南方红豆杉水提物通过氧化应激和线粒体损伤影响口腔鳞癌细胞CAL27的增殖和凋亡

Aqueous Extract of Taxus Chinensis var.Mairei Affects Proliferation and Apoptosis of Oral Squamous Cell Carcinoma CAL27 Cells through Oxidative Stress and Mitochondrial Damage

目的研究南方红豆杉水提物(AETC)对口腔鳞癌(OSCC)细胞CAL27增殖、凋亡的影响及机制。方法CCK-8法检测不同质量浓度AETC(0、0.125、0.250、0.500、1.000、2.000、4.000和6.000 mg·mL^(-1))处理CAL27细胞的存活率并筛选后续浓度;克隆形成实验检测细胞增殖;qRT-PCR检测增殖相关指标Ki67和p21 mRNA的表达;流式细胞术检测细胞凋亡;DCF染色法检测活性氧(ROS)含量;ELISA试剂盒检测氧化应激指标[超氧化物歧化酶(SOD)、丙二醛(MDA)和乳酸脱氢酶(LDH)]的水平;JC-1染色法评估线粒体膜电位变化;Western blot法检测增殖、凋亡和线粒体损伤相关蛋白(p21、Ki67、Caspase-3、Caspase-9、Bcl-2和Bax)的表达。结果与0 mg·mL^(-1) AETC组比较,CAL27细胞的存活率随着AETC质量浓度的增加而降低,后续选择0.250、0.500、1.000 mg·mL^(-1) AETC;与0 mg·mL^(-1) AETC组比较,0.500 mg·mL^(-1)组和1.000 mg·mL^(-1)组的AETC细胞克隆形成率显著降低、Ki67表达降低、p21表达增强、细胞凋亡率增加、活化Caspase-9和Caspase-3表达及Bax/Bcl-2增加、SOD水平降低、ROS、MDA和LDH水平增加、线粒体膜电位显著降低(均P<0.05)。结论AETC可抑制CAL27细胞增殖,促进细胞凋亡,其机制可能与细胞氧化应激和线粒体损伤增加有关。

Objective To study the effects of aqueous extract of Taxus chinensis var.mairei(AETC)on proliferation and apoptosis of oral squamous cell carcinoma(OSCC)CAL27 cells and its mechanisms.Methods CAL27 cells were treated with different concentrations of AETC(0,0.125,0.250,0.500,1.000,2.000,4.000 and 6.000 mg·mL^(-1)).Cell viability,proliferation,apoptosis and mitochondrial damage were determined by CCK-8 assay,colony formation test and flow cytometry,respectively.The mRNA levels of Ki67 and p21 were measured by qRT-PCR.The production of reactive oxygen species(ROS)was detected by DCF staining.The levels of superoxide dismutase(SOD),malondialdehyde(MDA)and lactic acid dehydrogenase(LDH)were measured by ELISA.Mitochondrial membrane potential(MMP)was assessed by JC-1 staining.The expression of proliferation-and apoptosis-related proteins(p21,Ki67,Caspase-3,Caspase-9,Bcl-2 and Bax)was determined by Western blot.Results Compared with 0 mg·mL^(-1) AETC group,the viability of CAL27 cells decreased with the increase of AETC concentration.Then,the doses of AETC at 0.250,0.500 and 1.000 mg·mL^(-1) were selected for the following experiments.Compared with 0 mg·mL^(-1) AETC group,treatment with 0.500 and 1.000 mg·mL^(-1) AETC reduced colony formation rate,Ki67 expression,SOD level and MMP,and increased the expression of p21,cleaved Caspase-9 and cleaved Caspase-3,apoptotic rate,Bax/Bcl-2 ratio and levels of ROS,MDA and LDH(all P<0.05).Conclusion AETC can inhibit proliferation and promote apoptosis in CAL27 cells,which may be involved in oxidative stress and mitochondrial damage.