Effect Of Curcumin And Glu-GNPs On Enhancing Radiosensitivity Of Breast Cancer Stem-like Cells

ObjectiveIn this study,human breast cancer cell lines MCF-7 and MDA-MB-231 adherent cells and mammospheres were studied.MCF-7 and MDA-MB-231 mammospheres were cultured in serum-free suspension-ball culture method,and the number of CD44+CD24-/low subsets was identified.The phagocytosis and distribution of nanoparticles-gold and glucose-coupled nanoparticles-gold(Glu-GNPs)in MDA-MB-231 adherent cells and mammospheres were observed by transmission electron microscopy(TEM).CCK8 and colony formation ability were used to detect the radiation sensitization of curcumin and Glu-GNPs and the combination group under 6MV X-Ray irradiation.Cobalt chloride simulated hypoxia model of breast cancer in vivo.The effects of curcumin and Glu-GNPs and combination group on ROS level,cell apoptosis,cycle,HIF-1alpha and HSP90 mRNA and protein levels were detected to explore the radiosensitization mechanism of curcumin and Glu-GNPs on breast cancer stem cells(BCSCs).Methods1.Culture and identification of MCF-7 and MDA-MB-231 adherent cells and mammospheres2.Electro-coupled synthesis of Glu-GNPs and transmission electron microscopy(TEM)were used to observe the phagocytosis and distribution of GNPs and Glu-GNPs in MDA-MB-231 adherent cells and mammospheres.3.The effects of curcumin combined with Glu-GNPs assistant irradiation on the proliferation and cloning ability of MCF-7 and MDA-MB-231 adherent cells and mammospheres.4.The effects of curcumin combined with Glu-GNPs assistant radiation on the levels of reactive oxygen species(ROS)in MCF-7 and MDA-MB-231 adherent cells mammospheres.5.Intervention of curcumin combined with Glu-GNPs assistant radiation on the cycle distribution and apoptosis of MCF-7 and MDA-MB-231 adherent cells and mammospheres.6.Quantitative RT-PCR detection of the effects of curcumin combined with Glu-GNPs auxiliary radiation on the transcriptional levels of hypoxia-inducible factor-1α(HIF-1alpha)and heat shock protein 90(HSP90)7.Western-Blot assay of the effect of curcumin combined with Glu-GNPs auxiliary radiation on the translation level of hypoxia-inducible factor(HIF-1alpha)and heat shock protein 90(HSP90)Results1.Human breast cancer cell lines MCF-7 and MDA-MB-231 were cultured by serum-free suspension method.The number of CD44+CD24-/low cell subsets was measured by flow cytometry.Serum-free suspension culture could significantly increase the number of CD44+CD24-/low cell subsets in MCF-7 cells,and also increase the number of CD44+CD24-/low cell subsets in MDA-MB-231 cells(P<0.05).)2.Transmission electron microscopy showed that GNPs were mostly distributed on the cell membrane of MDA-MB-231 adherent cells and mammospheres,while Glu-GNPs were mostly distributed on the cytoplasm of MDA-MB-231 adherent cells and mammospheres,and the uptake of Glu-GNPs was more than that of GNPs.3.Curcumin inhibited the viability of MCF-7 and MDA-MB-231 breast cancer cells(P<0.05),but GNPs and Glu-GNPs had no significant effect on the viability of MCF-7 and MDA-MB-231 breast cancer cells(P>0.05).4.Curcumin and Glu-GNPs alone and combined groups significantly decreased the proliferation of MCF-7 and MDA-MB-231 adherent cells and mammospheres under 4Gy X-Ray irradiation,and the inhibition effect of combined group was more significant.Curcumin and curcumin combined with Glu-GNPs inhibited the proliferation of MCF-7 and MDA-MB-231 adherent cells and mammospheres without X-Ray irradiation.At the same time,the results of colony formation experiment showed that curcumin and Glu-GNPs alone and combined groups significantly reduced the clone formation ability and survival fraction(SF)of MCF-7 and MDA-MB-231 adherent cells and mammospheres under 4Gy X-Ray irradiation,and the combined group was more effective(P<0.05).5.The ROS levels of MCF-7 and MDA-MB-231 adherent cells and mammospheres increased after 4 Gy X-Ray irradiation.Both curcumin group and Glu-GNPs group showed elevated ROS levels,while curcumin combined with Glu-GNPs group increased most significantly.The elevated ROS levels promoted the apoptosis of cancer cells and helped kill cancer cells(P<0.05).6.Flow cytometry showed that curcumin and Glu-GNPs alone and combined groups increased the apoptotic levels of MCF-7 and MDA-MB-231 adherent cells and mammospheres under 4Gy X-Ray irradiation,and the combined group had the most significant relative effect,and the apoptotic rate was more frequent in late apoptosis.Curcumin and curcumin combined with Glu-GNPs increased the apoptotic level of cells without irradiation,but the apoptotic rate was mainly in the early stage(P<0.05).Under CoCl2 induced hypoxia,compared with DMSO group,curcumin and Glu-GNPs group had lower S phase and higher G0/G1 phase.Whether X-ray irradiation alone or curcumin,Glu-GNPs combined with X-ray irradiation,G0/G1 phase significantly increased,inducing apoptosis.7.The results of qRT-PCR and Western-Blot showed that curcumin and Glu-GNPs could significantly inhibit the expression of HIF-1α and HSP90 in MCF-7 and MDA-MB-231 adherent cells and mammospheres under 4Gy X-Ray irradiation,and the inhibition effect of curcumin and Glu-GNPs combined group was more significant.The expression of HIF-1α and HSP90 in Glu-GNPs group increased at transcriptional and translation levels(P<0.05)after X-ray irradiation alone and without X-ray irradiation.Conclusion:Curcumin combined with Glu-GNPs can significantly enhance the radiosensitivity of human breast cancer MCF-7 and MDA-MB-231 adherent and mammospheres.Its molecular mechanism may be related to inhibiting the expression of HIF-1a and HSP90,increasing ROS level and inducing apoptosis of cancer cells.