Department of Pathology, Dr, ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai, Tamilnadu 600113, India.
BMC Complement Altern Med. 2013 Nov 21;13:323. doi: 10.1186/1472-6882-13-323.
Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths reported worldwide. The incidence is higher in Asia and Africa, where there is greater endemic prevalence of hepatitis B and C. The devastating outcome of cancer can be minimized only by the use of potent therapeutic agents. Tridham (TD) has been acknowledged since olden days for its wide spectrum of biological properties and was used by traditional practitioners of Siddha and other indigenous systems of medicine. The present study aims at investigating the mechanistic action of TD by assessing the antiproliferative and pro-apoptotic effects on human hepatocellular carcinoma cell line (Huh7).
Cell viability and apoptosis assay using MTT analysis and trypan blue staining, DAPI staining, DNA fragmentation, cell cycle analysis, mitochondrial membrane potential, real-time reverse transcription-polymerase chain reaction, western blotting and immunofluorescence staining were determined in Huh7 cells.
Viability studies of TD treated Huh7 cells showed an inhibition in cell growth in time and dose dependent manner. Chromatin condensation, DNA fragmentation and apoptotic bodies, which are structural changes characteristic of apoptosis, were found following TD treatment of Huh7 cells. DAPI staining and agarose gel electrophoresis confirmed the induction of apoptosis by TD. Cell cycle analysis of Huh7 cells treated with TD exhibited a marked accumulation of cells in the sub-G1 phase of the cell cycle in a dose dependent manner. Immunofluorescent staining for Ki-67 showed a higher level of expression in untreated cells as compared to TD treated cells. We observed a significant loss in the mitochondrial membrane potential and the release of cytochrome c into the cytosol in TD treated cells. Down regulation of Bcl-2, up regulation of Bax and Bad as well as activation of caspases-3 and 9 were also observed. The p53 gene expression was found to be unaltered in TD treated cells.
These results suggest that TD induces apoptosis of Huh7 cells through activation of Bax and triggered caspase cascade, independent of p53 function. This study throws light on the mechanistic action of TD in triggering apoptosis in Huh 7 cells.
肝细胞癌（HCC）是全球报道的癌症死亡的第三大原因。亚洲和非洲的发病率更高，那里乙型肝炎和丙型肝炎的地方流行率更高。只有使用有效的治疗药物，才能将癌症的破坏性后果降到最低。自古以来，Tridham（TD）就因其广泛的生物学特性而得到认可，并被 Siddha 和其他本土医学系统的传统从业者使用。本研究旨在通过评估对人肝癌细胞系（Huh7）的抗增殖和促凋亡作用来研究 TD 的作用机制。
使用 MTT 分析和台盼蓝染色、DAPI 染色、DNA 片段化、细胞周期分析、线粒体膜电位、实时逆转录-聚合酶链反应、western blotting 和免疫荧光染色来确定 Huh7 细胞中的细胞活力和细胞凋亡。
TD 处理的 Huh7 细胞的活力研究表明，细胞生长呈时间和剂量依赖性抑制。在 Huh7 细胞中用 TD 处理后，发现染色质浓缩、DNA 片段化和凋亡小体，这是凋亡的特征结构变化。DAPI 染色和琼脂糖凝胶电泳证实了 TD 诱导的凋亡。用 TD 处理的 Huh7 细胞的细胞周期分析显示，细胞周期明显在亚 G1 期积聚，呈剂量依赖性。Ki-67 的免疫荧光染色显示，未处理细胞中的表达水平高于 TD 处理细胞。我们观察到 TD 处理的细胞中线粒体膜电位显著丧失，细胞色素 c 释放到细胞质中。还观察到 Bcl-2 下调、Bax 和 Bad 上调以及 caspase-3 和 9 的激活。发现 TD 处理的细胞中 p53 基因表达未改变。
这些结果表明，TD 通过激活 Bax 并触发 caspase 级联反应诱导 Huh7 细胞凋亡，这与 p53 功能无关。这项研究阐明了 TD 在触发 Huh7 细胞凋亡中的作用机制。
