白花丹素对涎腺腺样囊性癌细胞增殖、迁移、凋亡的干预作用及其机制

Intervention effects of plumbagin on proliferation,migration,and apoptosis of salivary gland adenoid cystic carcinoma cells

目的观察白花丹素(PLB)对涎腺腺样囊性癌细胞增殖、迁移、凋亡的干预作用,并探讨其作用机制。方法取涎腺腺样囊性癌SACC-83细胞进行体外培养,将细胞分为0、12.5、25、50μmol/L PLB组,分别加入含0、12.5、25、50μmol/L PLB的DMSO培养基培养24 h。采用乳酸脱氢酶(LDH)释放法检测细胞毒性;CCK-8实验观察细胞增殖能力;Transwell试验观察细胞迁移能力;TUNEL染色法观察细胞凋亡情况;荧光探针法检测活性氧(ROS)水平,微量法检测丙二醛(MDA)含量;荧光探针法检测线粒体膜电位;Western blotting法检测细胞凋亡相关蛋白Bcl-2、Bax、Cleaved Caspase-3、Caspase-3表达,计算Bcl-2/Bax、Cleaved Caspase-3/Caspase-3。结果12.5、25、50μmol/L PLB组细胞LDH释放量均高于对照组,其中25、50μmol/L PLB组高于12.5μmol/L PLB组,50μmol/L PLB组高于25μmol/L PLB组(P均<0.05)。不同浓度PLB组细胞增殖能力、细胞迁移率均较对照组下降,其中25、50μmol/L PLB组低于12.5μmol/L PLB组(P均<0.05),25μmol/L PLB组与50μmol/L PLB组比较无统计学差异(P>0.05)。不同浓度PLB组细胞凋亡率均高于对照组,其中25、50μmol/L PLB组高于12.5μmol/L PLB组,50μmol/L PLB组高于25μmol/L PLB组(P均<0.05)。25、50μmol/L PLB组细胞内ROS水平均高于对照组和12.5μmol/L PLB组,且50μmol/L PLB组高于25μmol/L PLB组(P均<0.05);25、50μmol/L PLB组细胞内MDA水平均高于对照组,且50μmol/L PLB组高于12.5、25μmol/L PLB组(P均<0.05)。25、50μmol/L PLB组线粒体膜电位低于对照组,50μmol/L PLB组低于12.5μmol/L PLB组(P均<0.05)。不同浓度PLB组细胞Bcl-2/Bax均低于对照组,Cleaved Caspase-3/Caspase-3均高于对照组(P均<0.05),不同浓度PLB组间比较差异无统计学意义(P均>0.05)。结论PLB可抑制涎腺腺样囊性癌细胞增殖和迁移,促进其凋亡;PLB可能是通过诱导线粒体氧化应激以及ROS介导的细胞凋亡发挥抗肿瘤作用。

Objective To explore the intervention effects of plumbagin(PLB)on the proliferation,migration,and apoptosis of salivary gland adenoid cystic carcinoma cells,and to investigate its mechanism of action.Methods Salivary gland adenoid cystic carcinoma SACC-83 cells were cultured in vitro,and were divided into 0,12.5,25,and 50μmol/L PLB groups,which were treated with 0,12.5,25,and 50μmol/L PLB(dissolved in DMSO)media for 24 h,respectively.Lactate dehydrogenase(LDH)release assay was used to detect cytotoxicity;CCK-8 assay was used to observe cell proliferation ability;Transwell assay was used to observe cell migration ability;TUNEL staining was used to observe the apoptosis;fluorescent probe was used to detect reactive oxygen species(ROS)level;malondialdehyde(MDA)content was detected by trace method;mitochondrial membrane potential was detected by fluorescent probe;the expression levels of apoptosis-related proteins Bcl-2,Bax,Cleaved Caspase-3/Caspase-3,and Caspase-3 were detected by Western blotting,and the ratios of Bcl-2/Bax,and Cleaved Caspase-3/Caspase-3 were calculated.Results The release of LDH in the 12.5,25,and 50μmol/L PLB groups was higher than that in the control group,which in the 25,and 50μmol/L PLB groups was higher than that in the 12.5μmol/L PLB group,and that in the 50μmol/L PLB group was higher than that in the 25μmol/L PLB group(all P<0.05).The OD values of cell proliferation and cell migration rates in different concentrations of PLB groups were lower than those in the control group,which in the 25,and 50μmol/L PLB groups were lower than in the 12.5μmol/L PLB group(all P<0.05),whereas there was no statistical difference between the 25 and 50μmol/L PLB groups(P>0.05).The apoptosis rates of different concentrations of PLB groups were higher than those of the control group,which were higher in the 25,and 50μmol/L PLB groups than in the 12.5μmol/L PLB group,and that in the 50μmol/L PLB group was higher than that in the 25μmol/L PLB group(all P<0.05).The intracellular ROS levels in the 25,and 50μmol/L PLB groups were higher than those in the control group and the 12.5μmol/L PLB group,and the intracellular ROS level in the 50μmol/L PLB group was higher than that in the 25μmol/L PLB group(all P<0.05).The intracellular MDA levels in the 25,and 50μmol/L PLB groups were higher than those in the control group,and that in the 50μmol/L PLB group was higher than those in the 12.5,and 25μmol/L PLB groups(all P<0.05).The mitochondrial membrane potential of the 25,and 50μmol/L PLB groups was lower than that of the control group,and that of the 50μmol/L PLB group was lower than that of 12.5μmol/L PLB group(all P<0.05).The ratios of Bcl-2/Bax in different concentrations of PLB groups were lower than that in the control group,and the ratio of Cleaved Caspase-3/Caspase-3 in the different concentrations of PLB groups were higher than that in the control group(all P<0.05),while there was no significant difference among the different concentrations of PLB groups(all P>0.05).Conclusion Plumbagin can inhibit the proliferation and migration,and promote apoptosis of salivary gland adenoid cystic carcinoma cells;the mechanism in antitumor effects of PLB may involve mitochondrial oxidative stress and ROS-mediated apoptosis.