δ-生育三烯酚诱导结肠癌细胞SW620死亡的机制研究

STUDY ON THE MECHANISM OF δ-TOCOTRIENOL-INDUCED CELL DEATH IN COLON CANCER CELLS SW620

目的研究δ-生育三烯酚诱导人结肠癌细胞SW620死亡方式,及其与内质网应激之间的关系。方法 (1)以不同剂量的δ-生育三烯酚作用于人结肠癌细胞SW620,DAPI染色,观察对SW620细胞形态学改变。(2)Caspase-3的活性测定,不同浓度δ-生育三烯酚作用SW620细胞,检测细胞中caspase-3活性。(3)DNA Ladder法检测典型凋亡阳性药物紫杉醇(PTX)及δ-生育三烯酚作用后DNA的降解情况。(4)免疫组织化学方法检测内质网膜钙连蛋白表达。(5)蛋白免疫印迹方法,测定δ-生育三烯酚对内质网应激标记蛋白(Bip)表达的影响。结果 (1)δ-生育三烯酚处理后的SW620细胞经DAPI染色,在荧光显微镜下观察,各组细胞均未出现以细胞核的半月状改变及凋亡小体出现为特征的典型凋亡现象。但可见到细胞内空泡。(2)不同浓度的δ-生育三烯酚(0、5、10、15和20·mol/L)对SW620细胞作用24h,经caspase-3分光光度法检测,随作用剂量增加caspase-3活性未显著增强(P>0.05)。(3)PTX作用24 h,可使结肠癌细胞SW620核内DNA发生梯度化降解,而20·mol/L的δ-生育三烯酚作用相同时间无同样现象。(4)20·mol/L的δ-生育三烯酚对SW620细胞作用24h,钙连蛋白表达量增多且成空泡状。内质网应激标记蛋白Bip表达量增加。结论(1)δ-生育三烯酚诱导人结肠癌细胞SW620死亡未出现caspase依赖性的经典凋亡现象。(2)δ-生育三烯酚诱导结肠癌细胞发生caspase非依赖性死亡可能与内质网应激有关。

Objective To study the process of δ-tocotrienol-induced cell death in human colon cancer cells SW620, and its mechanism. Methods （1） Colon cancer cells SW620 were treated with different doses of δ-tocotrienol. Morphological changes were observed by DAPI staining. （2） Cellular caspase-3 activity in SW620 cells treated with different concentrations of δ-tocotrienol was examined by the caspase-3 colorimetric substrate assay kit. （3） DNA degradation of SW620 cells treated with typical apoptotic positive drug paclitaxel （PTX） and 8-tocotrienol was determined by DNA ladder. （4） The protein expression of calnexin was detected by immunohistochemistry. （5） The protein expressions level of Bip was detected by Western blotting. Results （1） No apoptotic bodies were noted after δ-tocotrienol treatment, （2） There was no significant difference in the caspase-3 activity after δ-toeotrienol treatment for 24 h （P〉0.05）. （3） After 24 h, PTX induced nuclear DNA degradation in SW620 colon cancer cells, and δ-tocotrienol did not show the same phenomenon. （4） The protein expression of calnexin and Bip was upregutated by δ-tocotrienol. Conclusion δ-Tocotrienol did not induce caspase-dependent programmed cell death （CD-PCD） in human colon cancer cells SW620 and its effect may be related to endoplasmic reticulum stress.