姜黄素通过β-连环蛋白/B细胞淋巴瘤/白血病-9通路调控胃癌细胞自噬的研究

Study of curcumin regulation of autophagy in gastric cancer cells by the catenin/B cell lymphoma/leukemia-9 pathway

目的:探讨姜黄素通过β-连环蛋白(β-catenin)/B细胞淋巴瘤/白血病-9(bcl-9)通路调控胃癌细胞自噬的机制。方法:动物水平上,利用小鼠胃癌细胞(MFC)接种于裸鼠右侧腋下成瘤,建立胃癌造模,分为模型组、低姜黄素组、中姜黄素组、高姜黄素组,每组6只。计算各组小鼠抑瘤率,采用苏木精-伊红(HE)染色法观察瘤体病理组织形态学,采用蛋白质免疫印迹法检测各组肿瘤组织自噬标志蛋白及β-catenin/bcl-9信号通路相关蛋白的表达水平。细胞水平上,利用氯化锂(LiCl)激活β-catenin/bcl-9信号通路,用0、10、20、40μmol/L姜黄素处理BGC-823细胞48 h,通过噻唑蓝(MTT)法、流式细胞术、吖啶橙染色及蛋白质印迹法(Western blot)检测细胞活力、凋亡、自噬水平及相关蛋白的表达。结果:动物水平,低姜黄素组、中姜黄素组、高姜黄素组抑瘤率高于模型组[(17.54±3.88)%、(26.87±5.12)%、(37.98±7.09)%比(0.00±0.00)%,F=162.010,P<0.01];低姜黄素组、中姜黄素组、高姜黄素组LC-Ⅱ表达高于模型组(1.54±0.09、1.89±0.12、2.75±0.28比1.00±0.04,F=5.983,P<0.05),p62表达低于模型组[(0.62±0.05、0.47±0.05、0.39±0.04比1.00±0.07,F=6.774,P<0.05),β-catenin表达低于模型组[(0.52±0.08、0.48±0.06、0.35±0.04比0.99±0.12,F=7.092,P<0.01),BCL9表达低于模型组[(0.54±0.06、0.46±0.05、0.31±0.02比0.99±0.10,F=7.365,P<0.01)。细胞水平,10、20、40μmol/L姜黄素BGC-823细胞活自噬水平高于对照组[(8.97±0.16)%,(17.68±0.55)%,(34.45±1.32)%比(5.65±0.12)%,F=52.932,P<0.05],LC-Ⅱ水平高于对照组[(0.23±0.00、0.32±0.00、0.95±0.01比0.18±0.00,F=83.028,P<0.05)、p62水平高于对照组(0.24±0.00、0.38±0.02、0.90±0.02比0.19±0.00,F=74.355,P<0.05),β-catenin水平高于对照组[(0.23±0.01、0.19±0.00、0.11±0.00比0.05±0.00,F=98.623,P<0.05)、BCL9水平高于对照组[(0.37±0.00、0.30±0.01、0.21±0.01比0.09±0.00,F=129.746,P<0.05)水平。β-catenin/BCL9信号通路激活剂LiCl预先处理细胞后,姜黄素诱导的BGC-823细胞自噬水平低于空白对照组[(1.47±0.22)%比(5.64±0.13)%,F=8.669,P<0.05]。结论:姜黄素能够通过下调β-catenin/bcl-9通路的表达,进而促进胃癌细胞自噬的发生。

Objective:To study the mechanism of curcumin regulating autophagy of gastric cancer cells throughβ-catenin/B cell lymphoma/leukemia-9(bcl-9)pathway.Methods:At the animal level,mouse gastric cancer MFC cells were inoculated into the right armpit of nude mice to establish gastric cancer models.The mice were divided into model group,low curcumin group,medium curcumin group and high curcumin group,with 6 mice in each group.The tumor inhibition rate of each group was calculated,and the pathological histomorphology of the tumor was observed by hematoxylin and eosin(HE)staining.The expression levels of autophagy marker proteins andβ-catenin/bcl-9 signaling pathway-related proteins in tumor tissues were detected by Western blotting.Cellular level,LiCl was used to activateβ-catenin/bcl-9 signaling pathway,BGC-823 cells were treated with 0,10,20,40μmol/L curcumin for 48 h,and cell viability,apoptosis,autophagy level and expression of related proteins were detected by methyl thiazolyl tetrazolium(MTT)assay,flow cytometry,acridine orange staining and Western blotting.Results:At the animal level,compared with the model group,the tumor inhibition rate of the low curcumin group,middle curcumin group and high curcumin group was significantly increased[(17.54±3.88)%,(26.87±5.12)%,(37.98±7.09)%vs.(0.00±0.00)%,F=162.010,P<0.01].Compared with model group,low the curcumin group,curcumin,high curcumin group in LC-Ⅱexpression increased(1.54±0.09,1.89±0.12,2.75±0.28 vs.1.00±0.04,F=5.983,P<0.05),and p62 expression was significantly decreased(0.62±0.05,0.47±0.05,0.39±0.04 vs.1.00±0.07,F=6.774,P<0.05),and the expression ofβ-catenin was significantly decreased(0.52±0.08,0.48±0.06,0.35±0.04 vs.0.99±0.12,F=7.092,P<0.01),and bcl-9 expression was significantly decreased(0.54±0.06,0.46±0.05,0.31±0.02 vs.0.99±0.10,F=7.365,P<0.01).At the cellular level,Compared with the control group,10,20,and 40μmol/L curcumin increased the level of active autophagy in BGC-823 cells[(8.97±0.16)%,(17.68±0.55)%,(34.45±1.32)%vs.(5.65±0.12)%,F=52.932,P<0.05],up-regulated LC-Ⅱ(0.23±0.00,0.32±0.00,0.95±0.01 vs.0.18±0.00,F=83.028,P<0.05),p62(0.24±0.00,0.38±0.02,0.90±0.02 vs.0.19±0.00,F=74.355,P<0.05)levels,while down-regulatedβ-catenin(0.23±0.01,0.19±0.01,0.19±±0.00,0.11±0.00 vs.0.05±0.00,F=98.623,P<0.05),bcl-9(0.37±0.00,0.30±0.01,0.21±0.01 vs.0.09±0.00,F=129.746,P<0.05)levels.Compared with the blank control group,the level of curcumin-induced autophagy in BGC-823 cells was significantly reduced after pretreatment withβ-catenin/bcl-9 signaling pathway activator LiCl[(1.47±0.22)%vs.(5.64±0.13)%,F=8.669,P<0.05].Conclusion:Curcumin can promote autophagy of gastric cancer cells by down-regulating the expression ofβ-catenin/bcl-9 pathway.This discovery provides a new idea and method for the treatment of gastric cancer,and also provides a theoretical basis for the application of curcumin in the treatment of gastric cancer.