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
- 上海中医药大学学报 2022年36卷第4期 页码：41-51
- 作者机构：
1.辽宁中医药大学中西医结合学院（辽宁 沈阳 110847）
- 作者简介：
潘鹏宇，男，在读硕士生，主要从事中西医结合防治肿瘤耐药的基础研究
王淳，教授，博士生导师；E-mail：1205055348@qq.com
- 基金信息：国家自然科学基金资助项目(81973735;82174254)
DOI：10.16306/j.1008-861x.2022.04.007
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潘鹏宇,周欢,徐铭等.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[J].上海中医药大学学报,2022,36(04):41-51.
PAN Pengyu,ZHOU Huan,XU Ming,et al.Study on Kang’ai injection regulating Beclin 1⁃dependent autophagy⁃apoptosis interaction and improving cisplatin resistance in A549/DDP cells[J].Academic Journal of Shanghai University of Traditional Chinese Medicine,2022,36(04):41-51.
潘鹏宇,周欢,徐铭等.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[J].上海中医药大学学报,2022,36(04):41-51. DOI： 10.16306/j.1008-861x.2022.04.007.
PAN Pengyu,ZHOU Huan,XU Ming,et al.Study on Kang’ai injection regulating Beclin 1⁃dependent autophagy⁃apoptosis interaction and improving cisplatin resistance in A549/DDP cells[J].Academic Journal of Shanghai University of Traditional Chinese Medicine,2022,36(04):41-51. DOI： 10.16306/j.1008-861x.2022.04.007.
摘要
目的,2,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,2,①应用Kaplan-Meier Plotter在线工具分析Beclin 1与肺癌患者生存期的关系。②CCK-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。③Western 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,2,①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）。②A549/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,2,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。
Abstract
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,2,①Kaplan-Meier Plotter online tool was used to analyze the relationship between Beclin 1 and suvival time of patients with lung cancer. ②The 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.③The expression levels of autophagy-related proteins （Beclin1， ATG7， LC3） and apoptosis-related proteins （Bcl-2， Bax， cleaved caspase-3） were detected by Western blot.,Results,2,①The 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）. ②The expression of Beclin 1 protein in A549/DDP cells was higher than that in A549 cells （,P,<,0.05）. ③Compared 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. ④Compared 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）. ⑤Compared 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,2,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.
关键词
康艾注射液非小细胞肺癌Beclin1自噬凋亡
Keywords
Kang’ai injectionnon-small cell lung cancerBeclin1autophagyapoptosis
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