低氧环境下蟾毒灵联合顺铂抗食管癌ECA109细胞研究

Effect of bufalin combined with cisplatin on the growth of esophageal cancer cell ECA109 under hypoxic environment

目的观察低氧环境下蟾毒灵联合顺铂对人食管癌ECA109细胞生长的影响,探讨蟾毒灵增加顺铂抗食管癌ECA109细胞抗肿瘤作用的可能机制。方法四甲基偶氮唑蓝(MTT)法测定顺铂(2.5、5.0、7.5、10.0 mg·L-1)在低氧及常氧环境下对人食管癌ECA109细胞增殖的影响,低氧环境下顺铂(2.5、5.0、7.5、10.0 mg·L-1)与蟾毒灵(10 nmol·L-1)联合对人食管癌ECA109细胞增殖的影响;Western blot法检测顺铂单药及蟾毒灵联合顺铂作用于人食管癌ECA109后凋亡相关蛋白表达的变化情况。结果常氧环境下,不同浓度的顺铂对人食管癌ECA109细胞生长抑制率均显著高于空白对照组(P<0.05),且呈浓度依赖性(P<0.05);低氧环境下,不同浓度的顺铂对人食管癌ECA109细胞生长抑制率均高于空白对照组(P<0.05),也呈浓度依赖性(P<0.05),但同一浓度顺铂对人食管癌ECA109细胞生长的抑制作用在低氧环境较常氧环境下显著减弱(P<0.05);低氧环境下,不同浓度的顺铂+蟾毒灵对人食管癌ECA109细胞生长抑制率均高于空白对照组(P<0.05),呈浓度依赖性(P<0.05),且同一浓度顺铂+蟾毒灵10 nmol·L-1对人食管癌ECA109细胞生长的抑制作用较不加蟾毒灵和常氧环境下显著增强(P<0.05)。低氧条件下,顺铂单药组随着顺铂药物浓度的增加,Bcl-2蛋白表达轻度下调,Bax表达轻度升高;联合蟾毒灵后,Bcl-2表达随着顺铂药物浓度的增加明显下调,Bax表达明显升高。结论低氧环境使人食管癌ECA109细胞对顺铂产生耐药,而蟾毒灵可逆转ECA109细胞对顺铂的耐药。

Objective To investigate the effect of bufalin combined with cisplatin on the growth of human esophageal cancer cell ECA109 and explore the potential anti-tumor mechanisms of bufalin combined with cisplatin in the hypoxic environment. Methods The effect of cisplatin（ 2. 5,5. 0,7. 5,10. 0 mg · L^- 1） on cell proliferation of human esophageal cancer ECA109 cell under hypoxic and normoxia environment was evaluated by methylthiazolyl tetrazolium（ MTT） assay; the effect of bufalin（ 10 nmol·L^- 1） combined with cisplatin（ 2. 5,5. 0,7. 5,10. 0 mg·L^- 1） on cell proliferation of human esophageal cancer ECA109 cell under hypoxic environment was evaluated by MTT. Expressions of apoptosis related proteins in ECA109 cells after given cisplatin only or bufalin combined with cisplatin were detected by Western blot. Results Under normoxic and hypoxic environment,the ECA109 cell growth inhibition rate in different concentrations of cisplatin group was significantly higher than that in the control group（ P 〈0. 05）; and the inhibition rate increased with the increasing concentration of cisplatin（ P 〈0. 05）. Under the same concentration cisplatin,the ECA109 cell growth inhibition rate in hypoxic environment was lower than that in normoxic environment（ P 〈0. 05）. Under hypoxic environment,the ECA109 cell growth inhibition rate in different concentration of cisplatin plus bufalin group was significantly higher than that in the control group（ P 〈0. 05）; and the inhibition rate increased with the increasing concentration of cisplatin（ P 〈0. 05）. The ECA109 cell growth inhibition rate in cisplatin plus bufalin group was higher than that in cisplatin group under the same concentration cisplatin（ P 〈0. 05）. Under hypoxic environment,the expression of Bcl-2 decreased and the expression of Bax increased with the increasing concentration of cisplatin. After cisplatin combined with bufalin,the expression of Bcl-2 was significantly decreased,and the expression of Bax was significantly increased with increasing concentrations of cisplatin. Conclusion Under hypoxic environment,the ECA109 cells are resistant to cisplatin,but bufalin can inverse the resistance.