| Objective1.To explore the effect of Fuzheng Kang-Ai(FZKA)decoction,a Chinese Herbal Medicine(CHM),on non-small cell lung cancer cells(A549,PC9 and H1650)invasion and migration,and to uncover its molecular mechanism at the level of gene and protein.2.To explore the effect on gefitinib primary and secondary resistant cells(A549 and H1650)proliferation,by which the FZKA decoction and combined with gefitinib,and to uncover its molecular mechanism at the level of gene and protein.3.To verify the mechanism by establishing the subcutaneous xenograft model of nude mice.Methods1.Transwell assay and wound healing assay were performed to show the cell migration and invasion ability by treating FZKA decoction.The protein expre-ssion of Vimentin,N-cadherin and STAT3 were carried out using western blot assay after FZKA decoction treatment.MMP9 activity assay and western blot assay were used to detect the MMP9 activity and expression treated with FZKA decoction.Transient transfection assay was used to overexpress STAT3,and then detected the activity of MMP9.2.MTS assay was used to detect lung cancer cell viability treated with FZKA decoction and combined with gefitinib.The apoptosis was carried out using Annexin V-FITC/PI flow cytometry after FZKA decoction treatment.The protein expression of p-Akt,p50,p65 and MUC1 were carried out using western blot assay treated with FZKA decoction.qRT-PCR assay was used to detect the MUC1 mRNA expression after FZKA decoction treatment.Dual-luciferase reporter gene assay was used to detect the MUC1 promotor expression after FZKA decoction treatment.Transient transfection assay was used to overexpress Akt,p65 and MUC1 respectively,and then detected others protein expression and the cell viability treated with FZKA decoction,by which to explore the relationships between them.The protein expression of p-Akt,p65 and MUC1 were carried out using western blot assay treated with FZKA decoction and gefitinib,by which to explore the synergistic mechanism of them.3.The control group,FZKA decoction group,gefitinib group,and combined group were set up by establishing the subcutaneous xenograft model of nude mice,and then measured the tumour volume and weight,the nude mice weight of each group.The fluorescence intensity of each group was measured by boilum-inescence imaging.The protein expression of p-Akt,p65 and MUC1 were carried out using western blot assay.Results1.Compared with the control group,the number of cells passing through the transwell chamber was significantly decreased treated with 2 mg/mL FZKA decoction(P<0.05).Compared with the control group,the wound was wider treated with 2 mg/mL FZKA decoction after 12 hours or 24 hours cell culture(P<0.05).2.The protein expression of Vimentin,N-Cadherin and MMP-9 were inhibited after FZKA decoction treatment,and the inhibition gradually increased with the increase of dose(P<0.05).The MMP9 activity was decreased treated with FZKA decoction,and the inhibition gradually increased with the increase of dose(P<0.05).The protein expression of p-STAT3(Tyr705)was inhibited after FZKA decoction treatment,detected at 2,4,8,24 hours(P<0.05).The MMP9 activity was increased after overexpress STAT3(P<0.05).3.The cell viability was decreased treated with FZKA decoction at 24,48,72 hours(P<0.05).The cell viability inhibition was gradually increased with the increase of dose,and with the increase of time(P<0.05).The cell apoptosis of early stage and late stage were increased,and the normal cell was decreased with the increase of dose(P<0.05).4.The protein expression of p-Akt(Ser473)was inhibited after 2 mg/mL FZKA decoction treatment,detected at 0.5,2,4,8,24 hours(P<0.05).The protein expression of MUC1 and p65 were inhibited treated with FZKA decoction,and the inhibition gradually increased with the increase of dose(P<0.05).The protein expression of MUC1 mRNA and promotor were inhibited after 2 mg/mL FZKA decoction treatment(P<0.05).The protein expression of MUC1 was increa-sed after overexpress Akt(P<0.05).The protein expression of p65 was basi-cally unchanged after overexpress MUCI(P>0.05).The protein expression of p-Akt(Ser473)was increased after overexpress MUCI(P<0.05).The cell activi-ty was increased after overexpress MUCI(P<0.05).5.Compared with the gefitinib group,the cell activity was inhibited treated with FZKA decoction and gefitinib(P<0.05).And the protein expression of p-Akt(Ser473),p65 and MUC1 were inhibited treated with FZKA decoction and gefitinib(P<0.05).6.Compared with the control group,the tumour volume,weight and fluore-scence intensity were basically unchanged treated with gefitinib(P>0.05).But the tumour volume,weight and fluorescence intensity were decreased trea-ted with FZKA decoction(P<0.05).Compared with the gefitinib group,the tumour volume,weight and fluorescence intensity were decreased treated with FZKA decoction and gefitinib(P)<0.05).Compared with the control group,the protein expression of p-Akt(Ser473),p65 and MUC1 were inhibited treated with FZKA decoction(P<0.05).Compared with the gefitinib group,the protein expression of p-Akt(Ser473),p65 and MUC1 were inhibited treated with FZKA decoction and gefitinib(P<0.05).Conclusions1.The ablity of lung cancer cell migration and invasion were significantly inhibited by FZKA decoction in a dose-dependent manner.The protein expression of mesenchymal markers including Vimentin and N-Cadherin were inhibited by FZKA decoction in a dose-dependent manner,which indicated that FZKA decoction could inhibit cell metastasis by inhibiting epithelial-mesenchymal transition(EMT).The activity and expression of MMP-9 protein was inhibited by FZKA decoction in a dose-dependent manner,and the expression of p-STAT3(Tyr705)was inhibited in a time-dependent manner,and overexpress of STAT3 could rescue MMP9 activity under FZKA treatment.It indicated that FZKA decoction could inhibit cell metastasis through STAT3-MMP9 pathway.2.The lung cancer cell proliferation was significantly inhibited by FZKA decoction in dose-dependent and time-dependent manners,and the cell apoptosis was significantly inhibited in a dose-dependent manner.The protein expression of p-Akt(Ser473)was inhibited by FZKA decoction in a time-dependent manner,and the protein expression of MUC1 and p65 were inhibited in a dose-dependent manner.Overexpress of Akt could rescue p65 expression,and overexpress of p65 could rescue MUC1 expression,and overexpress of MUC1 could rescue p-Akt(Ser473)expression,and overexpress of MUC1 could rescue cell activity.It indicated that FZKA decoction could inhibit cell proliferation through Akt-p65-MUC1 pathway.3.The inhibition on cell proliferation after FZKA decoction combined with gefitinib treatment was better than that of gefitinib monotherapy,which mec-hanism was also through Akt-p65-MUC1 pathway.4.The vivo experimentation verified that FZKA decoction and combined with gefitinib could inhibit cell proliferation through Akt-p65-MUC1 pathway. |
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