3-6-dihydroxyflavone and Breast-Neoplasms

Breast cancer is the common cancer in China. In previous study, we determined that 3,6-dihydroxyflavone (3,6-DHF) increases miR-34a significantly in breast carcinogenesis, but the mechanism remains unclear.. We used qRT-PCR to analyze miR-34a and ten-eleven translocation (TET)1, TET2, TET3 levels in breast cancer cells. With a cellular breast carcinogenesis model and an experimental model of carcinogenesis in rats, TET1 levels were evaluated by western blot analysis and immunofluorescence. TET1 and 5hmC (5-hydroxymethylcytosine) levels were evaluated by immunofluorescence in nude mouse xenografts of MDA-MB-231 cells. Chromatin immunoprecipitation(ChIP) assayed for TET1 on the TET1 promoter, and dot blot analysis of DNA 5hmC was performed in MDA-MB-231 cells. We evaluated the mechanism of 3,6-DHF on the expression of tumor suppressor miR-34a by transfecting them with DNA methyltransferase (DNMT)1 plasmid and TET1 siRNA in breast cancer cells. Methylation-specific PCR detected methylation of the miR-34a promoter.. First, we found that 3,6-DHF promotes the expression of TET1 during carcinogen-induced breast carcinogenesis in MCF10A cells and in rats. 3,6-DHF also increased TET1 and 5hmC levels in MDA-MB-231 cells. Further study indicated that TET1 siRNA and pcDNA3/Myc-DNMT1 inhibited the 3,6-DHF reactivation effect on expression of miR-34a in breast cancer cells. Methylation-specific PCR assays indicated that TET1 siRNA and pcDNA3/Myc-DNMT1 inhibit the effect of 3,6-DHF on the demethylation of the miR-34a promoter.. Our study showed that 3,6-DHF effectively increases TET1 expression by inhibiting DNMT1 and DNA hypermethylation, and consequently up-regulates miR-34a in breast carcinogenesis.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Transformation, Neoplastic; DNA (Cytosine-5-)-Methyltransferase 1; DNA Methylation; Female; Flavonoids; Gene Expression Regulation, Neoplastic; Humans; Mice; MicroRNAs; Mixed Function Oxygenases; Promoter Regions, Genetic; Proto-Oncogene Proteins; Rats; RNA Interference; RNA, Small Interfering

The epithelial to mesenchymal transition (EMT) is a critical developmental program in cancer stem cell (CSC) maintenance and in cancer metastasis. Here, our study found that 3,6-DHF could effectively inhibit EMT in BC cells in vitro and in vivo. 3,6-DHF effectively inhibits the formation and proliferation of BCSCs, and consequently reduces the tumor-initiating capacity of tumor cells in NOD/SCID mice. Optical in vivo imaging of cancer metastasis showed that 3,6-DHF administration suppresses the lung metastasis of BC cells in vivo. Further studies indicated that 3,6-DHF down-regulates Notch1, NICD, Hes-1 and c-Myc, consequently decreasing the formation of the functional transcriptional unit of NICD-CSL-MAML, causing Notch signaling inactivation in BC cells. Over-expression of Notch1 or inhibition of miR-34a significantly reduced the inhibitory effects of 3,6-DHF on EMT, CSCs, as well as cells migration and invasion in BC cells. These data indicated that 3,6-DHF effectively inhibits EMT and CSCs, as well as cells migration and invasion in BC cells, in which miR-34a-mediated Notch1 down-regulation plays a crucial role.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Down-Regulation; Epithelial-Mesenchymal Transition; Female; Flavonoids; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Mammary Neoplasms, Animal; MCF-7 Cells; Mice; Mice, Inbred BALB C; Mice, Inbred NOD; Mice, Nude; Mice, SCID; MicroRNAs; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Neoplastic Stem Cells; Receptor, Notch1; Receptors, Notch; Signal Transduction; Wound Healing

Our previous study selected a promising chemopreventive agent 3,6-dihydroxyflavone (3,6-DHF) and found that 3,6-DHF significantly upregulates miR-34a and downregulates miR-21 in breast carcinogenesis, yet the upstream and downstream events of the anticancer mechanism remain unclear. The present study showed that 3,6-DHF cotreatment effectively inhibits carcinogens-induced breast carcinogenic transformation in human breast epithelial MCF10A cells. The data revealed the significant downregulation of miR-34a and upregulation of miR-21 in breast carcinogenesis, which could be mitigated by 3,6-DHF treatment. Methylation-specific PCR detections showed that 3,6-DHF inhibits the hypermethylation of the miR-34a promoter. Further studies indicated that 3,6-DHF is an effective methyltransferase (DNMT)1 inhibitor, docking to the putative cytosine pocket of the protein, and thus decreases the DNMT activity in a dose-dependent manner. Moreover, the ChIP-qPCR analysis for histone modifications showed that 3,6-DHF treatment significantly lowers the H3K9-14ac on the miR-21 promoter. In addition, our study revealed that 3,6-DHF represses the PI3K/Akt/mTOR signaling pathway in breast carcinogenesis in vitro and in vivo. Inhibition of miR-34a or overexpression of miR-21 significantly reduced the effects of 3,6-DHF on Notch-1 and PTEN, and consequently weakened the suppression of 3,6-DHF on PI3K/Akt/mTOR. We concluded that 3,6-DHF upregulates miR-34a via inhibiting DNMT1 and hypermethylation, whereas downregulates miR-21 by modulating histone modification, and consequently suppresses the PI3K/Akt/mTOR signaling pathway in breast carcinogenesis.

Topics: Animals; Apoptosis; Blotting, Western; Breast Neoplasms; Cell Proliferation; Cell Transformation, Neoplastic; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; Epigenomics; Female; Flavonoids; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Humans; Immunoenzyme Techniques; Mice; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; TOR Serine-Threonine Kinases; Tumor Cells, Cultured; Xenograft Model Antitumor Assays