Biomolecular Research Group, Biochemistry Program, Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia.
BMC Complement Altern Med. 2014 Aug 15;14:299. doi: 10.1186/1472-6882-14-299.
Annona muricata leaves have been reported to have antiproliferative effects against various cancer cell lines. However, the detailed mechanism has yet to be defined. The current study was designed to evaluate the molecular mechanisms of A. muricata leaves ethyl acetate extract (AMEAE) against lung cancer A549 cells.
The effect of AMEAE on cell proliferation of different cell lines was analyzed by MTT assay. High content screening (HCS) was applied to investigate the suppression of NF-κB translocation, cell membrane permeability, mitochondrial membrane potential (MMP) and cytochrome c translocation from mitochondria to cytosol. Reactive oxygen species (ROS) formation, lactate dehydrogenase (LDH) release and activation of caspase-3/7, -8 and -9 were measured while treatment. The western blot analysis also carried out to determine the protein expression of cleaved caspase-3 and -9. Flow cytometry analysis was used to determine the cell cycle distribution and phosphatidylserine externalization. Quantitative PCR analysis was performed to measure the gene expression of Bax and Bcl-2 proteins.
Cell viability analysis revealed the selective cytotoxic effect of AMEAE towards lung cancer cells, A549, with an IC50 value of 5.09 ± 0.41 μg/mL after 72 h of treatment. Significant LDH leakage and phosphatidylserine externalization were observed in AMEAE treated cells by fluorescence analysis. Treatment of A549 cells with AMEAE significantly elevated ROS formation, followed by attenuation of MMP via upregulation of Bax and downregulation of Bcl-2, accompanied by cytochrome c release to the cytosol. The incubation of A549 cells with superoxide dismutase and catalase significantly attenuated the cytotoxicity caused by AMEAE, indicating that intracellular ROS plays a pivotal role in cell death. The released cytochrome c triggered the activation of caspase-9 followed by caspase-3. In addition, AMEAE-induced apoptosis was accompanied by cell cycle arrest at G0/G1 phase. Moreover, AMEAE suppressed the induced translocation of NF-κB from cytoplasm to nucleus.
Our data showed for the first time that the ethyl acetate extract of Annona muricata inhibited the proliferation of A549 cells, leading to cell cycle arrest and programmed cell death through activation of the mitochondrial-mediated signaling pathway with the involvement of the NF-kB signalling pathway.
已报道番荔枝叶具有抗多种癌细胞系增殖的作用。然而，其详细机制尚未明确。本研究旨在评估番荔枝叶乙酸乙酯提取物（AMEAE）对肺癌 A549 细胞的分子机制。
通过 MTT 分析检测 AMEAE 对不同细胞系细胞增殖的影响。应用高内涵筛选（HCS）研究 NF-κB 易位、细胞膜通透性、线粒体膜电位（MMP）和细胞色素 c 从线粒体向细胞质的易位抑制情况。检测细胞处理后活性氧（ROS）形成、乳酸脱氢酶（LDH）释放以及 caspase-3/7、-8 和 -9 的激活情况。还进行了 Western blot 分析以确定裂解的 caspase-3 和 -9 的蛋白表达。通过流式细胞术分析确定细胞周期分布和磷脂酰丝氨酸外化。进行定量 PCR 分析以测量 Bax 和 Bcl-2 蛋白的基因表达。
细胞活力分析显示 AMEAE 对肺癌细胞 A549 具有选择性细胞毒性作用，处理 72 小时后 IC50 值为 5.09±0.41μg/mL。荧光分析显示，AMEAE 处理的细胞中观察到明显的 LDH 渗漏和磷脂酰丝氨酸外化。用 AMEAE 处理 A549 细胞显著增加了 ROS 的形成，随后通过上调 Bax 和下调 Bcl-2 来减弱 MMP，同时伴有细胞色素 c 释放到细胞质中。用超氧化物歧化酶和过氧化氢酶孵育 A549 细胞可显著减弱 AMEAE 引起的细胞毒性，表明细胞内 ROS 在细胞死亡中起关键作用。释放的细胞色素 c 触发了 caspase-9 的激活，随后是 caspase-3 的激活。此外，AMEAE 诱导的细胞凋亡伴随着细胞周期停滞在 G0/G1 期。此外，AMEAE 抑制了 NF-κB 从细胞质向细胞核的诱导易位。
我们的数据首次表明，番荔枝叶的乙酸乙酯提取物通过激活线粒体介导的信号通路，抑制 A549 细胞的增殖，导致细胞周期停滞和程序性细胞死亡，该通路涉及 NF-κB 信号通路。
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