The Reversal Of Drug-resistance Of HCT-8/V Cells By Vincristine Combined With DDP,rhIL-24 And Que And Its Mechanism

Objective:1.To investigate the inhibitory and apoptosis effect on HCT-8/V cells treated with VCR,DDP,rhIL-24,quercetin respectively and VCR combination with DDP,rhIL-24 and quercetin;2.To explore the possible reversal mechanism of drug-resistance treated with VCR,DDP,quercetin respectively and VCR combination with DDP and quercetin in HCT-8/V cells,to provide experimental and theoretical support for clinical treatment.Methods:1.Human colorectal cancer HCT-8 cells and vincristine-resistant HCT-8/V cells were cultured and revealed by IncuCyte ZOOM real dynamic live cell imaging platform,screening for appropriate cell density;2.HCT-8/V cells were treated with VCR,DDP,rhIL-24,quercetin respectively and VCR in combination with DDP and quercetin by IncuCyte ZOOM real dynamic live cell imaging platform.ThevincristineresistantHCT-8/Vcellsweredetectedbycell proliferation inhibition,including growth patterns,inhibition rates at different times and different concentration.3.The apoptosis effect on vincristine-resistant HCT-8/V cells,which were affected by VCR,DDP and quercetin,were detected by IncuCyte ZOOM real dynamic live cell imaging platform.4.Western blotting was used to detect the expression of P-gp and mTOR protein in drug resistant HCT-8/V cells after single and double factors.Results:1.The Real dynamic living cell imaging platform was used to culture HCT-8 and HCT-8/V cells for detecting the proliferative rate of tumor cells.When the density of HCT-8 cells was 1×10~3/ml,the cells proliferated in the logarithmic growth phase at 64h;and when the cell density was 1×10~4/ml,the cells proliferated into the logarithmic growth phase at 30h,with the cell fusion degree reaching 80%.When the density of HCT-8/V cells was1×10~3/ml,the cell fusion degree was only 10%at 120h;and when the cell density was1×10~4/ml,the cell fusion degree was 40%at 64h.At 144h,the cells reached logarithmic growth phase.Thus,selecting the density of 1×10~4/ml for HCT-8/V cell planting was more suitable for real-time dynamic observation.2.After treated with VCR,DDP and quercetin,the cell inhibitory rates of HCT-8/V cell proliferation by IncuCyte ZOOM real dynamic live cell imaging platform:VCR,DDP,Que alone and combined application inhibited HCT-8/V cells,the inhibition rate(%)were 24.3,31.2,20.7,55.3,45.4 respectively,in which the inhibition rate of the combination group was significantly higher than that of the individual drug groups.After combined treatment VCR with DDP or quercetin,the inhibitory rate of the tumor cells increased from 24.3%to55.3%(2.28 times)in the VCR+DDP group and increased from 24.3%to 45.4%(1.89times)in the VCR+Que group.On the other hand,there was no significant changes in cell proliferation rate after HCT-8/V cells were treated with rhIL-24.3.The effects of VCR,DDP and quercetin on HCT-8/V cell apoptosis were observed by IncuCyte ZOOM real dynamic live cell imaging platform:VCR and DDP acted alone on HCT-8/V cells,and induced apoptosis was not obvious.However,Que(200?M)alone in HCT-8/V cells showed obvious apoptosis in 48h(p<0.05).4.The protein expression of each group was measured by western blotting:The Que group reduced mTOR protein expression levels;the VCR+DDP group decreased P-gp protein expression levels.Conclusion:1.Dynamic observation of real-time living cell growth with HCT-8 cells and HCT-8/V cells at the concentration of 1×10~4/ml.The logarithmic growth phase time of HCT-8 cells is 30 hours,while HCT-8/V cells are 144 hours;2.The effect of VCR/DDP and VCR/Que on the proliferation of HCT-8/V cells proliferation was better than that of each drug alone.rhIL-24 has no inhibitory effect on HCT-8/V cell proliferation;3.VCR?DDP had no significant effect on HCT-8/V cell induce apoptosis,while Que can induce apoptosis of HCT-8/V cells;4.It may be one of DDP sensitization mechanisms to VCR by expression of inhibition P-gp protein.