Study On The Role Of Differential Regulations Of Estrogen Metabolism In The Effects Of Different PUFAs On The Proliferation Of MDA-MB-231Cell

Breast cancer is the most frequently diagnosed cancer and is the leading cause of cancerdeath among women worldwide. Breast cancer incidence rates have been rising in manydeveloping countries including China most likely due to lifestyle changes associated withwesternization. Breast cancer risk is associated with some potentially modifiable factors suchas dietary and nutritional factors. Above all, consumption of dietary fat is closely related toincrease risk of breast cancer. However, research on the relationship between dietary fattyacids and breast cancer risk has been done a lot. Results of epidemiological studies can not beconsistent partly due to differences in dietary pattern, research methods and quality control.Researches in vitro/vivo agree that different types of fatty acids produce different effects oncarcinogenesis. The role of n-6polyunsaturated fatty acids (PUFA) and n-3PUFA played incarcinogenesis is just the opposite. The former can promote growth of cancer cells, while thelatter inhibits growth and induces apoptosis of cancer cells. But the molecular mechanisms arestill unclear.Breast cancer is a kind of hormone–dependent tumors. Estrogen is linked to breastcancer incidence. The molecular mechanisms underlying the estrogen–inducedcarcinogenesis are to stimulate cell proliferation via estrogen receptor (ER)–mediatedhormonal activity and to generate metabolites which lead to increased mutation rates orchromosome abnormalities via non ER–mediated pathways. Because breast cancer lackingER is usually more advanced and more difficult to treat than ER+breast cancer, to study themechanism of estrogen carcinogenesis via ER–independent pathways is very important.Aromatase (cytochrome P-45019, CYP19) is the rate-limiting enzyme in estrogenbiosynthesis. Its activity directly affects the generation of endogenous estrogen. Estrogensundergo phase I and II metabolism by which they are biotransformed into metabolites with different characteristics. Among them,4-hydroxyestradiol (4-OH E2) is considered to be oneof the most important endogenous carcinogens.4-OH E2can generate quinone andsemiquinone in vivo redox which can covalently bind to DNA and cause DNA damage.Catechol-O-methyl transferase (COMT) catalyzes the methoxylation of4-OH E2to form itsO-methyoxy estrogens (4-MeO E2) which is water-soluble and can be excreted. Estrogenmetabolism in different sites of reaction can directly affect the characters of the metabolites.Therefore, the regulation of key enzymes in estrogen metabolism may affect estrogencarcinogenesis.Studies suggest that n-6and n-3PUFA may regulate the expression of estrogenmetabolic enzymes. But these researches are only limited to PUFA metabolites ProstaglandinE2(PGE2)/PGE3or endometrial stromal cells. The mechanisms of fatty acids influencebreast cancer through the estrogen pathway are still unclear.To explore the relationship between n-6/n-3PUFA promoting/inhibiting proliferation ofbreast cancer cells and estrogen metabolic pathways, we used estrogen receptor-negative (ER-)breast cancer MDA-MB-231cells to analyze the effects of arachidonic acid (AA, n-6PUFA)and eicosapentaecnoic acid (EPA, n-3PUFA) on proliferation of breast cancer cells andexpressions of estrogen metabolic enzymes through CCK-8cell counting assay, qPCR(real-time fluorescence quantitative PCR), Western Blotting and RNA interference methods.Then we observed COMT mRNA expression and cell proliferation after RNA interferenceand PUFA treatment. Finally, we drew the conclusion that PUFAs effected proliferation ofbreast cancer cells through different regulations on estrogen metabolic enzyme CYP1B1expression.The main results and final conclusions were summarized as followings:1. Breast cancer cells MDA-MB-231were treated with EPA and AA in differentconcentrations (20,40,60,80and100μmol/L). The cell proliferation was detected by cellcounting kit-8(CCK-8) assay. We found that EPA inhibited MDA-MB-231cell proliferationdoes-dependently, while AA promoted cell proliferation.2. We observed that AA notably increased CYP19and CYP1B1mRNA expressions anddecreased COMT mRNA expression in MDA-MB-231cells. These different regulations willincrease the production of estrogen metabolism-related carcinogens. We also found that EPAcould decrease mRNA expressions of CYP19and CYP1B1and increase COMT mRNA expression. These results indicated that these two different PUFAs EPA and AA coulddifferently regulate expressions of estrogen metabolic enzymes.3. MDA-MB-231cells were transfected with CYP1B1siRNA and treated with EPA andAA. We discovered that our transfection can efficiently decrease CYP1B1mRNA and proteinlevels by (80±3.67)%and (85±2.58)%respectively. We also found that expression ofCOMT mRNA and protein levels were increased significantly, which could increase theinactivation of endogenous carcinogens such as4-OH E2.4. We observed that the cell number of transfected group was reduced significantly. Theinhibition ratio was (30±6.76)%、(33±6.91)%and (35±5.87)%at24h、36h and48hrespectively. In negatively-transfected cells, EPA treatment could inhibit breast cancer cellproliferation while AA increased cell proliferation. In CYPB1siRNA transfected cells, cellproliferation inhibition of EPA was reversed and AA’ promotion was strongly decreased.These results indicated that EPA might inhibit breast cancer cell proliferation throughdown-regulating expression of CYP1B1.In all, we have used EPA and AA to analyze the effects of different PUFAs on breastcancer cell proliferation and expressions of estrogen metabolic enzymes. The results suggestthat PUFA might affect proliferation of breast cancer cells through different regulations onestrogen metabolic enzymes. Our study provides reliable experimental evidences forelucidating the molecular mechanisms of PUFA affecting breast cancer and preventing breastcancer through dietary intervention.