【摘要】：目的：已有研究發(fā)現(xiàn)ER-a36可通過非經(jīng)典的雌激素膜信號傳導(dǎo)通路,使激素受體陽性的乳腺癌對內(nèi)分泌治療產(chǎn)生耐藥。拉帕替尼(Lapatinib)是一種小分子表皮生長因子酪氨酸激酶抑制劑,可通過阻斷下調(diào)信號抑制乳腺癌細(xì)胞增殖。本研究將探索拉帕替尼是否能通過下調(diào)ER-a36相關(guān)非經(jīng)典的雌激素膜信號傳導(dǎo)通路,逆轉(zhuǎn)激素受體陽性乳腺癌細(xì)胞的他莫昔芬(tamoxifen, TAM)耐藥,并初步探索其逆轉(zhuǎn)耐藥的分子機(jī)制,為乳腺癌內(nèi)分泌治療及靶向治療提供新的理論支持。 方法：運(yùn)用MTT、AnnexinV-FITC/PI雙染法流式細(xì)胞術(shù)、Western Blot等技術(shù)檢測經(jīng)拉帕替尼單藥或加用4-羥基他莫昔芬(4-hydroxy-tamoxifen,4OHT)處理后乳腺癌細(xì)胞株的增殖情況及相關(guān)蛋白水平的變化。 結(jié)果：1、MTT檢測可發(fā)現(xiàn)拉帕替尼對ER陽性乳腺癌細(xì)胞存在明顯抑制作用,在MCF-7、MCF-7/ER-a36兩細(xì)胞系中,存在劑量依賴效應(yīng)；2、AnnexinV-FITC/PI雙染法流式細(xì)胞儀檢測發(fā)現(xiàn)拉帕替尼可顯著增強(qiáng)40HT誘導(dǎo)激素受體陽性、ER-a36過表達(dá)的乳腺癌細(xì)胞凋亡(P0.001)；3、拉帕替尼預(yù)處理或與40HT聯(lián)合作用均可加強(qiáng)凋亡起始、執(zhí)行階段關(guān)鍵Caspase家族蛋白剪切活化、抑制抗凋亡Bcl-2蛋白表達(dá),下調(diào)細(xì)胞周期正性調(diào)控蛋白Cyclin D1、Cyclin D3、CDK2、CDK4等的表達(dá)；4、拉帕替尼顯著抑制ER-a36相關(guān)非經(jīng)典的雌激素膜信號傳導(dǎo)通路中AktT308位點(diǎn)及S6K1蛋白的磷酸化活化,發(fā)揮增敏、部分逆轉(zhuǎn)MCF-7/ER-a36對40HT耐藥的作用。 結(jié)論：拉帕替尼可逆轉(zhuǎn)ER陽性、ER-a36過表達(dá)的乳腺癌細(xì)胞對他莫昔芬的耐藥,其主要通過(1)誘導(dǎo)細(xì)胞周期停滯與凋亡,(2)抑制ER-a36相關(guān)的非經(jīng)典的Akt-mTOR信號傳導(dǎo)通路過度活化,發(fā)揮逆轉(zhuǎn)他莫昔芬耐藥作用。拉帕替尼可能成為激素受體陽性、ER-a36過表達(dá)的他莫昔芬耐藥乳腺癌治療的新靶點(diǎn)。
[Abstract]:Aim: it has been found that ER-a36 can make hormone receptor-positive breast cancer resistant to endocrine therapy through non-classical estrogen membrane signaling pathway. Rapatini (Lapatinib) is a small molecular inhibitor of epidermal growth factor tyrosine kinase, which inhibits breast cancer cell proliferation by blocking down-regulation signals. This study will explore whether Rapatinib can reverse tamoxifen (tamoxifen, TAM) resistance in steroid-receptor-positive breast cancer cells by down-regulating the non-classical estrogen membrane signaling pathway associated with ER-a36. The molecular mechanism of reversal of drug resistance was preliminarily explored to provide new theoretical support for endocrine therapy and targeted therapy of breast cancer. Methods: MTT,AnnexinV-FITC/PI double staining flow cytometry (, Western Blot) and other techniques were used to detect 4-hydroxy-tamoxifenin (4-hydroxy-tamoxifen). After 4OHT treatment, the proliferation of breast cancer cell lines and the changes of related protein levels were observed. Results: (1) ER positive breast cancer cells were significantly inhibited by Rapatinib, and there was a dose-dependent effect in two MCF-7,MCF-7/ER-a36 cell lines. 2Annexin V-FITC / Pi double staining flow cytometry showed that Rapatinib could significantly enhance the apoptosis of breast cancer cells induced by 40HT and overexpression of ER-a36 (P0.001). 3, pretreatment with Rapatinib or combined with 40HT can enhance the initiation of apoptosis, shear activation of key Caspase family proteins, inhibit the expression of anti-apoptotic Bcl-2 protein, and down-regulate the cell cycle positive regulatory protein Cyclin D1 cyclin D3 CDK2. The expression of CDK4 et al. 4. Rapatini significantly inhibited the phosphorylation of AktT308 site and S6K1 protein in the non-classical estrogen membrane signal transduction pathway associated with ER-a36, and partially reversed the effect of MCF-7/ER-a36 on 40HT resistance. Conclusion: Rapatinib can reverse the drug resistance of breast cancer cells with ER positive and ER-a36 overexpression to tamoxifen mainly through (1) inducing cell cycle arrest and apoptosis. (2) inhibiting the overexpression of non-classical Akt-mTOR signal transduction pathway associated with ER-a36 and reversing tamoxifen resistance. Rapatinib may be a new target for tamoxifen-resistant breast cancer with hormone receptor positive and ER-a36 overexpression.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R737.9

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