Study On Inhibition Effect And Mechanism Of Tan ⅡA In Human Esophageal Carcinoma Cell Line Eca-109

Background: According to the report,the number of deaths from ephageal cancer is about 210000 every year in our country.The main treatment is suigicalresection,radiotherapy and chemotherapy,etc, single method is not ideal,therefore,we must take the comprehensive treatment and use chemotherapy after the surgery.Now the new chemotherapy drugs is very more,but the side effect is still very large,and the drug resistance is increasing,the 5-year survival of patients is low.Therefore chinese medicine and the active ingredient is the one of the driection in the future in research of cancer.This study investigates the possible mechanism of esophageal cancer and the effection of the volatile oil of Salvia---TanⅡA in human esophaeal carcinoma cell line Eca-109,this can provide experimental date for TanⅡAtreatment of esophageal cnacer. TanⅡA is the main component of Salvia,Tan has been widely used in non-tumor areas of the clinically,mainly for the treatment of cardiovascular disease.In recent years,scholar generated great interest about antitumor activity of TanⅡA, but the anti-caner effect of esophageal cancer was rarely reported.Objects: By cultured human esophageal carcinoma Eca-109,different concentration of TanⅡA interfere the cell growth to explore mechanisms that TanⅡA promotes Eca-109 apoptosis.Methods: 1. By cultured human esophageal carcinoma Eca-109,different doses of TanⅡA intervene Eca-109 cell,incubate cell for 12、24、36、48 hours, MTT method measure absorbance(OD) of each group,every group cell proliferation rate is calculated,the proliferation inhibition rate curve is drawn.2. Drug group and the normal group are cultured for 24 hours,observe the cell growth and morphological of normal group and drug intervention. 3. Drug group and the normal group are cultured for 24 hours,fluorescenc e microscope observe two group of morphological change of cells in A O/EB staining. 4. Select TanⅡA of 140μg/ml and 160μg/ml intervention cell for 24 and 48 hours,setting a control,using FCM detector cell cycle and apoptosis. 5. Using TanⅡA of 140μg/ml and 160μg/ml intervention cell 24 hours,using Western blot techniques detect Caspase-3、Caspase-9、Cyclin A and CDK2 expression,β-actin as control,using BIO-RAD software analysis the gray value.Results: 1.MTT assay show:cell viability is significant higher than the drug group,the viable cell decline when the concentration of the drug is increased in a certain rang,the highest inhibition rate is 66.164%,P value is less than 0.05 between group. 2.Microscopy observation show: After TanⅡA treat the cell,cells have significant morphology change, cells appear floating,nuclear condensation,membrane damage appears “vesicles”phenomenon. 3.Fluorescence microscopy observation show:cell nuclear is green and conde nsation,some cells are appear to be dyed orange. 4.FCM result show:TanⅡA 140μg/ml interven Eca-109 24 hour,the number of cells in S phase increase to 54.47%,the number of GO/G1 phase decline from 51.317% to 41.787%,p<0.05. 5.Western blot result show:Comparing with normal group,drug treat Eca-109 24 hours,Cyclin A,CDK2 expression decrease,Caspase-3,Caspase-9 expression incr ease.Conclusion: 1. TanⅡA can significantly inhibit the activity of Eca-109,and this inhibit effect with increasing concentrations of the drug will be more obvious,but the concentration exceeding 160μg/ml,the inhibition will not rise. 2. In the process of TanⅡA acting on Eca-109,as the drug concentration increases,the proportion of cells in S phase gradually increase, Cyclin A,CDK2 decrease. 3. After TanⅡA intervention human esophageal cancer cell line Eca-109,the expression of apoptosis index and Caspase-3,Caspase-9 increase. 4. The possible mechanism that TanⅡA inhibits cell proliferation and promotes cells apoptosis is decline Cyclin A and CDK2 cell cycle is blocked in S phase and rise Caspase-3,Caspase-9 promoting cell apoptosis.