摘要
目的探讨V-ATP酶抑制剂巴弗洛霉素A1(Baf-A1)对食管鳞癌(ESCC)细胞增殖、凋亡和放射敏感性的影响。方法通过CCK-8法检测ESCC细胞Eca109和KYSE150经Baf-A1处理后的细胞活力并计算20%抑制浓度(IC 20)和50%抑制浓度(IC 50),采用BCECF-AM探针检测IC 20浓度的Baf-A1处理后的细胞质pH水平,IC 20浓度的Baf-A1联合8 Gy辐射剂量处理后行流式细胞术和γ-H2AX共聚焦显像实验观察Baf-A1对ESCC细胞凋亡的影响,基于克隆形成实验观察Baf-A1对ESCC细胞放射敏感性的影响,Western blotting和实时定量PCR检测ESCC细胞增殖和凋亡相关因子的水平。结果Eca109和KYSE150细胞经Baf-A1(0、1、10、100、1000 nmol/L)处理后的细胞活力均呈浓度依赖的方式下降,对应IC 50分别为163.4和175.3 nmol/L;经Baf-A1(0、5、10、20、40、80、160 nmol/L)处理48 h后的IC 20约为60 nmol/L。Baf-A1处理ESCC细胞48 h后的ATP6V0C水平降低且细胞内BCECF绿色荧光减弱。8 Gy照射后,Baf-A1处理ESCC细胞的凋亡率升高,γ-H2AX信号延长且凋亡抑制因子Bcl-2和DNA损伤修复因子PARP水平降低而促凋亡因子Bax水平升高,上述差异均有统计学意义(P<0.05)。单击多靶模型拟合曲线显示,Baf-A1处理的Eca109和KYSE150细胞与对应对照细胞相比,增敏比分别为1.23和1.29。结论通过V-ATP酶抑制剂Baf-A1改变肿瘤细胞的酸环境可提高ESCC细胞的放射敏感性,这为ESCC的治疗提供了潜在策略。
Objective To investigate the effects of V-ATPase inhibitor bafilomycin A1(Baf-A1)on the proliferation,apoptosis and radiosensitivity of esophageal squamous cell carcinoma(ESCC)cells.Methods Cell viability of ESCC cells Eca109 and KYSE150 treated with Baf-A1 was detected by CCK-8,and the 20%inhibition concentration(IC 20)and 50%inhibition concentration(IC 50)were calculated.BCECF-AM probe was used to detect the cytoplasmic pH level after treatment with Baf-A1 at IC 20 concentration.After Baf-A1 treatment with IC 20 concentration plus 8 Gy radiation dose,the effect on apoptosis was observed by flow cytometry andγ-H2AX confocal imaging.Radiosensitivity of Baf-A1 on ESCC cells was observed via clone formation experiment.Western blotting and real-time quantitative PCR were used to detect the levels of factors related to proliferation and apoptosis of ESCC cells after exposure to Baf-A1 with 8 Gy irradiation.Results Cell viability of Eca109 and KYSE150 cells treated with Baf-A1(0,1,10,100,1000 nmol/L)decreased in a concentration-dependent manner,and the corresponding IC 50 were 163.4 and 175.3 nmol/L,respectively.IC 20 of Eca109 and KYSE150 cells after treatment with Baf-A1(0,5,10,20,40,80,160 nmol/L)for 48 h was approximately 60 nmol/L.After 48 h of Baf-A1 treatment,level of ATP6V0C decreased and the green fluorescence of BCECF weakened.After 8 Gy irradiation,there were increased apoptotic rates,prolongedγ-H2AX signal,decreased levels of anti-apoptotic factor Bcl-2 and DNA damage repair factor PARP but increased level of proapoptotic factor Bax in ESCC cells treated with Baf-A1(P<0.05).Multi-target single-hit model fitting curve showed that the sensitization ratios of Eca109 and KYSE150 cells treated with Baf-A1 were 1.23 and 1.29 compared with the corresponding control cells.Conclusion Changing the acid environment of tumor cells by V-ATPase inhibitor Baf-A1 can improve the radiosensitivity of ESCC cells,which provides a potential strategy for the treatment of ESCC.
作者
姚希娟
陈婷婷
陈慧
徐冰
鲁璟
孙新臣
YAO Xijuan;CHEN Tingting;CHEN Hui;XU Bing;LU Jing;SUN Xinchen(Department of Radiation Oncology,the First Affiliated Hospital of Nanjing Medical University,Nanjing 210029,China)
出处
《临床肿瘤学杂志》
CAS
2021年第8期673-679,共7页
Chinese Clinical Oncology
基金
国家自然科学基金资助项目(81703028,81703027,82073344,82102831)。
关键词
食管鳞癌
巴弗洛霉素A1
增殖
凋亡
放射敏感性
Esophageal squamous cell carcinoma
Bafilomycin A1
Proliferation
Apoptosis
Radiosensitivity
