Influences of Lupinol on the Proliferation,Apoptosis and Invasion of Cervical Cancer Cells by Regulating PI3K/AKT/mTOR Pathway-Mediated Autophagy / 实用妇产科杂志

ABSTRACT

Objective:To investigate the influences of lupinol on the proliferation,apoptosis and invasion of cer-vical cancer cells by regulating autophagy mediated by phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)/mammalian target of rapamycin(mTOR)pathway.Methods:The proliferation rate of human cervical cancer cell line HeLa cells treated with 0,10,25,50,70,90 μmol/L lupinol was determined,and the appropriate concentration of lupinol was screened out.HeLa cells cultured in vitro were randomly grouped into control group,low-dose lupinol group,high-dose lupinol group,740 Y-P group(PI3K activator),and high-dose lupinol+740 Y-P group.After group intervention with lupinol and 740 Y-P,MDC fluorescence staining was used to detect the forma-tion of autophagic vacuolation of HeLa cells in each group;western blot was used to detect the expression of au-tophagy and PI3K/AKT/mTOR pathway-related proteins in HeLa cells in each group.HeLa cells cultured in vitro were randomly grouped into control group,low-dose lupinol group,high-dose lupinol group,high-dose lupinol+rapamycin(Rapa),and high-dose lupinol+3-methyladenine(3-MA)group.After the intervention of high dose of lupinol,Rapa and 3-MA,the proliferation of HeLa cells in each group was detected by MTT assay and plate colony formation assay;flow cytometry was used to detect the apoptosis of HeLa cells in each group;transwell assay was used to detect the invasion of HeLa cells in each group;western blot was used to detect the expressions of proliferation,apoptosis and epithelial-mesenchymal transition-related proteins in HeLa cells in each group.Re-sults:Compared with the control group,the relative content of autophagic vacuoles,the protein expressions of Mi-crotubule-associated protein 1A/1 B-light chain 3(LC3)Ⅱ/LC3Ⅰ,and Beclin-1 in the low and high dose lupinol groups were all increased(P＜0.05),the phosphorylated PI3K(p-PI3K)/PI3K,phosphorylated AKT(p-AKT)/AKT,and phosphorylated mTOR(p-mTOR)/mTOR decreased(P＜0.05);the relative content of autophagic vac-uoles,the protein expressions of LC3Ⅱ/LC3Ⅰ,and Beclin-1 in the high-dose lupinol group were further increased compared with the low-dose lupinol group(P＜0.05),the p-PI3K/PI3K,p-AKT/AKT,and p-mTOR/mTOR were further decreased(P＜0.05);the relative content of autophagic vacuoles,the protein expressions of LC3Ⅱ/LC3Ⅰ,and Beclin-1 in 740 Y-P group decreased compared with the control group(P＜0.05),the p-PI3K/PI3K,p-AKT/AKT,and p-mTOR/mTOR increased(P＜0.05).Compared with the high-dose lupinol group,the relative content of autophagic vacuoles,the protein expressions of LC3Ⅱ/LC3Ⅰ,and Beclin-1 in the high-dose lupinol+740 Y-P group decreased(P＜0.05),the p-PI3K/PI3K,p-AKT/AKT,and p-mTOR/mTOR increased(P＜0.05).Com-pared with the control group,the cell proliferation rate,colony formation rate,invasion number,and the protein ex-pressions of proliferating cell nuclear antigen(PCNA),B cell lymphoma 2(Bcl-2)and Vimentin in the low and high dose groups of lupinol were all decreased(P＜0.05),the apoptosis rate,and the protein expressions of Bcl-2 as-sociated x protein(Bax)and zonula occludens protein 1(ZO-1)were all increased(P＜0.05);compared with the low-dose lupinol group,the cell proliferation rate,colony formation rate,invasion number,and the protein expres-sions of PCNA,Bcl-2 and Vimentin in the high-dose lupinol group were further decreased(P＜0.05),the apopto-sis rate,and the protein expressions of Bax and ZO-1 were further increased(P＜0.05).Compared with the high-dose lupinol group,the cell proliferation rate,colony formation rate,invasion number,and the protein expres-sions of PCNA,Bcl-2 and Vimentin in the high-dose lupinol+Rapa group were increased(P＜0.05),the apopto-sis rate,and the protein expressions of Bax and ZO-1 were decreased(P＜0.05);the cell proliferation rate,colo-ny formation rate,invasion number,and the protein expressions of PCNA,Bcl-2 and Vimentin in the high-dose lu-pinol+3-MA group were decreased(P＜0.05),the apoptosis rate,and the protein expressions of Bax and ZO-1 were increased(P＜0.05).Conclusions:Lupinol induces protective autophagy by inhibiting the PI3K/AKT/mTOR pathway,thereby promoting the apoptosis of cervical cancer cells and inhibiting their proliferation and inva-sion.Activation of autophagy attenuates the effects of lupinol on the proliferation,apoptosis and invasion of cervi-cal cancer cells.