氯化两面针碱对人食管癌Eca109细胞抑制作用及机制研究

Inhibitory effect and mechanism of nitidine chloride on human esophageal cancer Eca109 cells

目的探讨氯化两面针碱对人食管癌Eca109细胞的抑制作用及其诱导凋亡的分子机制。方法采用CCK-8法检测不同浓度的氯化两面针碱不同的干预时间对Eca109细胞的增殖抑制率。依据CCK-8法的结果将实验设4组,各组氯化两面针碱的终浓度分别为0、5、10、15μmol/L,药物作用时间为48 h。以AnnexinV-FITC/PI双染法流式细胞术检测凋亡率;以实时荧光定量PCR(q RT-PCR)法检测Caspase-3、Caspase-9和Noxa的m RNA表达;采用Westernblotting法检测cleaved Caspase-3、Bcl-2、p53和Noxa的蛋白表达水平。结果氯化两面针碱对Eca109细胞具有增殖抑制作用,一定范围内呈时间和浓度依赖性。流式细胞术结果显示,5μmol/L的氯化两面针碱主要诱发早期凋亡(P<0.01);10、15μmol/L的氯化两面针碱主要诱发的是晚期凋亡(P<0.01)。q RT-PCR结果显示,Caspase-3、Caspase-9和Noxa的m RNA表达均随氯化两面针碱的浓度增加而升高,其中10、15μmol/L组升高比较显著。Western blotting法结果显示,cleaved Caspase-3、p53和Noxa的蛋白水平均随氯化两面针碱浓度的增加而升高(P<0.01),但Noxa的升高在5μmol/L组不显著,在10、15μmol/L组升高明显(P<0.01);Bcl-2蛋白的表达随着氯化两面针碱浓度的升高而降低,10、15μmol/L组较为显著(P<0.05、0.01)。结论氯化两面针碱对Eca109细胞的增殖抑制作用主要是通过诱导凋亡实现的,氯化两面针碱诱导Eca109细胞的凋亡与p53和Noxa的表达升高和Bcl-2的下调以及Caspase-3的活化有关。

Objective To investigate the inhibitory effect of nitidine chloride(NC) on human esophageal carcinoma cell line Eca109 and the molecular mechanism of its induction. Methods CCK-8 method was used to detect the inhibition rate of human esophageal cancer Eca109 cells with different concentrations of NC and different intervention time. According to the result of CCK-8 method, the experiment was divided into four groups, and the concentrations of NC in each group were 0, 5, 10, and 15 μmol/L, respectively, and the drug action time was 48 h. The apoptotic rate was detected by flow cytometry with Annexin V-FITC/PI double staining. The mRNA expressions of Caspase-3, Caspase-9, and Noxa were detected by qRT-PCR. The expressions of cleaved Caspase-3 and Bcl-2, p53, and Noxa protein levels were detected by Western blotting. Results NC had inhibitory effect on Eca109 cells in a certain range of time and dose-dependent manner. Flow cytometry showed that NC at 5 μmol/L mainly induced early apoptosis(P < 0.01);NCs at 10 and 15 μmol/L mainly induced late apoptosis(P < 0.01). qRT-PCR results showed that the mRNA expression of Caspase-3, Caspase-9 and Noxa was increased with the increase of NC concentration, of which 10 and 15 μmol/L group increased significantly. The results of Western blotting showed that the protein levels of cleaved Caspase-3, p53, and Noxa were both increased with the increase of NC concentration(P < 0.01), but the increase of Noxa was not significant in 5 μmol/L group(P < 0.01). The expression of Bcl-2 protein was decreased with the increase of NC concentration, and which was significantly higher in 10 and 15 μmol/L groups(P < 0.05, P < 0.01). Conclusion The inhibitory effect of NC on human esophageal carcinoma Eca109 cells is mainly through apoptosis. The apoptosis of NC induced of Eca109 cells is associated with increased expression of p53 and Noxa, downregulation of Bcl-2, and activation of Caspase-3.