溴苯基姜黄素对胆管癌细胞凋亡、迁移和侵袭的影响及机制研究
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篇名: 溴苯基姜黄素对胆管癌细胞凋亡、迁移和侵袭的影响及机制研究
TITLE: Study on the Effect and Its Mechanism of Bromophenylcurcumin on Apoptosis ,Migration and Invasion of Cho- langiocarcinoma Cells
摘要: 目的:研究溴苯基姜黄素(GL63)对人胆管癌RBE细胞凋亡、迁移和侵袭的影响,并基于Janus激酶(JAK)/信号转导及转录激活因子(STAT)信号通路探讨其作用机制。方法:采用MTT法检测不同浓度GL63[0(空白对照,下同)、1.25、2.5、5、10、20、40μmol/L]作用48h对RBE细胞增殖的影响,并计算其半数抑制浓度(IC50);采用结晶紫染色法检测不同浓度GL63(0、5、10、20μmol/L)作用48h对细胞集落形成的影响;分别采用流式细胞术、Hoechst33342染色法、细胞划痕实验、Transwell小室侵袭实验检测不同浓度GL63(0、5、10、20μmol/L)作用24h对细胞周期分布、凋亡情况以及细胞迁移和侵袭能力的影响;采用Westernblotting法检测不同浓度GL63(0、5、10、20μmol/L)作用24h对细胞中JAK2/STAT3信号通路肿瘤相关蛋白表达的影响。结果:不同浓度GL63组(1.25~40μmol/L)RBE细胞的增殖抑制率均较空白对照组显著升高(P<0.01),并呈剂量依赖趋势;其IC50为(8.46±1.30)μmol/L。与空白对照组比较,不同浓度GL63组(5、10、20μmol/L)RBE细胞的集落形成抑制率均显著降低(P<0.01);G0/G1期细胞占比均显著升高,S期细胞占比均显著降低(P<0.01);细胞凋亡率均显著升高(P<0.01),且细胞核呈浓染致密的固缩形态并可见凋亡小体;细胞迁移愈合率均显著降低(P<0.01),穿过基底膜的侵袭细胞数均显著减少(P<0.01);细胞中磷酸化JAK2、磷酸化STAT3、B淋巴细胞瘤-2(Bcl-2)、基质金属蛋白酶2(MMP-2)、MMP-9、前体胱天蛋白酶9(Pro-caspase-9)、Pro-caspase-3蛋白表达水平均显著降低,Bcl-2相关x蛋白(Bax)、细胞色素C、裂解Caspase-9(Cleaved-caspase-9)、Cleaved-caspase-3蛋白表达水平均显著升高(P<0.01)。结论:GL63能通过抑制JAK2/STAT3信号通路来实现对人胆管癌RBE细胞增殖、迁移和侵袭的抑制并诱导其发生凋亡。
ABSTRACT: OBJECTIVE:To study t he effects of bro mophenylcurcumin(GL63)on the apoptosis ,migration and invasion of human cholangiocarcinoma RBE cells ,and to investigate its mechanism based on JAK/STAT signaling pathway. METHODS :MTT assay was used to detect the effects of different concentrations of GL 63 [0(blank control ,similarly hereinafter ),1.25,2.5,5,10, 20,40 μ mol/L] on the proliferation of RBE cells after 48 h treatment ;the IC 50 was calculated. The effects of different concentrations of GL 63(0,5,10,20 μmol/L)on colony formation were detected by crystal violet staining after 48 h treatment. Flow cytometry ,Hoechst 33342 staining,cell scratch test and Transwell chamber invasion test were used to detect the effects of different concentrations of GL 63(0,5,10,20 μmol/L)on cell cycle distribution ,apoptosis,migration and invasion ability after 24 h treatment. Western blotting assay was adopted to detect the effects of different concentration of GL 63(0,5,10,20 μmol/L) on the expression of JAK 2/STAT3 signal pathway associated proteins. RESULTS :The proliferation inhibition rates of RBE cells in different concentrations of GL 63 groups(1.25-40 μmol/L)were significantly increase d,compared with blank control group (P< 0.01),and showed a dose-dependent trend ,with IC 50 of (8.46±1.30)μmol/L. Compared with blank control group, 85917439。E-mail:zhaoji-an-88@163.com inhibition rates of RBE cell colony formation were significantly decreased in different concentrations (5,10,20 μmol/L)of GL 63 groups(P<0.01). The percentage of RBE cells at G 0/G1 phase increased significantly ,while that at S phase decreased significantly (P<0.01). The apoptotic rate increased significantly(P<0.01),and the nucleus showed dense pyknosis and apoptotic bodies. The rate of cell migration and healing was significantly decreased (P<0.01),and the number of invasive cells through basement membrane was significantly decreased (P< 0.01). The protein expression of p-JAK 2, p-STAT3, Bcl-2, MMP-2, MMP-9, Pro-caspase-9 and P ro-caspase-3 were down-regulated significantly while the expression of Bax ,Cyt-c,Cleaved-caspase-9 and Cleaved-caspase- 3 were up-regulated significantly(P<0.01). CONCLUSIONS :GL63 may inhibit the proliferation ,migration and invasion of RBE cells and promote its apoptosis by inhibiting JAK 2/STAT3 signal pathway.
期刊: 2021年第32卷第04期
作者: 赵冀安,马艳荣,聂文佳,董梁,刘文聪,谷敬锋
AUTHORS: ZHAO Ji ’an,MA Yanrong ,NIE Wenjia ,DONG Liang,LIU Wencong ,GU Jingfeng
关键字: 溴苯基姜黄素;胆管癌;RBE细胞;Janus激酶2/信号转导及转录激活因子3信号通路;迁移;侵袭;凋亡
KEYWORDS: Bromophenylcurcumin;Cholangiocarcinoma;RBE cell ;JAK2/STAT3 signal pathway ;Migration;Invasion;
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