Adina Anugerah Budipratama, Goenadi Fina Aryani, Handoko Franciscus Feby, Nawangsari Dwi Ana, Hermawan Adam, Jenie Riris Istighfari, Meiyanto Edy
Cancer Chemoprevention Research Center Faculty of Pharmacy, University of Gadjah Mada Sekip Utara II, Yogyakarta, 55281, Indonesia.
Iran J Pharm Res. 2014 Summer;13(3):919-26.
New approach of breast cancer therapy is developed toward combination therapy with agents that have a specific molecular target. Our previous study showed that Citrus aurantifolia lime peels ethanolic extract (CPE) increased the sensitivity of MCF-7 cells againts doxorubicin. This study aims to observe the mechanism of combination CPE and doxorubicin in cell cycle modulation and apoptosis on MCF-7 cells. The assays were performed in the study were cell cycle assay, apoptosis induction, and immunocytochemistry of MCF-7 cells.The effect on the modulation of cell cycle and apoptosis were observed by flowcytometry assay in both single dose of CPE and its combination with Doxorubicin. Cell cycle distribution were observed with flowcytometer FACS-Calibur and its data was analyzed by Cell Quest program. Apoptotic induction in MCF-7 cells was examined using acrydine orange-ethidium bromide (AO-EtBr) double staining. Immunocytochemistry assay was done to observe the expression of apoptotic regulation protein p53 and Bcl-2. The result showed that CPE 6 μg/mL induced apoptosis and cell accumulation at G1 phase, while CPE 15 μg/mL induced apoptosis and cell accumulation at G2/M phase. The combination of doxorubicin 200 nM with CPE 6 μg/mL increased apoptosis induction than their single treatment, and cell accumulation at G2/M phase. Evidence of apoptosis and protein expression of p53 and Bcl-2 indicated that both single applications and combinations of CPE and doxorubicin are able to increase apoptotic bodies of MCF-7 cells by increasing the proteins expression. This result suggested that CPE could perform as co-chemotherapeutic agent with doxorubicin on breast cancer cells.
乳腺癌治疗的新方法是朝着与具有特定分子靶点的药物联合治疗方向发展。我们之前的研究表明，酸橙果皮乙醇提取物（CPE）可增加MCF-7细胞对阿霉素的敏感性。本研究旨在观察CPE与阿霉素联合作用对MCF-7细胞周期调控和凋亡的机制。本研究中进行的检测包括细胞周期检测、凋亡诱导以及MCF-7细胞的免疫细胞化学检测。通过流式细胞术检测观察单剂量CPE及其与阿霉素联合作用对细胞周期调控和凋亡的影响。使用流式细胞仪FACS-Calibur观察细胞周期分布，并通过Cell Quest程序分析其数据。使用吖啶橙-溴化乙锭（AO-EtBr）双重染色检测MCF-7细胞中的凋亡诱导情况。进行免疫细胞化学检测以观察凋亡调节蛋白p53和Bcl-2的表达。结果表明，6μg/mL的CPE诱导凋亡并使细胞在G1期积累，而15μg/mL的CPE诱导凋亡并使细胞在G2/M期积累。200 nM阿霉素与6μg/mL CPE联合使用比单独使用诱导凋亡的效果更强，且使细胞在G2/M期积累。凋亡证据以及p53和Bcl-2的蛋白表达表明，CPE单独应用及其与阿霉素联合应用均能够通过增加蛋白表达来增加MCF-7细胞的凋亡小体。该结果表明，CPE可作为阿霉素的辅助化疗药物用于乳腺癌细胞。
