甲氟奎对食管鳞癌细胞增殖、迁移和侵袭的影响及其机制

Mechanism of mefloquine regulating proliferation, migration and invasion of esophageal squamous carcinoma cells

目的研究甲氟奎对食管鳞癌细胞增殖、迁移和侵袭的影响及其机制。方法运用四甲基偶氮唑盐微量酶反应比色法(MTT法)法检测甲氟奎(5、10、15、30μmol/L)对食管鳞癌细胞KYSE140的增殖的调控,筛选最适浓度30μmol/L;用β连环素(β-catenin)信号通路激活剂Wnt3a或二甲基亚砜(DMSO)与30μmol/L甲氟奎共处理KYSE140细胞48 h。Transwell法检测细胞的迁移和侵袭;蛋白质印迹法(Western blotting)检测细胞中β-catenin、周期素依赖激酶抑制剂p21、增殖细胞核抗原(PCNA)、神经钙黏素(N-cadherin)、上皮钙黏素(E-cadherin)的蛋白表达;实时荧光定量逆转录聚合酶链反应(qRT-PCR)检测细胞中β-catenin的表达。结果甲氟奎可呈浓度依赖性抑制食管鳞癌细胞KYSE140的增殖,最适浓度为15μmol/L;在24、48和72 h时对照组细胞吸光度分别为(0.21±0.02)、(0.52±0.04)、(1.19±0.10),15μmol/L甲氟奎组细胞吸光度分别为(0.21±0.01)、(0.34±0.03)、(0.68±0.06);对照组迁移和侵袭细胞数分别为(120±8)个、(80±6)个,甲氟奎组迁移和侵袭细胞数分别为(62±5)个、(45±4)个,甲氟奎可明显的抑制KYSE140细胞的增殖、迁移和侵袭,上调p21、E-cadherin,下调PCNA、N-cadherin;最重要的是,甲氟奎可明显的抑制β-catenin信号通路的关键基因β-catenin的表达,且激活β-catenin信号通路可逆转甲氟奎对KYSE140细胞的增殖、迁移和侵袭的抑制。结论甲氟奎可抑制食管鳞癌细胞的增殖、迁移和侵袭,其机制与抑制β-catenin信号通路的活性相关,将可为甲氟奎治疗食管鳞癌提供更充分的依据。

Objective To study the effect and mechanism of mefloquine on proliferation,migration and invasion of esophageal squamous carcinoma cells.Methods The methyl thiazolyl tetrazolium(MTT)method was used to detect the regulation of mefloquine(5,10,15,30μmol/L)on the proliferation of esophageal squamous cell carcinoma KYSE140,and the optimal concentration was 30μmol/L.β-catenin signaling pathway activator Wnt3a or dimethyl sulfoxide(DMSO)and 30μmol/L mefloquine co-treated KYSE140 cells for 48 h.Transwell method was used to detect cell migration and invasion;Western blotting was used to detect the expression levels ofβ-catenin,cyclin-dependent kinase inhibitor p21,proliferating cell nuclear antigen(PCNA),neural cadherin(N-cadherin),and epithelial cadherin(E-cadherin).Real-time fluorescent quantitative polymerase chain reaction(qRT-PCR)was adopted to detect the expression ofβ-catenin in cells.Results Mefloquine inhibited the proliferation of esophageal squamous cell carcinoma KYSE140 in a concentration-dependent manner.The optimal concentration was 15μmol/L.The A450 nm values of cells in the control group at 24,48 and 72 h were(0.21±0.02),(0.52±0.04),(1.19±0.10),and the A450 nm values of cells in the 15μmol/L mefloquine group were:(0.21±0.01),(0.34±0.03),(0.68±0.06).The number of migrant and invasive cells in the control group were(120±8)and(80±6),respectively,while the number of migrant and invasivecells in the mefloquine group were(62±5)and(45±4).Mefloquine significantly inhibited the proliferation,migration and invasion of KYSE140 cells,up-regulated p21 and E-cadherin,and down-regulated PCNA and N-cadherin.What was most important is thatmefloquine could significantly inhibit the expression ofβ-catenin,a key gene in theβ-catenin signaling pathway,and activation of theβ-catenin signaling pathway could reverse the inhibition of proliferation,migration and invasion of KYSE140 cells by mefloquine.Conclusion Mefloquine can inhibit the proliferation,migration and invasion of esophageal squamous carcinoma cells,and its mechanism is related to the inhibition ofβ-catenin signaling pathway,which will provide a more sufficient basis for the treatment of esophageal squamous carcinoma.