Inhibitory Effects Of Ampelopsin On Breast Cell Carcinogenesis Induced By Chemical Carcinogens And Its Correlation With Methylation Of ATM Gene

Breast cancer is reported as the most common malignancy and the leading cause ofcancer-related death among women worldwide. Some tumor-suppressor genes can be lost orinactivated in a variety of ways during tumor development, ranging from chromosomaldeletions, insertions or rearrangements to single base mutations, or epigenetic promoteralterations such as DNA hypermethylation. Aberrant patterns and dysregulation of DNAmethylation cause stable, heritable transcriptional silencing of the associated gene duringbreast carcinogenesis. It has been accepted that DNA methylation plays an important role intranscriptional regulation and is commonly altered during malignant transformation.Moreover, deviant DNA methylation is an early event in tumorigenesis, which was evenearlier than some visible lesions. The cytosine DNA methyltransferases (DNMT)1,3a, and3b have been implicated to different extents in initiating gene silencing through de novomethylation and recruitment of chromatin remodeling proteins. Dnmt1maintains themethylation in mouse embryonic development and thus is considered to be the majormaintenance methyltransferase enzyme. A number of previous reports suggest a direct role ofDNMT1in the restoration of epigenetic information during DNA repair. Specifically, DNMT1and proliferating cell nuclear antigen are binding partners that provide a means to recruitDNMT1to DNA repair sites.The ataxia telangiectasia mutated (ATM) protein belongs to the core components ofDNA-damage-response machinery and acts as an intracellular sensor by recognizingdouble-strand breaks (DSBs), and it has also been implicated in breast cancer susceptibility.However, deletions of ATM alleles are much more common in breast tumors indicating theimportant role of the gene in breast cancer development. The aberrant DNA methylationstatus of ATM in breast cancer tissues has previously been analyzed. Accordingly, the ATMpromoter in non-small cell lung cancers (NSCLCs) and the colon cancer cell line Hct-116 have been previously reported to be hypermethylated. The epigenetic silencing of ATMexpression occurred in locally advanced breast tumors suggests a link between reduced ATMfunction and sporadic breast cancer.Accumulating evidences indicate that dietary chemopreventive agents can prevent orreverse these alterations by affecting global DNA methylation, reexpressing tumor suppressorgenes by altering methylation. A naturally occurring dietary pigment from the tender stem andleaves of the plant species Ampelopsis grossedentata, ampelopsin (AMP, also known asDihydromyricetin), has been reported to exhibit a wide range of biological andpharmacological activities, such as anti-inflammatory, antihypertension as well ashepotoprotective and anticancer activities. However, the underlying molecular mechanism ofanti-cancer effect has not been clearly defined and studies focused on tumorigenesis haverarely reported yet. Indeed, investigating the effects of AMP on breast tumorigenesis andexploring the potential epigenetic mechanisms seems to be more meaningful. Here, wehypothesize that AMP could possess hypermethylation of the ATM gene by DNMT1is amolecular cause for ATM activation and suppression of breast tumorigenesis. The focus ofthis study is to evaluate the chemopreventive effects of AMP in breast cell carcinogenesisinduced by environmental carcinogens in vitro，and explore the underlying mechanisms.The main results and conclusions were summarized as follows:1. Breast cancer MCF-7cells were treated with5μM,10μM,20μM and40μM of AMPfor48h, cell viability assay showed that10μM,20μM and40μM AMP treatment significantlydecreased MCF-7cell viability in a dose-dependment manner (P <0.05), while5μM AMPtreatment had no significant effect, and IC50of AMP is about20μM.2. Exposure of MCF-10A cells to100pmol/L NNK and100pmol/L B[a]P result inprogressive acquisition of various cancer-associated properties such as reduced dependenceon growth factors, anchorage-independent growth, increased capacity of scratch healing,and increased DNA damage as evidenced by the increased number of γH2AX foci.However, these cancer-associated properties inuced by NNK and B[a]P could be inhibited byAMP treatment at the concentration of20μM.3. In the progression of chronic breast MCF-10A cell carcinogenesis induced bychemical carcinogen-, the whole genome DNA methylation levels significantly decreasedafter exposures to NNK and B[a]P for10and20cycles (were named P10and P15 respectively)(P <0.05), whereas20μM AMP treatment significantly suppressed thedecreased whole genome DNA methylation levels induced by NNK and B[a]P treatment.4. The activity and expression of DNMT1in NNK and B[a]P-treated MCF-10A cellsincreased in a time-dependent manner, but20μM AMP treatment inhibited NNK andB[a]P-induced increase in the activity and expression of DNMT1.5. Molecular modeling results indicated that AMP may be docked to the putativecytosine pocket of DNMT1, thereby inhibiting its activity and expression.6. The expression of ATM was increased significantly in MCF-10A cells treated with20μM AMP and carcinogens, while it was decreased in cells treated with carcinogens only.However, the expressions of CHK2, RAD51and p53have no significant change in thecarcinogens group, but were increased in the AMP and carcinogens combined treatment groupin a time-dependent manner.7. MSP detections showed that AMP treatment significantly inhibits the methylation ofATM promoter in carcinogens-treated MCF10A cells, which indicated that AMP couldrestrain the hypermethylation of ATM promoter in breast cancer and carcinogenesis,consequently increases the expression of ATM.In conclusion, these results indicate that AMP can inhibit the activity and expression ofDNMT1by reducing promoter methylation of tumor suppressor gene-ATM, and thusincrease the expression of the ATM gene, thereby inhibiting chemical carcinogens inducedcarcinogenesis on breast MCF-10A cells.