TET对舌鳞癌细胞SCC9生长、运动和裸鼠体内成瘤的影响及机制研究

Effects of TET on growth, movement of tongue squamous cell SCC9 and tumorigenesis in nude mice and its mechanism

目的探究汉防己甲素(TET)对舌鳞癌细胞SCC9生长、运动和裸鼠体内成瘤的影响及机制。方法体外培养人舌鳞癌细胞SCC9,分为空白对照组、低剂量TET组(TET 2.5μmol/L)、中剂量TET组(TET 5μmol/L)、高剂量TET组(TET 10μmol/L),分别使用对应剂量的TET预处理。使用克隆形成实验检测细胞生长;使用流式细胞术检测细胞凋亡情况;使用Transwell检测细胞侵袭情况;使用Western blot检测Ki67、CaspasE-3、血管内皮细胞生长因子(VEGF)、PI3K、p-PI3K、AKT、p-AKT、mTOR、p-mTOR蛋白表达水平。选取裸鼠60只,采用0.002%4-硝基喹啉-1-氧化物自然饮水喂养36周诱发建立舌鳞癌动物模型,分别使用低剂量TET 12.5 mg/(kg·d)、中剂量TET 25 mg/(kg·d)和高剂量TET 50 mg/(kg·d)灌胃处理,29 d后检测瘤子重量,免疫组化染色检测舌鳞癌组织Ki67、Caspase-3和VEGF的表达。结果与空白对照组比较,使用TET处理后舌鳞癌细胞克隆形成率、单位面积内侵袭细胞数目,p-PI3K、p-AKT、p-mTOR蛋白表达降低,且高剂量TET组低于中剂量TET组,中剂量TET组低于低剂量TET组;与空白对照组比较,其他各组PI3K、AKT及mTOR蛋白表达水平差异无统计学意义;与空白对照组比较,使用TET处理后Caspase-3蛋白表达水平升高,且高剂量TET组高于中剂量TET组,中剂量TET组高于低剂量TET组。小鼠体内实验可以看出,与空白对照组相比,使用TET灌胃处理的裸鼠肿瘤质量、Ki67蛋白表达水平及阳性率、VEGF蛋白表达水平及阳性率降低(P<0.05),且灌胃浓度越高上述指标越低;与空白对照组比较,使用TET灌胃处理的裸鼠Caspase-3蛋白表达水平及阳性率升高(P<0.05),且灌胃浓度越高上述指标越高。结论 TET可以通过抑制PI3K/AKT/mTOR信号通路抑制舌鳞癌细胞的生长、运动,促进舌鳞癌细胞的凋亡,在一定浓度范围内呈浓度依赖性。

Objective To explore effects of tetrandrine(TET) on growth, movement of tongue squamous cell SCC9 and tumorigenesis in nude mice and its mechanism. Methods Human tongue squamous cells SCC9 were cultured in vitro. They were divided into blank group, low-dose TET group(TET 2.5 μmol/L), medium-dose TET group(TET 5μmol/L) and high-dose TET group(TET 10 μmol/L). And these groups were pretreated with corresponding doses of TET. Cell growth was detected by colony formation assay. The apoptosis was detected by flow cytometry. Cell invasion was detected by Transwell. The expression levels of Ki67, Caspase-3, vascular endothelial cell growth factor(VEGF), PI3 K, p-PI3 K, AKT, p-AKT, mTOR and p-mTOR proteins were detected by Western blot. A total of 60 nude mice were selected. 0.002% 4-nitroquinoline-1-oxide natural drinking water was applied to feed them for 36 weeks to establish animal models of tongue squamous cell carcinoma. They were given low-dose TET[12.5 mg/(kg·d)], medium-dose TET [25 mg/(kg·d)] and high-dose TET [50 mg/(kg·d)] for gavage treatment. 29 d later, tumor weight was detected. The expression of Ki67, caspase-3 and VEGF in tongue squamous tissues was detected by immumohistochemical staining. Results Compared with those in blank group, colony formation rate of tongue squamous cells, number of invasive cells per unit area, expression of p-PI3 K, p-AKT and p-mTOR protein were significantly decreased after treated with TET. The above indexes were the highest in high-dose TET group, followed by medium-dose TET group and low-dose TET group. Compared with those in blank group, there was no significant difference in expression levels of PI3 K, AKT and mTOR protein among the other three groups. Compared with those in blank group, expression level of Caspase-3 protein was significantly increased after treated with TET. The above index was the highest in high-dose TET group, followed bymedium-dose TET group and low-dose TET group. In vivo experiments of mice showed that compared with those in blank group, tumor mass of nude mice, expression level and positive rate of Ki67 protein and VEGF were significantly decreased after gavage treatment with TET(P<0.05), and the higher the gavage concentration, the lower the above indexes. Compared with those in blank group, expression level and positive rate of Caspase-3 protein in nude mice were significantly increased after gavage treatment with TET(P<0.05), and the higher the gavage concentration, the higher the above indexes. Conclusion TET can inhibit growth and movement of tongue squamous cells by inhibiting PI3 K/AKT/mTOR signaling pathway, and promote apoptosis of tongue squamous cells, which shows concentration-dependence within certain concentration range.