摘要目的 探讨电化学疗法(ECT)对人乳腺癌MDA-MB231细胞株抗侵袭迁移的作用机制.方法 采用噻唑蓝(MTT)法检测1～15 C的ECT和(0.05～ 1.00) μmol/L MK2206作用MDA-MB231细胞24h后的生长抑制率;Transwell实验检测5 C ECT对细胞侵袭、迁移能力改变;用反转录-聚合酶链反应(RT-PCR)、Western blot法检测1～10 C ECT作用MDA-MB231细胞24h后血管内皮生长因子(VEGF)、基质金属蛋白酶(MMP)-2、第10号染色体上缺失与张力蛋白同源的磷酸酯酶基因(PTEN)、蛋白激酶B(Akt)基因mRNA和蛋白的表达.结果 Transwell实验证实5 C ECT作用后MDA-MB231细胞侵袭、迁移能力下降;RT-PCR结果显示5C、10C的ECT组与空白对照组比较,随着ECT电量的提高,侵袭基因VEGF、MMP-2的表达量逐渐降低,5 C ECT组VEGF基因的相对表达量为0.591±0.038,MMP-2基因为0.601 ±0.045,与空白对照组比较差异有统计学意义(P＜0.01);磷酸肌醇3激酶(PI3 K)/Akt信号通路中的PTEN基因表达量逐渐增高而Akt基因的表达量逐渐降低;相同抑制率的ECT组(1 C)和MK2206组(0.05 μmol/L)比较,两组降低VEGF、MMP-2、Akt基因表达量的差异无统计学意义(P＞0.05),MK2206组能增高PTEN基因的表达量.Western blot检测结果显示3C、5C、10C不同库仑剂量的ECT治疗组与空白对照组比较,随着ECT库仑剂量的提高VEGF、MMP-2蛋白表达量逐渐降低[3 C ECT组VEGF的相对表达量为0.426±0.072,MMP-2的相对表达量为0.214±0.016,与空白对照组比较差异有统计学意义(P＜0.01)],PI3K/Akt信号通路中的磷酸化Akt (p-Akt)蛋白表达量逐渐降低,PTEN蛋白表达量逐渐增高,总Akt基因表达量无明显变化;相同抑制率的ECT组1C和MK2206组0.05 μmol/L比较,ECT组更能降低VEGF、MMP-2的蛋白表达量且更能增高PTEN的蛋白表达量,但MK2206组更能降低p-Akt的蛋白表达量,对总Akt蛋白表达量两组差异无统计学意义(P＞0.05).结论 ECT具有抗乳腺癌侵袭性转移的作用,其机制可能是与抑制PI3K/Akt信号通道相关.

abstractsObjective To explore the mechanism of electrochemical therapy (ECT) through phosphatidylinositol 3 kinase(PI3K)/protein kinase B (Akt) signal pathway inhibiting migration and invasion of breast cancer cell lines.Methods MTT assay was used to measure the inhibtion rate of 1-15 C ECT coulomb and (0.05-1.00) μmol/L MK2206 on breast cancer MDA-MB231 cell lines after 24 h.Transwell migration and matrigel invasion assays were performed to evaluate the ability of 5 C ECT migration and matrigel invasion respectively.reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting were used to evaluate the mRNA and protein expression levels of vascular endothelial growth factor (VEGF),matrix metalloproteinase (MMP)-2,phosphatase and tensin homologue deleted on chromosometen (PTEN),Akt and phosphorylated Akt (p-Akt) in MDA-MB231 cells.Methods Transwll showed ECT could inhibit the ability of cell migration and invasion obviously.RT-PCR showed the relative expression levels of VEGF and MMP-2 in 5 C and 10 C ECT groups were reduced gradually (for VEGF:0.591 ±0.038,and for MMP-2:0.601 ±0.045 in 5 C ECT group;for VEGF,0.453 ±0.003,and for MMP-2:0.410 ±0.064 in 10 C ECT group,P ＜0.01).The relative gene expression of Akt was reduced gradually,but PTEN increased gradually.The expression levels of VEGF,MMP-2 and Akt in the cells treated separately by 1 C ECT and 0.05 μmol/L MK2206 showed no significant difference at the same inhibition rate.Western blotting showed expression levels of VEGF and MMP-2 in the 3 C,5 C and 10 C ECT groups were reduced gradually (for VEGF,0.426 ±0.046 and for MMP-2,0.223 ±0.018 in 5 C ECT group,P ＜0.01).The protein expression of p-Akt was also reduced gradually,but PTEN increased.The total Akt protein expression was unchanged (P ＞ 0.05).At the same inhibitory rate of 1 C ECT and MK2206 (0.05 μmol/L),the ECT group was more effective than MK2206 group in reducing the protein expression of VEGF,MMP-2 and p-Akt and increasing the protein expression of PTEN.The total Akt protein expression showed no significant difference between two groups (P ＞ 0.05).Conclusion The effects of ECT inhibiting migration and invasion of breast cancer cells probably involve PI3K/Akt signal pathway.