Ahani Narges, Sangtarash Mohammad Hossein, Houshmand Massoud, Eskandani Majid Alipour
Department of Biology, Faculty of Sciences, University of Sistan and Baluchestan, Zahedan, Iran.
Department of Medical Genetics, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran.
J Cell Biochem. 2019 Feb;120(2):2047-2057. doi: 10.1002/jcb.27512. Epub 2018 Aug 30.
Genipin, a compound derived from Gardenis jasminoides Ellis fruits, was demonstrated to be the specific uncoupling protein 2 (UCP2) inhibitor. UCP2 is a mitochondrial carrier protein that creates proton leaks across the inner mitochondrial membrane, thus uncoupling oxidative phosphorylation from adenosine triphosphate (ATP) synthesis. Several studies revealed that UCP2 is broadly over-expressed in leukemia, colorectal, lung, ovarian, prostate, testicular, and bladder cancers. However, the effect of genipin still needs to be elucidated in neurological malignancies. In this study, we investigated the anticancer effect of genipin in U87MG and A172 cell lines. The anticancer effect of genipin on these cell lines was measured by microculture tetrazoliumtest (MTT), Trypan blue exclusion, and colony formation assays, in the presence of various concentrations of genipin at different time intervals. We assessed apoptosis and measure intracellular reactive oxygen species (ROS) by flow cytometry. Expression of UCP2 and some of the genes involved in apoptosis was analyzed by real-time quantitative polymerase chain reaction (PCR). Results of the MTT assay showed that genipin moderately reduced metabolic activity of both cell lines in dose- and time-dependent manner. Result of Trypan blue exclusion test indicated that the viable cell count decreased in the treated group in a concentration-dependent manner. Genipin also significantly decreased colony formation ability of these cells in a concentration-dependent manner. Result of morphological changes showed that there were significant differences in cell number and morphology in treated groups as compared with the untreated groups. Flow cytometric analysis of U87MG and A172 cells with annexin V/propidium iodide staining, 48 hours after treatment with genipin, displays 22.4% and 26.1% apoptotic population, respectively, in treated cells, in comparison to 7.42% and 9.31% apoptotic cells of untreated cells. After treatment, UCP2 and B-cell lymphoma 2 (BCL ) genes are downregulated, and BCL associated X protein, BCL antagonist/killer, BCL interacting killer, and Cytochrome c genes are upregulated. Genipin treatment increased mitochondrial ROS levels and also induced apoptosis through caspase-3 upregulation. In conclusion, the antiproliferative effects of genipin on the growth of both glioblastoma cell lines have been shown in all of these assays, and genipin profoundly induced apoptosis in both cell lines via the UCP2-related mitochondrial pathway through the induction of intracellular ROS.
京尼平是一种从栀子果实中提取的化合物，已被证明是特异性解偶联蛋白2（UCP2）抑制剂。UCP2是一种线粒体载体蛋白，可导致质子跨线粒体内膜泄漏，从而使氧化磷酸化与三磷酸腺苷（ATP）合成解偶联。多项研究表明，UCP2在白血病、结直肠癌、肺癌、卵巢癌、前列腺癌、睾丸癌和膀胱癌中广泛过度表达。然而，京尼平在神经恶性肿瘤中的作用仍有待阐明。在本研究中，我们调查了京尼平对U87MG和A172细胞系的抗癌作用。通过微培养四唑盐试验（MTT）、台盼蓝排斥试验和集落形成试验，在不同时间间隔存在不同浓度京尼平的情况下，测定京尼平对这些细胞系的抗癌作用。我们通过流式细胞术评估细胞凋亡并测量细胞内活性氧（ROS）。通过实时定量聚合酶链反应（PCR）分析UCP2和一些参与细胞凋亡的基因的表达。MTT试验结果表明，京尼平以剂量和时间依赖性方式适度降低了两种细胞系的代谢活性。台盼蓝排斥试验结果表明，处理组活细胞计数以浓度依赖性方式下降。京尼平还以浓度依赖性方式显著降低了这些细胞的集落形成能力。形态学变化结果表明，与未处理组相比，处理组的细胞数量和形态存在显著差异。用膜联蛋白V/碘化丙啶染色对U87MG和A172细胞进行流式细胞术分析，在京尼平处理48小时后，处理组细胞的凋亡率分别为22.4%和26.1%，而未处理组细胞的凋亡率分别为7.42%和9.31%。处理后，UCP2和B细胞淋巴瘤2（BCL）基因下调，而BCL相关X蛋白、BCL拮抗剂/杀手、BCL相互作用杀手和细胞色素c基因上调。京尼平处理增加了线粒体ROS水平，并通过上调半胱天冬酶-3诱导细胞凋亡。总之，在所有这些试验中均显示了京尼平对两种胶质母细胞瘤细胞系生长的抗增殖作用，并且京尼平通过诱导细胞内ROS，经由UCP2相关的线粒体途径在两种细胞系中均显著诱导了细胞凋亡。
