目的 研究1-磷酸鞘氨醇(S1P)信号激活对乳腺癌BT549细胞增殖的影响.方法 将细胞分为对照组和实验组.实验组用0.1、1.0和10.0 μmol·L-1 S1P受体激动药SEW2871处理乳腺癌细胞72 h;对照组用含0.1％胎牛血清培养基培养.用噻唑蓝(MTT)法检测细胞增殖情况.建立乳腺癌BT549细胞过表达S1P受体的细胞模型,将转染的细胞分为空白质粒组(LUC)、野生型S1P受体组(WT)、S1P受体磷酸化位点突变组(MUT).用MTT法检测细胞增殖情况,并计算细胞增殖率;用克隆集落形成实验检测细胞克隆形成能力.用S1P受体拮抗药W146(10 μmol·L-1)及蛋白激酶B(AKT)信号通路抑制药MK2206(90 nmol·L-1)检测S1P信号在乳腺癌BT549细胞增殖中的作用,用蛋白质印迹法检测磷酸化信号传导与转录激活因子-3(p-STAT3)蛋白、原癌基因c-Myc蛋白的表达情况.结果 对照组和0.1、1.0和10.0μmol·L-1实验组的细胞增殖率分别为 1.00±0.03、1.13±0.06、1.06±0.10 和 1.07±0.03,SEW2871在0.1 μmol·L-1时可促进细胞增殖(P＜0.05).过表达S1P受体后,WT组、MUT组与LUC组相比,细胞增殖率和克隆集落形成数量均明显增加(均P＜0.05).用W146处理后,LUC组、MUT组、WT组的细胞相对增殖率分别为1.25±0.12、1.31±0.03 和 1.43±0.14;用 MK2206 处理后 LUC 组、MUT 组、WT组的细胞相对增殖率分别为0.87±0.15、0.77±0.03和0.88±0.02;WT组、MUT组与相应DSMO组比较,在统计学上差异均有统计学意义(均P＜0.01).用W146处理后,LUC组、MUT组、WT组的细胞克隆形成数量分别为(65.65±5.12),(141.48±5.63)和(93.64±5.14)个,WT 组、MUT 组与相应DSMO组比较,在统计学上差异均有统计学意义(均P＜0.05).MK2206处理后,LUC组、MUT组、WT组的p-STAT3蛋白相对表达水平分别为0.67±0.04、0.69±0.08和0.81±0.06,原癌基因c-Myc蛋白相对表达水平分别为1.69±0.03、0.70±0.10和 0.67±0.07,WT 组、MUT 组的上述指标与 DMSO 组比较,在统计学上差异均有统计学意义(均P＜0.05).结论 S1P信号激活可促进乳腺癌BT549细胞增殖,其机制可能与AKT及STAT3信号通路有关.
Objective To study the role of sphingosine-1-phosphate(S1P)signal on the proliferation of breast cancer BT549 cells.Methods Cells were divided into control group and experimental group,experimental group were treated with 0.1,1.0,10.0 μmol·L-1 S1P receptor agonist SEW2871 for 72 h.Control group was cultured with 0.1％fetal bovine serum.Cell proliferation was detected by methyl thiazolyl tetrazolium(MTT)assay.Cell models of overexpressing S1P receptors in BT549 were divided into three groups:blank plasmid group(LUC),wild type S1P receptor overexpression group(WT),S1P receptor phosphorylation site mutation overexpression group(MUT);the proliferation ratio was detected by MTT,the number of cell clones was counted by colony formation experiment.S1P antagonist W146(10 μmol·L-1)and protein kinase(AKT)signaling inhibitor MK2206(90 nmol·L-1)were used to detect the role of S1P signaling in the proliferation of breast cancer cells.The expression of phosphorylate signal transducer and activator of transcription 3(p-STAT3),c-Myc proteins were detected by Western blot.Results The growth ratio of BT549 cells in control group and 0.1,1.0,10.0 μmol·L-1experimental groups were 1.00±0.03,1.13±0.06,1.06±0.10 and 1.07±0.03,0.1 μmol·L-1 SEW2871 promot the cell proliferation(P＜0.05).Compared between WT group,MUT group and LUC group,the growth rate and the number of clonal colonies were increased after overexpression of S1P receptor(all P＜0.05).The growth ratio of BT549 cells after treatment with W146 and MK2206 in the LUC group,WT group and MUT group were 1.25±0.12,1.31±0.03,1.43±0.14 and 0.87±0.15,0.77±0.03,0.88±0.02.Compared between MUT group,WT group and corresponding DMSO group,the differences were statistically significant(all P＜0.01).The number of cell clony formation number after treatment with W146 were 65.65±5.12,141.48±5.63 and 93.64±5.14;compared between MUT,WT group and corresponding DMSO group,the differences were statistically significant(all P＜0.05).The relative protein expression levels of p-STAT3 in LUC group,WT group and MUT group were 0.67±0.04,0.69±0.08 and 0.81±0.06,the relative protein expression levels of proto-oncogene c-Myc were 1.69±0.03,0.70±0.10 and 0.67±0.07,compared between WT group,MUT group and corresponding DMSO group,the difference was statistically significant(P＜0.05).Conclusion S1P signaling can promote proliferation in breast cancer BT549 cells,and the mechanism could be related to AKT and STAT3 signaling pathway.
宋娟;王明;刘欣洋;张浩天;张奇;赵学梅;刘颖;崔红霞
齐齐哈尔医学院药学院,黑龙江齐齐哈尔 161006
药学
1-磷酸鞘氨醇受体激动药和拮抗药;蛋白激酶B信号通路抑制药;1-磷酸鞘氨醇信号;乳腺癌细胞;增殖
sphingosine 1-phosphate receptor agonist and antagonist;protein kinase B inhibitor;sphingosine 1-phosphate signaling;breast cancer cell;proliferation
《中国临床药理学杂志》 2024 (011)
1578-1582 / 5
黑龙江省省属高等学校基本科研业务费科研基金资助项目(2019-KYYWF-1240);齐齐哈尔市科技计划联合引导基金资助项目(LHYD-202019)
10.13699/j.cnki.1001-6821.2024.11.008
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