Reserch Into The Effect And Mechanism Of WCWE On HepG2 Cells

Background and objective:Malignant tumor is a major public health problem in the world.Especially in china,the number of new cases of liver cancer in China is 366 thousand people each year,ranking the country’s cancer incidence of the top third,the annual death rate of liver cancer was 321 thousand,ranking second of the national cancer death rate.The incidence of HCC is a result of multiple factors,its etiology is not clear,the hepatitis virus infection(HBV/HCV),alcoholic liver,fatty liver,aflatoxin is its recognized carcinogensgy6 h.Surgical excision and minimally invasive treatment for liver cancer therapy are the likely treatment at present,but for the most advanced liver cancer are unsuitable for operation,combined therapy of TACE,PEI,RFA,radiotherapy and Traditional Chinese Medcine are used to improve the quality of life of patients and prolong survival time.It is valuable to research the traditional Chinese medicine,especially how to inhibit the proliferation and invasion of tumor cells.Finding the medicine inhibiting the proliferation and invasion of tumor cells and elucidating the mechanism became a new direction of new drug research.As the medicine of clearing away the heat and toxins,Wild Chrysanthemum are Widely used in the clinical anti-tumor therapy,and many clinical experience about Wild Chrysanthemum have been got in the anti-tumor therapy.However,there are a few researches about the mechanisms of inhibiting the proliferation and invasion on tumor cells of Wild Chrysanthemum,which cannot reflect the inhibiting effect of Wild Chrysanthemum on hepatocellular carcinoma cells directly.This study is to define the effect of water extract of wild chrysanthemum on HepG2 cells,including cell proliferation,cell apoptosis,cell migration and invasion,and pursue the potential mechanism,to consolidate an experimental basis for the treatment of liver cancer by Wild Chrysanthemum.Methods:1.Prepare water extract of Wild Chrysanthemum,using High Performance Liquid Chromatography(HPLC)to determine the fingerprints of water extract of Wild Chrysanthemum,in order to provide an effective reference and repeatability for the experiments afterwards.2.HepG2 cells and LO2 cells were treated with different concentration of WCWE(Wild Chrysanthemum Water Extracts)(1μg/ml,5μg/ml,10μg/ml,20μg/ml,50μg/ml,100μg/ml,250μg/ml and 500μg/ml),MMT assay was used to detect the inhibitory effect of drugs on HepG2 cells and LO2 cells,and calculate the IC50 and IC30 of WCWE on HepG2 cells.3.HepG2 cells were divided into three groups:blank control group,negative control group(DMSO),WCWE(22μg/ml,IC30)group,MTT assay was used to draw the cell growth curve of HepG2 cells in four days by measuring OD value of HepG2 cells.4.HepG2 cells were divided into three groups:blank control group,negative control group(DMSO),WCWE(22μg/ml,IC30)group,HepG2 cells were cultivated for 24 hours.EdU Cell proliferation assay was used to detect the cell proliferation of HepG2 cells,and flow cytometry methods(PI stain)was used to measure the cell cycle distribution;5.HepG2 cells were divided into three groups:blank control group,negative control group(DMSO),WCWE(50μg/ml,IC50)group,HepG2 cells were cultivated for 24 hours,cell apoptosis was detected by CSFE/ PI and Annexin V-FITC/PI double staining method;6.HepG2 cells were divided into three groups:blank control group,negative control group(DMSO),WCWE(22μg/ml,IC30)group,Wound Healing Assay and Transwell Invasion Assay to detect the migration and invasion of HepG2 cells;7.HepG2 cells were divided into three groups:blank control group,negative control group(DMSO),WCWE(22μg/ml,IC30)group,HepG2 cells were cultivated for 24 hours,real-time fluorescence quantitative PCR and semi quantitative Western blot were used to analyses the differences of Cell proliferation,migration and invasion related genes Cyclin D1,Cyclin E2,COX2、MMP2、MMP9 expression.8.HepG2 cells were divided into three groups:blank control group,negative control group(DMSO),WCWE(50μg/ml,IC50)group,HepG2 cells were cultivated for 24 hours,real-time fluorescence quantitative PCR and semi quantitative Western blot were used to analyses the differences of expression of apoptosis related genes Bcl-2,Bax and Fas.Results:1.There were 7 common peaks among Wild Chrysanthemum,and the peak3 was similar to Chlorogenic acid with the similarity of greater than 0.90,the peak4 was similar to Luteolin-7-O-glucoside with the similarity of greater than 0.90.2.Wild Chrysanthemum Water Extracts can inhibit the proliferation of HepG2 cells,with increasement of drug concentration,the inhibition of cell growth was significantly increased(P<0.01).The IC30 and IC50 of Wild Chrysanthemum Water Extracts on HepG2 cells were 21.45±1.08μg/ml and 52.72±l.64μg/ml.The toxicity of Wild Chrysanthemum Water Extracts on LO2 was less than HepG2 cells.3.The growth curve of Wild Chrysanthemum Water Extracts on HepG2 cells showed the inhibition effect of Wild Chrysanthemum Water Extracts on the growth of HepG2 cells.4.EdU Cell Proliferation Assay showed that cells of S phase of WCWE group was 24.61±1.08 %,the cells of S phase of negative control group(DMSO)and blank control group was 36.15±3.21% and 39.28±4.29%,cells of S phase of WCWE group was significantly decreased(P<0.01);the flow cytometry methods(PI stain)was used to detect the cell cycle,showed that cells of G0/G1 phase and S phase of WCWE group was 68.1% and 28.3%,cells of G0/G1 phase and S phase of the negative control group(DMSO)was 53.8% and 38.2%,cells of G0/G1 phase and S phase of blank control group was 53.5% and 38.4%.Compared to the negative control group(DMSO)and blank control group,HepG2 cells in G0/G1 phases of WCWE group was increased,cells of S phases was decreased(P<0.01).5.CSFE/PI double labeling method showed that the percent of CSFE/PI double positive cells of WCWE group was 23.6±2.35%,the percent of CSFE/PI double positive cells of negative control group(DMSO)and blank control group was 4.28±1.02% and 2.64±1.16%,the rate of the CSFE/PI double positive cells of WCWE group was significantly increased when compared to the negative control group(DMSO)and blank control group(P<0.01).Annexin V-FITC/PI double staining method the cell apoptosis of the WCWE group was 20.16±2.17%,the cell apoptosis of the negative control group(DMSO)and blank control group was 4.36±0.48% and 3.92±0.61%.Compared to the negative control group(DMSO)and blank control group,the cell apoptosis of WCWE group was significantly increased(P<0.01).6.Wound Healing Assay showed that the relative scratch healing rate of the WCWE group was 20.57±1.78%,the relative scratch healing rate of the negative control group(DMSO)and blank control group was 32.91±1.93 % and 33.92±2.18 %.Compared to the negative control group(DMSO)and blank control group,the relative scratch healing rate of the WCWE group was decreased(P<0.01).Transwell Invasion Assay showed that the number of cells through the Transwell chamber of the basement membrane of the WCWE group was 103.25±14.38,the number of cells through the Transwell chamber of the basement membrane of the negative control group(DMSO)and blank control group was 196.81±18.25 and 204.76±30.94.Compared to the negative control group(DMSO)and blank control group,the number of cells through the Transwell chamber of the basement membrane of the WCWE group was decreased(P<0.01).7.Real time fluorescent quantitative PCR and Semi quantitative Western blot showed that the expression of Cyclin D1,COX2,MMP2,MMP9,Bcl-2 genes of HepG2 cells were down regulated by WCWE compared to the blank control group and negative control group(DMSO),the expression of Bax gene of HepG2 cells was up regulated by WCWE compared to the blank control group and negative control group(DMSO),the expression of Fas gene and Cyclin E2 gene was not regulated by WCWE.Conclusion:1.WCWE can inhibit the proliferation of HepG2 cells and induce the apoptosis of HepG2 cells,and also,WCWE can inhibit the cell migration and invasion of HepG2 cells.2.The molecular mechanism of the inhibition of cells proliferation and induction of apoptosis of WCWE is related to the down-regulation expression of Cyclin D1,Bcl-2 genes and up-regulation expression of Bax gene;3.The molecular mechanism of the inhibition of cells migration and invasion of WCWE is related to the down-regulation expression of COX2,MMP2 and MMP9 genes.