BEZ235和多西他赛协同抑制乳腺癌机制及Mortalin蛋白的标志物作用研究

发布时间：2018-03-25 10:03

本文选题：乳腺癌　切入点：BEZ235　出处：《延边大学》2016年博士论文

【摘要】：研究目的：通过体内外实验检测BEZ235和多西他赛联合用药对乳腺癌细胞生物学行为的影响,分析其协同抑制肿瘤的作用及机制,并探讨Mortalin蛋白在乳腺癌中的作用机制及其预后评估作用,为乳腺癌靶向治疗提供新的药物选择。材料与方法：1)体外实验：MTT及平板克隆实验检测BEZ235和多西他赛单独或联合用药对乳腺癌细胞的增殖抑制作用；细胞划痕实验,细胞迁移实验,细胞侵袭实验观察单独或联合用药对细胞迁移的影响；Hoechst33342荧光染色法,Western blot实验检测药物处理后的乳腺癌细胞凋亡的变化；Western blot实验检测BEZ235和多西他赛单独或联合用药后PI3K/Akt/mTOR信号通路和EMT标志物分子的改变。RT-PCR实验检测不同乳腺癌细胞中Mortalin蛋白的差异表达,进一步以siMortalin方法沉默Mortalin的表达后检测诱导EMT的能力,明确Mortalin蛋白与EMT的相关性。2)体内实验：首先,建立MDA-MB231乳腺癌细胞裸鼠移植瘤模型,随机分成对照组、单独用药组和联合用药组(每组5只)。连续给药4周后检测肿瘤大小,并将肿瘤组织制成病理切片,免疫组化分析E-cadherin, p-AKT, p-S6, p-4ebp1, cleaved caspase 3蛋白的表达情况；其次,应用免疫组化方法检测Mortalin蛋白在155例乳腺浸润性导管癌中的表达情况,并分析其异常表达与乳腺癌临床病理学特点之间的关系,继而分析Mortalin蛋白表达对患者生存期的影响。结果：MTT结果显示,BEZ235与多西他赛可协同抑制乳腺癌细胞株MDA-MB231和MDA-MB468的增殖；平板克隆实验显示,BEZ235与多西他赛可协同抑制乳腺癌的克隆形成能力；细胞划痕实验,细胞迁移实验,细胞侵袭实验结果表明联合用药可明显抑制乳腺癌细胞的迁移能力；Hoechst33342荧光实验表明联合用药可明显诱导乳腺癌细胞的凋亡;Western blot实验和免疫荧光实验结果显示BEZ235与多西他赛联合用药可下调PI3K/Akt/mTOR信号通路蛋白,上调PARP蛋白,下调Bcl2, Bax蛋白,促进caspase3,8,9蛋白的活化诱导凋亡的发生；此外,Western blot实验和免疫荧光实验结果也显示BEZ235与多西他赛联合用药可抑制EMT的发生,下调snail, slug, twist和vimentin蛋白的表达,上调E-cadherin蛋白的表达；RT-PCR结果显示,MDA-MB231细胞Mortalin的mRNA水平明显高与其它乳腺癌细胞系；且Western blot实验结果显示, siMortalin方法沉默Mortalin的表达后可抑制EMT的发生；BEZ235与多西他赛可协同抑制MDA-MB231细胞的裸鼠移植瘤成瘤能力,而且联合用药组肿瘤组织坏死明显增多,p-AKT, p-S6, p-4ebp1的表达明显下调且cleaved caspase3, E-cadherin的表达明显上调。免疫组织化学结果显示,Mortalin蛋白主要定位于细胞浆,且在乳腺癌中的阳性率63.9%(99/155)明显高于导管原位癌34.6%(18/52)及正常组织15.6%(7/45),差异具有显著性,并与患者的组织分级、临床分期和有无淋巴结转移密切相关；生存期分析结果表明Mortalin蛋白的异常表达与乳腺癌的生存期密切相关。此外,多因素分析结果表明,Mortalin蛋白,临床分期与HER2表达在乳腺癌中是重要的独立风险因素。结论：1)BEZ235与多西他赛联合应用可以协同抑制乳腺癌细胞的增殖；2)BEZ235与多西他赛联合应用与诱导细胞凋亡密切相关,而且可以通过EMT抑制乳腺癌细胞的迁移；3) Mortalin蛋白在乳腺癌形成EMT过程中起着重要的作用,而且在乳腺癌中异常高表达,可以作为乳腺癌不良预后的分子标志物。
[Abstract]:Objective: To study effects of in vivo detection of the combination of BEZ235 and docetaxel on the biological behaviors of breast cancer cells, analysis of the synergistic effect and mechanism of tumor suppression, and to explore the mechanism of Mortalin protein in breast cancer and its prognostic role for breast cancer target to provide new drug treatment options. Materials and methods: 1) in vitro: inhibition of MTT proliferation and clonogenic assay for detection of BEZ235 and docetaxel alone or in combination on breast cancer cells; cell scratch assay, cell migration assay, cell invasion assay observed alone or combined effect on cell migration; Hoechst33342 fluorescence staining, the changes of drug Western blot assay after treatment apoptosis of breast cancer cells; Western blot assay, BEZ235 and docetaxel alone or in combination after PI3K/Akt/mTOR signal pathway The expression and change of.RT-PCR assay of EMT marker Mortalin protein in breast cancer cells to detect differences, the ability to induce EMT further to siMortalin silencing Mortalin expression, Mortalin protein clear correlation with EMT.2) in vivo: first, to establish the xenograft model of breast cancer MDA-MB231 cells in nude mice were randomly divided into control group., single drug group and combination group (n = 5). After 4 weeks of continuous administration of detection of tumor size and tumor tissue sections were prepared, immunohistochemical analysis of E-cadherin, p-AKT, p-S6, p-4ebp1, cleaved expression of caspase 3 protein; secondly, immunohistochemical method was used to detect the expression of Mortalin protein the situation of ductal carcinoma of the breast in 155 cases, and analyze the relationship between the expression and clinicopathological characteristics of breast cancer, and analysis of Mortalin protein expression in patients The effects of survival. Results: MTT results showed that BEZ235 and docetaxel match synergistic inhibition of MDA-MB231 and MDA-MB468 in breast cancer cell proliferation; experimental plate cloning, cloning BEZ235 and docetaxel competitively inhibit breast cancer collaborative forming ability; cell scratch test, cell migration assay, cell invasion assay results showed that migration the ability of combined drugs can inhibit breast cancer cells; Hoechst33342 fluorescence experiments show that combination therapy can induce apoptosis of breast cancer cells; Western blot assay and immunofluorescence results showed that BEZ235 and docetaxel combination therapy can regulate PI3K/Akt/mTOR signaling pathway protein, up-regulated PARP protein, down Bcl2, Bax protein, caspase3,8,9 protein activation induced promotion in addition, Western apoptosis; blot assay and immunofluorescence experiment showed that the combination of BEZ235 and docetaxel Occurrence of medication can inhibit EMT down-regulation of snail, slug, twist and the expression of vimentin protein, up-regulated E-cadherin protein expression; RT-PCR results showed that MDA-MB231 cell Mortalin mRNA levels were significantly higher with other breast cancer cell lines; Western and blot results showed that the expression siMortalin silencing Mortalin could inhibit EMT; BEZ235 and his West match synergistic inhibition of MDA-MB231 cells in nude mice tumorigenicity, and the combination group of tumor tissue necrosis increased significantly, p-AKT, p-S6, p-4ebp1 and cleaved significantly decreased expression of Caspase3 significantly up-regulated the expression of E-cadherin. Immunohistochemistry showed that Mortalin protein was mainly localized in the cytoplasm, and in the breast carcinoma the positive rate was 63.9% (99/155) was significantly higher than that of ductal carcinoma in situ (18/52) and 34.6% normal tissues 15.6% (7/45), the difference was significant, and with the patient The histological grade, clinical stage and lymph node metastasis; survival analysis showed that abnormal expression of Mortalin protein in breast cancer and survival are closely related. In addition, the results of multivariate analysis showed that Mortalin protein, clinical stage and the expression of HER2 in breast cancer is an important independent risk factor. Conclusion: 1) BEZ235 combined with docetaxel can inhibit the proliferation of breast cancer cells; 2) BEZ235 combined with docetaxel is closely related with the induction of apoptosis, and can inhibit the migration of breast cancer cells by EMT; 3) Mortalin protein plays an important role in the process of EMT in breast cancer, and high abnormal expression in breast cancer that can be used as molecular marker for poor prognosis of breast cancer.

【学位授予单位】：延边大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.9

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