Lutein Inhibits Proliferation,invasion And Migration Of Hypoxic Breast Cancer Cells Via Down-regulation Of HES1

BackgroundLutein（LUT）,a plant-derived compound and a non-vitamin A carotenoids,was shown to possess multiple protective properties including anti-inflammation,anti-oxidative stress and anti-tumor effects.An intratumoral hypoxic microenvironment is frequently observed in solid tumors,including breast cancer.Hypoxic and necrotic areas of a tumor in turn produce proinflammatory mediators that recruit more immune cells resulting in suppression of the immune response at the tumor site as well as tumor cell proliferation,angiogenesis and metastasis.Levels of Intracellular reactive oxygen species（ROS）paradoxically increase under hypoxia where mitochondria appear to be their main source of production.Hypoxia inducible factor-1α（HIF-1α）is involved in both intrinsic and extrinsic activation of tumor-associated inflammatory signaling,angiogenesis and cancer invasion.Under hypoxic conditions,ROS can also activate signalling components upstream of the HIF-1,such as ERK and p38 MAP kinase pathways.Nuclear HIF-1αinduce NOTCH to upregulate a subset of target genes for Notch and this occurs via a physical interaction between HIF1αand NOTCH.HES1 is a critical transcriptional factor and affects tumorgenesis,cell differentiation,maintenance of stem cells,and the malignancy and maintenance of tumor cells.HES1 is activated by both canonical and non-canonical path-ways and NOTCH represents one of the prominent canonical pathways.Since hairy and enhancer of split 1（HES1）promotes epithelial-mesenchymal transition（EMT）related alterations and EMT is considered to be the mechanism that promotes invasion and metastasis of tumor,HES1 play crucial roles in tumor invasion and metastasis.ObjectiveThe main objective of the present research was to elucidate the roles of lutein in the production of ROS under hypoxia,the activation of HES1,proliferation,invasion and migration of breast cancer cells.Methods1.The human breast cancer cell lines,MDA-MB-157 and MCF-7,were exposed to a hypoxic or normoxic condition and various concentrations of lutein（5,10,20,40,80,and 120μM）for 24 h;0μM lutein was used as negative control.The MTT assay was performed to examine cell growth and The IC50 for the effects on breast cancer cell proliferation of lutein was calculated respectively.2.AnnexinV-FITC/Propidium iodide（PI）staining was performed by flow cytometry to analyze the apoptosis ratio of MDA-MB-157 and MCF-7 cells treated with lutein（0,20,40,and 80μM）for 24 h under hypoxia or normoxia.3.The mRNA and protein expression levels of HIF-1α,NOTCH signaling molecules,HES1 and epithelial-mesenchymal transition-related factors were examined by qRT-PCR and western blot analysis in MDA-MB-157 and MCF-7 cells treated with 40μM lutein or transfected with HES1 expression vector under hypoxia.4.Transwell invasion assay was employed to detect the invasion of MDA-MB-157 and MCF-7 cells treated with 40μM lutein or transfected with HES1expression vector under hypoxia.5.Wound healing assay was employed to detect the migration of MDA-MB-157and MCF-7 cells treated with 40μM lutein or transfected with HES1 expression vector under hypoxia.6.The intracellular ROS levels of MDA-MB-157 and MCF-7 cells treated withlutein,hydrogen peroxide（H2O2）or N-acetylcysteine（NAC）were examined using DCFH-DA by flow cytometry.7.The mRNA and protein levels of HIF-1α,NOTCH and HES1 of MDA-MB-157 and MCF-7 cells treated with lutein,H2O2 or NAC were examined using qRT-PCR and western blot analysisResults1.The MTT data showed that MDA-MB-157 and MCF-7 cells proliferation was clearly inhibited with lutein in a dose-dependent manner compared to the 0μM lutein was used as negative control.2.The apoptosis ratio of MDA-MB-157 and MCF-7 cells gradually increased with the treatment of lutein under hypoxia compared to groups with treatment of 0μM lutein as evident from flow cytometry-based analysis.3.Exposure to lutein inhibited hypoxia-mediated activation of HIF-1α,decreased the mRNA and protein levels of NOTCH signalings and HES1 expression,and suppressed the expression of hypoxia-induced expression of EMT-related factors in MDA-MB-157 and MCF-7 cells.Downregulation of EMT-related factors induced by lutein was notably inhibited by overexpression of HES14.Lutein markedly inhibited the invasion of breast cancer cells under hypoxia.lutein-induced decrease in invasion was notably inhibited by overexpression of HES1.5.Lutein markedly inhibited the migration of breast cancer cells under hypoxia.lutein-induced decrease in migration was notably inhibited by overexpression of HES1.6.Hypoxia-induced production of ROS was also decreased by lutein and ROS scavenger NAC,but increased by H₂O₂.7.HIF-1αand HES1 expression was also suppressed by the NAC in breast cancer cells during hypoxia,but increased by H₂O₂.Conclusion1.Lutein significantly suppressed proliferation,invasion,and migration of breast cancer cells under hypoxia in vitro.2.Lutein markedly suppressed hypoxia-induced ROS levels,which is essential for the stability of HIF-1α.3.These results provide insight into the molecular mechanisms underlying significant suppression of proliferation,invasion,and migration of breast cancer cells.Lutein inhibited HES1 expression through HIF-1αand NOTCH signaling pathway,which contributed to inhibition of EMT in breast cancer cells.