| ObjectiveIn this study,triple negative breast cancer cells were taken as the research object.Firstly,the effect of curcumin on the proliferation,invasion and migration of triple negative breast cancer cells was studied,and then the characteristics of stem cells and the effect of curcumin on the formation of stem cells were discussed.Base on that,we studied further whether curcumin can reduce the expression of key factors of Gli nuclear molecular by mediating the Hedgehog/Gli signaling.We expect this research can provide experimental basis for the application of curcumin in the prevention and treatment of recurrence and metastasis of triple negative breast cancer.Methods1.CCK8 assay and cell colony assay were used to detect the effect of curcumin on the cell viability and proliferation of MDA-MB-231 and MDA-MB-468 adherent cells.2.Transwell assay and wound healing assay were performed to evaluate the effect of curcumin on the invasion and metastasis ability of MDA-MB-231 adherent cells.3.Breast cancer stem cells were cultured by serum-free suspension method.The markers of stem cells were detected by flow cytometry and the stemness was identified by subcutaneous tumorigenesis in nude mice.4.The effect of curcumin on the formation ability of stem cell of triple negative breast cancer was detected by the formation of mammosphere assay.5.qRT-PCR and Western blot were used to detect the changes of Hedgehog signaling pathway related genes in triple negative breast cancer cells treated with curcumin.6.The changes of the nuclear localization of Glil and Gli2 in triple negative breast cancer cells treated with curcumin were observed by confocal.Results1.Curcumin and GANT61 significantly inhibited the viability of MDA-MB-231 and MDA-MB-468 adherent cells of triple negative breast cancer.According to the CCK8 assay,for MDA-MB-231 adherent cells,the concentration of curcumin was 20 μM,the concentration of GANT61 was 10 μM;for MDA-MB-468 cells,the concentration of curcumin was 10 μM,the concentration of GANT61 was 10 μM.In cell clone assay,compared to DMSO group,the number of colony in curcumin group,GANT61 group and Cur+GANT61 group decreased significantly(P<0.001).2.The results of Transwell assay in MDA-MB-231 adherent cells showed that the invasion rate of GANT61 group was(56.25±6.61)%,that of curcumin group was(3.36±0.83)%,and that of curcumin combined with GANT61 group was(0.97±0.83)%.Compared to DMSO group,the number decreased,especially curcumin and Cur+GANT61 group(P<0.01).The results of wound healing assay in MDA-MB-231 adherent cells showed that the healing rate of DMSO group could be completely healed 24 hours after treatment,while the healing rates of GANT61 group,curcumin group and Cur+GANT61 group were(63.94±5.48)%,(22.98±1.65)%,(12.76±4.38)%.And the difference between the administration group and DMSO group was statistically significant(P<0.05).3.The results of flow cytometry showed that the sternness markers CD44+CD24-/low of breast cancer mammosphere cultured by serum-free suspension method reached 98.2%,which was higher than that of adherent cells.Consistently,the results of subcutaneous transplantation in nude mice showed that MDA-MB-231 stem cells only needed 5×105 cells to develope tumor in a short period of time,and the tumor growth rate was much faster.However,MDA-MB-231 adherent cells needed 2×106 cells to form tumor.4.The inhibition rates of GANT61 group,curcumin group and Cur+GANT61 group on the mammospheres formation of MDA-MB-231 cells were(38.48±1.36)%,(92.18±2.16)%,(95.56±4.88)%respectively after 24 hours of treatment.And the inhibition rates on the mammospheres formation of MDA-MB-468 cells were(54.82±3.68)%,(72.09±5.60)%and(91.37±3.68)%respectively,compared to DMSO group(P<0.01).5.1n MDA-MB-231 cells,curcumin group and Cur+GANT61 group can effectively inhibit the transcription levels of Glil,Gli2,PTCH1,SHH and SMO genes on the Hh pathway.And they also reduced the expression of Gli1,Gli2 and PTCH1 proteins.6.The confocal observation of MDA-MB-231 cells and MDA-MB-468 cells showed that Glil and Gli2 proteins were mainly expressed in the nucleus and a small amount in the cytoplasm of DMSO group,while the expression of Glil and Gli2 was significantly reduced in the nucleus and cytoplasm in GANT61 group and curcumin group.In Cur+GANT61 group,Glil and Gli2 were hardly expressed.ConclusionsCurcumin can inhibit the proliferation and metastasis of triple negative breast cancer cells,which may be related to the inhibition of stem cell characteristics and the mediating of Hedgehog pathway;Gli1 and Gli2 are expected to be effective targets for gene therapy of triple negative breast cancer.Curcumin can inhibit of invasion and metastasis of triple negative breast cancer by partially mediating Gli. |
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