番泻苷A对胆囊癌细胞恶性生物学行为的影响及相关机制

The effect of Sennoside A on malignant biological behavior of gallbladder cancer cells and the related mechanism

目的探讨番泻苷A(SA)对胆囊癌细胞增殖、迁移、侵袭和糖酵解等恶性生物学行为的影响,并对其相关机制进行分析。方法体外培养人胆囊癌NOZ和SGC-996细胞,并分别将其分为空白对照组、SA低剂量组(25μmol/L)、SA中剂量组(50μmol/L)、SA高剂量组(100μmol/L)、H-SA+磷脂酰肌醇3激酶/蛋白激酶B(PI3K/AKT)信号通路激活剂(740Y-P)组。利用细胞计数实验、Transwell、流式细胞术和糖酵解试剂盒检测各组细胞的增殖、迁移、侵袭、凋亡和糖酵解能力;蛋白免疫印迹实验检测各组细胞中PI3K、AKT、p-PI3K、p-AKT的蛋白水平。使用NOZ细胞构建裸鼠成瘤模型,分别用生理盐水和10 mg/kg SA腹腔注射,比较两组小鼠的成瘤能力,免疫组化检测肿瘤中Ki-67的表达水平。结果与空白对照组细胞相比,各SA处理组细胞的增殖、迁移、侵袭和糖酵解能力降低,而凋亡水平显著上调,差异均有统计学意义(均P<0.05)。SA处理胆囊癌细胞使p-PI3K和p-AKT水平明显降低;而740Y-P激活PI3K/AKT通路后细胞的增殖、迁移、侵袭和糖酵解能力上调,凋亡水平降低,差异均有统计学意义(均P<0.05)。体内成瘤实验标明,第28天时与空白对照组相比,SA处理组小鼠的肿瘤体积降低[(1051.32±130.29)mm 3比(575.07±170.54)mm 3,P=0.0003],肿瘤重量减轻[(1.04±0.24)g比(0.58±0.13)g,P=0.0019],Ki-67表达平均光密度值降低[(77.00±7.00)比(33.33±7.51),P=0.0018]。结论SA通过调控PI3K/AKT通路抑制胆囊癌细胞的增殖、迁移、侵袭和糖酵解等恶性生物学行为。

ObjectiveTo investigate the effects of Sennoside A(SA)on the proliferation,migration,invasion,glycolysis and other malignant biological behaviors of gallbladder carcinoma cells,and to analyze the related mechanisms.MethodsHuman gallbladder carcinoma cell lines,NOZ and SGC-996,were cultured in vitro and divided into control group,SA low dose group(L-SA,25μmol/L),SA medium dose group(M-SA,50μmol/L)and SA high dose group(H-SA,100μmol/L),and H-SA+phosphatidylinositol 3-kinase/protein kinase B(PI3K/AKT)signaling pathway activator 740Y-P group,respectively.The proliferation,migration,invasion,apoptosis and glycolysis of gallbladder cancer cells in each group were detected by cell counting assay,Transwell,flow cytometry and glycolysis kit.The protein levels of PI3K,p-PI3K,AKT and p-AKT were detected by Western blot assay.NOZ cells were used to construct tumor model of nude mice,and the mice were divided into saline treatment group and 10 mg/kg SA treatment group.The tumor formation ability of the two groups of mice was compared,and the expression level of Ki-67 in tumor of the two groups was detected by immunohistochemical assay.ResultsCompared with control group,the proliferation,migration,invasion,glycolysis ability,the expression levels of p-PI3K and p-AKT were significantly decreased in SA treatment groups,while the apoptosis level was significantly up-regulated,all differences were statistically significant(P<0.05).Compared with H-SA group,the proliferation,migration,invasion and glycolysis of H-SA+740Y-P group cells were up-regulated,while the apoptosis level was significantly decreased,all differences were statistically significant(P<0.05).In vivo tumorigenesis experiments showed that,compared with the control group,the tumor volume of the SA-treated mice was reduced at day 28[(1051.32±130.29)mm 3 vs(575.07±170.54)mm 3,P=0.0003],the tumor weight was reduced[(1.04±0.24)g vs(0.58±0.13)g,P=0.0019],and the average optical density of Ki-67 expression was reduced[(77.00±7.00)vs(33.33±7.51),P=0.0018].ConclusionSA can inhibit the proliferation,migration,invasion and glycolysis of gallbladder carcinoma cells by regulating PI3K/AKT pathway.