抑制上皮-間葉轉化調控因子Slug促進doxorubicin在MCF-7/ADR抗藥性細胞株中誘導的細胞凋亡

Abstract

目的:腫瘤細胞對於結構不同的化療藥物具有抗藥性，稱為多重抗藥性(multidrug resistance)。近來，上皮-間葉轉化(epithelial-mesenchymal transition)在研究多重抗藥性的領域中漸被重視。因此，本研究主要的目的是探討上皮-間葉轉化調控因子Slug對於doxorubicin引起細胞的抗藥性所扮演的角色。 方法:透過顯微鏡與免疫螢光染色分別觀察MCF-7/WT與MCF-7/ADR型態差異與上皮-間葉轉化的標記蛋白。藉由即時定量-聚合連鎖酶反應法和西方墨點法分析對doxorubicin敏感的MCF-7/WT與具doxorubicin抗藥性的MCF-7/ADR中上皮-間葉轉化標記蛋白包括E-cadherin, N-cadherin與vimentin與其主要調控因子如Snail, Slug and ZEB1的表現量。抑制Slug表現則用以探討上皮-間葉轉化調控因子對於doxorubicin抗性的調控角色，並透過流式細胞儀、caspase-3/7測定與caspase-8測定探討Slug siRNA引起的細胞凋亡。同樣地，藉由即時定量-聚合連鎖酶反應法分析抑制Slug後引起細胞凋亡相關分子如caspase-6, DFF40, bcl-2 family 及p53的基因表現。 結果:間葉細胞標記蛋白N-cadherin與vimentin的表現在MCF-7/ADR中相對高於MCF-7/WT，但是上皮細胞標記蛋白E-cadherin在MCF-7/ADR細胞中則較MCF-7/WT細胞中的表現量低。相較於MCF-7/WT，Slug在MCF-7/ADR中的表現則顯著增加(P value=7.21×10-6)。抑制MCF-7/ADR中Slug的表現能夠促進doxorubicin引起的細胞凋亡，包括cleaved PARP與sub-G1細胞族群的增加。且抑制Slug能增加Bad與p53的表現，降低Bcl-2的表現，以及活化caspase-3/7。 結論:在MCF-7/ADR中表現上皮-間葉轉化的現象可能與多重抗藥性的有關，上皮-間葉轉化調控因子Slug具對抗doxorubicin引起的細胞凋亡的能力，於乳癌治療中或許能夠當作是doxorubicin引起的抗藥性的治療標的。

Purpose: The resistance of tumor cells to a variety of structurally different chemotherapeutics is termed multidrug resistance (MDR). Recently, epithelial-mesenchymal transition (EMT) has been shown to be associated with multidrug resistance. The aim of the present study is to examine the role of EMT regulator Slug in cells resistant to doxorubicin treatment. Methods: Differences in morphology and EMT markers between MCF-7/WT and MCF-7/ADR were observed by microscopy and immunofluorescence staining, respectively. The expression levels of EMT markers (i.e. E-cadherin, N-cadherin and vimentin) and EMT key regulators (i.e. Snail, Slug and ZEB1) in MCF-7/WT and MCF-7/ADR were determined using quantitative real-time polymerase chain reaction and Western blot analysis. Slug silencing was performed to investigate the effects of Slug on doxorubicin-induced resistance. Apoptosis upon Slug siRNA treatment was evaluated by flow cytometry, caspase-3/7 assay and caspase-8 assay. The gene expressions of apoptosis-related molecules (i.e. caspase-6, DFF40, bcl-2 family and p53) caused by Slug silencing were analyzed by quantitative real-time polymerase chain reaction. Results: The expressions of mesenchymal markers N-cadherin and vimentin were higher in MCF-7/ADR than that in MCF-7/WT, but the expression of epithelial marker E-cadherin decreased in MCF-7/ADR, compared to MCF-7/WT. Slug expression significantly increased in MCF-7/ADR (P value=7.21×10-6). Silencing of Slug in MCF-7/ADR enhanced doxorubicin-induced apoptosis such as high levels of cleaved PARP protein, sub-G1 population, Bad and p53 expressions, and caspase-3/7 activity and lower level of Bcl-2. Conclusion: EMT was observed in MCF-7/ADR cells indicating the association of EMT and multidrug resistance. The EMT regulator Slug may be against doxorubicin-induced apoptosis and turns out as a potential therapeutic target for treating doxorubicin-induced multidrug resistance in cancers.