Lawal Akeem O, Marnewick Jeanine L, Ellis Elizabeth M
Oxidative Stress Research Centre, Faculty of Health and Wellness Sciences, Cape Peninsula University of Technology, Bellville Campus, Bellville, 7535, South Africa.
Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, G1 1XW, Glasgow, UK.
BMC Pharmacol Toxicol. 2015 Dec 15;16:41. doi: 10.1186/s40360-015-0040-y.
Cadmium (Cd) is a well known environmental and industrial toxicant causing damaging effects in numerous organs. In this study, we examined the role of heme oxygenase-1 (HO-1) in modulating the Cd-induced apoptosis in human hepatoma (HepG2) cells after 24 h exposure.
HepG2 cells were exposed to 5 and 10 μM Cd as CdCl2 for 24 h while other sets of cells were pre-treated with either 10 μM Cobalt protoporphyrin (CoPPIX) or 10 μM Tin protoporphyrin (SnPPIX) for 24 h, or 50 μM Z-DEVD-FMK for 1 h before exposure to 5 and 10 μM CdCl2 for 24 h. Expressions of caspase 3, cytosolic cytochrome c, mitochondrial Bax and anti-apoptotic BCL-xl proteins were assessed by western blot. Intracellular reactive oxygen species (ROS) production was determined using the dihydrofluorescein diacetate (H2DFA) method. Cell viability was assessed by MTT assay, while a flow cytometry method was used to assess the level of apoptosis in the cell populations.
Our results show that there were a significant increase in the expression of cytosolic cytochrome c, mitochondrial Bax protein, and caspase 3 at 5 and 10 μM compared to the control, but these increases were attenuated by the presence of CoPPIX. The presence of SnPPIX significantly enhanced Cd-induced caspase 3 activities. CoPPIX significantly decreased the level of ROS production by 24.6 and 22.2 % in 5 and 10 μM CdCl2, respectively, but SnPPIX caused a significant increase in ROS production in the presence of CdCl2. HepG2 cell viability was also significantly impaired by 13.89 and 32.53 % in the presence of 5 and 10 μM CdCl2, respectively, but the presence of CoPPIX and Z-DEVD-FMK significantly enhanced cell survival, while SnPPIX enhanced Cd-impaired cell viability. The presence of CoPPIX and Z-DEVD-FMK also significantly decreased the population of apoptotic and necrotic cells compared with Cd.
In summary, the present study showed that HO-1 attenuates the Cd-induced caspase 3 dependent pathway of apoptosis in HepG2 cells, probably by modulating Cd-induced oxidative stress.
镉（Cd）是一种广为人知的环境和工业毒物，会对多个器官造成损害。在本研究中，我们检测了血红素加氧酶-1（HO-1）在人肝癌（HepG2）细胞暴露于镉24小时后调节镉诱导的细胞凋亡中的作用。
将HepG2细胞暴露于5和10μM氯化镉（CdCl2）中24小时，而其他细胞组先用10μM钴原卟啉（CoPPIX）或10μM锡原卟啉（SnPPIX）预处理24小时，或在暴露于5和10μM CdCl2之前用50μM Z-DEVD-FMK预处理1小时。通过蛋白质印迹法评估半胱天冬酶3、细胞溶质细胞色素c、线粒体Bax和抗凋亡BCL-xl蛋白的表达。使用二氢荧光素二乙酸酯（H2DFA）法测定细胞内活性氧（ROS）的产生。通过MTT法评估细胞活力，同时使用流式细胞术方法评估细胞群体中的凋亡水平。
我们的结果表明，与对照组相比，5和10μM时细胞溶质细胞色素c、线粒体Bax蛋白和半胱天冬酶3的表达显著增加，但CoPPIX的存在减弱了这些增加。SnPPIX的存在显著增强了镉诱导的半胱天冬酶3活性。CoPPIX分别使5和10μM CdCl2中的ROS产生水平显著降低24.6%和22.2%，但SnPPIX在存在CdCl2的情况下导致ROS产生显著增加。在存在5和10μM CdCl2的情况下，HepG2细胞活力也分别显著受损13.89%和32.53%，但CoPPIX和Z-DEVD-FMK的存在显著提高了细胞存活率，而SnPPIX提高了镉损害的细胞活力。与镉相比，CoPPIX和Z-DEVD-FMK的存在也显著减少了凋亡和坏死细胞的数量。
总之，本研究表明HO-1可能通过调节镉诱导的氧化应激来减弱镉诱导的HepG2细胞中依赖半胱天冬酶3的凋亡途径。
