Miltirone induced mitochondrial dysfunction and ROS-dependent apoptosis in colon cancer cells

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• 作者：Lin Wang^a ; ^b ; ^{wanglinster@gmail.com" class="auth_mail" title="E-mail the corresponding author} ; Tao Hu^a ; Jing Shen^a ; Lin Zhang^a ; Long-fei Li^a ; Ruby Lok-Yi Chan^a ; Ming-xing Li^a ; William Ka-Kei Wu^c ; Chi -Hin Cho^a ; ^{chcho@cuhk.edu.hk" class="auth_mail" title="E-mail the corresponding author}
• 关键词：ROS ; reactive oxygen species ; FCM ; flow cytometry ; MMP ; mitochondrial membrane potential ; NQO1 ; quinone oxidoreductase1 ; NAC ; N-acetyl cysteine
• 刊名：Life Sciences
• 出版年：2016
• 出版时间：15 April 2016
• 年：2016
• 卷：151
• 期：Complete
• 页码：224-234
• 全文大小：1925 K

文摘

To study the characteristics of miltirone-induced anti-colon cancer effects.

Materials and methods

Cell viability was detected using MTT assay. LDH (lactate dehydrogenase) leakage was detected using CytoTox96® non-radioactive cytotoxicity kit. Apoptosis was detected by FCM (flow cytometry). Caspase activation was determined by chemiluminescence or western blotting. AIF (apoptosis-inducing factor) expression in the cell fraction was determined by western blotting. ROS (reactive oxygen species), MMP (mitochondrial membrane potential) and mitochondrial mass were determined by confocal microscope. Intracellular calcium was detected by both FCM and confocal microscope. To determine the roles of ROS and Ca^2 + in the pro-apoptotic activity of miltirone, colon cancer cells were pretreated with kinds of antioxidants, dicoumarol, calpeptin or BAPTA-AM in some cases.

Key findings

Miltirone exhibited potent cytotoxicity on colon cancer cells with a better selectivity than that of dihydrotanshinone. The pro-apoptotic activity of miltirone was p53- and ROS-dependent. In detail, miltirone induced direct mitochondrial damage, including significant decrease of mitochondrial ROS, MMP, mass and increase of intracellular ROS and Ca^2 +. NQO1 (quinone oxidoreductase1) was supposed to be a defender for the cytotoxicity induced by miltirone in colon cancer cells. Furthermore, miltirone induced time- and concentration-dependent translocation of AIF and activation of caspases.

Significance

In this study, ROS- and p53-dependent apoptosis induced by miltirone on colon cancer cells was firstly revealed. Strong positive feedback between mitochondrial dysfunction and accumulation of intracellular Ca^2 + was suggested to be the characteristic of the anti-colon cancer activity of miltirone.