Aloperine Promotes Autophagy And Apoptosis In Prostate Cancer Cells By Inhibiting The PI3K/Akt/mTOR Signaling Pathway

Objective:To investigate the effect of Aloperine on the inhibition of prostate cancer cells in vitro and its possible molecular mechanism.Methods: 1.Human prostate cancer cells(LNCaP and PC3)were purchased and cultured,subcultured and cryopreserved.The experimental groups were: control group,ALO(25 μM)group,ALO(50 μM)group,ALO(100 μM)group,ALO(200μM)group,ALO(400 μM)group,ALO(800 μM)group and ALO(1600 μM)group.The cell viability was detected by CCK8 method to screen the concentration of ALO.2.Experimental groups: control group,ALO(100 μM)group,ALO(200 μM)group,observed the morphological changes of cells by light microscope,observed cell clones by colony formation test,detected cell cycle and apoptosis by flow cytometry,detected the expression of autophagy index LC3Ⅱ by immunofluorescence technique,and detected the expression levels of key proteins of autophagy(LC3 Ⅱ,Beclin1,p62)and apoptosis(Caspase3,Bcl-2,Bax)by Western blot.3.Experimental groups: control group,ALO(100 μM)group,HCQ(20mg/mL)group,ALO(100μM)+ HCQ(20mg/mL)group.PC3 and LNCaP cells were pretreated with autophagy inhibitor chloroquine(HCQ,20mg/mL)for 1 hour,then PC3 and LNCaP cells were treated with ALO for 48 hours.,Western blot was used to detect the protein expression of LC3Ⅱ,Beclin-1,p62,Caspase3,Bcl-2 and Bax.4.Experimental groups: control group,ALO(50 μM)group,ALO(100 μM)groupand ALO(200 μM)group were used to detect the expression of key proteins in PI3K/AKT/ mTOR signal pathway by Western blot.Results: After different concentrations of ALO were given to PC3 and LNCaP,CCK8 and colony formation experiments indicated that ALO inhibited the cell viability of PC3 and LNCaP in a concentration dependent manner,and IC50 values of PC3 and LNCaP were calculated to be 180 ± 10 μM and 140 ± 10 μM respectively.When PC3 and LNCaP were treated with AlO(100 or 200 μM)for 24,48,72 and 96 hours,ALO also inhibited the cell viability of PC3 and LNCaP in a time-dependent manner(P < 0.05).PC3 and LNCaP were treated with AlO(100 or 200 μM)for 48 hours.Immunofluorescence detection showed that the number and intensity of green fluorescent dots in the cytoplasm of alo 100 and 200 μM group were higher than those of control group.Western blot showed that compared with control group,the expression levels of LC3Ⅱ,Beclin1,caspase 3 and Bax in ALO 100 and 200 μM group were higher,The expression of p62 and Bcl-2 protein decreased(P < 0.05).After pretreatment with chloroquine(HCQ,20mg/mL)for 1h,PC3 and LNCaP were treated with AlO(100 μ m)for 48 h.Compared with AlO(100 μM)group,the expression levels of LC3Ⅱ and Beclin1 in the two cell lines of AlO(100 μM)+HCQ(20mg/mL)group decreased(P < 0.05),while the expression levels of Caspase3 and Bax were significantly increased(P < 0.01).In addition,there was no significant difference in the protein expression of PI3 K,Akt and mTOR between the two groups(P > 0.05).There was no significant difference in the protein expression of p-PI3 K,p-Akt and p-mTOR between the two groups(P > 0.05).There was no significant difference in the protein expression of p-PI3 K,p-Akt and p-mTOR between the two groups(P > 0.05)P-PI3 K,p-Akt and p-mTOR protein expression of PC3 and LNCaP cells in 200 μM group decreased significantly(P < 0.01).Conclusion: 1.Aloperine inhibited the growth of PC3 and LNCaP by promotingapoptosis and autophagy;2.Inhibition of autophagy can reduce apoptosis activity of PC3 and LNCaP induced by aloperine;3.Aloperine may inhibit the proliferation of PC3 and LNCaP by inhibiting PI3 K / Akt / mTOR signaling pathway and promoting apoptosis and autophagy.