Qingjie Fuzheng Granules Inhibits The Growth Of Colorectal Cancer Via PI3K/AKT And ERK Pathways

Aim: To explore the effect and mechanism of Qingjie Fuzheng Granules(QFG)on the growth of colorectal cancer(CRC)cells in vitro and in vivo,and to provide experimental basis for its clinical application.Methods:In vitro study:(1)After treatment with different concentration of Qingjie Fuzheng Granules(QFG),MTT and LDH assays were used to detect the effect of QFG on cell viability and cytotoxicity;colony formation and cell cycle assays were used to detect the effect of QFG on cell proliferation;Hoechst33258 and AnnexinV-PI assays were used to detect the effect of QFG on cell apoptosis;Western Blot assays to detected the expression of relative proteins of proliferation,apoptosis,and angiogenesis(Survivin,Cyclin D1,CDK4,p21,cleaved-caspase-3/-8/-9,Bax,Bcl-2,Fas,FasL,and VEGF-A).(2)Inhibitors of caspase-3/-8/-9 were also used to elucidate the exact apoptosis pathway induced by QFG in CRC cells.(3)After treatment with different concentration of QFG,MTT assay was used to detect the effect of QFG on cell viability of HUVEC cells;Transwell and Tube Formation assays were used to detect the effect of QFG on HUVEC cell migration and angiogenesis ability;exogenous-VEGF-A stimulating factor was used to further detect the effect of QFG on VEGF-A-mediated angiogenesis.(4)Western Blot assay was used to detect the effect of QFG on the expression of PI3K/AKT and ERK signaling pathway-related proteins(p-PI3 K,p-AKT and p-ERK).In vivo study: The nude mice model of HCT-116 subcutaneously transplanted tumors was constructed and randomly divided into control group and QFG group according to the size of tumors in nude mice.The QFG group(n=10)was given QFG(1 g/kg)by gavage,and the control group(n=10)was given the same volume of saline by gavage for 6 days a week.The size and weight of nude mice tumors were measured every two days.One month later,the nude mice were sacrificed and the tumors were removed for weighing,photographing and follow-up experiments;TUNEL assay was used to detect the effect of QFG on tumor tissueapoptosis;IHC staining assay was performed to determine the expression of proliferation-related proteins,apoptosis-related proteins,and angiogenesis-related proteins(Ki-67,Survivin,Cyclin D1,CDK4,p21,cleaved-caspase-3,Bax,Bcl-2,CD31,VEGF-A,and VEGFR-2);Western Blot assay was used to detect the effect of QFG on the expression of PI3K/AKT and ERK signaling pathway-related proteins(p-PI3 K,p-AKT,and p-ERK).Results:In vitro study:(1)MTT and LDH assays revealed that QFG can inhibit cell viability and increased cytotoxicity in CRC cells(P<0.05);colony formation and cell cycle assays showed that QFG inhibited proliferation in CRC cells(P<0.05);Hoechst33258 and AnnexinV-PI assays showed that QFG induced apoptosis in CRC cells(P<0.05);Western Blot assays showed that QFG inhibited the expression of Survivin,Cyclin D1,CDK4,and VEGF-A,while increased the expression of p21,cleaved-caspase-3/-8/-9,Bax,Fas,and FasL(P<0.05).(2)After inhibitors of caspase-3/-8/-9 were used,the suppression effect of QFG on cell viability and the induction effect on cell apoptosis were markedly inhibited by these three inhibitors(P<0.05).(3)MTT assay was showed that QFG can inhibit the cell viability of HUVEC(P<0.01);Transwell and Tube Formation assays showed that QFG can inhibit the cell migration and angiogenesis ability of HUVEC;when exogenous-VEGF-A stimulated,QFG can inhibit the VEGF-A-mediated angiogenesis.(4)Western Blot assays showed that the ratio of p-PI3K/PI3 K,p-AKT/AKT,and p-ERK/ERK in CRC cells were significantly down-regulated by QFG(P<0.05).In vivo study: QFG inhibited the rise of tumor volume and weight while had no effect on mice body weight.TUNEL assay showed QFG treatment increased the percentage of TUNEL-positive cells in xenograft mice tumor tissues(P<0.01).IHC results showed that the expressions lever of Ki-67,Survivin,Cyclin D1,CDK4,Bcl-2,CD31,and VEGF-A in QFG treatment group were significantly decreased(P<0.05),while the expressions lever of p21,Bax,and cleaved-caspase-3 were significantly increased(P<0.05).Western Blot showed that the ratio of p-PI3K/PI3 K,p-AKT/AKT,and p-ERK/ERK in QFG group decreased significantly(P<0.01).Conclusions: QFG can inhibit the proliferation and induce apoptosis of CRC cells and inhibit angiogenesis of CRC both in vitro and in vivo.QFG can inhibit the activation of PI3K/AKT and ERK signaling pathways to play the anti-CRC effect.