| Objective: To investigate the synergistic effects and mechanism of Qingjie Fuzheng Granule(QFG)in combination with 5-fluorouracil(5-FU)on colorectal cancer(CRC)through the PI3K/AKT pathway in vitro and in vivo,and in order to provide a new evidence for its clinical application.Methods: In vitro experiment: 1.Chou-Talalay calculated the combined QFG and 5-FU indices of colorectal cancer cell lines HCT-8 and HCT-16 at different times.2.Cell viability effects of QFG in combination with 5-FU were measured using CCK8.Cell morphology was observed using inverted microscope.The effect of QFG in combination with 5-FU on cell proliferation was measured by colony formation.Western Blot measured p53,p21,CDK4 and Cyclin D1 protein expression in cells.3.Apoptosis was observed by Annexin V/PI and DAPI staining.Western Blot measured the expression of Bax,Bcl-2 and cleaved-caspase-3 proteins in cells.4.Cell metastasis was detected by migration and invasion assays.Western Blot measured the expression of E-cadherin,N-cadherin and Vimentin proteins in cells.5.Western Blot measured DPD and TS protein expression levels in cells to evaluate the drug’s effect on5-FU metabolism.6.Western Blot measured PI3 K,p-PI3 K,AKT,and p-AKT protein expression levels in cells,and 10% of FBS activated the PI3 K / AKT pathway,which was observed by microscopy,colony assays,DAPI staining,migration,and invasion assays to observe the effects of QFG in combination with 5-FU.In vivo experiment: Subcutaneous xenograft tumor models: nude mice were subcutaneous xenograft tumor models using HCT-8 cells and randomly classified into Control,QFG,5-FU,and QFG+5-FU groups based on tumor volume.QFG group was given intragastric administration at 1 g/kg/ day,5-FU group was injected at 25 mg/kg/ day,QFG+5-FU group was given QFG 1 g/kg/ day,and 5-FU was injected at 25 mg/kg/ day.Tumor volume and weight were measured at 1-day intervals,and after 3 weeks of continuous administration,nude mice were executed and stripped of tumor tissue for weighing.Immunohistochemistry(IHC)was used to detect Ki67 expression in tumor tissues.TUNEL staining detected apoptosis in tumor tissue.Western Blot detected the expression of p53,p21,CDK4,Cyclin D1,Bax,Bcl-2,cleaved-caspase-3,E-cadherin,N-cadherin,Vimentin,DPD,TS,PI3 K,p-PI3 K,AKT,and other proteins in tumor tissues.Results: In vitro experiment: 1.The combination index of QFG combined with 5-FU on HCT-8 and HCT-16 cells at different time periods was less than 2.The cell viability of HCT-8 cells treated with QFG combined with 5-FU at different time periods was significantly different from that in the single drug group(P<0.05),colony formation test suggested that QFG combined with 5-FU had more inhibitory effect on cell proliferation than single drug(P<0.05).At the same time,compared with single drug,QFG combined with 5-FU could inhibit the expression of CDK4 and Cyclin D1 and enhance the expression of p53 and p21protein(P<0.05).3.Annexin V/PI and DAPI staining experiments showed that QFG combined with 5-FU could promote apoptosis more than single drug(P<0.05).At the same time,QFG combined with 5-FU could inhibit the expression of Bcl-2 and enhance the expression of Bax and cleaved-caspase-3 protein compared with single drug(P<0.05).4.Migration and invasion experiments showed that QFG combined with 5-FU had more inhibitory effect on cell metastasis than single drug(P<0.05).At the same time,compared with single drug,QFG combined with 5-FU could inhibit the expression of N-cadherin and Vimentin and enhance the expression of E-cadherin protein(P<0.05).5.Compared with single drug,QFG combined with 5-FU could inhibit the expression of DPD and TS protein(P<0.05).6.Compared with single drug,QFG combined with 5-FU could inhibit the activation of PI3K/AKT signal pathway,inhibit the proliferation and metastasis of HCT-8cells activated by PI3K/AKT pathway,and promote the apoptosis of HCT-8 cells(P<0.05).In vivo experiment,QFG combined with 5-FU could inhibit the tumor volume(P<0.05),QFG combined with 5-FU could inhibit the expression of Ki67(P<0.05)and increase the positive expression of TUNEL(P<0.05).Compared with single drug,QFG combined with 5-FU could inhibit the expression of CDK4,Cyclin D1,Bcl-2,N-cadherin,Vimentin,DPD,TS(P<0.05),enhance the expression of p53,p21,Bax,cleaved-caspase-3,E-cadherin protein(P<0.05),and inhibit the activation of PI3K/AKT pathway(P<0.05).Conclusion: QFG combined with 5-FU can play a synergistic role in the treatment of colorectal cancer by inhibiting PI3 K / AKT signaling pathway... |
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