新型多靶点抑制剂CUDC-101与多西紫杉醇联合对前列腺癌的抗肿瘤作用及分子机制

发布时间：2018-03-03 22:35

本文选题：前列腺癌　切入点：CUDC-101　出处：《延边大学》2017年博士论文　论文类型：学位论文

【摘要】：研究背景:前列腺癌是男性最常见的恶性肿瘤之一,其死亡率占男性癌症死亡第三位。多西紫杉醇是一种经典的广谱抗肿瘤药物,广泛用于多种癌症的治疗,已成为治疗晚期前列腺癌的一线化疗药物。然而因患者自身耐受差、药物选择性弱等原因亦导致化疗失败或复发,使其疗效欠佳。因此,寻找有效的治疗方法或靶向药物是改善目前前列腺癌治疗失败的重要任务。CUDC-101作为一种新型的多靶点抑制剂,可以同时抑制组蛋白去乙酰化酶(HDAC)、表皮生长因子受体(EGFR)以及表皮生长因子受体2(HER2)。目前,对于CUDC-101的药理特征,治疗效果以及作用机制尚未完全阐明,并且与多西紫杉醇等经典化疗药物的联合抗肿瘤效果也未完全明确。目的:通过体内外实验,探讨CUDC-101对前列腺癌细胞增殖及转移的作用,进一步阐明CUDC-101与多西紫杉醇联合用药对前列腺癌的杀伤作用及机制。材料和方法:1)临床标本以及数据库资料采集:免疫组织化学染色,观察HDACs蛋白在前列腺癌以及癌旁良性前列腺组织中的表达。进一步通过HPA和Oncomine数据库检索和分析HDACs蛋白及mRNA在前列腺癌组织中的表达水平。2)体外实验:采用MTT实验,细胞克隆形成以及CFSE细胞增殖检测实验,观察CUDC-101与多西紫杉醇单独或联合用药对前列腺癌细胞的抗增殖作用。FACS流式细胞术检测药物治疗前后细胞凋亡率,以及细胞周期分布的变化。进一步的蛋白免疫印迹实验检测CUDC-101和多西紫杉醇处理后,细胞凋亡和周期相关蛋白的表达情况。采用划痕实验、细胞免疫荧光、细胞侵袭和转移、微管生成,以及蛋白免疫印记实验,检测CUDC-101和多西紫杉醇单独或联合用药对前列腺癌迁移和EMT进程的影响。蛋白免疫印迹实验检测PI3K/AKT/mTOR和ERK信号通路相关蛋白在前列腺癌细胞中的表达。3)体内实验:首先,建立PC-3前列腺癌细胞裸鼠移植瘤模型,待肿瘤形成随即进行随机分组,分别是:对照组,CUDC-101用药组,多西紫杉醇用药组,以及联合用药组,每组5只。经腹腔注射分别给予生理盐水(对照组),CUDC-101(60mg/kg/d)和/或多西紫杉醇(5 mg/kg),每3天一次共5次,并且每3-4天检测裸鼠体重以及肿瘤大小。待实验结束后处死裸鼠,剥离瘤体,进行取材、固定,制成组织切片,进一步进行HE和免疫组织化学染色。显微镜下观察不同给药组中HDAC 4、增殖相关蛋白ki-67、凋亡相关蛋白survivin以及EMT相关蛋白E-cadherin和vimentin的表达情况。结果:1)在前列腺癌(PCa)组织中HDAC 4和HDAC 6蛋白表达呈强阳性,而在癌旁良性组织以及前列腺上皮内瘤变(PIN)组织中表达呈阴性或弱阳性。根据HPA和Oncomine数据库检索结果显示,与正常前列腺组织相比,HDACs蛋白及mRNA表达水平在前列腺癌组织中呈明显高表达。2)MTT结果显示:CUDC-101和多西紫杉醇单独用药均可抑制PC-3和DU145前列腺癌细胞的增殖。与单用药组相比,二者联合用药对前列腺癌细胞的抗增殖效果更显著,具有协同抑制作用。3)细胞克隆形成实验和CFSE细胞增殖检测结果显示:CUDC-101联合多西紫杉醇可协同抑制前列腺癌细胞的克隆形成能力以及细胞增殖能力。4)流式细胞术检测结果显示:CUDC-101上调PC-3和DU145前列腺癌细胞的凋亡。而CUDC-101与多西紫杉醇联合显著诱导前列腺癌细胞的凋亡以及G2/M期细胞周期阻滞。蛋白免疫印迹实验结果显示:CUDC-101与多西紫杉醇联合用药组Bax,cleaved-caspase-3,-8,-9,-PARP以及p21蛋白的表达上调,而Bcl-2,CyclinB1蛋白表达下调。并且,与单用药组相比,联合用药后Bax/Bcl-2比值显著增加。5)划痕实验结果显示:与单用药组相比,CUDC-101联合多西紫杉醇可以明显抑制细胞的愈合能力。细胞迁移和侵袭实验,以及微管生成实验结果也显示:与多西紫杉醇相比,CUDC-101具有更好的抑制前列腺癌细胞迁移、侵袭以及微管形成的能力。联合用药时抑制效果更显著。蛋白免疫印迹实验结果显示:CUDC-101和多西紫杉醇联合处理后,上皮细胞相关标志物E-cadherin和ZO-1蛋白表达明显上调,相反间质细胞相关标志物vimentin,Snail,Slug和MMP-9蛋白表达则减少。细胞免疫荧光实验进一步证实上述实验结果。6)蛋白免疫印迹实验结果显示:PI3K/AKT/mTOR和ERK信号通路参与CUDC-101和多西紫杉醇联合用药对前列腺癌的抗肿瘤作用机制。7)PC-3前列腺癌细胞裸鼠移植瘤模型体内实验结果显示:与对照组和单用药组相比,联合用药组的肿瘤细胞生长明显受到抑制,肿瘤体积明显减少。免疫组织化学染色结果显示:联合用药组的E-cadherin蛋白表达显著增多,相反HDAC4、vimentin、ki-67和survivin蛋白的表达减少。结论:1)CUDC-101和多西紫杉醇均可抑制前列腺癌细胞的增殖,两药联合具有协同作用。2)CUDC-101与多西紫杉醇联合用药可诱导前列腺癌细胞的凋亡以及细胞周期阻滞,显著抑制前列腺癌细胞增殖及EMT进程。3)PI3K/AKT/mTOR和ERK信号通路参与CUDC-101与多西紫杉醇联合用药抑制前列腺癌的作用机制。
[Abstract]:Background: prostate cancer is one of the most common malignancy in men, the mortality rate of male cancer deaths accounted for third. Anticancer drug docetaxel is a broad-spectrum classic, widely used for the treatment of various cancers, has become the first-line chemotherapy in the treatment of advanced prostate cancer. However, because the patient self tolerance, drug selective weak and other reasons also lead to the failure of chemotherapy or relapse, the curative effect is poor. Therefore, looking for effective treatment or drug targeting is an important task to improve the current.CUDC-101 prostate cancer treatment failure as a novel multitargeted inhibitor, can inhibit histone deacetylase (HDAC), epidermal growth factor receptor (EGFR) and the epidermal growth factor receptor 2 (HER2). At present, the pharmacological characteristics of CUDC-101, therapeutic effect and mechanism of action has not been fully elucidated, and docetaxel classic Antitumor effect of combination chemotherapy is not entirely clear. Objective: in vitro and in vivo, to investigate the effect of CUDC-101 on proliferation and metastasis of prostate cancer cells, to further clarify the killing effect of CUDC-101 and docetaxel combined with medication on prostate cancer and its mechanism. Material and method: 1) clinical specimens and database data acquisition: immunohistochemistry staining to observe the expression of HDACs protein in prostatic carcinoma and benign prostatic tissue. Further HPA and Oncomine database retrieval and analysis of.2 expression level of HDACs protein and mRNA in prostate cancer tissues) in vitro by MTT experiments, cell clone formation and CFSE cell proliferation assay, the anti proliferative effect of.FACS on CUDC-101 alone or in combination with docetaxel on prostate cancer cell apoptosis rate of flow cytometry before and after drug treatment, in order to And the change of cell cycle distribution. Further western blot assay of CUDC-101 and docetaxel treatment, the expression of apoptosis and cell cycle related proteins. Using scratch test, immunofluorescence, invasion and metastasis, microtubule formation, and Western blot experiments, the detection of CUDC-101 and effects of docetaxel alone or in combination for prostate cancer the process of migration and EMT. The expression of.3 by Western blot detection of PI3K/AKT/mTOR and ERK signaling pathway related protein in prostate cancer cells) in vivo: first, establish PC-3 prostate cancer cell xenografts in nude mice, when the tumor formation then were randomly divided into two groups, respectively: control group, CUDC-101 treatment group and docetaxel treatment group, and the combination group, 5 rats in each group were given normal saline by intraperitoneal injection (control group), CUDC-101 (60mg/kg/d) and / Or docetaxel (5 mg/kg), every 3 days for a total of 5 times, and every 3-4 days to detect the weight of nude mice and tumor size. To the end of the experiment were sacrificed and stripping tumors, were fixed, paraffin sections and HE immunohistochemical staining. Further microscopy to observe the different drugs for HDAC in group 4, proliferation related protein Ki-67, apoptosis related protein survivin and expression of EMT related protein E-cadherin and vimentin. Results: 1) in prostate cancer (PCa) expression of HDAC 4 and HDAC 6 protein was strongly positive in the paracancerous tissues and benign prostatic intraepithelial neoplasia (PIN) expression in negative or weak positive tissues. According to the HPA and Oncomine database retrieval results show that compared with normal prostate tissue, the expression of HDACs protein and mRNA levels in prostate cancer tissues showed high expression of.2) MTT results showed: CUDC-101 and docetaxel Alcohol alone can inhibit PC-3 and DU145 prostate cancer cell proliferation. Compared with the single drug group, two drug combination of prostate cancer cells by anti proliferation effect is more significant, has a synergistic inhibitory effect of.3) cell clone formation assay and CFSE cell proliferation assay showed that CUDC-101 combined with docetaxel can synergistically inhibit prostate cancer cell clones the formation ability and the ability of cell proliferation).4 flow cytometry showed that the apoptosis of CUDC-101 upregulated PC-3 and DU145 prostate cancer cells. CUDC-101 and docetaxel significantly induce prostate cancer cell apoptosis and G2/M cell cycle arrest. Western blot results showed that CUDC-101 treatment group and docetaxel combined with Bax and cleaved-caspase-3. -8, -9, -PARP and expression of p21 protein and Bcl-2 CyclinB1 protein expression decreased. And, with the single drug group Compared with the combination of the ratio of Bax/Bcl-2 increased significantly in.5) scratch test results showed that: compared with the single drug group, CUDC-101 combined with docetaxel can inhibit healing ability. The invasion and migration of cells, and microtubule generating experimental results also show that: compared with docetaxel, CUDC-101 has better inhibition of prostate cancer cell migration. The ability of invasion and microtubule formation. Combination inhibition effect is more significant. Western blot results showed that CUDC-101 and docetaxel treatment, epithelial cell markers E-cadherin and ZO-1 protein expression was significantly up-regulated in contrast, stromal cell associated markers vimentin, Snail, Slug and MMP-9 protein expression decreased by immunofluorescence. Experiments further confirmed the above results.6) Western blot results showed that PI3K/AKT/mTOR and ERK signaling pathway. The mechanism of anti tumor effect of.7 on prostate cancer with CUDC-101 and docetaxel combination) PC-3 prostate cancer cells in nude mice model in vivo experiment results show that: compared with the control group and single drug group, the combination group was significantly inhibited the growth of tumor cells, the tumor volume was significantly reduced. The results of immunohistochemical staining showed that the expression of drug combination group of E-cadherin protein was significantly increased, whereas HDAC4, vimentin, reduce the expression of Ki-67 and survivin protein. Conclusion: 1) CUDC-101 and docetaxel can inhibit the proliferation of prostate cancer cells, the combination of two drugs have a synergistic effect of.2 CUDC-101) and docetaxel combined with medication can induce prostate cancer cell apoptosis and cell cycle arrest, inhibit the proliferation and the prostate cancer cell EMT process.3) PI3K/AKT/mTOR and ERK signaling pathway is involved in CUDC-101 and docetaxel combined with medication Inhibition of the mechanism of prostate cancer.

【学位授予单位】：延边大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.25

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