Goker Bakiye, Caliskan Cansu, Onur Caglar Hasan, Kayabasi Cagla, Balci Tugce, Erbaykent Tepedelen Burcu, Aygunes Duygu, Yilmaz Susluer Sunde, Mutlu Zeynep, Selvi Gunel Nur, Korkmaz Mehmet, Saydam Guray, Gunduz Cumhur, Biray Avci Cigir
Department of Medical Biology, School of Medicine, Ege University, Izmir, Turkey.
J BUON. 2014 Oct-Dec;19(4):992-8.
Ponatinib (P) has been used for the treatment of chronic myeloid leukemia (CML) and it is known that inhibition of BCR-ABL fusion protein by ponatinib induces apoptosis of CML cells. Epigallocatechin-3-gallate (EGCG), which is a polyphenol in green tea, induces apoptosis in different types of cancer cells. The purpose of this study was to determine the cytotoxic and apoptotic effects of ponatinib and EGCG combination in K562 CML cell line. This study also aimed to detect alterations of the expression levels of cell cycle-regulation related genes after ponatinib and EGCG combination in K562 CML cell line.
The cytotoxic effects of the compounds on K562 cells were determined in a time-and dose-dependent manner by using WST-1 analysis. The combination index (CI) isobologram was used to analyze the data. Apoptotic effects of P-EGCG were defined by flow cytometry and gene expressions were detected by RT-qPCR.
IC50values of ponatinib and EGCG were 87.13 nM and 50μM, respectively. CI value of the P-EGCG was 0.658 and the combination showed synergistic effect (ED90 value: 28.39 nM ponatinib, 117.12 μg/ml EGCG). Ponatinib, EGCG and P-EGCG induced apoptosis compared to control cells. CyclinD1 and CDC25A were downregulated by P-EGCG by 2.49 and 2.63-fold, respectively. TGF-β2 was upregulated by 4.57-fold.
EGCG possesses cytotoxic and apoptotic properties and may cooperate with the growth inhibiting activity of ponatinib synergistically against CML cells. P-EGCG mediated apoptosis might be associated with upregulation of TGF-β2 gene and downregulation of cyclinD1 and CDC25A genes.
波纳替尼（P）已被用于治疗慢性粒细胞白血病（CML），已知波纳替尼抑制BCR-ABL融合蛋白可诱导CML细胞凋亡。表没食子儿茶素-3-没食子酸酯（EGCG）是绿茶中的一种多酚，可诱导不同类型癌细胞凋亡。本研究的目的是确定波纳替尼与EGCG联合使用对K562 CML细胞系的细胞毒性和凋亡作用。本研究还旨在检测波纳替尼与EGCG联合作用于K562 CML细胞系后细胞周期调控相关基因表达水平的变化。
采用WST-1分析法，以时间和剂量依赖性方式测定化合物对K562细胞的细胞毒性作用。用组合指数（CI）等效线图分析数据。通过流式细胞术确定P-EGCG的凋亡作用，并通过RT-qPCR检测基因表达。
波纳替尼和EGCG的IC50值分别为87.13 nM和50μM。P-EGCG的CI值为0.658，该组合显示出协同作用（ED90值：28.39 nM波纳替尼，117.12μg/ml EGCG）。与对照细胞相比，波纳替尼、EGCG和P-EGCG均诱导细胞凋亡。P-EGCG使细胞周期蛋白D1（CyclinD1）和细胞周期蛋白依赖性激酶25A（CDC25A）分别下调2.49倍和2.63倍。转化生长因子-β2（TGF-β2）上调4.57倍。
EGCG具有细胞毒性和凋亡特性，可能与波纳替尼协同抑制CML细胞生长活性。P-EGCG介导的细胞凋亡可能与TGF-β2基因上调以及CyclinD1和CDC25A基因下调有关。
