本文選題：前列腺癌　切入點(diǎn)：CUDC-101　出處：《延邊大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：研究背景:前列腺癌是男性最常見的惡性腫瘤之一,其死亡率占男性癌癥死亡第三位。多西紫杉醇是一種經(jīng)典的廣譜抗腫瘤藥物,廣泛用于多種癌癥的治療,已成為治療晚期前列腺癌的一線化療藥物。然而因患者自身耐受差、藥物選擇性弱等原因亦導(dǎo)致化療失敗或復(fù)發(fā),使其療效欠佳。因此,尋找有效的治療方法或靶向藥物是改善目前前列腺癌治療失敗的重要任務(wù)。CUDC-101作為一種新型的多靶點(diǎn)抑制劑,可以同時(shí)抑制組蛋白去乙�；�(HDAC)、表皮生長因子受體(EGFR)以及表皮生長因子受體2(HER2)。目前,對(duì)于CUDC-101的藥理特征,治療效果以及作用機(jī)制尚未完全闡明,并且與多西紫杉醇等經(jīng)典化療藥物的聯(lián)合抗腫瘤效果也未完全明確。目的:通過體內(nèi)外實(shí)驗(yàn),探討CUDC-101對(duì)前列腺癌細(xì)胞增殖及轉(zhuǎn)移的作用,進(jìn)一步闡明CUDC-101與多西紫杉醇聯(lián)合用藥對(duì)前列腺癌的殺傷作用及機(jī)制。材料和方法:1)臨床標(biāo)本以及數(shù)據(jù)庫資料采集:免疫組織化學(xué)染色,觀察HDACs蛋白在前列腺癌以及癌旁良性前列腺組織中的表達(dá)。進(jìn)一步通過HPA和Oncomine數(shù)據(jù)庫檢索和分析HDACs蛋白及mRNA在前列腺癌組織中的表達(dá)水平。2)體外實(shí)驗(yàn):采用MTT實(shí)驗(yàn),細(xì)胞克隆形成以及CFSE細(xì)胞增殖檢測(cè)實(shí)驗(yàn),觀察CUDC-101與多西紫杉醇單獨(dú)或聯(lián)合用藥對(duì)前列腺癌細(xì)胞的抗增殖作用。FACS流式細(xì)胞術(shù)檢測(cè)藥物治療前后細(xì)胞凋亡率,以及細(xì)胞周期分布的變化。進(jìn)一步的蛋白免疫印跡實(shí)驗(yàn)檢測(cè)CUDC-101和多西紫杉醇處理后,細(xì)胞凋亡和周期相關(guān)蛋白的表達(dá)情況。采用劃痕實(shí)驗(yàn)、細(xì)胞免疫熒光、細(xì)胞侵襲和轉(zhuǎn)移、微管生成,以及蛋白免疫印記實(shí)驗(yàn),檢測(cè)CUDC-101和多西紫杉醇單獨(dú)或聯(lián)合用藥對(duì)前列腺癌遷移和EMT進(jìn)程的影響。蛋白免疫印跡實(shí)驗(yàn)檢測(cè)PI3K/AKT/mTOR和ERK信號(hào)通路相關(guān)蛋白在前列腺癌細(xì)胞中的表達(dá)。3)體內(nèi)實(shí)驗(yàn):首先,建立PC-3前列腺癌細(xì)胞裸鼠移植瘤模型,待腫瘤形成隨即進(jìn)行隨機(jī)分組,分別是:對(duì)照組,CUDC-101用藥組,多西紫杉醇用藥組,以及聯(lián)合用藥組,每組5只。經(jīng)腹腔注射分別給予生理鹽水(對(duì)照組),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天檢測(cè)裸鼠體重以及腫瘤大小。待實(shí)驗(yàn)結(jié)束后處死裸鼠,剝離瘤體,進(jìn)行取材、固定,制成組織切片,進(jìn)一步進(jìn)行HE和免疫組織化學(xué)染色。顯微鏡下觀察不同給藥組中HDAC 4、增殖相關(guān)蛋白ki-67、凋亡相關(guān)蛋白survivin以及EMT相關(guān)蛋白E-cadherin和vimentin的表達(dá)情況。結(jié)果:1)在前列腺癌(PCa)組織中HDAC 4和HDAC 6蛋白表達(dá)呈強(qiáng)陽性,而在癌旁良性組織以及前列腺上皮內(nèi)瘤變(PIN)組織中表達(dá)呈陰性或弱陽性。根據(jù)HPA和Oncomine數(shù)據(jù)庫檢索結(jié)果顯示,與正常前列腺組織相比,HDACs蛋白及mRNA表達(dá)水平在前列腺癌組織中呈明顯高表達(dá)。2)MTT結(jié)果顯示:CUDC-101和多西紫杉醇單獨(dú)用藥均可抑制PC-3和DU145前列腺癌細(xì)胞的增殖。與單用藥組相比,二者聯(lián)合用藥對(duì)前列腺癌細(xì)胞的抗增殖效果更顯著,具有協(xié)同抑制作用。3)細(xì)胞克隆形成實(shí)驗(yàn)和CFSE細(xì)胞增殖檢測(cè)結(jié)果顯示:CUDC-101聯(lián)合多西紫杉醇可協(xié)同抑制前列腺癌細(xì)胞的克隆形成能力以及細(xì)胞增殖能力。4)流式細(xì)胞術(shù)檢測(cè)結(jié)果顯示:CUDC-101上調(diào)PC-3和DU145前列腺癌細(xì)胞的凋亡。而CUDC-101與多西紫杉醇聯(lián)合顯著誘導(dǎo)前列腺癌細(xì)胞的凋亡以及G2/M期細(xì)胞周期阻滯。蛋白免疫印跡實(shí)驗(yàn)結(jié)果顯示:CUDC-101與多西紫杉醇聯(lián)合用藥組Bax,cleaved-caspase-3,-8,-9,-PARP以及p21蛋白的表達(dá)上調(diào),而Bcl-2,CyclinB1蛋白表達(dá)下調(diào)。并且,與單用藥組相比,聯(lián)合用藥后Bax/Bcl-2比值顯著增加。5)劃痕實(shí)驗(yàn)結(jié)果顯示:與單用藥組相比,CUDC-101聯(lián)合多西紫杉醇可以明顯抑制細(xì)胞的愈合能力。細(xì)胞遷移和侵襲實(shí)驗(yàn),以及微管生成實(shí)驗(yàn)結(jié)果也顯示:與多西紫杉醇相比,CUDC-101具有更好的抑制前列腺癌細(xì)胞遷移、侵襲以及微管形成的能力。聯(lián)合用藥時(shí)抑制效果更顯著。蛋白免疫印跡實(shí)驗(yàn)結(jié)果顯示:CUDC-101和多西紫杉醇聯(lián)合處理后,上皮細(xì)胞相關(guān)標(biāo)志物E-cadherin和ZO-1蛋白表達(dá)明顯上調(diào),相反間質(zhì)細(xì)胞相關(guān)標(biāo)志物vimentin,Snail,Slug和MMP-9蛋白表達(dá)則減少。細(xì)胞免疫熒光實(shí)驗(yàn)進(jìn)一步證實(shí)上述實(shí)驗(yàn)結(jié)果。6)蛋白免疫印跡實(shí)驗(yàn)結(jié)果顯示:PI3K/AKT/mTOR和ERK信號(hào)通路參與CUDC-101和多西紫杉醇聯(lián)合用藥對(duì)前列腺癌的抗腫瘤作用機(jī)制。7)PC-3前列腺癌細(xì)胞裸鼠移植瘤模型體內(nèi)實(shí)驗(yàn)結(jié)果顯示:與對(duì)照組和單用藥組相比,聯(lián)合用藥組的腫瘤細(xì)胞生長明顯受到抑制,腫瘤體積明顯減少。免疫組織化學(xué)染色結(jié)果顯示:聯(lián)合用藥組的E-cadherin蛋白表達(dá)顯著增多,相反HDAC4、vimentin、ki-67和survivin蛋白的表達(dá)減少。結(jié)論:1)CUDC-101和多西紫杉醇均可抑制前列腺癌細(xì)胞的增殖,兩藥聯(lián)合具有協(xié)同作用。2)CUDC-101與多西紫杉醇聯(lián)合用藥可誘導(dǎo)前列腺癌細(xì)胞的凋亡以及細(xì)胞周期阻滯,顯著抑制前列腺癌細(xì)胞增殖及EMT進(jìn)程。3)PI3K/AKT/mTOR和ERK信號(hào)通路參與CUDC-101與多西紫杉醇聯(lián)合用藥抑制前列腺癌的作用機(jī)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.

【學(xué)位授予單位】：延邊大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.25

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