Zhao Jin-yan, Chen Zhi-hong, Lin Wei, Zhong Xiao-yong, Chen Xu-zheng, Peng Jun, Hong Zhen-feng
Fujian Academy of Integrative Medicine, Fuzhou, 350108, China.
Chin J Integr Med. 2014 Feb;20(2):123-9. doi: 10.1007/s11655-013-1581-9. Epub 2014 Mar 6.
To evaluate the effect of Bear Bile Powder(, BBP) on the growth and apoptosis of HepG2 human hepatocellular carcinoma cells, and investigate the possible molecular mechanisms mediating its anti-cancer activity.
HepG2 cells were treated with 0.4-1.0 mg/mL of BBP for 24, 48 and 72 h. The viability of HePG2 cells was determined by MTT assay. Cellular morphology was observed via phase-contrast microscopy. Fluorescence-activated cell sorting analysis with Annexin-V/propidium idodide and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazol-carbocyanine iodide (JC-1) staining was performed to determine cell apoptosis and the loss of mitochondrial membrane potential, respectively. Activation of caspase-9 and -3 was evaluated by a colorimetric assay.
The treatment with 0.4-1 mg/mL of BBP for 24, 48, or 72 h respectively reduced cell viability significantly by 7%-60%, 20%-90% or 25%-98%, compared with the untreated control cells (P<0.01). In addition, BBP treatment induced morphological changes in HepG2 cells. Furthermore, after treated with 0, 0.4, 0.6, 0.8 and 1.0 mg/mL of BBP, apoptosis cells (including early and late apoptotic cells) were 18.0%±1.3%, 34.9%±2.2%, 33.9%±2.8%, 37.4%±2.8% and 46.0%±2.5%, respectively (P<0.05); and the percentage of cells with reduced JC-1 red fluorescence were 6.6%±0.8%, 8.5%±0.8%, 13.5%±1.6%, 17.6%±2.3% and 46.7%±3.6%, respectively (P<0.01). Finally, BBP treatment significantly and dose-dependently induced activation of both caspase-9 and caspase-3 in HepG2 cells (P<0.05).
BBP could inhibit the growth of HepG2 hepatocellular cancer cells through mitochondrion-mediated apoptosis, which may, in part, explain its anti-cancer activity. BBP may be a potential novel therapeutic agent for the treatment of hepatocellular carcinoma.
评估熊胆粉（BBP）对人肝癌HepG2细胞生长和凋亡的影响，并探讨其抗癌活性的可能分子机制。
用0.4 - 1.0 mg/mL的BBP处理HepG2细胞24、48和72小时。采用MTT法测定HepG2细胞的活力。通过相差显微镜观察细胞形态。分别用膜联蛋白V/碘化丙啶和5,5',6,6'-四氯-1,1',3,3'-四乙基苯并咪唑羰花青碘化物（JC-1）染色进行荧光激活细胞分选分析，以确定细胞凋亡和线粒体膜电位的丧失。通过比色法评估半胱天冬酶-9和-3的激活情况。
与未处理的对照细胞相比，分别用0.4 - 1 mg/mL的BBP处理24、48或72小时，细胞活力显著降低7% - 60%、20% - 90%或25% - 98%（P<0.01）。此外，BBP处理诱导了HepG2细胞的形态变化。此外，用0、0.4、0.6、0.8和1.0 mg/mL的BBP处理后，凋亡细胞（包括早期和晚期凋亡细胞）分别为18.0%±1.3%、34.9%±2.2%、33.9%±2.8%、37.4%±2.8%和46.0%±2.5%（P<0.05）；JC-1红色荧光降低的细胞百分比分别为6.6%±0.8%、8.5%±0.8%、13.5%±1.6%、17.6%±2.3%和46.7%±3.6%（P<0.01）。最后，BBP处理显著且剂量依赖性地诱导HepG2细胞中半胱天冬酶-9和半胱天冬酶-3的激活（P<0.05）。
BBP可通过线粒体介导的凋亡抑制HepG2肝癌细胞的生长，这可能部分解释了其抗癌活性。BBP可能是一种潜在的治疗肝细胞癌的新型治疗药物。
