* Biochemistry and Molecular Biology, Department of Preclinical Science, Faculty of Medicine, Thailand.
† Microbiology and Immunology, Department of Preclinical Science, Faculty of Medicine, Thailand.
Am J Chin Med. 2016;44(7):1473-1490. doi: 10.1142/S0192415X16500828. Epub 2016 Oct 25.
5,6-dihydroxy-2,4-dimethoxy-9,10-dihydrophenanthrene (HMP) is an active compound isolated from the rhizome extracts of Dioscorea membranacea Pierre, a Thai medicinal plant. This study aimed to investigate the growth-inhibitory and apoptosis-inducing effects of HMP in human lung cancer A549 cells. The antiproliferative and cytotoxic effects of HMP were analyzed by a Sulforhodamine B assay. Cell division, cell cycle distribution and membrane asymmetry changes were each performed with different fluorescent dyes and then analyzed by flow cytometry. Real-time PCR and immunoblotting were used to detect cell cycle- and apoptosis-related mRNA levels and proteins, respectively. The nuclear morphology of the cells stained with DAPI and DNA fragmentation were detected by fluorescence microscopy and gel electrophoresis, respectively. The results showed that HMP exerted strong antiproliferative and cytotoxic activities in A549 cells with the highest selectivity index. It halted the cell cycle in [Formula: see text]/M phase via down-regulation of the expression levels of regulatory proteins Cdc25C, Cdk1 and cyclinB1. In addition, HMP induced early apoptotic cells with externalized phosphatidylserine and subsequent apoptotic cells in sub-[Formula: see text] phase. HMP increased caspase-3 activity and levels of the cleaved (active) form of caspase-3 whose actions were supported by the cleavage of its target PARP, nuclear condensation and DNA apoptotic ladder. Moreover, HMP significantly increased the mRNA and protein levels of proapoptotic Bax as well as promoted subsequent caspase-9 activation and BID cleavage, indicating HMP-induced apoptosis via both intrinsic and extrinsic pathways. These data support, for the first time, the potential role of HMP as a cell-cycle arrest and apoptosis-inducing agent for lung cancer treatment.
5,6-二羟基-2,4-二甲氧基-9,10-二氢菲（HMP）是从泰国药用植物盾叶薯蓣根茎提取物中分离得到的一种活性化合物。本研究旨在探讨 HMP 对人肺癌 A549 细胞的生长抑制和诱导凋亡作用。采用磺酰罗丹明 B 法分析 HMP 的抗增殖和细胞毒性作用。用不同荧光染料检测细胞分裂、细胞周期分布和膜不对称性变化，然后通过流式细胞术进行分析。实时 PCR 和免疫印迹分别用于检测细胞周期和凋亡相关的 mRNA 水平和蛋白质。用 DAPI 染色的细胞核形态和 DNA 片段化通过荧光显微镜和凝胶电泳分别检测。结果表明，HMP 对 A549 细胞表现出强烈的增殖抑制和细胞毒性作用，具有最高的选择性指数。它通过下调调节蛋白 Cdc25C、Cdk1 和 cyclinB1 的表达水平，将细胞周期阻滞在 [Formula: see text]/M 期。此外，HMP 诱导具有膜外翻磷脂酰丝氨酸的早期凋亡细胞，随后在亚 [Formula: see text] 期出现凋亡细胞。HMP 增加了 caspase-3 活性和 cleaved（活性）形式 caspase-3 的水平，其作用得到其靶蛋白 PARP 切割、核浓缩和 DNA 凋亡梯的支持。此外，HMP 显著增加了促凋亡 Bax 的 mRNA 和蛋白水平，并促进了随后的 caspase-9 激活和 BID 切割，表明 HMP 通过内在和外在途径诱导凋亡。这些数据首次支持 HMP 作为一种细胞周期停滞和诱导凋亡剂用于肺癌治疗的潜力。
