3,3'-二乙基-9-甲基硫代碳菁碘化物对人肝癌BEL-7402细胞DNA引发酶的抑制作用及诱导凋亡的研究

Inhibition of DNA primase and induction of apoptosis by 3,3'-diethyl-9-methylthia-carbocyanine iodide in hepatocellular carcinoma BEL-7402 cells.

作者信息

Li Zhi-Ming, Liu Zong-Chao, Guan Zhong-Zhen, Zhu Xiao-Feng, Zhou Jun-Min, Xie Bing-Fen, Feng Gong-Kan, Zhu Zhen-Yu, Jiang Wen-Qi

机构信息

Cancer Hospital, Cancer Center, Sun Yat-sen University, Guangzhou 510060, Guangdong Province, China.

出版信息

World J Gastroenterol. 2004 Feb 15;10(4):514-20. doi: 10.3748/wjg.v10.i4.514.

DOI:10.3748/wjg.v10.i4.514

PMID:14966908

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC4716971/

Abstract

AIM

To evaluate the effects of 3,3'-diethyl-9-methylthia-carbocyanine iodide (DMTCCI) on DNA primase activity and on apoptosis of human hepatocellular carcinoma BEL-7402 cells.

METHODS

DNA primase assay was used to investigate DNA primase activity. MTT assay was applied to determine cell proliferation. Flow cytometric analysis, transmission electron microscopy, DNA fragmentation assay were performed to detect DMTCCI-induced apoptosis. Expression levels of p53, Bcl-2, Bcl-xL, Bad, Bax, survivin, Caspase-3 and poly (ADP-ribose) polymerase (PARP) were evaluated by immunoblot analysis. Caspase-3 activity was assessed with ApoAlert Caspase-3 colorimetric assay kit.

RESULTS

DMTCCI had inhibitory effects on eukaryotic DNA primase activity with IC(50) value of 162.2 nmol/L. It also inhibited proliferation of human hepatocellular carcinoma BEL-7402 cells with IC(50) value of 2.09 micromol/L. Furthermore, DMTCCI-induced BEL-7402 cell apoptosis was confirmed by DNA fragmentation (DNA ladders and sub-G1 formation) and transmission electron microscopy (apoptotic bodies formation). During the induction of apoptosis, expression of Bcl-2, Bcl-xL and survivin was decreased, and that of p53, Bad and Bax was increased. Caspase-3 was activated and poly (ADP-ribose) polymerase (PARP) was cleaved in BEL-7402 cells treated with DMTCCI.

CONCLUSION

The present data suggest that DMTCCI has inhibitory effects on eukaryotic DNA primase and can induce apoptosis of BEL-7402 cells. The modulation of expression of p53 and Bcl-2 family proteins, and activation of Caspase-3 might be involved in the induction of apoptosis.

摘要

目的

评估3,3'-二乙基-9-甲基硫代羰花青碘化物（DMTCCI）对人肝癌BEL-7402细胞DNA引发酶活性及细胞凋亡的影响。

方法

采用DNA引发酶测定法研究DNA引发酶活性。应用MTT法检测细胞增殖。通过流式细胞术分析、透射电子显微镜观察、DNA片段化检测来检测DMTCCI诱导的细胞凋亡。采用免疫印迹分析评估p53、Bcl-2、Bcl-xL、Bad、Bax、生存素、半胱天冬酶-3和聚（ADP-核糖）聚合酶（PARP）的表达水平。使用ApoAlert半胱天冬酶-3比色测定试剂盒评估半胱天冬酶-3活性。

结果

DMTCCI对真核DNA引发酶活性具有抑制作用，IC(50)值为162.2 nmol/L。它还抑制人肝癌BEL-7402细胞的增殖，IC(50)值为2.09 μmol/L。此外，通过DNA片段化（DNA梯形条带和亚G1峰形成）和透射电子显微镜观察（凋亡小体形成）证实了DMTCCI诱导的BEL-7402细胞凋亡。在凋亡诱导过程中，Bcl-2、Bcl-xL和生存素的表达降低，而p53、Bad和Bax的表达增加。在DMTCCI处理的BEL-7402细胞中，半胱天冬酶-3被激活，聚（ADP-核糖）聚合酶（PARP）被切割。