3,6-dihydroxyflavone Suppresses The Epithelial-mesenchymal Transition In Breast Cancer Cells

Breast cancer,the most common malignant tumor,has been a serious threat to the physical and mental health of women in world,and gives a heavy burden to society and family.Therefore,the research about breast cancer is particularly important.Epidemiological studies have found that dietary factors is closely related to the development of breast cancer.We used pharmacodynamics experiment method to screen common flavonoid compounds.We found 3,6-dihydroxyflavone(3,6-DHF)can inhibit breast carcinogenesis.3,6-DHF widely exist in fruits and vegetables,but the specific mechanism is still not very clear.The epithelial-mesenchymal transition(EMT)is a critical developmental program in cancer progress,which induced down-regulation of E-cadherin and up-regulation of Snail,Slug,Twist,N-cadherin.Many studies have showned that the activation of EMT can promote growth and metastasis of many tumors(breast cancer,gastric carcinoma,prostate cancer,cervical cancer and so on).It is well known that breast cancer stem cells(BCSCs)are pluripotent cells with self-renewal and multi-directional differentiation ability,which are related to tumor recurrence,resistance and metastasis.The breast cancer cells with ALDH+ phenotype or CD44+ CD24-phenotype of cells showed stem cell properties.Recent studies have shown that EMT has played a key role in CSCs(cancer stem cells)characteristics.EMT can promote the generation of CSCs,CSCs can promote the EMT of tumor cells,which depend on each other and promote each other.Our study found that 3,6-DHF can effectively inhibit the EMT of breast cancer cells in vivo and in vitro.And 3,6-DHF significantly inhibited the formation and growth of BCSCs.Optical in vivo imaging of cancer metastasis showed that 3,6-DHF asministration suppresses the lung metastasis of BC cells in vivo.There are many kinds of signaling pathways in regulation of EMT,that Notch signal pathway has the vital significance.Recent studies have shown that activation of Notch signaling pathway can effectively promote EMT and the formation of BCSCs.Notch signaling pathways was activated after the binding of Notch receptor and Jagged ligand.NICD(Notch1 intracellular domain)was released with ?-secretase.After Notch activation,NICD binds to CSL,displacing corepressors and recruiting MAML.Notch signaling is often over-expressed in many cancers to promote the EMT.mi RNAs play critical roles in many human diseases,including malignancy,and may function as both oncogenes and tumor suppressors.mi R-34 a is a direct transcriptional target of p53,a potent tumor suppressor.Moreover,Notch1 is a direct target of mi R-34 a which can negatively regulate the Notch signaling pathway to inhibit the growth of tumor cells.Therefore,we hypothesized that 3,6-DHF could regulate EMT in breast cancer by Notch signaling pathway.that may occur through the Notch signaling pathways regulate breast cancer.Our study intended to explore whether 3,6-DHF can regulate the related protein expression of Notch signaling pathway by enhancing the expression of mi R-34.To clarify the mechanism that 3,6-DHF regulate the Notch signaling pathway to inhibit EMT of breast cancer.Our study analyzed the effect of 3,6-DHF on EMT and Notch related proteins by Western Blotand immunofluorescence analysis in breast cancer cells.We used fluorescent reporter gene transfection of MDA-MB-231 cells(Luc-MDA-MB-231)to build nude mouse transplantation tumor model.An in vivo luminescence imaging system(IVIS)allows for the lon-gitudinal monitoring of tumor luciferase of 3,6-DHF.We analyze the effect of 3,6-DHF on EMT and Notch related proteins by immunohistochemical analysis(IHC).We analyze the inhibitory effect of 3,6-DHF on BCSCs by ALDEFLOUR analysis,CCK-8 assay and microspere formation assay.Using Transwell invasion and migration experiment and wound-healing assay to analyze the inhibition of 3,6-DHF on breast cancer cells in vitro.We used Luc-MDA-MB-231 cells to build nude mice in vivo pulmonary metastasis model.And observed the inhibitory effect of 3,6-DHF administration on metastasis of breast cancer cells by IVIS and HE staining.We investigated the effect of 3,6-DHF on NICD-CSL-MAML transcription complex by WB.We further investigated the effect of 3,6-DHF on Notch signaling pathway and EMT related proteins by WB,transfection assay,Traswell invasion and migration assay,thereby inhibiting the EMT of in breast cancer.Results1.3,6-DHF inhibits EMT and BCSCs in breast cancer cells in vitro and in vivo.(1)WB results showed that 3,6-DHF(0?M,5?M,10?M and 20?M)inhibited the expression of EMT related protein-E-cadherin and strengthened the expression of N-cadherin,Twist,Snail and Slug in the breast cancer cell line MDA-MB-231 cells and MCF-7 cells.TGF-?(10ng/ml)can significantly enhance the expression of EMT related proteins.Immunofluorescence results showed that Snail expression reduced and E-cadherin expression increased with the increasing dose of 3,6-DHF.(2)Luc-MDA-MB-231 was injected into the mammary fat pad of female BALB/c mice by subcutaneous inoculation to establish nude mouse transplantation tumor model.After 1,2 and 4 weeks for inoculation,We used IVIS to analyze fluorescent quantitative.The results showed that the nude mouse fluorescent quantity in 3,6-DHF(20mg/kg)group is higher than the control group.The tumors were isolated from the mouse after 4 weeks to weigh and measure the volume.The results showed that the volume and weight of 3,6-DHF(20mg/kg)are much smaller than the control group.Immunohistochemical staining showed that 3,6-DHF(20mg/kg)significantly reduced the expression of Snail,Twist and Slug,increased the expression of E-cadherin.(3)Flow cytometry analysis combined with ALDEFLOUR analysis to detect the ratio of ALDH+ cells in breast cancer cell line MDA-MB-231 cells.The results indicated that the proportion of the ALDH+ cells decreased with the increase of concentration of 3,6-DHF.Microspheres formation assay showed 3,6-DHF reduced the number and volume of microspheres.CCK-8 kit was used to detect vitality of ALDH+ cell in breast cancer cells MDA-MB-231 and SUM-159.The results showed that with the increase of concentration of 3,6-DHF,the vitality of breast cancer stem cells decreased obviously.(4)Flow cytometry analysis was used to detect the proportion of BCSCs in BALB/c nude mice transplantation tumor.the proportion of BCSCs in 3,6-DHF(20mg/kg)group was lower significantly than the control group.The tumors isolating from the mouse in 3,6-DHF(20mg/kg)group and the control group were cultured to tumor cells,subcutaneous inoculation to NOD/SCID mice.At last,we observe the tumor formation in the NOD/SCID mice.Results showed that five mice form tumor in six mice of the control group.However,there is only 1 mice tumor formation 3,6-DHF group.2.3,6-DHF inhibits metastasis and invasion in breast cancer cells in vitro and in vivo.(1)Transwell migration assay results showed that 3,6-DHF obviously decreased the number of tumor cells through the polycarbonate membrane,and the number of MDAMB-231 cells through the polycarbonate membrane is higher than the number of MCF-7 cells.Similarly,transwell invasion assay showed same results.Wound healing assay results showed that cell scratches healing rate of breast cancer cells MDA-MB-231 and MCF-7 decreased with 3,6-DHF treatment.(2)We injected Luc-MDA-MB-231 cells(1×106)to BALB/c nude mice by the tail vein to build in vivo pulmonary metastasis model.The results showed that five nude mice of the control group all form lung metastases with 3,6-DHF(20mg/kg)treatment after 5 weeks.However,there is only one mice forming lung metastases in 3,6-DHF group.The lung tissue of nude mice were isolated from 3,6-DHF(20mg/kg)group and control group.HE staining results showed that the control group appeared obvious lung metastasis nodule,the other is the opposite.3.Notch signaling pathway plays critical roles in inhibiting EMT in breast cancer cells with 3,6-DHF treatment.(1)WB results showed that 3,6-DHF could inhibit the Notch signaling pathway and the expression of related protein Notch1,NICD,Hes-1 and c-Myc in the breast cancer cell line MDA-MB-231 cells and MCF-7 cells.Similarly,immunofluorescence results showed that 3,6-DHF could inhibit the expression of related protein Notch1 and Hes-1.Immune coprecipitation experiment(CO-IP)and WB showed that 3,6-DHF could inhibit the expression of NICD when IP protein is CSL or MAML respectively.WB results showed that the 3,6-DHF(20 mg/kg,i.g.)administration could decrease Notch1 and NICD expression significantly in transplanted tumor.Similarly,immunohistochemical experiment results showed the same results.(2)MDA-MB-231 cells were transfected pc DNA3.1-Notch1 plasmid(TCNotch1)or anti-mi R-34 a oligonucleotide(TCanti-34a),WB results showed that 20?M 3,6-DHF could notreduce the protein expression levels of Notch1 and NICD in TCNotch1 and TCanti-34 a cells.Notch1 over-expression in TCNotch1 and TCanti-34 a cells,could reduce the inhibitory effect of 3,6-DHF on Twist,Slug and Snail;and the upregulation of E-cadherin.Similarly,mi R-34 a silence could also reduce the down-regulation of 3,6-DHF on Snail,Twist and Slug;and upregulation on E-cadherin.Transwell invasion and migration assay results showed that the 3,6-DHF decreased the number of tumor cells through the polycarbonate membrane in TCNotch1 and TCanti-34 a cells,but still significantly higher than the UTC cells.ALDEFLOUR analysis results showed that 3,6-DHF inhibits ALDH+ cells TCNotch1 and TCanti-34 a cells.Microspheres formation assay results showed that the 3,6-DHF reduced the number of microspheres in TCNotch1 and TCanti-34 a cells.Conclusions(1)3,6-DHF inhibited EMT and BCSCs in breast cancer cells in vivo and in vitro.(2)3,6-DHF could reduce breast cancer cells invasion and migration ability in vivo and in vitro.(3)3,6-DHF could inhibit Notch related proteins expression and the combination of NICD-CSL-MAML transcription complex,to inhibit Notch signaling pathway;3,6-DHF could regulate EMT related protein by regulating mi R-34 a and Notch signaling pathway with plasmid or oligonucleotide transfection,further inhibition of breast cancer development and metastasis.