| Research purpose and significance:In this study,we aimed to evaluate the inhibitory effect of DHA on cancer stemlike cells(CSLCs)and the enhancement effect of DHA on oxaliplatin sensitivity in colorectal cancer(CRC),further to explore the potential mechanism.It will further expand the understanding of DHA in the field of anti-tumor,and provide a certain scientific basis for clinical research and application.Methods:1.The effects of DHA(0,2.5,5,10,20,40,80 μM)on the cell viability of HCT116 and SW620 cells were analyzed by CCK-8 assay and clonal formation assay;HCT116 and SW620 cells were treated with different concentrations of oxaliplatin(LOHP)(0.75,1.5,3,6,12,24 μM)and 10 μM DHA,and the effects of DHA on sensitivity to L-OHP were detected by CCK-8 assay.2.Sphere forming assay was used to detect the effect of DHA on the ability of cell suspension sphere formation;Western blotting was used to detect the effect of DHA on the expression of cancer stem surface marker CD133,CD44 and cancer stem cell key transcription factor Nanog,c-Myc and OCT4;3.The effect of DHA on AKT/mTOR signaling was evaluated.The protein levels of mTOR,p-mTOR,AKT and p-AKT were determined by Western blotting in HCT116 and SW620 cells treated with different concentrations of DHA.CCK-8 assay and clonal formation assay were used to detect the change of cell survival rate and clone formation number after cotreated with SC79 and DHA;Cells were treated with AKT agonist SC79 and DHA,sphere formation and Western blotting were used to detect the changes in the characteristics of cancer stem-like cells.4.The effect of DHA on tumorigenicity of colorectal cancer cells in nude mice:nude mice vaccinated with HCT116 cells were randomly divided into control group and the drug treated group.The drug treated group was injected with DHA 40 mg/kg daily,and the tumor size was measured every two days;Result:1.CCK-8 analysis and the results of colony formation showed that DHA inhibited the viability of CRC cells.It was found that the inhibition rate of L-OHP in CRC cells treated with DHA was higher than that without DHA treatment,and the IC50 of L-OHP was significantly reduced;2.Tumor sphere formation assay showed that DHA decreased the size and number of spheroids compared with the control group.DHA down-regulated the expression of stem cell characteristics related proteins CD133,CD44,Nanog,c-Myc and OCT4 in CRC cells;3.Acquired data showed that the protein expressions of mTOR,p-mTOR,AKT and p-AKT were decreased in a dose-dependent manner with the increase of DHA concentration.SC79 inhibited the declines of cell viability,colony formation and tumor sphere mediated by DHA in CRC cells,while impaired DHA-increased chemosensitivity of CRC cells to L-OHP.DHA-decreased expressions of Nanog,c-Myc,OCT4 were rescued by SC79 treatment in HCT116 and SW620 cells.4.According to the measurement results,the growth rate of tumors in DHA treatment group was generally lower than that in the control group.DHA treatment significantly suppressed xenograft tumor size and weight compared to the control;Conclusion:DHA has the effect of inhibiting the cell growth,tumorigenicity and CSLCs properties of CRC,and improving the L-OHP chemosensitivity;DHA can reduce cancer stem-like cells properties through AKT/mTOR signaling pathway.DHA may act as a potential therapeutic agent for colorectal cancer. |
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