绿原酸通过Bax/Bcl-2/Caspase-3信号通路对喉癌Hep-2细胞增殖、凋亡的影响

Effects of Chlorogenic Acid on Proliferation and Apoptosis of Laryngeal Cancer Hep-2 Cells Through Bax/Bcl-2/Caspase-3 Signaling Pathway

目的:探讨绿原酸(CGA)对喉癌Hep-2细胞增殖、凋亡及对Bcl-2相关X蛋白(Bax)/B淋巴细胞瘤2(Bcl-2)/胱天蛋白酶-3(Caspase-3)信号通路的影响。方法:分别用含有0、250、500、750和1 000μmol/L的CGA培养液处理喉癌Hep-2细胞24、48 h,寻找出最适宜CGA的浓度和处理时间;将喉癌Hep-2细胞分为对照组(正常培养的喉癌Hep-2细胞)、CGA低浓度组(750μmol/L CGA培养液)、CGA高浓度组(1 000μmol/L CGA培养液)和CGA高浓度+通路抑制剂组(1 000μmol/L CGA培养液+50μmol/L Bax/Bcl-2/Caspase-3通路抑制剂Bax inhibitor peptide V5)。采用细胞计数试剂盒-8(CCK-8)法测定喉癌Hep-2细胞增殖抑制率,通过流式细胞仪检测喉癌Hep-2细胞凋亡率,采用蛋白质印迹法检测喉癌Hep-2细胞中增殖相关蛋白(ki-67)、凋亡相关蛋白(p53)及Bax/Bcl-2/Caspase-3信号通路相关蛋白的表达情况。结果:与对照组相比,CGA低、高浓度组喉癌Hep-2细胞增殖抑制率、细胞凋亡率,p53、Bax和Caspase-3蛋白表达水平显著升高,ki-67、Bcl-2蛋白表达水平显著降低,差异均有统计学意义(P<0.05);且CGA高浓度组喉癌Hep-2细胞增殖抑制率、细胞凋亡率,p53、Bax和Caspase-3蛋白表达水平高于CGA低浓度组,ki-67、Bcl-2蛋白表达水平低于CGA低浓度组,差异均有统计学意义(P<0.05)。与CGA高浓度组相比,CGA高浓度+通路抑制剂组喉癌Hep-2细胞增殖抑制率、细胞凋亡率,p53、Bax和Caspase-3蛋白表达水平显著降低,ki-67、Bcl-2蛋白表达水平显著升高,差异均有统计学意义(P<0.05)。结论:CGA可显著抑制喉癌Hep-2细胞的增殖,并促进其凋亡,可能与Bax/Bcl-2/Caspase-3信号通路激活有关。

OBJECTIVE: To probe into the effects of chlorogenic acid(CGA) on proliferation and apoptosis of laryngeal cancer Hep-2 cells through Bcl-2-associated X protein(Bax)/B cell lymphoma-2(Bcl-2)/cysteinyl aspartate specific proteinase-3(Caspase-3). METHODS: Laryngeal cancer Hep-2 cells were treated with CGA culture medium of 0, 250, 500, 750 and 1 000 μmol/L for 24 and 48 h respectively to find out the most suitable concentration and treatment time of CGA. Laryngeal cancer Hep-2 cells were divided into the control group(normally cultured laryngeal cancer Hep-2 cells), CGA low concentration group(750 μmol/L CGA medium), CGA high concentration group(1 000 μmol/L CGA medium), and CGA high concentration+pathway inhibitor group(1 000 μmol/L CGA medium+50 μmol/L Bax/Bcl-2/Caspase-3 pathway inhibitor Bax inhibitor peptide V5). Cell counting kit-8(CCK-8) method was used to determine the proliferation inhibition rate of laryngeal cancer Hep-2 cells in each group, flow cytometry was used to detect the apoptosis rate of laryngeal cancer Hep-2 cells in each group, Western blotting was used to determine the expression of proliferation related protein(ki-67), apoptosis related protein(p53) and Bax/Bcl-2/Caspase-3 signaling pathway related proteins in laryngeal cancer Hep-2 cells. RESULTS: Compared with the control group, the proliferation inhibition rate, apoptosis rate, p53, Bax and Caspase-3 protein expression levels of laryngeal cancer Hep-2 cells in CGA low and high concentration group were significantly higher, and the ki-67 and Bcl-2 protein expression levels were significantly lower, with statistically significant differences(P<0.05);and the proliferation inhibition rate, apoptosis rate, p53, Bax and Caspase-3 protein expression levels of laryngeal cancer Hep-2 cells in CGA high concentration group were higher than those in CGA low concentration group, the of ki-67 and Bcl-2 protein expression levels were lower than those in CGA low concentration group, with statistically significant differences(P<0.05). Compared with the CGA high concentration group, the proliferation inhibition rate, apoptosis rate, p53, Bax and Caspase-3 protein expression levels of laryngeal cancer Hep-2 cells in the CGA high concentration+pathway inhibitor group were significantly lower, and the ki-67 and Bcl-2 protein expression levels were significantly higher, with statistically significant differences(P<0.05). CONCLUSIONS: CGA can significantly inhibit the proliferation of laryngeal cancer Hep-2 cells and promote the apoptosis, which may be related to the activation of Bax/Bcl-2/Caspase-3 signaling pathway.