【作者】 胡亦汪；

【导师】 张苏展；

【作者基本信息】 浙江大学 ， 肿瘤学（专业学位）， 2014， 博士

【摘要】 目的：紫檀芪是一种最初在蓝莓中被发现的抗氧化剂,同时它被认为是一种抗乳腺癌的药物,不管雌激素受体-a66(ER-a66)的状态如何,它都能通过半胱天冬酶(casepase)依赖或非caspase依赖的方式诱导乳腺癌细胞凋亡。然而,在ER-a66阴性的乳腺癌细胞中,紫檀芪诱导的凋亡率远远高于ER-a66阳性的乳腺癌细胞。雌激素受体-α36(ER-a36)是ER-a66的一种变体,在ER-a66阴性的乳腺癌细胞中广泛表达。ER-a36的高表达能调节ER-a66阳性乳腺癌患者他莫昔芬耐药。紫檀芪在乳腺癌细胞中的抗增殖能力是否与ER-a36表达有关至今还不得而知。本实验探讨ER-a36在紫檀芪引起的乳腺癌细胞凋亡中的作用。方法：首先,我们用RT-PCR和western blot的方法检测乳腺癌细胞系Mb231、Mb231/Si36、MCF-7和MCF-7/ER36中ER-a36的表达情况。然后通过MTT实验确定乳腺癌细胞对紫檀芪的敏感性是否与ER-a36表达有关。细胞凋亡实验和体内实验验证ER-α36对紫檀芪引起乳腺癌细胞凋亡的影响。结果：相比ER-α36阳性的Mb231乳腺癌细胞,ER-α36阴性的Mb231/Si36乳腺癌细胞对紫檀芪的敏感性显著下降；而ER-α36高表达的MCF-7/ER36乳腺癌细胞对紫檀芪的敏感性高于ER-α6低表达的MCF-7乳腺癌细胞。细胞凋亡实验发现相比Mb231细胞,ER-α36敲除的Mb231/Si36乳腺癌细胞的紫檀芪诱导的凋亡比率有所下降(26.6±1.9%versus13.6±3.9%,p=0.006);相比ER-α36低表达的MCF-7细胞,紫檀芪引起ER-α36高表达的MCF-7/ER36出现更多的细胞凋亡(25.7±2.2%versus10.5±1.5%,p=0.006)。体内实验发现紫檀芪处理能显著降低Mb231乳腺癌的大小；而紫檀芪对Mb231/Si36乳腺的生长速率的影响并不大。免疫组化验证Mb231/Si36和Mb231乳腺癌的ER-α36的表达情况。在Mb231移植瘤中,我们能发现经紫檀芪处理后出现大片坏死。而紫檀芪处理后的Mb231/Si36移植瘤的坏死面积小而零散,与对照组没有显著差异。结论：ER-α36是乳腺癌的一个重要治疗靶点,紫檀芪是作用于ER-α36的潜在抑制剂,能用于ER-α36阳性乳腺癌的治疗。更多还原

【Abstract】 Objective:Pterostilbene (trans-3,5-dimethoxy-4’-hudroxystilbene) is an anti-oxidant primarily found in blueberries and has also been considered as a agent against breast cancer regardless of conventional estrogen receptor (ER-a66) status through inducing both caspase dependent and caspase independent apoptosis. However, the pterostilbene induced apoptosis rate in ER-a66negative breast cancer cells was much higher than ER-a66positive breast cancer cells. Estrogen receptor ER-a36(ER-a36), a variant of ER-a66, is widely expressed in ER-a66negative breast cancer and its high expression could mediate ER-a66positive breast cancer patients resistant to Tamoxifen therapy. However, whether the antiproliferation activity of pterostilbene in breast cancer cells was.related.with.ER-a36.expression.remains.unknown. Here, we report the role of ER-a36in breast cancer apoptosis induced by pterostilbene.Methods:RT-PCR and western blot showed the expression of ER-α36in Mb231、 Mb231/Si36、MCF-7and MCF-7/ER36breast cancer. We use MTT trial to determine whether the sensitivity of breast cancer cells to pterostilbene was depended on ER-a36expressions. Apoptosis and our in vivo finding show ER-a36plays an important role in pterostilbene-induced apoptosis in breast cancer cells.Results:Mb231/Si36with negative expression of ER-a36exhibited dramatically decreased sensitivity to pterostilbene, compared to parental Mb231cells. Moreover, MCF-7/ER36cells with ER-a36overexpressed were more sensitive to pterostilbene than MCF-7cells. Compared to parental Mb231cells, the apoptotic percentage of Mb231/Si36with ER-a36knocked down induced by pterostilbene was decreased (26.6±1.9%versus13.6±3.9%, p=0.006). The apoptosis induced by pterostilbene was promoted in MCF-7/ER36cells with higher expression of ER-a36, compared to the parental MCF-7cells (25.7±2.2%versus10.5±1.5%,p=0.006). Pterostibene treatment significantly reduced the growth rate of the Mb231tumors compared to physiological saline treatment. However, there was no significantly reduction in the growth rate of Mb231/Si36tumors after pterostilbene treatment compared to vehicle control group. Immunohistochemical staining investigates whether the reduction of tumor growth is associated with the downregulation of ER-a36expression. A large area of necrosis was found in Mb231xenograft tumors after pterostilbene treatment. However, the areas of necrosis in Mb231/Si36tumors after pterostilbene treated was small and scattered, which was similar to that of vehicle.Conclusions:ER-a36is a potential therapeutic target in ER-a36-positive breast cancer that has resistance to TAM. And, pterostilbene could be considered as a selective inhibitor targeting ER-a36in the future therapy against ER-a36-positive breast cancer.更多还原