川楝素抑制AKT/GSK-3β/β-catenin通路对恶性黑色素瘤细胞增殖、凋亡和上皮间质转化的影响

Effect of toosendanin on proliferation,apoptosis and epithelial-mesenchymal transition of malignant melanoma cells by inhibiting AKT/GSK-3β/β-catenin pathway

目的探讨川楝素(TSN)对恶性黑色素瘤(MM)细胞增殖、凋亡和上皮间质转化(EMT)的影响及其作用机制。方法将MM细胞分为对照(NC)组、TSN低剂量组(10μmol/L)、TSN中剂量组(20μmol/L)、TSN高剂量组(40μmol/L)、SC79组[TSN+蛋白激酶B(AKT)激活剂(SC79)]、SC79+CP21R7组[TSN+SC79+糖原合成酶激酶3β(GSK-3β)抑制剂(CP21R7)]。采用CCK-8法测定细胞活力,流式细胞术检测细胞凋亡,划痕实验及Transwell检测迁移和侵袭,蛋白质印迹法(Western blot)检测B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、酶切含半胱氨酸的天冬氨酸蛋白水解酶-3(C-caspase-3)、波形蛋白(Vimentin)、上皮钙黏蛋白(E-cadherin)、神经钙黏蛋白(N-cadherin)、AKT、磷酸化AKT(p-AKT)Ser^(473)、GSK-3β、磷酸化GSK-3β(p-GSK-3β)Ser^(9)、β-链环蛋白(β-catenin)水平。结果MM细胞活力随着TSN浓度的升高逐渐降低(P<0.05)。与NC组比较,TSN低剂量组、TSN中剂量组、TSN高剂量组细胞活力、划痕愈合率、侵袭能力及Bcl-2、N-cadherin、Vimentin、p-AKT Ser^(473)、p-GSK-3β Ser^(9)、β-catenin水平降低,凋亡率及Bax、C-caspase-3、E-cadherin水平升高(P<0.05);与TSN高剂量组比较,SC79组细胞活力、划痕愈合率、侵袭能力及Bcl-2、N-cadherin、Vimentin水平升高,凋亡率及Bax、C-caspase-3、E-cadherin水平降低(P<0.05)。CP21R7可降低SC79对TSN治疗的抑制作用。结论TSN能抑制MM细胞增殖,促进凋亡,抑制EMT发生,其作用机制可能与TSN抑制AKT/GSK-3β/β-catenin通路有关。

Objective To investigate the effects of toosendanin(TSN)on the proliferation,apoptosis and epithelial-mesenchymal transition(EMT)of malignant melanoma(MM)cells and its mechanism.Methods The MM cells were divided into control(NC)group,TSN low dose group(10μmol/L),TSN medium dose group(20μmol/L),TSN high dose group(40μmol/L),SC79 group[TSN 40μmol/L+protein kinase B(AKT)activator(SC79)10 nmol/L],SC79+CP21R7 group[TSN 40μmol/L+SC7910 nmol/L+glycogen synthase kinase 3β(GSK-3β)inhibitor(CP21R7)10 nmol/L].CCK-8 assay was used to detect cell viability,flow cytometry was used to detect cell apoptosis,scratch test and Transwell were used to detect cell migration and invasion.The expression of B-cell lymphoma-2(Bcl-2),Bcl-2 associated X protein(Bax),enzyme digestion of cysteinyl aspartate specific proteinase-3(C-caspase-3),Vimentin,E-cadherin,N-cadherin,AKT,phosphorylated AKT(p-AKT)Ser^(473),GSK-3β,phosphorylated GSK-3β(p-GSK-3β)Ser^(9),β-catenin were detected by Western blot.Results The viability of MM cells decreased gradually with the increase of TSN concentration(P<0.05).Compared with NC group,the cell viability,scratch healing rate,invasion ability,Bcl-2,N-cadherin,Vimentin,p-AKT Ser^(473),p-GSK-3β Ser^(9) and β-catenin levels were decreased,and the apoptosis rate,Bax,C-caspase-3,E-cadherin levels were increased in TSN low dose group,TSN medium dose group and TSN high dose group(P<0.05).Compared with the TSN high dose group,the cell viability,scratch healing rate,invasion ability,Bcl-2,N-cadherin and Vimentin levels were significantly increased,and the apoptosis rate,Bax,C-caspase-3,E-cadherin levels were decreased in the SC79 group(P<0.05).CP21R7 reduced the inhibitory effect of SC79 on TSN treatment.Conclusion TSN can inhibit the proliferation of MM cells,promote apoptosis and reduce the occurrence of EMT.The mechanism may be related to the inhibition of AKT/GSK-3β/β-catenin pathway.