的表达特征。方法:采用RT-PCR技术,检测20例正常子宫内膜、20例子宫内膜癌及癌旁组织中TRAIL受体DR4、DR5和假受体DcR1、DcR2阳性表达情况。结果:TRAIL受体DR4、DR5的mRNA在人正常子宫内膜组织、子宫内膜癌组织和癌旁组织中均表达,诱骗受体DcR1的mRNA在子宫内膜各组织中的表达率不同,但差异无统计学意义(P>0.05),而诱骗受体DcR2的mRNA在子宫内膜癌组织中的表达率明显低于正常子宫内膜组织和癌旁组织,差异具有统计学意义(P<0.005),癌旁组织细胞中DcR2表达和正常子宫内膜组织相近(P>0.05)。结论:TRAIL诱骗受体DcR1、DcR2在子宫内膜癌中的低表达可能与其发病机制有关,可能对防止子宫内膜癌变具有一定作用。     

重组腺相关病毒介导TRAIL对卵巢癌裸鼠肝转移的抑制作用的实验研究

目的探讨重组腺相关病毒介导TRAIL对卵巢癌裸鼠肝转移的抑制作用。方法构建卵巢癌裸鼠肝转移模型。选择重组腺相关病毒rAAV—PEG—sTRAIL治疗，将裸鼠分为TRAIL治疗组和对照组，治疗6w后，观察肝转移率和肝转移结节数；Tunnel法分析TRAIL对肿瘤细胞的凋亡诱导情况。结果全身系统性给予重组腺相关病毒介导TRAIL后，治疗组和对照组相比转移率低，肝转移结节数目少，差异有统计学意义（p〈0．05）；Tunnel法分析TRAIL可诱导肿瘤细胞凋亡。结论重组腺相关病毒介导TRAIL可抑制卵巢癌裸鼠肝的转移生长，TRAIL用于卵巢癌肝转移的基因治疗中具有较好的应用前景。     

CD13 inhibition augments DR4-induced tumor cell death in a p-ERK1/2-independent manner

Objective:Death receptor 4 (DR4; TRAIL-R1) critically mediates extrinsic apoptosis cascades via binding to TNF-related apoptosis-inducing ligand (TRAIL). However, intrinsic and/or acquired resistance are observed in the clinical application of TRAIL. The aim of this study was to investigate the function and molecular mechanism of CD13 in the TRAIL/DR4 pathway against tumor cells, and provide a new strategy for improving therapeutic efficacy or overcoming TRAIL-resistance.Methods:TRAIL protein was expressed as a secretory protein in a Pichia pastoris expression system and was isolated and purified by affinity chromatography. The cell viability and apoptosis were evaluated with MTT (thiazolyl blue tetrazolium bromide) assays and annexin V-FITC/PI staining with flow cytometry analysis, respectively. Western blot analysis was used to detect the levels of the indicated proteins in tumor cells. DR4 degradation or stability was examined with cycloheximide chase assays, and cell surface DR4 was assessed with flow cytometric analysis after staining with a FITC-conjugated antibody. The effects of cell migration were determined with Transwell and gelatin zymography assays. A xenograft nude mouse model was used to detect the anti-tumor effect in vivo, and the proliferation in tumor tissues was examined with immunohistochemical staining.Results:CD13 inhibition potently sensitized tumor cells to TRAIL-induced killing, including proliferation inhibition, increased apoptosis, and migration suppression. In addition, the inhibition of CD13 elevated both total cellular expression and cell surface DR4 through stabilizing DR4 by suppressing its degradation. DR4 siRNA attenuated the enhanced anti-tumor effects of TRAIL plus CD13 inhibition. Interestingly, these phenomena were p-ERK1/2 independent, although p-ERK1/2 down-regulation was tightly correlated with the cooperation of TRAIL and CD13 inhibition. Moreover, a synergistic decrease in tumor growth was surprisingly achieved in the xenograft model by treatment of TRAIL with a CD13 inhibitor (**P < 0.01, CDI = 0.47).Conclusions:CD13 inhibition cooperates with TRAIL in enhancing DR4-mediated cell death, through the up-regulation and stabilization of DR4 in a p-ERK1/2-independent manner. Thus CD13 inhibition has emerged as an effective strategy for TRAIL/DR4-based therapy.     

The angular structure of ONC201, a TRAIL pathway-inducing compound,determines its potent anti-cancer activity

We previously identified TRAIL-inducing compound 10 (TIC10), also known as NSC350625 or ONC201, from a NCI chemical library screen as a small molecule that has potent anti-tumor efficacy and a benign safety profile in preclinical cancer models. The chemical structure that was originally published by Stahle, et. al. in the patent literature was described as an imidazo[1,2-a]pyrido[4,3-d]pyrimidine derivative. The NCI and others generally accepted this as the correct structure, which was consistent with the mass spectrometry analysis outlined in the publication by Allen et. al. that first reported the molecule''s anticancer properties. A recent publication demonstrated that the chemical structure of ONC201 material from the NCI is an angular [3,4-e] isomer of the originally disclosed, linear [4,3-d] structure. Here we confirm by NMR and X-ray structural analysis of the dihydrochloride salt form that the ONC201 material produced by Oncoceutics is the angular [3,4-e] structure and not the linear structure originally depicted in the patent literature and by the NCI. Similarly, in accordance with our biological evaluation, the previously disclosed anti-cancer activity is associated with the angular structure and not the linear isomer. Together these studies confirm that ONC201, produced by Oncoceutics or obtained from the NCI, possesses an angular [3,4-e] structure that represents the highly active anti-cancer compound utilized in prior preclinical studies and now entering clinical trials in advanced cancers.