仙客来皂苷对肝癌细胞体外增殖和凋亡的选择性作用及其机制

目的：探讨紫金牛科植物紫金牛仙客来皂苷对肝癌细胞体外增殖和凋亡的选择性作用，并阐明作用靶点及其相关机制。方法：按照不同处理方式将人肝癌细胞HepG2或人正常肝细胞HL-7702分为仙客来皂苷组（加入0、2.5、5.0和10.0 μmol·L^-1仙客来皂苷）、胆固醇干预组（仙客来皂苷+20.0 mg·L^-1胆固醇）和对照组（0.5% DMSO），MTT法检测各组细胞存活率，Filipin标记法检测细胞中胆固醇水平，乳酸脱氢酶（LDH）释放法检测细胞LDH释放水平，流式细胞术检测细胞凋亡率，Western blotting法检测细胞中Fas、FADD和caspase-8蛋白表达情况。结果：5.0和10.0 μmol·L^-1仙客来皂苷组HepG2细胞存活率明显低于HL-7702细胞（P<0.05）。Filipin标记后HepG2细胞中胆固醇水平明显高于HL-7702细胞（P<0.05）。仙客来皂苷组HepG2细胞LDH释放水平高于HL-7702细胞（P<0.05）。与5.0μmol·L^-1仙客来皂苷组比较，胆固醇干预组（5.0μmol·L^-1仙客来皂苷+20.0mg·L^-1胆固醇） LDH释放水平和细胞凋亡率均明显降低（P<0.05）。Western blotting法检测，与对照组比较，仙客来皂苷组HepG2细胞中Fas和PADD蛋白和caspase-8蛋白表达水平明显升高（P<0.05）；与仙客来皂苷组比较，胆固醇干预组HepG2细胞中Fas、FADD和caspase-8蛋白表达水平明显降低（P<0.05）。结论：仙客来皂苷对肝癌细胞有选择性增殖抑制作用，其分子机制可能与通过肝癌细胞膜上胆固醇成分改变细胞膜通透性、进而以配体非依赖的方式激活凋亡相关Fas信号通路有关。

Selective effects of cyclamin on proliferation and apoptosis of liver cancer cells and their mechanisms

Objective: To explore the selective effects of cyclamin on the proliferation and apoptosis of the liver cancer cells in vitro, and to clarify the molecular target and the underlying mechanisms.Methods: The human liver cancer HepG2 cells or normal human HL-7702 liver cells were divided into cyclamin groups (added with 0, 2.5, 5.0 and 10.0 μmol·L^-1 cyclamin), cholesterol intervention group (cyclamin + 20.0 mg·L^-1 cholesterol) and control group (0.5% DMSO only). MTT assay was performed to determine the survival rates of cells in various groups. The cellular cholesterol level was measured by Filipin staining. Lactate dehydrogenase (LDH) release assay was used to determine the LDH realease levels in cells. Flow cytometry was used to detect the apoptotic rates of cells. The expressions of Fas, FADD and caspase-8 proteins were measured by Western blotting method.Results: The survival rates of HepG2 cells in 5.0 and 10.0 μmol·L^-1 cyclamin groups were significantly lower than those of HL-7702 cells (P<0.05).The Filipin staining results showed that the cholesterol level in HepG2 cells was higher than that in HL-7702 cells (P<0.05).The LDH release levels in HepG2 cells in cyclamin groups were higher than those in than HL-7702 cells(P<0.05). Compared with 5.0 μmol·L^-1 cyclamin group, the LDH release level and the apoptotic rate in cholesterol intervention group were decreased(P<0.05).The Western blotting results showed that the expression levels of Fas,FADD and caspase-8 proteins in HepG2 cells in cyclamin groups were increased compared with control group(P<0.05);compared with cyclamin group, the expression levels of Fas, FADD and caspase-8 proteins in cholesterol intervention group were decreased(P<0.05).Conclusion: Cyclamin can selectively inhibit the proliferation of liver cancer cells, and its molecular mechanisms may be related to the increased cell membrane permeabilization via targeting membrane cholesterol component and the consequent ligand-independent activation of Fas signaling pathway.