Department of Traditional Chinese Medicine, Provincial Hospital affiliated with Shandong University, Jinan 250021, China.
J Ethnopharmacol. 2010 Apr 21;128(3):654-61. doi: 10.1016/j.jep.2010.02.022. Epub 2010 Mar 1.
Cinobufacini (Huachansu), an aqueous extract from the skin and parotid venom glands of Bufo bufo gargarizans Cantor, is a traditional Chinese medicine widely used in clinical cancer therapy in China. The present study sought to investigate the possible signaling pathway implicated in cinobufacini-induced apoptosis in the hepatocellular carcinoma cell lines HepG(2) and Bel-7402.
The effects of cinobufacini on cell proliferation of HepG(2) and Bel-7402 cells were evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assays. Cell apoptosis was detected by Hoechst 33258 staining and flow cytometry analysis. The mitochondrial membrane potential (Deltapsim) and caspase-9 and -3 activity were detected using MitoCapture reagent staining and colorimetric assays, respectively. The expression of apoptosis-related proteins and release of cytochrome c were assessed by Western blot analysis.
Cinobufacini significantly inhibited cell proliferation of both cell lines in a dose- and time-dependent manner. Marked changes in apoptotic morphology and apoptosis rates were clearly observed after cinobufacini treatment. The protein expression of Bax increased whereas that of Bcl-2 decreased, leading to an increase in the Bax/Bcl-2 ratio. Subsequently, cinobufacini disrupted the mitochondrial membrane potential (Deltapsim) and resulted in the release of cytochrome c, activation of both caspase-9 and -3, and cleavage of poly (ADP-ribose) polymerase (PARP).
The present study indicated that cinobufacini can induce apoptosis of HepG(2) and Bel-7402 cells through a mitochondria-mediated apoptosis pathway.
华蟾素是从中华大蟾蜍的皮肤和腮腺毒液腺中提取的一种水溶性提取物,是一种在中国广泛用于临床癌症治疗的传统中药。本研究旨在探讨华蟾素诱导肝癌细胞系 HepG(2)和 Bel-7402 细胞凋亡的可能信号通路。
通过 3-[4,5-二甲基噻唑-2-基]-2,5-二苯基四氮唑溴盐(MTT)检测华蟾素对 HepG(2)和 Bel-7402 细胞增殖的影响。通过 Hoechst 33258 染色和流式细胞术分析检测细胞凋亡。通过 MitoCapture 试剂染色和比色法检测线粒体膜电位(Deltapsim)和 caspase-9 和 -3 活性。通过 Western blot 分析评估凋亡相关蛋白的表达和细胞色素 c 的释放。
华蟾素呈剂量和时间依赖性显著抑制两种细胞系的细胞增殖。华蟾素处理后,明显观察到凋亡形态和凋亡率的明显变化。Bax 蛋白表达增加,Bcl-2 蛋白表达减少,导致 Bax/Bcl-2 比值增加。随后,华蟾素破坏线粒体膜电位(Deltapsim),导致细胞色素 c 释放,caspase-9 和 -3 激活,并切割多聚(ADP-核糖)聚合酶(PARP)。
本研究表明,华蟾素可以通过线粒体介导的凋亡途径诱导 HepG(2)和 Bel-7402 细胞凋亡。