没食子乙醇提取物对结肠癌细胞JAK2/STAT3信号通路的调控作用研究

Study on regulation of ethanol extract of Turkish Galls on JAK2/STAT3 signaling pathway in colorectal cancer cells

目的基于蛋白酪氨酸激酶2(JAK2)/信号传导及转录活化因子3(STAT3)信号通路研究没食子乙醇提取物对结肠癌细胞HCT-116、Caco-2增殖、迁移的调控机制。方法采用CCK-8法检测0.05、0.1、0.2、0.3、0.4、0.5 mg/mL没食子乙醇提取物作用12、24、48、72 h后对HCT-116、Caco-2细胞增殖的影响。以0.1、0.3、0.5 mg/mL没食子乙醇提取物作用于HCT-116、Caco-2细胞24 h后,采用划痕实验测定细胞的迁移情况,采用荧光探针法检测细胞内活性氧(ROS)水平,采用酶联免疫吸附法检测细胞上清液中白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)水平,采用Western blot法检测细胞中JAK2、STAT3磷酸化水平和B细胞淋巴瘤2(Bcl-2)、Bcl-2相关X蛋白(Bax)的表达水平。结果与空白对照比较,0.05、0.1、0.2、0.3、0.4、0.5 mg/mL没食子乙醇提取物作用12、24、48、72 h后均可显著抑制细胞增殖(P<0.05)。0.1、0.3、0.5 mg/mL没食子乙醇提物作用24 h后,2种细胞的划痕愈合率和细胞上清液中IL-6、TNF-α水平,以及细胞中JAK2、STAT3磷酸化水平和Bcl-2蛋白表达水平均显著降低(P<0.05);细胞内ROS水平、Bax蛋白表达水平均显著升高(P<0.05)。结论没食子乙醇提取物可抑制结肠癌细胞HCT-116、Caco-2的增殖和迁移,其机制可能与增加细胞内ROS的积累,下调肿瘤微环境中炎症因子IL-6、TNF-α的表达和JAK2/STAT3信号通路中JAK2、STAT3的磷酸化水平,进而下调Bcl-2蛋白表达、上调Bax蛋白表达有关。

OBJECTIVE To study the regulatory mechanism of ethanol extract of Turkish Galls on proliferation and migration of colorectal cancer cells HCT-116 and Caco-2 based on janus kinase 2(JAK2)/signal transducer and activator of transcription 3(STAT3)signaling pathway. METHODS CCK-8 method was used to detect the effects of 0.05,0.1,0.2,0.3,0.4 and 0.5 mg/mL ethanol extract of Turkish Galls on the proliferation of HCT-116 and Caco-2 cells after treated for 12,24,48 and 72 h. After treated with 0.1,0.3,0.5 mg/mL ethanol extract of Turkish Galls for 24 h,the migrations of HCT-116 and Caco-2 cells were detected by scratch test;the level of reactive oxygen species(ROS) was detected by fluorescent probe method. The levels of interleukin-6(IL-6) and tumor necrosis factor-α (TNF-α) in cell supernatant were detected by ELISA. The phosphorylations of JAK2 and STAT3 as well as the expressions of B-cell lymphoma-2(Bcl-2)and Bcl-2 associated protein X(Bax)were detected by Western blot assay. RESULTS Compared with blank control,0.05,0.1,0.2,0.3,0.4 and 0.5 mg/mL ethanol extract of Turkish Galls could significantly inhibit cell proliferation after treated for 12,24,48,72 h(P<0.05). After treated with 0.1,0.3 and 0.5 mg/mL ethanol extract of Turkish Galls for 24 h,the scratch healing rate of 2 kinds of cells,the levels of IL-6 and TNF-α in the cell supernatant,the phosphorylation of JAK2 and STAT3 as well as the expression of Bcl-2 protein were all significantly decreased(P<0.05);the level of ROS and protein expression of Bax were increased significantly(P<0.05). CONCLUSIONS The ethanol extract of Turkish Galls can inhibit the proliferation and migration of HCT-116 and Caco-2 cells. The mechanism may be related with down-regulation of protein expression of Bcl-2 and up-regulation of protein expression of Bax by increasing the accumulation of intracellular ROS,down-regulating the expressions of inflammatory factors IL-6 and TNF-α and the phosphorylation of JAK2 and STAT3 in JAK2/STAT3 signaling pathway.