康艾注射液调节Beclin 1依赖的自噬-凋亡互作改善A549/DDP细胞顺铂耐药性的研究

Study on Kang’ai injection regulating Beclin 1-dependent autophagy-apoptosis interaction and improving cisplatin resistance in A549/DDP cells

目的:基于自噬-凋亡互作蛋白Beclin 1,探究康艾注射液逆转人肺腺癌A549/DDP细胞顺铂耐药性的分子机制。方法:(1)应用Kaplan-Meier Plotter在线工具分析Beclin 1与肺癌患者生存期的关系。(2)CCK-8法检测顺铂、康艾及康艾联合顺铂对A549(顺铂敏感的亲本细胞)和A549/DDP(顺铂耐药细胞)细胞增殖活性的影响。(3)Western blot法检测自噬相关蛋白(Beclin 1、ATG7、LC3)和凋亡相关蛋白(Bcl-2、Bax、cleaved caspase-3)的表达水平。结果:(1)应用Kaplan-Meier Plotter在线工具对BECN1 mRNA表达水平和临床病理学指标分析显示,BECN1高表达组生存期显著高于BECN1低表达组,肺腺癌组织BECN1表达水平与患者预后呈显著正相关(P<0.001)。(2)A549/DDP细胞的Beclin 1蛋白表达量比A549细胞高(P<0.05)。(3)与对照组比较,顺铂上调了A549和A549/DDP细胞的Beclin 1、ATG7、LC3Ⅰ、LC3Ⅱ表达(P<0.05,P<0.01,P<0.001)。在A549细胞中,顺铂干预后Bax、cleaved caspase-3表达增加(P<0.01,P<0.001),Bcl-2表达下降(P<0.01),且Bax/Bcl-2比值上调(P<0.01);在A549/DDP细胞中,顺铂干预后Bax、Bcl-2表达增加(P<0.05,P<0.01),但Bax/Bcl-2比值下降(P<0.05)。(4)与顺铂组比较,低、中、高浓度康艾注射液联合顺铂均能显著抑制A549/DDP细胞增殖活性(P<0.01,P<0.001),顺铂+低浓度康艾组Beclin 1表达降低(P<0.001),顺铂+中、高浓度康艾组Beclin 1表达增加(P<0.001),且顺铂+高浓度康艾组表达增加量最多(P<0.001)。(5)与顺铂组比较,低、中、高浓度康艾注射液联合顺铂均可上调ATG7(P<0.05,P<0.01)、LC3Ⅰ(P<0.01,P<0.001)、LC3Ⅱ(P<0.01,P<0.001)表达水平,且与康艾注射液的浓度升高呈正相关。Bax表达在顺铂+康艾低浓度组中降低(P<0.05),在顺铂+康艾中、高浓度组中增加(P<0.01);Bcl-2表达在顺铂+康艾低、中浓度组中降低(P<0.05),在顺铂+高浓度康艾组中增加(P<0.05);cleaved caspase-3表达与Bax/Bcl-2比值在顺铂+康艾低、中、高浓度组中均上调(P<0.05,P<0.01,P<0.001),且cleaved caspase-3表达与康艾注射液浓度升高呈正相关。结论:康艾注射液能有效改善A549/DDP细胞顺铂耐药性,其机制可能为上调Beclin 1蛋白表达水平,调控Beclin1介导下的细胞自噬与凋亡互作,从而导致A549/DDP细胞自噬性死亡与凋亡。

Objective:To explore the molecular mechanism of Kang’ai injection reversing cisplatin resistance in human lung adenocarcinoma cell line A549/DDP based on autophagy-apoptosis interaction protein Beclin 1.Methods:(1)Kaplan-Meier Plotter online tool was used to analyze the relationship between Beclin 1 and suvival time of patients with lung cancer.(2)The effects of cisplatin,Kang’ai and Kang’ai combined with cisplatin on the proliferation of A549(cisplatin-sensitive parent cells)and A549/DDP(cisplatin-resistant cells)cells were analyzed by CCK-8 method.(3)The expression levels of autophagy-related proteins(Beclin1,ATG7,LC3)and apoptosisrelated proteins(Bcl-2,Bax,cleaved caspase-3)were detected by Western blot.Results:(1)The analysis of BECN1 mRNA expression level and clinicopathological indexes by Kaplan-Meier Plotter online tool showed that the survival time of BECN1 high expression group was significantly higher than that of BECN1 low expression group,and the expression level of BECN1 in lung adenocarcinoma tissues was positively correlated with the prognosis of patients(P<0.001).(2)The expression of Beclin 1 protein in A549/DDP cells was higher than that in A549 cells(P<0.05).(3)Compared with the control group,cisplatin upregulated the expression of Beclin 1,ATG7,LC3Ⅰ and LC3Ⅱ in A549 and A549/DDP cells(P<0.05,P<0.01,P<0.001). In A549 cells,the expression of Bax and cleaved caspase-3 increased(P<0.01,P<0.001),the expression of Bcl-2 decreased(P<0.01)and the ratio of Bax/Bcl-2 increased(P<0.01)after cisplatin intervention. In A549/DDP cells,the expression of Bax and Bcl-2 increased(P<0.05,P<0.01),but the ratio of Bax/Bcl-2 decreased(P<0.05)after cisplatin intervention.(4) Compared with the cisplatin group,low,middle and high concentration of Kang’ai injection combined with cisplatin significantly inhibited the proliferation of A549/DDP cells(P<0.01,P<0.001). The expression of Beclin1 decreased in cisplatin+low concentration Kang’ai group(P<0.001),increased in cisplatin+medium and high concentration Kang’ai groups(P<0.001),and cisplatin+high concentration Kang’ai group has the largest increase(P<0.001).(5) Compared with cisplatin group,low,middle and high concentration of Kang’ai injection combined with cisplatin could upregulate the expression of ATG7(P<0.05,P<0.01),LC3Ⅰ(P<0.01,P<0.001)and LC3Ⅱ(P<0.01,P<0.001),which was positively correlated with the concentration of Kang’ai injection. The expression of Bax decreased in cisplatin+low concentration Kang’ai group(P<0.05),and increased in cisplatin+middle and high concentration of Kang’ai groups(P<0.01). The expression of Bcl-2 decreased in cisplatin+low and middle concentration Kang’ai groups(P<0.05),and increased in cisplatin+high concentration Kang’ai group(P<0.05). The expression of cleaved caspase-3 and Bax/Bcl-2 ratio increased in cisplatin+low,middle and high concentration Kang’ai groups(P<0.05,P<0.01,P<0.001). And there was a positive correlation between the expression of cleaved caspase-3 and the concentration of Kang’ai injection.Conclusions:Kang’ai injection can effectively improve the cisplatin resistance of A549/DDP cells. The mechanism may be that it can upregulate the expression of Beclin 1 protein and regulates the interaction between autophagy and apoptosis mediated by Beclin1,which leads to autophagic death and apoptosis of A549/DDP cells.