Copper Iron Nanoparticles Induce Apoptosis In Prostate Cancer Cells Via Generation Of ROS

Objective:The inhibitory effect of Cu@Fe₃O₄ NPs on human prostate cancer cells was investigated,and the mechanism of Cu@Fe₃O₄ NPs inducing apoptosis of PC-3 cells through ROS generation was clarified.Methods:1.The effects of Cu@Fe₃O₄ NPs on the proliferation viability of human prostate cancer PC-3 and C4-2 cells were investigated using the CCK8 method,and the cell proliferation inhibition rate and half inhibition concentration were calculated to clarify the dose-effect relationship and time-effect relationship of Cu@Fe₃O₄ NPs on the inhibition of human prostate cancer cells.2.Microscopic observation of the effect of Cu@Fe3O4 NPs on the morphology of PC-3 cells.3.The effect of Cu@Fe₃O₄ NPs on the migration ability of PC-3 cells was investigated by cell scratching assay.4.To study the effect of Cu@Fe₃O₄ NPs on the invasion ability of PC-3 cells using Transwell assay.5.To study the effect of Cu@Fe₃O₄ NPs on ROS generation in PC-3 cells using ROS kit.6.To study the effect of Cu@Fe₃O₄ NPs on apoptosis of PC-3 cells using Annexin V-FITC kit.7.To clarify the changes of Bcl-2 and Bax genes in Cu@Fe₃O₄ NPs-induced PC-3 cell regulation using Western blot technique.Results:1.Cu@Fe₃O₄ NPs were able to inhibit the proliferation of PC-3 and C4-2 cells in a dose-and time-dependent manner.The onset of inhibition of cell proliferation in PC-3 cells treated with different concentrations of Cu@Fe₃O₄ NPs（0.03125-4μM）for 24 h was the inhibition rate was10.03%±9.8%at the concentration of 0.03125μM and 60.73%±7.1%at the maximum concentration.The different concentrations of Cu@Fe₃O₄ NPs（0.03125-4μM）treated PC-3 cells for 48 h inhibited cell proliferation at a starting concentration of0.03125μM,with an inhibition rate of 12.34%±7.7%and a maximum concentration corresponding to an inhibition rate of 67.34%±7.2%,suggesting that different concentrations of Cu@Fe₃O₄ NPs have a dose-dependent effect on PC-3 cells.dependence.The IC50 of Cu@Fe₃O₄ NPs inhibiting PC-3 at 24 h and 48 h was 2.174μM with 95%confidence interval（1.704-2.933μM）and 1.595μM with 95%confidence interval（1.181-2.035μM）,respectively,suggesting that the inhibition of Cu@Fe₃O₄ NPs on PC-3 cells was time-dependent.The starting concentration of Cu@Fe₃O₄ NPs（0.15625-10μM）treated C4-2 cells for 24 h was 0.15625μM,and the initial concentration of Cu@Fe₃O₄ NPs inhibited cell proliferation.The initial concentration of Cu@Fe₃O₄ NPs（0.15625-10μM）was0.15625μM,and the inhibition rate was%8.77±4.8%,while the maximum concentration corresponded to 53.18%±0.66%.Different concentrations of Cu@Fe₃O₄NPs（0.15625-10μM）treated C4-2 cells for 48 h inhibited cell proliferation at the starting concentration of 0.15625μM with an inhibition rate of 3.49%±2.6%,and the maximum concentration corresponded to an inhibition rate of 71.67%±2.7%,suggesting that different concentrations of Cu@Fe₃O₄ NPs have a dose-dependent effect on C4-2 cells.dependence.The IC50 of Cu@Fe₃O₄ NPs inhibiting C4-2 cells at24 h and 48 h was 4.982μM with 95%confidence interval（3.395-8.461μM）and 4.608μM with 95%confidence interval（3.169-7.348μM）,respectively,suggesting that the inhibition of C4-2 cells by Cu@Fe₃O₄ NPs is time-dependence.2.After treatment of PC-3 cells with different concentrations of Cu@Fe₃O₄ NPs（0μM,2μM,4μM）,the cells became sphere-like with reduced refractive index and fewer normal morphology cells compared with the control group.Moreover,after 48 h of action in the drug administration group,cell shedding increased and the number of adherent cells decreased as the drug concentration increased.3.Different concentrations of Cu@Fe₃O₄ NPs（0μM,2μM,4μM）treated PC-3 cells for 48 h.The scratch healing rate of PC-3 cells under the effect of Cu@Fe₃O₄NPs was slowed down compared with the control group,and the scratch healing rate of the cells decreased with the increase of Cu@Fe₃O₄ NPs concentration.It was suggested that Cu@Fe₃O₄ NPs inhibited the migration of PC-3 cells.4.PC-3 cells treated with different concentrations of Cu@Fe₃O₄ NPs（0μM,2μM,4μM）for 48 h.The number of cells invading through the chambers in the administered group decreased with the increase of the administered concentration compared with the control group.It indicates that Cu@Fe₃O₄ NPs can inhibit the invasion of PC-3 cells.5.Cu@Fe₃O₄ NPs can induce ROS generation.Different concentrations of Cu@Fe₃O₄ NPs（0μM,2μM,4μM）treated PC-3cells for 48 h.The green fluorescence produced by the administered cells was gradually enhanced compared with the control group.It indicates that Cu@Fe₃O₄ NPs increased the ROS level of PC-3 cells in a concentration-dependent manner.6.Cu@Fe₃O₄ NPs could induce apoptosis in prostate cancer PC-3 cells.Annexin V-FITC/PI staining showed that treatment of PC-3 cells with different concentrations of Cu@Fe₃O₄ NPs（0μM,2μM,4μM）for 48 h revealed that the apoptotic levels were all higher than those in the non-administered group after drug action and increased the apoptotic cell content in a dose-dependent manner.It indicates that Cu@Fe₃O₄ NPs can induce apoptosis in PC-3 cells.7.Western blot assays showed that 4μM Cu@Fe₃O₄ NPs inhibited the production of Bcl-2 and increased the level of Bax.Conclusions:1.Cu@Fe₃O₄ NPs had an inhibitory effect on the proliferation of human prostate cancer cells.2.Cu@Fe₃O₄ NPs inhibited the migration and invasion of human prostate cancer cells.3.Cu@Fe₃O₄ NPs were able to induce apoptosis in PC-3 cells by generating ROS,activating Bax,and inhibiting Bcl-2.