内质网应激在二十二碳六烯酸提高食管癌EC9706细胞顺铂化疗敏感性中的作用

Docosahexaenoic acid enhances the sensitivity of esophageal carcinoma cell line EC9706 to cisplatin viainhibiting endoplasmic reticulum stress

目的探讨二十二碳六烯酸（DHA）对食管癌细胞EC9706顺铂（DDP）化疗敏感性的影响及其机制。方法不同药物干预将实验组分为：空白对照组、DHA组、DDP组、DHA＋DDP组、DHA＋DDP＋应激诱导剂衣霉素（TM）组（DHA＋DDP＋TM组）。四甲基偶氮唑盐比色法（MTT法）检测各组食管癌EC9706细胞增殖抑制率；流式细胞仪检测各组食管癌EC9706细胞凋亡率；Westernblot法检测各组细胞凋亡相关因子（半胱氨酸蛋白酶-3、Bcl-2）和内质网应激相关因子[葡萄糖调节蛋白78（GRP78）、磷酸化肌醇需要酶1（IRE-1）]蛋白的表达。结果MTr检测显示浓度〉25μmol／LDHA对食管癌细胞EC9706的增殖抑制呈时间和浓度依赖性（P=0．00）。DHA预处理增强DDP对EC9706细胞增殖的抑制，促进细胞凋亡。与DDP组相比，DHA＋DDP组对EC9706细胞的增殖抑制率明显升高[（60．19±5．05）％比（36．72±3．52）％，P=0．02]，凋亡率也明显升高[（54．88±4．94）％比（39．74±4．64）％，P=0．03]。Westernblot检测显示，与DDP组相比，DHA＋DDP组抗凋亡因子Bcl-2明显下降，凋亡因子半胱氨酸蛋白酶．3表达增加，内质网相关因子（GRP78和IRE-1）蛋白表达明显减少（P均=0．01）；内质网应激诱导剂TM抵消DHA抑制内质网应激的作用，逆转DHA提高食管癌细胞DDP化疗敏感性的作用（P=0．02）。结论DHA预处理增强DDP对EC9706细胞增殖的抑制，促进细胞凋亡，提高食管癌细胞对DDP的化疗敏感性，其机制可能是通过抑制癌细胞内质网应激起作用。

Objective To investigate the effect of docosahexaenoic acid （DHA） on the sensitivity of e- sophageal carcinoma cell line EC9706 to cisplatin and the mechanism behind this effect. Methods EC9706 cells were randomly divided into 5 groups ： control group, DHA group, cisplatin （DDP） group, DHA ± DDP group and endoplasmic reticulum stress （ERS） activation tunicamycin （TM） group （DHA±DDP±TM group）. MTY method was used to evaluate inhibition ratio of cell proliferation. The apoptotic ratio was examined by flow eytometry. Western blot was used to detect the protein expressions of apoptosis cytokines （caspase-3 and Bcl-2） and ERS cytokines [ glucose-regulated protein 78 （GRP78） and inositol-requiring enzyme 1 （IRE-1 ） t. Results DHA causes concentration-dependent and time-dependent inhibition of the proliferation of EC9706 cells （P= 0. 00）. DHA significantly enhanced the sensitivity of esophageal carcinoma cell line EC9706 to cisplatin. Compared to DDP treatment alone, the inhibition ratio [ （60. 19±5.05）% vs. （36. 72±3.52）%, P=0. 02] and apoptotic ratio [ （54. 88±4.94）% vs. （39.74±4.64）%, P=O. 03] of EC9706 cells were enhanced by DHA± DDP treatment. Western blot showed that the expression of apoptotic factor caspase-3 protein was increased by DHA±DDP treatment. Meanwhile, the protein expressions of anti-apoptotic factor （Bcl-2） and ERS-related factors（GRP78 and IRE-1 ） were significantly inhibited by DHA＋DDP treatment （P= 0. O1 ）. However, the salutary effects of DHA were reversed by ERS activation tunicamycin. Conclusion DHA enhances the sensitivity of esophageal car- cinoma cell line EC9706 to cisplatin, the mechanism of which may be the suppression of ERS response.