Study On The Drug Resistance And Reversion In Ovarian Cancer Stem-like Cells

ObjectiveTumor stem cell is a minority subpopulation of stem-like cells in cancer tissues.It is highly related to the relapse and metastasis oftumor.lt has the characteristics of stem cells,able to be self-renewal and forming resemble cancer cells through growing in the body.Tumor stem cell has the natural drug resistance;it can pump drugs out of the membrane.Therefore,we have enough evidence to suspect whether it is the reason which limits the effect in traditional cancer chemotherapy.There are all kinds of drugs used in treating ovarian cancer in clinic,however,no matter with single or combining,the effect is not so good.Traditional drugs we often use are doxorubicin,cisplatinum,cytoxan and so on.They are non-specific drugs in the cell cycle and toxic to the DNA of cancer cell.They have the limited effect in killing cancer stem cells because there is a kind of membrane transporter ABCG2 in the membrane of cancer stem cells.It's ATP-binding transporter family,be able to pump the drugs out of the cell in order to have natural multi-drug resistance.Some researches have found that CD 133+cells existing in the neuroglia tumor tissue can resist the toxin of doxorubincin,so we choose doxorubicin and traditional drugs cisplatinum to be the representatives to complete this experimentation.Calciphorin blocker verapamil is the inhibitor of ABC family.lt can reverse the multi-drug resistance of the cells which express ABCG2,in order to make the cancer stem cell dead rapidly.Therefore,we try to use verapamil to block ABCG2,to observe whether it can reverse the drug resistance.Materials and MethodsOvarian cancer cell lines:OVCAR3 cell line was privided by Professor Zhou Xin (Department of Gynecology and Obstetrics,Shengjing Hospital Affiliated China Medicin University,Shenyang China).Ovarian cancer stem-like cell:OVCAR3 cell cultured in the FBS-free nutrient solution.Cell culturePick up the cancer stem-like enriching agglomerates in ovarian cancer:take OVCAR3 cells out of the nutrient solution which contains 15%FBS,put them into the 6cm culure plate extended with agar.Add FBS-free DMEM/F12 solution to the plate,changing the half solution interval,forming cell agglomerates after 4-5 days.Repeat the method above several times to form cancer stem-like enriching agglomerates.The method of methyl thiazolyl tetrazolium(MTT) was used to determine the inhibition ratio of OVCAR3 cells and ovarian cancer stem-like cells with traditional chemotherapy drugsAdd OVCAR3 cells or ovarian cancer stem-like cells 100μL to each hole,containing 6000 cells.Put 10μL drug,DMEM 90μL into each hole.There are no drugs in negative control,200μL solution in blank.Repeat it three times,examinate the IR.The method of soft agar clone was used to determine the inhibition ratio of OVCAR3 cells and ovarian cancer stem-like cells with traditional chemotherapy drugsThere are no cells in the 0.6%underlayer agar.The 0.3%upperlayer agar contains OVCAR3 cells or ovarian cancer stem-like cells.Put condition solution on it.Calcualate the colons after 3 weeks.Repeat it 3 times.Results1.The ovarian cancer stem-like cells we cultured have the characterastics of stem cell.They are suspended in the solution and forming stem-like cell agglomerates.2.The consequences of MTTThe ovarian cancer stem-like cell have the drug resistence of doxorubicin and cisplatinum comparing to the OVCAR3 cells.Verapamil can reverse the drug resistance of ovarian cancer stem-like cells to a certain extent.3.The consequences of soft agar clone.The ovarian cancer stem-like cell have the drug resistence of doxorubicin and cisplatinum comparing to the OVCAR3 cells.Verapamil can reverse the drug resistance of ovarian cancer stem-like cells to doxorubicin to a certain extent,but not obvious to cisplatinum. ConclusionThe ovarian cancer stem cell have the drag resistence of doxorabicin and cisplatinum comparing to the OVCAR3 cells.In MTT test verapamil can reverse the drag resistance of ovarian cancer stem-like cells to a certain extent.In soft agar clone test verapamil can reverse the drag resistance of ovarian cancer stem-like cells to doxorabicin to a certain extent,but not obvious to cisplatinum.