Laboratory of Molecular Biomedicine, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.
BMC Complement Altern Med. 2014 Jun 19;14:197. doi: 10.1186/1472-6882-14-197.
Dillenia suffruticosa root dichloromethane extract (DCM-DS) has been reported to exhibit strong cytotoxicity towards breast cancer cells. The present study was designed to investigate the cell cycle profile, mode of cell death and signalling pathways of DCM-DS-treated human caspase-3 deficient MCF-7 breast cancer cells.
Dillenia suffruticosa root was extracted by sequential solvent extraction. The anti-proliferative activity of DCM-DS was determined by using MTT assay. The mode of cell death was evaluated by using inverted light microscope and Annexin-V/PI-flow cytometry analysis. Cell cycle analysis and measurement of intracellular reactive oxygen species (ROS) were performed by using flow cytometry. MCF-7 cells were co-treated with antioxidants α-tocopherol and ascorbic acid to evaluate whether the cell death was mainly due to oxidative stress. GeXP-based multiplex system was employed to investigate the expression of apoptotic, growth and survival genes in MCF-7 cells. Western blot analysis was performed to confirm the expression of the genes.
DCM-DS was cytotoxic to the MCF-7 cells in a time-and dose-dependent manner. The IC50 values of DCM-DS at 24, 48 and 72 hours were 20.3 ± 2.8, 17.8 ± 1.5 and 15.5 ± 0.5 μg/mL, respectively. Cell cycle analysis revealed that DCM-DS induced G0/G1 and G2/M phase cell cycle arrest in MCF-7 cells at low concentration (12.5 and 25 μg/mL) and high concentration (50 μg/mL), respectively. Although Annexin-V/PI-flow cytometry analysis has confirmed that DCM-DS induced apoptosis in MCF-7 cells, the distinct characteristics of apoptosis such as membrane blebbing, chromatin condensation, nuclear fragmentation and formation of apoptotic bodies were not observed under microscope. DCM-DS induced formation of ROS in MCF-7 cells. Nevertheless, co-treatment with antioxidants did not attenuate the cell death at low concentration of DCM-DS. The pro-apoptotic gene JNK was up-regulated whereby anti-apoptotic genes AKT1 and ERK1/2 were down-regulated in a dose-dependent manner. Western blot analysis has confirmed that DCM-DS significantly up-regulated the expression of pro-apoptotic JNK1, pJNK and down-regulated anti-apoptotic AKT1, ERK1 in MCF-7 cells.
DCM-DS induced cell cycle arrest and apoptosis in MCF-7 cells via multiple signalling pathways. It shows the potential of DCM-DS to be developed to target the cancer cells with mutant caspase-3.
据报道，吊灯果根的二氯甲烷提取物（DCM-DS）对乳腺癌细胞具有很强的细胞毒性。本研究旨在探讨 DCM-DS 处理人 caspase-3 缺陷 MCF-7 乳腺癌细胞的细胞周期谱、细胞死亡方式和信号通路。
采用连续溶剂萃取法从吊灯果根中提取。用 MTT 法测定 DCM-DS 的抗增殖活性。通过倒置相差显微镜和 Annexin-V/PI-流式细胞术分析评估细胞死亡方式。通过流式细胞术进行细胞周期分析和细胞内活性氧（ROS）的测量。用抗氧化剂 α-生育酚和抗坏血酸共同处理 MCF-7 细胞，以评估细胞死亡是否主要归因于氧化应激。采用 GeXP 多重系统检测 MCF-7 细胞中凋亡、生长和存活基因的表达。通过 Western blot 分析证实基因的表达。
DCM-DS 对 MCF-7 细胞呈时间和剂量依赖性细胞毒性。DCM-DS 在 24、48 和 72 小时的 IC50 值分别为 20.3±2.8、17.8±1.5 和 15.5±0.5μg/mL。细胞周期分析显示，DCM-DS 以低浓度（12.5 和 25μg/mL）和高浓度（50μg/mL）分别诱导 MCF-7 细胞的 G0/G1 和 G2/M 期细胞周期阻滞。尽管 Annexin-V/PI-流式细胞术分析证实 DCM-DS 诱导 MCF-7 细胞凋亡，但在显微镜下未观察到凋亡的明显特征，如细胞膜起泡、染色质浓缩、核碎裂和凋亡小体的形成。DCM-DS 诱导 MCF-7 细胞中 ROS 的形成。然而，用抗氧化剂共同处理不能减轻低浓度 DCM-DS 引起的细胞死亡。促凋亡基因 JNK 被上调，而抗凋亡基因 AKT1 和 ERK1/2 则呈剂量依赖性下调。Western blot 分析证实，DCM-DS 显著上调 MCF-7 细胞中促凋亡 JNK1、pJNK 的表达，下调抗凋亡 AKT1、ERK1 的表达。
DCM-DS 通过多种信号通路诱导 MCF-7 细胞周期阻滞和凋亡。这表明 DCM-DS 有可能被开发用于靶向具有突变 caspase-3 的癌细胞。
