| OBJECTIVE:Studying the reversal effect of Nitidine Chloride on KB/ADM cells mediated by Topo ?? and the impact on PTEN/PI3K/AKT signaling pathway,to provide a theoretical basis for NC as a Topo ?? inhibitor application in clinical multidrug resistance.METHOD:1 The reversal effects of NC on MDR cells evaluated by cells models:MTT was used to detect the growth inhibition to determine the reversal concentration of NC,and a suiTab cells was selected from six MDR cells.HE staining was used to obsever the morphology,Transmission Electron Microscope was used to obsever the microstructure,AO/EB staining was used to obsever apoptosis,Scratch Test and Transwell were used to detect the migration and invasion ability,Flow Cytometry was used to detect the apoptosis rate.2 The reversal effect of NC on MDR cells evaluated by nude mice and zebrafish model:KB/ADM cells was injected into nude mice,tumor volum and weight were record,HE staining was used to obsever the morphology,Transmission Electron Microscope was used to obsever the microstructure,Hoechst33342 and TUNEL staining were used to obsever the apoptosise.CM-Dil labeled KB/ADM cells were injected into zebrafish by microinjection,the proliferation of KB/ADM cells was observed by fluorescence microscopy.3 The reversal effect of NC on MDR cells mediated by Topo ?? was study.Computer Aided Drug Design(CADD)was used to study the molecular docking to comfirm the binding and interaction between NC and Topo ??.DNA relaxation and DNA embedding experiments were used to detect the inhibition activity and action mode of NC on Topo ??.RT-PCR and Western Blot were used to detecte the gene and protein expression of Topo ?? in MDR cells,nude mice and zebrafishs.4 The reserval effect of NC on Topo ?? over expression cells:Topo ??over expression cells was established by lentivirus infection.MTT was used to detect the growth inhibition rate,HE staining was used to obsever the morphology,Hoechst33342 staining used to obsever apoptosis,Scratch Test and Transwell were used to detect the migration and invasion ability,Flow Cytometry wsa used to detect the apoptosis rate,RT-PCR and Western Blot were used to detecte the gene and protein expression of Topo ??.5 The impact on PTEN/PI3K/AKT signaling pathway in KB/ADM cells:Western Blot was used to detecte the ptoteins expression of PTEN,PI3K,AKT,p-AKT and Caspase-3 in KB/ADM cells in each group.RESULTS:1 The reversal effect on KB/ADM cells:(1)The reversal concentration of NC on KB/ADM cells was 1.5?g.mL-1 and NC showed a best reversal effect on KB/ADM cells.Cells number reduced,membrane damaged and nuclear condensed were observe in VP 16 combined with VRP or NC group while there were no significant change in VP 16 group in HE staining.Condensation of chromatin at margins of nuclei,disintegration of nucleolus and vacuoles in cytoplasm were observed VP 16 combined with VRP or NC group by transmission electron microscopy(TEM).Scratch healing speed was faster in VP 16 combined with VRP or NC group compare to VP 16 group.Green fluorescence was observed in VP 16 group,while cells number reduced,nuclear condensed,and orange fluorescence were observed in VP 16 combined with VRP or NC group by AO/EB staining.The apoptosis rate of KB/ADM cells was 5.8%,14.9%and 17.3%in VP16,VP16 combined with VRP or NC group respectively detected by Flow Cytomete.2 The reversal effect on nude mice and zebrafish transplant tumors(1)The KB/ADM cells can proliferation after injected into nude mice.The tumors volume and weight were reduced in VP 16 combined with VRP or NC group compare to VP 16 group,there was a statistically significant difference(p<0.05).Obvious necrosis and vacuolar changes were observed in VP 16 combined with VRP or NC group by HE staining.Cells number decreased,nucleus contracted and the nuclear staining increased were observed in VP 16 combined with VRP or NC group by Hoest33342 staining.The apoptosis index were 15.45%and 48.03%,and 60.13%respectively in VP 16,VP 16 combined with VRP or NC group with TUNEL staining.(2)CM-Dil labeled KB/ADM cells can proliferation after injected into zebrafish.The fluorescence intensity were 474934 ±36023,466046±28864,279622±17216 and 245544 ±16309 respectively in control,VP16,VP16 combined with VRP or NC group,and there was a statistically significant difference in VP 16 combined with VRP or NC group compare to VP 16 group(p<0.05),indicating that the combination of drugs could inhibit the growth of KB/ADM cells in zebrafish.3 Mechanism of the reversal activity of NC on MDR cells(1)Molecular docking showed that NC might reverse the drug resistance of VP 16 Topo ?? by enhancing the affinity between VP 16 and Topo ??.The supercoiled unwinding and embedding experiments showed that NC has inhibitory effect on Topo ??,and its mechanism may embedding into Topo ??.(2)compare to VP 16,Topo ?? proteins and mRNA was down-regulated in VP 16 combined with VRP or NC group in cells,nude mice transplanted tumor and zebrafish transplanted tumor,there was a statistically significant difference(p<0.05),the results showed that the reversal activity of NC on MDR cells may related to Topo ??.4 The inhibition effect of NC on Topo ?? over expression cells(1)1:5 dilution virus showed a best infection result;the sTab transfection of KB/2307 cells were selected with 0.5?g/ml puromycin for 4 days,RT-PCR and Western Blot assay showed that Topo ?? gene and protein were up-regulated in KB/2307 cells.(2)KB/2307 cells showed a stronger proliferation compare to KB cells.The IC50 were 52.20±5.75and 27.02±2.14?g.mL-1 after treated with VP16 and VP 16 combined with NC,there was a statistically significant difference(p<0.05).Scratch healing speed speed was slow in VP16 combined with NC group and cells death was observed in these group.Cells number of though membranes was significantly reduced in VP 16 combined with NC group in Transwell test.Cells number decreased,nucleus contracted and the nuclear staining increased were observed in VP 16 combined with NC group by Hoest33342 staining.The apoptosis rate of KB/ADM cells was 10.3%and 14.9%in VP16 and VP16 combined with NC group detected by Flow Cytometer.(3)Topo ?? gene and protein expression were observed down-regulated in VP 16 combined with NC group compare to VP 16 group by RT-PCR and Western Blot assay,there was a statistically significant difference(p<0.05).5 PTEN and.Caspase-3 proteins expression were up-regulated,PI3K and p-AKT proteins expression were down-regulated in VP 16 combined with VRP or NC group compare to VP 16 group(p<0.05),AKT proteins expression were no obvious change in each group.Conclusions:NC can reverse the drug risistance of KB/ADM cells showed a obvious reversion of drug resistanc on the MDR cells.Molecular docking,DNA relaxation,DNA embedding experiments,the gene and protein expression of Topo ?? in MDR cells,and the inhibition effect of NC on Topo ?? over expression cells,indicating that the reversal effect target of NC on KB/ADM cells may Topo ??,and the reverse effect of NC on KB/ADM cells may related to PTEN/PI3K/AKT signaling pathway for the proteins expression changes in these pathway.From the above results,we suspected that the mechanism action of NC on MDR cells were embedding into Topo ?? and enhancing the affinity between VP 16 and Topo ??,damaging cell DNA,and inducing cell apoptosis through PTEN/PI3K/AKT signaling pathway may. |
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