The Effect And Mechanism Of Aminoquinol,a Novel CDK4/6 Inhibitor,in The Treatment Of Prostate Cancer

Objective:To verify the anti-prostate cancer effect of the novel CDK4/6 inhibitor screened by computer and explore the underlying mechanisms.Methods:(1)The X-ray crystal structures of CDK4/6 protein domain were selected from PDB database.The compound database consists of 3,167 drugs that have been approved for marketing in many countries.Idock software was used to predict the binding structure of CDK4/6 with compound database.The more accurate rating RF scoring v3 software was used to reassess the intermolecular binding strength of all compounds.After excluding the compounds with antitumor activity reported by Pubmed and CNKI,we selected 6 compounds as candidate CDK4/6 inhibitors with the top binding affinity.(2)MTT assay was used to verify the anti-tumor effect of candidate CDK4/6 inhibitors at different concentrations(1,3,10,30μM)on prostate cancer cell lines PC-3 and DU 145,and the compounds with the highest activity were screened out.Palbociclib,a CDK4/6 inhibitor,was served as a positive control.(3)The effect of candidate CDK4/6 inhibitor(1μM)and Palbociclib(1μM)on the clone proliferation of prostate cancer cell lines PC-3 and DU 145 was tested by clonal formation assay.(4)The effect of candidate CDK4/6 inhibitors(5,10 μM)on the cell cycle distribution of PC3 and DU 145 was detected by flow cytometry.5μM Palbociclib was served as a positive control.(5)Flow cytometry was used to detect the effects of candidate CDK4/6 inhibitors(3,10,30μM)on apoptosis and necrosis of PC-3 and DU 145 cells.10μM Palbociclib was served as a positive control.(6)Western blotting was used to detect the changes of CDK4/6 signaling pathway in prostate cancer cells,including the changes of CDK4,CDK6,p-CDK4,p-CDK6,Rb,p-Rb,E2F-1 and Caspase-3.3μM Palbociclib was served as a positive control.(7)The xenografts models of prostate cancer in nude mice was established to verify the antitumor effect of the candidate CDK4/6 inhibitor(60mg/kg/d)in vivo.60mg/kg/d Palbociclib was served as a positive control.Results:(1)The 6 compounds with top binding affinity were:Aminoquinol,CYPERMETHRIN,Lifibrate,Permethrin,Miconazole nitrate,Econazole Nitrate.The idock scores ranged from-10.04 and-8.2kcal/mol,and the RF scores ranged from 7.56 and 7.65 pKd.(2)The selected 6 compounds were screened by MTT,and the results showed that Aminoquinol had the strongest inhibitory effect on prostate cancer cells,and different concentrations of Aminoquinol had different degrees effect on proliferation of PC-3 and Du 145 cells,showing a dose-dependent relationship(P<0.05).The effects of Aminoquinol on cells were tested at different times(24,48,72h),showing a time-dependent relationship.Aminoquinol achieved the best inhibitory effect at 72h(PC-3 IC50=3.2μM,DU145 IC50=4.3 μM).Compared with the positive control CDK4/6 inhibitor Palbociclib(PC-3 IC50=3.0μM,DU 145 IC50=5.6μM),there was no statistical difference between two compounds.(3)Aminoquinol and Palbociclib(1μM)reduced the proliferation of prostate cancer cells in the clone formation assay(P<0.05).Aminoquinol had better effect than Palbociclib(P<0.05).(4)Flow cytometry assay showed that Aminoquinol could induce cell cycle G0/G1 phase arrest.And the proportion of G0/G1 phase increased gradually with the increase of Aminoquinol concentration,showing a dose-dependent relationship(P<0.05).At the same concentration(5μM),the positive control drug Palbociclib had a slightly better effect in blocking G0/G1 phase,and there was no statistical difference between two drugs.(5)The Annexin-FITC/PI double staining method was used to detect the apoptosis of cells after Aminoquinol treatment.It was found that with the increase of concentration of Aminoquinol,the proportion of early and late apoptosis of prostate cancer cells gradually increased in a dose-dependent manner(P<0.05).Palbociclib(10μM)couldn’t cause cell apoptosis.(6)Western blotting showed that 0.3,1 and 3μM Aminoquinol and 3μM Palbociclib inhibited the phosphorylation of CDK4/6 protein in prostate cancer cell lines(PC-3 and DU 145),inhibited the expression of p-RB and E2F1.And Aminoquinol increased the cleavage of caspase-3 protein in cells.(7)BALB/C mouse model of PC-3 prostate cancer xenograft tumor was established,and Aminoquinol(60mg/kg/d)and Palbociclib(60mg/kg/d)was given by gavage for 21 days.Compared with the negative control,intragastric administration of Aminoquinol and Palbociclib could significantly inhibit the growth of tumors.There was no statistical difference between two groups.And no animals died due to the side effects of drugs.Conclusion:Aminoquinol can reduce the proliferation and cloning of prostate cancer cell lines,block the cell cycle by inhibiting the CDK4/6 cell cycle signaling pathway,induced the apoptosis of prostate cancer cells and inhibited the growth of xenograft tumor in nude mice.It is a novel CDK4/6 inhibitor.