Effects Of Dihydroartemisinin On UHRF1 In Human Prostate Cancer PC-3 Cells And Its Regulatory Mechanism

Objective: Our study aimed to investigate the effect of dihydroartemisinin(DHA)on the expression of ubiquitin-like with PHD and ring finger domains 1(UHRF1)in human prostate cancer PC-3 cells,and to futher explore its possible regulatory mechanism of methylation.Methods: The PC-3 cells were treated with different concentrations of DHA(0,25,50,and 100 ?mol/L)for 48 h,while PC-3 cells without DHA treatment were used as the control.Then the cell-cycle distribution,apoptosis rates and the levels of reactive oxygen species(ROS)in PC-3 cells were detected by FCM.Methylation level of p16INK4 A Cp G islands was determined by bisulfite genomic sequencing(BGS).RT-PCR was performed to examine UHRF1,DNA methyltransferase 1(DNMT1)and p16INK4 A expressions at m RNA levels,whereas the expressions at protein levels were detected by Western blotting.The cellular location and expression of p16INK4 A protein were detected by immunofluorescence staining.Results: 1.MTT and FCM results showed that DHA could significantly inhibit the proliferation of PC-3 cells by inducing G1/S cell-cycle arrest and apoptosis accompanied by elevated ROS levels.2.Decreased p16INK4 A promoter methylation levels in DHA-treated groups were observed in PC-3 cells by BGS method.3.RT-PCR and Western blotting results showed that the expressions of UHRF1 and DNMT1 at both m RNA and protein levels were decreased,while the expression of p16INK4 A was up-regulated.4.The result of immunofluorescence showed that p16INK4 A protein was expressed in both nucleus and cytoplasm.And the fluorescence intensity of p16INK4 A protein in DHA-treated groups was significantly increased.Conclusion: DHA may restore the expression of p16INK4 A by decreasing the methylation level of p16INK4 A through the down-regulation of UHRF1,further to induce G1/S arrest,apoptosis in PC-3 cells.The mechanism may be related to the regulation of UHRF1 on p16INK4 A.