Study On The Effect Of Curcumin On The Chemoresistance To Irinotecan In Human Colon Cancer Cells And Its Mechanism Involved

BackgroundResistance to conventional chemotherapeutic agents,such as irinotecan(CPT-11)and 5-fluorouracil(5-FU),is one of the major causes for therapeutic failure in colorectal cancer(CRC)patients.The response rate to irinitecan is only in the range of about 30-35%,and the 5-year survival rate is less than 10%.Increasing evidences have demonstrated that cancer cells with stem cell-like characteristics are related to the development of resistance to chemotherapeutic agents.As a plant polyphenol,curcumin has been demonstrated to have the ability to ameliorate resistance of CRC to chemotherapeutic agents,but the relationships among curcumin,cancer stem cell(CSC)and chemoresistance in CRC are still unclear.Part 1 The establishment and evaluation of irinotecan-resistant cells of human colon cancer,and the effect of curcumin on chemoresistance and characteristics of cancer stem cellsObjectivesTo investigate the relationship between the chemoresistance of human colon cancer cells and the characteristics of cancer stem cells,study the effect of curcumin(Cur)on the chemoresistance in human colon cancer cells and explore the mechanism involved.MethodsIrinotecan-resistant human colon cancer cell line(LoVo/CPT-11)was established by exposure to gradually increased concentration of CPT-11.Cell counting kit-8(CCK-8)assay was applied to detect the growth inhibition rate and figure up the resistance index.RT-PCR and western blot assays were used to examine the changes of multi-drug resistance protein ABCB1 and cancer stem cell(CSC)identification markers CD44?CD133?EpCAM and CD24 in mRNA and protein levels.The effect of Cur on resistance to CPT-11 was detected after treatment with the mixture of Cur(2.5,5?mol/L)and CPT-11.RT-PCR and western blot assays were used to examine the effect of Cur and CPT-11 on the expression of CSC identification markers.Results1.LoVo/CPT-11 cells were able to grow steadily in the culture containing 50?mol/L CPT-11.The half-maximal inhibitory concentration(IC50)of irinotecan in LoVo/CPT-11 cells was 268.72 ± 2.43 ?mol/L,significantly higher than that of LoVo cells(43.27 ± 1.64 ?mol/L).The resistance index was 6.21.2.The expression of multi-drug resistance protein ABCB1 and cancer stem cell identification markers CD44,CD133,EpCAM,and CD24 in mRNA and protein levels were increased markedly in LoVo/CPT-11 cells compared to the parental cells,and the result reached statistical significance.3.The growth inhibitory rates were significantly increased in the presence of Cur(2.5,5?mol/L),the half-maximal inhibitory concentration(IC50)of CPT-11 were significantly reduced,curcumin could down-regulated the resistance of LoVo/CPT-11 cells to CPT-11,and the result reached statistical significance.4.The expression of CD44,CD133,EpCAM and CD24 were reduced by curcumin in a concentration dependent manner in both mRNA and protein levels,whereas CPT-11 had no effect on the expression of them.ConclusionsThe chemoresistance of human colon cancer cells were related to the characteristics of CSC,Cur could reduce chemoresistance in human colon carcinoma cells by inhibiting the characteristics of CSC.Part 2 Study on the effect of curcumin on enriched colon cancer stem cells and its mechanism involvedObjectivesTo investigate the effect of curcumin on the biological characteristics of colon cancer stem cells and its mechanism involvedMethodsColon cancer stem cells were enriched by culturing sphere-forming cells in serum free medium.The percentages of CD 133-positive cells in LoVo cells,LoVo/CPT-11 cells and sphere-forming cells were measured by flow cytometry assay.Sphere-forming cells were treated with various concentrations of curcumin(0,2.5,5,10,20 ?M)and irinotecan(0,10,20,40,100 ?M)separately and in combination,the tumorspheres of each group were imaged by light microscope.The effect of curcumin on the expression of CSC identification makers in sphere-forming cells were detected by western blot assay.The effect of curcumin on tumor initiation capacity of colon CSC was detected by experimentation on animals with bearing cancer.The apoptosis distribution was detected by flow cytometry assay and the expression levels of proteins that are related to apoptosis were measured by western blot assay.Results1.LoVo/CPT-11 cells were plated in SFM containing several appropriate growth factors and then stable cell spheroids were formed after 1 week.The percentage of CD 133-positive cells was 63.3%in sphere-forming cells,much higher than that in LoVo cells(1.0%)and LoVo/CPT-11 cells(5.0%).2.Curcumin had a dose-dependent inhibitory effect on tumorsphere formation and there were almost no tumorsphere in SFM culture upon 10 ?M curcumin treatment.Whereas irinotecan alone had almost no effect on the formation of tumorsphere.What's more,for the treatment of the combination of curcumin and irinotecan,the inhibitory effect was stronger than that of curcumin alone.3.The expression of CSC identification makers was markedly inhibited by curcumin alone or combination with irinotecan compared to the control group,but irinotecan alone had almost no effect on the expression.4.Sphere-forming cells untreated with curcumin can formed larger tumor than treated cells,it indicated a significantly inhibitory effect of curcumin on tumor initiation capacity of colon CSC.5.The result of flow cytometry analysis indicated that cell apoptosis was induced in the presence of curcumin in sphere-forming cells.Besides,the expression level of anti-apoptosis protein was decreased in the presence of curcumin,and the levels of pro-apoptosis proteins were increased.But irinotecan alone had almost no effect on the apoptosis.ConclusionsCurcumin could effectively inhibited the biological characteristics of colon cancer stem cells,it might be related to the effect of inducing apoptosis.