银杏叶提取物通过JAK2/STAT3信号通路调控食管癌细胞EC9706增殖、凋亡、侵袭、迁移的研究

Ginkgo biloba extract regulates the proliferation, apoptosis, invasion and migration of esophageal cancer cell EC9706 through JAK2/STAT3 signaling pathway and its clinical significance

摘要:

目的 探讨银杏叶提取物对食管癌细胞EC9706增殖、凋亡、侵袭、迁移的影响及机制。方法 食管癌细胞EC9706分为对照组（不做任何处理）、不同浓度银杏叶提取物组（100 mg/L、200 mg/L、400 mg/L银杏叶提取物）、银杏叶提取物+白细胞介素-6（IL-6）组（20 ng/ml IL-6和400 mg/L银杏叶提取物）。采用CCK-8法检测各组细胞的光密度（OD）值；采用流式细胞术检测各组细胞凋亡率；Transwell实验检测各组细胞的迁移和侵袭；Western blotting检测细胞增殖核抗原Ki-67、裂解的半胱氨酸天冬氨酸蛋白酶-3（Cleaved-caspase-3）、基质金属蛋白酶-2（MMP-2）、基质金属蛋白酶-9（MMP-9）、磷酸化酪氨酸激酶2（p-JAK2）、磷酸化信号转导和转录激活因子3（p-STAT3）蛋白相对表达量。结果 对照组，100 mg/L、200 mg/L、400 mg/L银杏叶提取物组，银杏叶提取物+IL-6组细胞OD值比较，差异有统计学意义（P <0.05）；与对照组相比，不同浓度银杏叶提取物组EC9706细胞OD值均降低（P <0.05），细胞活性降低，且呈浓度依赖性；银杏叶提取物+IL-6组较400 mg/L银杏叶提取物组EC9706细胞活性升高（P <0.05）。5组的细胞凋亡率比较，差异有统计学意义（P <0.05）；与对照组相比，不同浓度银杏叶提取物组EC9706细胞凋亡率升高（P <0.05），且呈浓度依赖性；银杏叶提取物+IL-6组EC9706细胞凋亡率较400 mg/L银杏叶提取物组降低（P <0.05）。5组细胞的Ki-67、Cleaved-caspase-3蛋白相对表达量比较，差异有统计学意义（P <0.05）；不同浓度银杏叶提取物组Ki-67蛋白相对表达量较对照组均降低（P <0.05），银杏叶提取物+IL-6组Ki-67蛋白相对表达量较400 mg/L银杏叶提取物组升高（P <0.05）；不同浓度银杏叶提取物组Cleaved-caspase-3蛋白相对表达量较对照组均升高（P <0.05），银杏叶提取物+IL-6组Cleaved-caspase-3蛋白相对表达量较400 mg/L银杏叶提取物组降低（P <0.05）。对照组、400 mg/L银杏叶提取物组、银杏叶提取物+IL-6组细胞的MMP-2、MMP-9、p-JAK2、p-STAT3蛋白相对表达量比较，差异有统计学意义（P <0.05）；400 mg/L银杏叶提取物组较对照组均降低（P <0.05），银杏叶提取物+IL-6组较400 mg/L银杏叶提取物组均升高（P <0.05）。对照组、400 mg/L银杏叶提取物组及银杏叶提取物+IL-6组EC9706细胞迁移数、细胞侵袭数比较，差异有统计学意义（P <0.05）；400 mg/L银杏叶提取物组EC9706细胞迁移数、细胞侵袭数较对照组减少（P <0.05），银杏叶提取物+IL-6组EC9706细胞迁移数、细胞侵袭数较400 mg/L银杏叶提取物组增多（P <0.05）。结论 银杏叶提取物可能通过阻断JAK2/STAT3信号通路抑制食管癌细胞EC9706增殖、侵袭、迁移，促进细胞凋亡。

Abstract:

Objective To explore the effect and mechanism of Ginkgo biloba extract on proliferation, apoptosis, invasion and migration of esophageal cancer cell EC9706.Methods Esophageal cancer cells EC9706 were divided into control group (without any treatment), Ginkgo biloba extract group (400 mg/L Ginkgo biloba extract), and different concentrations of Ginkgo biloba extract group (100 mg/L, 200 mg/L, 400 mg/L Ginkgo biloba extract), Ginkgo biloba extract + IL-6 group (20 ng/mL IL-6 and 400 mg/L Ginkgo biloba extract). Western blot detection of cell proliferation nuclear antigen Ki-67 (Ki-67), cleaved cysteine-containing aspartate-specific proteases-3 (Cleaved-caspase-3), matrix metalloprotein (MMP) -2, MMP-9, phosphorylated tyrosine kinase (p-JAK2), phosphorylation signal transduction and transcription activator 3 (p-STAT3) protein expression; cell counting kit 8 (CCK-8) to detect cell activity; flow cytometry to detect apoptosis; Transwell detects cell migration and invasion.Results Compared with the NC group, the expression level of Ki-67 in EC9706 cells of different concentrations of Ginkgo biloba extract group was decreased (P < 0.05), cell activity was decreased (P < 0.05), and Cleaved-caspase-3 expression level was increased (P < 0.05), cell apoptosis rate was increased (P < 0.05), and was concentration-dependent. Compared with the NC group, the levels of MMP-2 and MMP-9 expression of EC9706 cells in the 400 mg/L Ginkgo biloba extract group were decreased (P < 0.05), the number of cell migration (P < 0.05), and the number of invasions were decreased (P < 0.05), p-JAK2 level and p-STAT3 expression level were decreased (P < 0.05). Compared with the Ginkgo biloba extract group, the EC9706 cell activity in the Ginkgo biloba extract + IL-6 group was increased (P < 0.05), the apoptosis rate was reduced (P < 0.05), the number of cell migration (P < 0.05), and the number of invasions increased (P < 0.05), Ki-67 level, MMP-2 level, MMP-9 expression level were increased (P < 0.05), Cleaved-caspase-3 level, p-JAK2 level, p-STAT3 expression level were decreased (P < 0.05)Conclusions Ginkgo biloba extract may inhibit the proliferation, invasion and migration of esophageal cancer cell EC9706 by blocking JAK/STAT3 signaling pathway and promote apoptosis.