The Effect And Mechanism Of Icariin On Colon Cancer Cell Proliferation And Metastasis Via Rap1GAP-mediated FAK/ERK Signaling

In 2018,statistics(the American Cancer Society)showed that colorectal cancer(CRC)is the third most common malignant neoplasm in the United State.Due to changes in the environment,diet,and lifestyle,the incidence and mortality of colon cancer in China have been rising in recent years.At present,the diagnosis and treatment of colon cancer emphasizes early diagnosis and surgical treatment,while the diagnosis of colon cancer mainly depends on colonoscopy and other means.Most patients are diagnosed at the advanced stage of cancer,and can only rely on radiotherapy,chemotherapy,palliative treatment to improve the 5-year survival rate.Therefore,it is important to look for new specific early diagnostic new biomarkers and molecular therapeutic targets.Rap is a small molecule G protein involved in many functions of cells.The disorder of Rap activity is related to the occurrence and development of malignant tumors.Rap1GAP is a Rap-specific GTPase-activating protein that catalyzes the conversion of Rap from an active GTP-bound form to an inactive GDP form,while galanin receptor(GALR)could activate Rap.Rap1GAP and GALR,which regulate Rap activity,are also closely related to tumors.The role of Rap1GAP and GALR in the development of normal tissue-colon adenoma-colon cancer is unknown.Focal Adhesion Kinase(FAK)is a key regulator of growth factor receptor and integrin-mediated signaling,which promotes malignant tumors through a highly coordinated signaling network that regulates tumorigenic and metastatic potential.These signaling networks coordinate various cellular processes such as cell survival,proliferation,invasion,and epithelial-mesenchymal transition.Extracellular signal kinase(ERK)is one of the major members of the mitogen-activated protein kinases(MAPKs)pathway,which can be activated by phosphorylation of various growth factors and cytokines,mainly regulating cell proliferation.And differentiation.Thus regulation of FAK/ERK activity is a potential target for cancer therapy.It is unclear whether Rap1GAP can exert anti-tumor effects by activating the FAK/ERK pathway in colon cancer.There is no the name "colon cancer" in Traditional Chinese Medicine(TCM).Its symptoms are close to "jiju","changxun ","suogangzhi" and so on.The total pathogenesis is deficiency of healthy qi and excessity of pathogenic qi.The kidney stores "jing",which is the natural foundation.The ancient book records:"if the jing is sufficient,it will not be sick,the jing is not sufficient,all the pathogenic will come”,"qi" of kidney deficiency is closely related to the occurrence of colon cancer,Chinese medicine emphasizes the kidney-reinforcing method in the treatment of colon cancer.Epimedium brevicomu Maxim is a commonly used kidney-reinforcing drug.Icariin(ICA)is a prenylated flavonol glycoside extracted from Epimedium brevicornu Maxim,which regulates cell cycle,inhibits angiogenesis,and inhibits metastasis through various mechanisms such as inducing apoptosis.It has an inhibitory effect on a wide range of tumor cells and is a potential drug for the treatment of cancer.The anti-cancer mechanism of icariin is mainly to inhibit tumor cell proliferation and activate apoptosis pathway in cancer cells.However,the mechanism by which icariin inhibits colon cancer is unclear,and its regulation of Rap 1 GAP has not been reported.Based on this,we used Rap 1 GAP as an entry point to investigate the effects and mechanisms of icariin on colon cancer cell proliferation and metastasis.Part I Expression and clinical significance of Rap1GAP and GALR in patients with colon adenoma and colon cancerOBJECTIVE:To investigate the expression of Rap1GAP and three galanin receptors(GALR1,GALR2,GALR3)in colon cancer,colon adenoma and normal human tissues and feces,and to analyze their relationship with pathological factors such as pathological type and differentiation degree of colorectal cancer.Methods:Paraffin-embedded specimens,40 colon cancer,40 colon adenoma and 13 normal colon tissue were collected respectively.Fecal specimens were collected respectively from 40 cases of colon cancer,40 colon adenoma and 10 healthy people.The expression of RaplGAP and GALR1,GALR2,GALR3 was detected by qRT-PCR.RESULTS:The expression of Rap1GAP in normal tissues of colorectal mucosa,adenoma tissues and colorectal cancer tissues showed a decreasing trend in turn,and the difference was statistically significant(P<0.001).Rap1GAP expression was decreased in adenomas.Rap1GAP expression was more severely decreased in colorectal cancer.In colon adenoma,the expression of Rap1GAP was not related to gender,age,and adenoma size.It was related to the pathological type,and the expression of Rap1GAP in the villous adenoma was lower than that in the tubular adenoma(P<0.05).It was related to the differentiation,and the expression of Rap1GAP in high-grade adenoma were lower than that in low-grade adenoma(P<0.05).In colon cancer tissues,the expression of Rap1GAP was not related to gender,age,and tumor size;it was related to tumor pathological type,which showed a decreasing trend correspondingly in the lump,ulcer and infiltrating tissues(P<0.05).It was related to the differentiation,and showed a decreasing trend correspondingly in the well differentiated,moderately differentiated and poorly differentiated tissues(P<0.05).It was related to the depth of infiltration,the expression of Rap1GAP in T1/T2 phase was higher than that in T3/T4 phase(P<0.05).It was associated with lymph node metastasis,the expression of Rap1GAP in sample with no lymph node metastasis was higher than that with lymphatic metastasis(P<0.05).It was related to distant metastasis,the expression of RaplGAP in sample without distant metastasis was higher than that with distant metastasis(P<0.05).The expression levels of RaplGAP in stools of healthy people,adenomas and colorectal cancer patients were decreased in turn,and the difference was statistically significant(P<0.001).RaplGAP expression was decreased in adenomas.RaplGAP expression was more severely decreased in colorectal cancer.In the stool of patients with colon adenoma,the expression of Rap1GAP was not related to gender,age,and adenoma size.It was related to the pathological type,and the expression of Rap1GAP in the villous adenoma was lower than that in the tubular adenoma(P<0.05).It was related to the differentiation,and the expression of RaplGAP in high-grade adenoma were lower than that in low-grade adenoma(P<0.05).In the feces of colon cancer patients,the expression of Rap1GAP was not related to gender,age,tumor size,tumor pathology and differentiation degree.It is related to the depth of infiltration,the expression of Rap1GAP in T1/T2 phase was higher than that in T3/T4 phase(P<0.05).It is associated with lymph node metastasis,the expression of Rap1GAP in sample with no lymph node metastasis was higher than that with lymphatic metastasis(P<0.05).It is related to distant metastasis,the expression of RaplGAP in sample without distant metastasis was higher than that with distant metastasis(P<0.05).GALR1,GALR2,and GALR3 showed no significant changes in normal colon,colon adenoma,and colon cancer tissues(P>0.05).GALR1,GALR2,and GALR3 showed no significant changes in feces of healthy people,colon adenomas,and colon cancer patients(P>0.05).Conclusion:The expression levels of Rap1GAP were gradually decreased in normal colon,colon adenoma and colon cancer,indicating that it may be involved in the early transformation of colon cells,and plays a role as tumor suppressor in the development of malignant tumors.The clinical finding provides a new idea for the early diagnosis of colon cancer.Part II Effect of icariin on proliferation and metastasis of colon cancer in vivo tumor model and its mechanismOBJECTIVE:To study the effect of icariin on the colorectal cancer cell growth and metastasis in vivo,and to explore the inhibitory mechanism of icariin on colon cancer cell growth and metastasis in vivo tumor model.METHODS:Logarithmic growth period of colon cancer HCT116 cells were injected subcutaneously into nude mice to establish a subcutaneous tumor pattern of nude mice.After tumor formation,they were randomly divided into normal group,control group(containing 0.5%normal saline)and Low-dose,middle-dose,high-dose icariin group(40,80,160 mg/kg)and the Xeloda group(267 mg/kg)with intragastric administration.The diet,the mind state,response and activity of each group of nude mice were administered by intragastric administration.The diet,spirit and activity of each group of nude mice were observed every day.The body weight was weighed every 3 days and the length and diameter of the tumor were measured.The body weight and tumor size curves were drawn.After the treatment,all the nude mice were sacrificed and the tumor tissues were taken out.The tumor inhibition rate of each group was compared.The changes of Rap1GAP,FAK,pFAK,ERK and pERK protein levels in tumor tissues were detected by Western blot.The log phase of colon cancer CT26 cells was injected into the spleen to establish a mouse colon cancer liver metastasis membrane.After tumor formation,they were randomly divided into normal group,control group(containing 0.5%normal saline)and Low-dose,middle-dose,high-dose icariin group(40,80,160 mg/kg)and the Xeloda group(267 mg/kg)with intragastric administration.The diet,mental,activity and abdomen of each group of mice were observed every day.After the treatment,all the mice were sacrificed.HE staining was used to observe the morphology of liver metastasis of each group.Immunohistochemical staining was used to detect Rap1GAP and pFAK,pERK protein levels of the liver tumors.RESULTS:The subcutaneous xenografts of nude mice and the colorectal cancer with liver metastasis were successfully established.In the subcutaneous xenograft model,no significant difference in diet,the mind state,response,activity,and body weight of the nude mice in each group was observed during the treatment.After the nude mice were sacrificed,it was found that icariin inhibited the volume and tumor weight of tumor-bearing mice in a dose-dependent manner(P<0.05).After treatment with icariin,Rap1GAP protein was up-regulated,FAK and ERK proteins were unchanged,and pFAK and pERK proteins were down-regulated in a dose-dependent manner.In the liver metastasis of colon cancer,the diet,the mind state,response and activity of the mice in the icariin group were better than those in the control group.After the mice were sacrificed,metastases were found in the liver of each group.Compared with the control group,the body weight,liver weight and tumor weight of the icariin group were lighter,the tumor/liver weight was smaller,and the lung and peritoneal metastasis were less,the difference was statistically significant(P<0.05).Icariin inhibited liver metastasis of colon cancer in a dose-dependent manner.HE staining showed that icariin can promote tumor tissue necrosis;immunohistochemistry results showed that icariin can up-regulate Rap 1 GAP and down-regulate pFAK and pERK.Conclusion:Icariin can inhibit the growth and metastasis of tumor-bearing mice,and this effect may be related to the regulation of Rap 1 GAP and FAK/ERK activities.Part III Rap 1 GAP regulates FAK/ERK pathway to inhibit proliferation,invasion and migration of colon cancer cellsOBJECTIVE:To investigate the effects of Rap 1 GAP on proliferation,invasion and migration of colon cancer cells,and to explore whether it is related to FAK/ERK pathway.METHODS:The expression of Rap1GAP in human normal colonic epithelial cells NCM460,human colon cancer cells HT29,HCT116 and DLD1 was compared.The lentiviral expression vector pLVPT-Rap1GAP was constructed,and the three-plasmid system virus package was transfected into 293T cells.The viral supernatant was collected for infecting HCT116 cells,positive monoclonal cell lines were selected for further experiment.PX459-sg-Rap1GAP plasmid was constructed for knockout of Rap1GAP in HT29 cells,positive monoclonal cell lines were selected for further experiment.MTT assay was performed to detect the effect of Rap1GAP on cell proliferation.Flow cytometry was used to detect Rap1GAP on cell cycle and apoptosis.Transwell assay was used to detect the effect of Rap1GAP on cell invasion and migration.Western blot was performed to detect FAK,pFAK,ERK,pERK,cyclin D1,cyclin E,MMP9,MMP2 protein expression.Western blot was performed to detect Rap activity and FAK/ERK signaling in HCT116 after treated with FAK inhibitor Y15 and EPAC2 activator 8-CPT-2'-O-Me-cAMP.Western blot was performed to detect Rap activity and FAK/ERK signaling after treated with of histone deacetylase inhibitor NaB or TSA,DNA methylation inhibitor 5-Aza.Results:HCT1 16-pLVPT-Rap1 GAP monoclonal cells and HT29-PX459-sg-Rap1GAP monoclonal cells were successfully screened.In HCT116-pLVPT-Rap 1 GAP cells,the proliferation of cells was significantly inhibited(P<0.05),and the proliferation of cells was significantly enhanced in HT29-PX459-sg-Rap1GAP cells(P<0.05).In HCT116-pLVPT-Rap1GAP cells,G0/G1 phase cells was increased(P<0.05),and in HT29-PX459-sg-Rap1GAP cells,G0/G1 phase cells was decreased(P<0.05).In HCT116-pLVPT-Rap 1 GAP cells,the invasion and migration ability were significantly inhibited(P<0.05).In HT29-PX459-sg-Rap1GAP cells,the invasion and migration ability of cells was significantly enhanced(P<0.05).In HCT 116-pLVPT-Rap 1 GAP cells,the expression of FAK and ERK was unchanged,pFAK,pERK,cyclin D1,cyclin E,MMP9 and MMP2 proteins were down-regulated(P<0.05).In HT29-PX459-sg-Rap1GAP cells,the expression of FAK and ERK was unchanged,pFAK,pERK,cyclin D1,cyclin E,MMP9,and MMP2 proteins were up-regulated(P<0.05).After 8-CPT-2'-O-Me-cAMP treatment,Rap2-GTP was increased in HCT116 cells,pFAK,pERK,Cyclin D1,Cyclin E,MMP9,and MMP2 were up-regulated(P<0.05),and FAK blocker Y15 completely inhibited this effect.In HCT116 and DLD1 cells,NaB up-regulated Rap1GAP,the expression of Rap2,FAK and ERK were unchanged,Rap2GTP,pFAK and pERK were down-regulated(P<0.05).TSA also up-regulated Rap1GAP(P<0.05),and 5-Aza had no significant effect.Treatment with NaB,TSA and 5-Aza had no effect on Rap 1 GAP expression in HT29 cells.Conclusion:Rap1GAP inhibits the proliferation,invasion and migration of colon cancer cells,and the mechanism was related to the inhibition of FAK/ERK signaling by Rap1GAP.Expression of Rap1GAP in colon cancer cells HCT116 and DLD1 is regulated by histone acetylation and is involved in the regulation of Rap2 activity and phosphorylation of ERK/FAK.Part IV Icariin inhibits colon cancer cell proliferation,invasion,and migration through the Rap1GAP-mediated FAK/ERK pathwayOBJECTIVE:To study the effects of icariin on the proliferation,invasion and migration of colon cancer cell line HCT 116,and to explore whether its mechanism is related to the regulation of Rap1GAP-mediated FAK/ERK pathway.METHODS:After HCT116 cells were treated with different dose of icariin(5,10,15,20,25?mol/L),effect of icariin on proliferation was detected by MTT assay.Effect of icariin on invasion and migration were compared with Transwell assay.Effect of icariin on apoptosis and cell cycle were detected by flow cytometry.The mRNA expression of Rap 1 GAP was detected by qRT-PCR.The protein expression of Rap 1 GAP,FAK;pFAK,ERK,pERK,cyclin D1,cyclin E,MMP9 and MMP2 in HCT116 was detected by Western Blot.RESULTS:Icariin inhibited the proliferation of HCT116 cells(P<0.05),which showed time and concentration dependence.The half-inhibitory concentration(IC50)of cells at 24h,48g and 72h was 21.04?mol/L,18.80?mol/L,17.26?mol/L.Icariin inhibited the invasion and migration of HCT116 cells in a dose-dependent manner(P<0.05).Icariin induced HCT116 cell apoptosis in a dose-dependent manner(P<0.05).The percentage of HCT116 cells in G0/G1 phase was increased in a dose-dependent manner(P<0.05).The mRNA and protein levels of Rap 1 GAP were up-regulated in HCT116 cells.The FAK and ERK proteins were unchanged,and the levels of pFAK,pERK,cyclin D1,cyclin E,MMP9,and MMP2 were down-regulated(P<0.05).Conclusion:Icariin can inhibit the proliferation and invasion of colon cancer cell,and its mechanism is related to the regulation of FAK/ERK pathway by Rap 1 GAP.In this study,we found for the first time that Rap 1 GAP was down-regulated in turn during the dynamic development of normal-adenomal-cancer,and its expression was negatively correlated with the higher degree of malignant classification.The alanin receptors(GALR1,GALR2,GALR3)were not significantly changed in different tissues and feces.By establishing model of subcutaneous xenografts and colorectal cancer with liver metastasis,we found that icariin inhibited colon cancer growth and metastasis in vivo,which was related to interventions of Rap 1 GAP and FAK/ERK activity.In addition,Rap 1 GAP could regulate cell proliferation and migration by interfering with FAK/ERK signaling pathway.By histone acetylation modification,Rap 1 GAP was up-regulated in HCT116 and DLD1 cells,which could modulate the activity of Rap2.Finally,experiments in vitro demonstrated that icariin could inhibit the proliferation and migration of colon cancer cells,which was related to the regulation of FAK/ERK activity by Rap 1 GAP.These results showed that icariin could target Rap 1 GAP and FAK/ERK signaling in colon cancer,supported the role of traditional Chinese medicine tonifying kidney for the treatment of colon cancer,and provided a theoretical basis for the pharmacological research and clinical treatment of Chinese medicine Epimedium.