Banjerdpongchai Ratana, Wudtiwai Benjawan, Khawon Patompong
Department of Biochemistry, Faculty of Medicine, Chiang Mai University, Chinag Mai, Thailand Email :
Asian Pac J Cancer Prev. 2016;17(7):3289-94.
Naringin, a bioflavonoid found in Citrus seeds, inhibits proliferation of cancer cells. The objectives of this study were to investigate the mode and mechanism(s) of hepatocellular carcinoma HepG2 cell death induced by naringin. The cytotoxicity of naringin towards HepG2 cells proved dosedependent, measured by MTT assay. Naringintreated HepG2 cells underwent apoptosis also in a concentration related manner, determined by annexin Vfluorescein isothiocyanate (FITC) and propidium iodide (PI) employing flow cytometry. Mitochondrial transmembrane potential (MTP) measured using 3,3'dihexyloxacarbocyanine iodide (DiOC6) and flow cytometer was reduced concentrationdependently, which indicated influence on the mitochondrial signaling pathway. Caspase3, 8 and 9 activities were enhanced as evidenced by colorimetric detection of paranitroaniline tagged with a substrate for each caspase. Thus, the extrinsic and intrinsic pathways were linked in human naringintreated HepG2 cell apoptosis. The expression levels of proapoptotic Bax and Bak proteins were increased whereas that of the antiapoptotic BclxL protein was decreased, confirming the involvement of the mitochondrial pathway by immunoblotting. There was an increased expression of truncated Bid (tBid), which indicated caspase8 proteolysis activity in Bid cleavage as its substrate in the extrinsic pathway. In conclusion, naringin induces human hepatocellular carcinoma HepG2 cell apoptosis via mitochondriamediated activation of caspase9 and caspase8mediated proteolysis of Bid. Naringin anticancer activity warrants further investigation for application in medical treatment.
柚皮苷是一种存在于柑橘种子中的生物类黄酮，可抑制癌细胞增殖。本研究的目的是探讨柚皮苷诱导肝癌HepG2细胞死亡的方式和机制。通过MTT法检测，结果表明柚皮苷对HepG2细胞的细胞毒性呈剂量依赖性。采用膜联蛋白V异硫氰酸荧光素（FITC）和碘化丙啶（PI），利用流式细胞术测定，经柚皮苷处理的HepG2细胞也以浓度相关的方式发生凋亡。使用3,3'-二己基氧杂羰花青碘化物（DiOC6）和流式细胞仪测量的线粒体跨膜电位（MTP）呈浓度依赖性降低，这表明对线粒体信号通路有影响。通过比色法检测各半胱天冬酶底物标记的对硝基苯胺，证实半胱天冬酶-3、8和9的活性增强。因此，在经柚皮苷处理的人HepG2细胞凋亡过程中，外源性和内源性途径相互关联。促凋亡蛋白Bax和Bak的表达水平升高，而抗凋亡蛋白Bcl-xL的表达水平降低，通过免疫印迹法证实了线粒体途径的参与。截短型Bid（tBid）的表达增加，这表明在外部途径中，半胱天冬酶-8作为底物对Bid具有蛋白水解活性。总之，柚皮苷通过线粒体介导的半胱天冬酶-9激活和半胱天冬酶-8介导的Bid蛋白水解诱导人肝癌HepG2细胞凋亡。柚皮苷的抗癌活性值得进一步研究以应用于医学治疗。
