The ROS-Mediated Activation Of STAT3/VEGF Signaling Is Involved In The 27-Hydroxycholesterol-Induced Angiogenesis In Human Breast Cancer

Breast cancer(BC)is one of most common malignant tumor of female worldwide,and there are about 1 million cases of breast cancer was diagnosed each year.Moreover,high fat,high cholesterol diet-induced obesity and hypercholesterolemia,which make breast cancer patients face higher risk.It is likely that obesity increases the circulating small moleculars then promotes the tumor progression.At present,studies indicate that,27-Hydroxycholesterol(27HC),a primary metabolite of cholesterol,is mainly from the circulatory system.Serum circulating levels of 27HC is positively correlated with cholesterol and hypercholesterolemia.27HC is an endogenous selective estrogen receptor modulator,playing estrogen-like effects and promoteing estrogen receptor(ER)-positive breast cancer growth.In mouse models of breast cancer,27HC can interact with liver X receptor(LXR),and promote breast cancer metastasis.In addition,the growth and metastasis of breast cancer depend on the formation of new blood vessels,however,the effects of 27HC on the angiogenesis in breast cancer,and the potential molecular mechanisms,remain largely uninvestigated.Therefore,the aim of the present study is to explore the potential molecular changes initiated by 27HC,and provide a better understanding of 27HC-induced breast cancer progression,which contribute to the prevention of 27HC-induced health hazards.It is great significant for the breast cancer patients,especially in obesity and hypercholesterolemia.Objection:In this study,human breast cancer ER positive cells(MCF7,T47D),and ER negative cells(MDA-MB-231)were used in vitro to investigate whether 27HC promote the angiogenesis ability of breast cancer,and its molecular mechanism.Methods:Cell Counting Kit-8(CCK-8)was used to detect the cell vitality of MCF7 and T47D breast cancer cells.The secretion of VEGF was tested by enzyme-linked immunosorbent assay(ELISA).Breast cancer cells were treated with 27HC,and then the cell culture medium was collected for the co-culture of human umbilical vein endothelial cells(HUVECs).Tube formation was used to test the angiogenesis ability of those HUVECs.siRNA transfection was used to knock down the expression of STAT3.The measurement of reactive oxygen species(ROS)was performed by fluorescent probes DCFH-DA and flow cytometry.The qRT-PCR and Western blot were utilized to test the mRNA levels and protein levels respectively.Results:MCF7 or T47D cells were exposed to different concentrations of 27HC.CCK-8 assay was used to test the cell vitalities,and there was no detectable cytotoxicity of 0.5,1.0,2.0,5.0 μM of 27HC.Then we treated MCF7,T47D cells with 0.0,2.0,5.0 μM 27HC for 48 h,and collected the cell culture medium.The results of ELISA showed that 27HC enhanced the secretion of VEGF;Tube formation showed that the conditioned medium improved the number of tubes,then the cells were treated with 27HC,and the medium VEGF neutralizing antibody,the ability of promoting blood vessel formation was inhibited.27HC and estradiol could interact with estrogen receptor,activate target genes PS2,and induce the mRNA expression of VEGF.The estrogen-like activity of 27HC was weaker than estradiol,while the 27HC-induced increased expression of VEGF was higher than that E2-treated group.When we used the estrogen receptor inhibitor ICI,estradiol-induced increased expression of VEGF was completely blocked,but 27HC-treated group was partly reduced.In MCF7,T47D and MDA-MB-231 cells,27HC could activate the expression of p-STAT3,HIF-1α,knockdown of STAT3.The 27HC-induced increased expression of VEGF mRNA and secretion were reduced;the angiogenesis ability of HUVECs that co-cultured with conditioned medium was reduced too.27HC increased the ROS levels in MCF7,T47D and MDA-MB-231 cells.In addition,blockage the generation of ROS via antioxidants n-acetyl cysteine(NAC)in breast cancer cells,reduced the expression of p-STAT3 and VEGF mRNA.ELISA and tube formation found that,the secretion of VEGF,angiogenesis ability of HUVECs were reduced.Conclusion:In breast cancer,27HC improved the angiogenesis via two independent manners:(1)the classical ERα/VEGF signaling in ER-positive breast cancer cells,and(2)the ROS/STAT-3/VEGF in both ER-positive and ER-negative breast cancer cells.These findings suggested that 27HC was a potential harmful factor in breast cancer,especially in the menopause patients.