Curcumin Inhibits Invasion And Metastasis Of Human Breast Cancer Stem Cells

ObjectTo investigate the effects of curcumin on the proliferation,cloning,invasion and migration of breast cancer adherent cells;to culture and identify MCF-7 and MDA-MB-231 tumor suspension cells in serum-free suspension culture;to study the effects of curcumin on stem cell pellets.To investigate the effects of curcumin on the expression of surface markers,stem cell genes and epithelial-mesenchymal transition(EMT)markers in breast cancer stem cells,and to explore the molecular mechanism of action.Methods1.CCK8 assay and cell clone formation assay were used to detect the effects of curcumin on proliferation of MCF-7 and MDA-MB-231 adherent cells.2.Trans well and cell scratch assays were used to detect the effects of curcumin on the invasion and migration of highly metastatic cell line MDA-MB-231.3.The serum-free suspension culture method was used to culture MCF-7 and MDA-MB-231 stem cell spheres.Flow cytometric analysis was used to identify the stem cells of tumor cells.Confocal laser scanning microscopy was used to observe the morphology and localization of the cells.4.Flow cytometry was used to detect the effect of curcumin on CD44+CD24-cell subsets of breast cancer stem cells.5.Curcumin interferes with the formation and differentiation of stem cells in breast cancer MCF-7 and MDA-MB-231 cells.6.qRT-PCR detected the effects of curcumin on stem cell gene and EMT marker gene expression at the transcriptional level;Western blot assay detected the effect of curcumin on stem cell gene expression at the translation level.ResuLts1.Curcumin inhibited the activity of MCF-7 and MDA-MB-231 cells,and the inhibitory effect increased with the increase of drug concentration.There was no statistically significant difference between the DMSO solvent control group and the blank control group,indicating that the concentration of DMSO required to dissolve curcumin did not affect the activity of breast cancer cells.The resuLts of cell colony formation experiments showed that curcumin inhibited the colony formation and proliferation of MCF-7 and MDA-MB-231 adherent cells,and with the increase of drug concentration,the lower the colony forming ability,the curcumin group Compared with the control group,the difference was statistically significant(P<0.05).2.Trans well experiment results showed that curcumin can reduce the number of cells that enter the lower compartment of the highly metastatic cell line MDA-MB-231;the cell scratches experimentally observed that the healing rate of the scratches of the curcumin group was lower than that of the control group,which was statistically significant,indicating that the curcumin It has a significant inhibitory effect on the invasion and migration of MDA-MB-231 cells.3.The serum-free suspension culture method was used to culture MCF-7 and MDA-MB-231 stem cells.They began to pellet from the first 3-5 days,began to pass on the 7th day,and were passaged on the 1st week to collect the 3rd generation suspension cells.FCM resuLts showed that CD44+CD24-cell subpopuLations of MCF-7 and MDA-MB-231 suspension breast cancer stem cells were significantly higher than adherent cells,indicating that the cultured floating sphere cells had enhanced dryness.CelluLar immunofluorescence experiments showed that after serum-free suspension culture,mesenchymal markers and stem cell genes increased,and cell adhesion decreased.4.Flow cytometric analysis showed that curcumin couLd reduce the expression of surface markers CD44+CD24-of MCF-7 and MDA-MB-231 suspension stem cells,indicating that curcumin inhibited the stemness of breast cancer stem cells.effect.5.The 3rd generation MCF-7 and MDA-MB-231 suspension stem cells were inocuLated into the uLtra-low adherence 6-well plate.After 5 days of drug treatment,the number of cell spheres in the curcumin group was significantly reduced compared with the control group.Compared with statistical significance.After counting,the cells in each well were differentiated and cultured with FBS-containing medium.The cells in the control group were still capable of adherent proliferation.After the concentration of curcumin exceeded 30 umol/L,no cell differentiation was adhered,indicating that curcumin can differentiate tumor stem cells.Ability has a significant inhibitory effect.6.The resuLts of qRT-PCR showed that MDA-MB-231 suspension cells were significantly higher than adherent stem cell genes and EMT mesenchymal marker expression.Curcumin(40 umol/L)treatment reduced Vimentin compared with DMSO group.Fibronection and β-catenin expression,but no inhibition of stem cell gene mRNA levels.The resuLts of Western blot showed that MCF-7 and MDA-MB-231 suspension cells had higher stem cell genes than adherent cells.Curcumin(40 umol/L)treatment reduced stem cell gene expression compared with DMSO group,indicating that curcumin has an effect on stem cells.Genes and EMT marker genes have reguLatory roles at transcriptional or translational levels.ConclusionsCurcumin can inhibit the proliferation and colony forming ability of MCF-7 and MDA-MB-231 cells,and inhibit the invasion and migration of MDA-MB-231 high metastatic cells.Further studies showed that curcumin can reduce the surface markers of breast cancer stem cells.CD44+CD24-cell subpopuLations inhibit the formation and differentiation of stem cells of MCF-7 and MDA-MB-231 stem cells,which may be related to the reguLation of stem cell genes and the expression of EMT markers by targeting breast cancer stem cells.Related reguLatory mechanisms are pending.discuss in depth.