Reversal Of Multidrug Resistance In Human Cholangiocarcinoma Cell By Ginsenoside Rg3

Objective: To investigate the effect of Ginsenoside Rg3 on multidrug resistance(MDR)of human cholangiocarcinoma cell line QBC939/ADM.Methods:The establishment of multidrug resistant human cholangiocarcinoma cell line QBC939/ADM,is mainly by adriamycin(ADM)concentration increasing continuous contact method,with high and low concentration alternating method induced by bile duct cancer cells QBC939 multidrug resistance(MDR)if necessary.Three different concentrations of chemotherapeutic drugs—Cyclophosphamide(CTX),Mitomycin(MMC)and 5-Fluorouracil(5-FU)respectively on QBC939 and QBC939/ADM,using CCK-8method to detect the cytotoxic effect of chemotherapeutic agents,to detect the cell apoptosis by flow cytometry;RT-PCR detection method the expression level of MDR gene.Collect all the data and use statistical analysis,finally draw the conclusions,which proves the establishment of multidrug resistant human cholangiocarcinoma cell line QBC939/ADM.The effective constituents of Ginsenoside Rg3 in ginseng as MDR reversal agents,to reverse the MDR of QBC939/ADM,by CCK-8 method、flow cytometry and RT-PCR method to detect the reversal effect.Results : To prove the establishment of resistant cells of human cholangiocar cinoma cell line multidrug QBC939/ADM:(1)Using CCK-8 to detect the cytotoxicity of the three chemotherapeutic drugs,respectively Cyclophosphamide(200 μg/ml),Mit omycin(2.0μg/ml),5-Fluorouracil(250μg/ml).Adopt the three drugs mentioned abo ve on QBC939 and observe 24 h,then get the OD date: Cyclophosphamide group 0.568±0.085,Mitomycin group 0.506±0.024,5-Fluorouracil group 0.574±0.093.W hen on QBC939/ADM after 24 h,the data are: Cyclophosphamide group 1.178±0.120,Mitomycin group1.037±0.073,5-Fluorouracil group 1.188 + 0.111;the activit y of QBC939/ADM cells was stronger than QBC939;the QBC939/ADM compared with the QBC939,P<0.05,the difference was statistically significant;(2)Using flow cyt ometry to observe the cell apoptosis of the three kinds of chemotherapeutic drugs.Adopt the drugs,respectirily Cyclophosphamide(200μg/ml),Mitomycin(2.0μg/ml)an d 5-Fluorouracil(250ug/ml),on QBC939 and QBC939/ADM,both oberving 48 h,th en record the data.The rate of cell apoptosis on QBC939: the cell apoptosis rates were: Cyclophosphamide group(69.16±1.97)%,Mitomycin group(59.45±1.74)%,5-Fluorouracil group(62.96±2.25)%;The rate of cell apoptosis on QBC939/ADM,the cell apoptosis rates were: Cyclophosphamide group(12.36±1.31)%,Mitomycin g roup(9.20±1.12)%,5-Fluorouracil group(11.89±1.50)%;the QBC939/ADM comp ared with the QBC939,P<0.05,the difference was statistically significant.The determination of the Ginsenoside Rg3 as reversal agents,reversal effect on QBC939/ADM:(1)The CCK-8 method for the determination of three kinds of chemotherapeutic drugs in the control group and the experimental group cytotoxicity.Control group: three kinds of chemotherapeutic drugs: Cyclophosphamide(200μg/ml),Mitomycin(2.0μg/ml),5-Fluorouracil(250μg/ml)respectively for 24 hours after QBC939/ADM,the OD value were: Cyclophosphamide group 1.153±0.158,Mitomycin group 1.202±0.107,5-Fluorouracil group 1.250±0.124;the OD data of three kinds of chemotherapeutic drugs were combined with Ginsenoside Rg3 in experimental group were:Cyclophosphamide + Ginsenoside Rg3 group 0.706 ± 0.056,Mitomycin + Ginsenoside Rg3 group 0.748 ± 0.105,5-Fluorouracil + Ginsenoside Rg3 group 0.779 ± 0.093;cell activity in the experimental group was significantly weaker than the control group;the experimental group compared with the control group,P<0.05,the difference was statistically significant;(2)The flow cytometry was used to detect apoptosis in the control group and the experimental group was three kinds of chemotherapeutic drugs.The three kinds of chemotherapeutic drugs: Cyclophosphamide(200μg/ml),Mitomycin(2.0μg/ml),5-Fluorouracil(250μg/ml),respectively for 48 hours after QBC939/ADM,the rate of cell apoptosis were Cyclophosphamide group(11.79 ± 1.06)%,Mitomycin group(9.22 ±1.25)%,5-Fluorouracil(10.75±1.28)% in the experiment;cell apoptosis the rate of three kinds of chemotherapeutic drugs were combined with Ginsenoside Rg3 respectively:Cyclophosphamide+Ginsenoside Rg3 group(22.51 ± 1.20)%,Mitomycin+Ginsenoside Rg3 group(26.56±1.67)%,5-Fluorouracil + Ginsenoside Rg3 group(21.90±1.60)%;the experimental group compare with the control group,P<0.05,the difference was statistically significant.Determination the MDR gene expression by RT-PCR: QBC939:0.23 ± 0.031;QBC939/ADM:8.17 ± 0.042;experimental group(added Ginsenoside Rg3 after the reversal of multidrug resistance of QBC939/ADM):0.55±0.029;compared with QBC939,P<0.05,the difference was statistically significant;the experimental group compare withQBC939/ADM,P<0.05,the difference was statistically significant.Conclusion: The resistant cell of human cholangiocarcinoma cell line multidrug QBC939/ADM is successfully established,and preliminary evidence that the Ginsenoside Rg3 of Panax Ginseng can effectively reverse the multidrug resistance,but its reversal mechanism has not been studied,which need the further explored.