斑蝥素对人恶性黑色素瘤A375细胞增殖和凋亡的影响及其机制

Influence of cantharidin on proliferation and apoptosis of malignant melanoma A375 Cells and its mechanism

目的探讨不同浓度斑蝥素(CTD)对人恶性黑色素瘤A375细胞增殖、凋亡的影响及其可能机制。方法体外培养A375细胞,分为对照组和不同浓度CTD处理组(0.625-5 mg/L CTD),分别培养24、48、72、96 h。MTS法检测CTD作用后A375细胞增殖情况,流式细胞术检测细胞周期及细胞凋亡变化,Westernblot检测信号通路蛋白ERK1/2、AKT磷酸化水平的改变。结果实验浓度下,CTD对A375细胞的增殖均具有明显抑制作用,呈时间和剂量依赖性(P<0.05)。细胞周期呈G0/G1期阻滞(P<0.05),G0/G1期细胞比例由对照组28.22%增加至CTD5 mg/L组69.61%,相应G2/M期和S期细胞比例明显下降(P<0.05)。A375细胞凋亡率显著增加,由对照组6.85%增加至CTD5 mg/L组69.76%。细胞周期阻滞和细胞凋亡具有剂量效应。Westernblot结果显示ERK1/2、AKT蛋白磷酸化水平在CTD 2.5 mg/L组、5 mg/L组明显下调,0.625 mg/L组、1.25 mg/L组未表现出显著性差异。结论 CTD对人恶性黑色素瘤A375细胞具有明显抑制作用,可能与抑制细胞增殖、诱导凋亡及G0/G1期细胞周期阻滞有关。另外,ERK和AKT信号通路可能参与了此调节作用。

Objective To investigate the effect of cantharidin（CTD） on proliferation and apoptosis of malignant melanoma A375 cells and its mechanism.Methods A375 cells were cultured in vitro for 24,48,72 and 96 h respectively.The cells were divided into control group and CTD groups（0.625-5 mg/L CTD）.MTS assay was employed to evaluate the inhibitory effect of CTD on A375 cells.Cell apoptosis and cell cycle were detected by Flow cytometry.The protein ERK1/2 and AKT phosphorylation expressions were detected by western blot.Results MTS results indicated that the proliferation of A375 cells were significantly inhibited by different treatment of CTD with obvious time-and dosage-dependent pattern（P〈0.05）.Cell apoptosis and G0/G1 phase arrest occurred in each group with CTD treated.The percentage of G0/G1 phase increased from 28.22% of control group to 69.61% of 5 mg/L CTD group（P〈0.05）.The percentage of G2/M and S phase had a significantly decrease.And the apoptosis rate of A375 cells increased from 6.85% to 69.76% accordingly（P〈0.05）.Cell cycle phase arrest and apoptosis rate increased in a dose-dependent manner.The protein expression of Perk1/2 and p AKT were down regulated at the dose of 2.5 mg/L and 5 mg/L.No significant change was found in 0.625 mg/L,1.25 mg/L CTD groups.Conclusions CTD has obvious inhibitory effects on melanoma A375 cells,which is related to proliferation inhibition,apoptosis induction and G0/G1 phase arrest.Moreover,ERK pathway and AKT pathway may be involved in the regulations.