Study On The Mechanism Of Eleanolactone Regulating MAPK-JNK/c-Jun Pathway To Inhibit Colorectal Cancer

Object: We made this research to formulate the pharmacological activity of anti-colorectal cancer(CRC)in vitro and in vivo of alantolactone(ATL),then explore its potential mechanism.In that case we can provide basic experimental data for the further search of the medical value of ATL.Metheds:Study on anti-CRC activity of ATL:In vitro: The cell viability of human colon cancer cell lines HCT-116,HT-29,LS174 t,SW480,SW620 and mouse colon cancer cell line CT26 was first tested by CCK-8 kit after treated with dosage ATL.Incubate HCT-116 cells with 0,7.5,15,30μM ATL for24 h,followed with the detection of cell apoptosis and cycle by Hoechst33258 stain,JC-1 stain,Flow Cytometry assay and clonogenic assay.Incubate SW480 cells with 0,2.5,5,10μM ATL for 24 h,followed with the detection of cell migration and invasion by wound-healing assay and Transwell invasion assay.In vivo: CT26 cells were subcutaneously injected into the right forelimb armpit of the mice to establish a subcutaneous tumor-bearing model.The mice were randomly divided into four groups including model control,ATL 25mg/kg,ATL 50mg/kg,ATL100mg/kg.The anti-tumor effect of ATL on this model was detected by tumor volume,tumor weight,PCNA-IHC and TUNEL analysis.The toxicity was evaluated on body weight analysis and H&E pathological section of liver and kidney.The experimental model of colitis-associated colorectal cancer(ca CRC)was established by azoxymethane(AOM)/ dextran sulfate sodium(DSS)co-treatment.The mice were randomly divided into four groups including vehicle control,ATL control,AOM/DSS model,ATL 50mg/kg.The body weight,bloody diarrhea score,colorectal length,spleen weight,tumour number,histological score analysis and Brd U assay were considered as the evaluation of anti-cancer effect of ATL on ca CRC model.Study on mechanism of ATL:In vitro: Incubate HCT-116 cells with 0,7.5,15,30μM ATL for 0,1,2,4,6h,then detected the expression of protein related to cell apotosis,cell cycle and MAPK signaling pathway by Western-Blot(WB)and q-PCR assay.In vitro: Dected the expression of apoptosis&cell cycle related proteins,JNK,c-Jun in tumour tissue of subcutaneous tumor-bearing model mice by WB,q-PCR and ICH assay.Results:In vitro activity & mechanism study: The results showed that ATL could inhibit the activity of various colon cancer cells in a dose-dependent and time-dependent manner.Firstly,ATL could induce HCT-116 cells nuclear pyknosis,mitochondrial membrane potential loss,G0/G1 phase arrest,as well as enhance the proportion of apoptosis cells and inhibite colony formation.The migration distance and invasion rate of SW480 cells was significantly reduced after treated with ATL.According to the WB and q-PCR assay results,we lead to the conclusion that the anti-proliferative and pro-apoptosis effect of ATL on colon cancer cells were caused by activating the phosphorylation of protein JNK1/2,c-Jun,p38,Erk1/2 of MAPK signaling pathway,then regulating the G0/G1 phase related proteins Cyclin D1,Cyclin E,CDK4,p21 and apoptosis related proteins Bcl-2,Bax,cleaved caspase3.In vivo effect & mechanism study: ATL can inhibit tumor growth of CT26tumor-bearing model mice,significantly reduce tumor weight and the positive staining area of PCNA-ICH.Body weight of mice in the ATL50mg/kg group was significantly lower than that in the control group,and showed hepatic steatosis according to H&E assay.While there was no significant difference between the mice of ATL25,50mg/kg and mice of control groups both in body weight and H&E assay.ATL can significantly improve the pathological phenomena in ca CRC mice such as weight loss,diarrhea,colon edema and shortening,significantly inhibit tumor formation and growth.The positive staining area of PCNA-ICH and Brd U-ICH were reduced compared with the model group.ATL can activat the phosphorylation of protein JNK1/2 and c-Jun,suppress Cyclin D1 according to WB,q-PCR and ICH assay.Conclusion:1.ATL can induce colon cancer cells apoptosis and inhibit its proliferation,migration and invasion.2.ATL can significantly inhibit tumor growth in CT26 tumor-bearing model mice and ca CRC model mice.3.The potential mechanism of the anti-colorectal cancer effect of ATL could be regulate MAPK-JNK/c-Jun signaling pathway,induce G0/G1 phase cycle arrest,and initiate mitochondrial mediated apoptosis pathway.