The Study Of Sex Hormone Metabolomics In Breast Cancer Patients And The Mechanism Of 4-hydroxyestradiol Inducing Malignant Transformation Of Cells

Background:During the last 20 years,laboratory and clinical studies have suggested several hypotheses about how estrogen metabolism（EM）might influence the risk of breast cancer.Accruing evidence,primarily laboratory based,suggests substantial differences in the genotoxic,mutagenic,and proliferative activities of various EM and their contributions to mammary carcinogenesis.Estrogenic hormones,estrone（E₁）,estradiol（E₂）,and estriol（E₃）,are metabolized in the C-2,C-4,or C-16pathways by a series of oxidizing enzymes in the cytochrome P450 family,resulting in 2-OH-E₁/E₂,4-OH-E₁/E₂,and 16α-OH-E₁,respectively.These catechol estrogens may has carcinogenic effect or not,and their carcinogenic effect can be deactivated by the process of methylation.These EMs can have stronger or weaker estrogenic activity and may often determines the mutagenic or carcinogenic potential of estrogen,and thus increase a woman’s risk of breast,uterine and other cancers.Material and method:All the premenopausal breast cancer patients were diagnosed in Jiangsu Province Hospital during 2012 and 2013.This study compares12 estrogen and estrogen metabolites,10 androgen metabolites,and 7 progestogen metabolites levels in urine of premenopausal breast cancer patients and normal healthy women using a quantitative gas chromatography-mass-mass spectrometry method（GC/MS/MS）.Result:In this study,all EMs levels in C-2 and C-4 pathway were increased in breast cancer group,but the 16α-OH-E₁ level came down.We also found elevated risks for high EMs levels of 2-pathway and 4-pathway but not in 16α-OH-E₁.The2-pathway EMs,which were not reported be carcinogenic,increased in our breast cancer group.In our study,the 2-OHE₁:16α-OHE₁ ratio was interestingly lower in healthy women.Besides,to our surprise,the 2-OHE₁:16α-OHE₁ radio increased with the breast cancer risk.2-pathway and 4-pathway were increased in breast cancer groups especially 4-OHE₁.2-OHE₁:4-OHE₁,Total 2-OH:Total 4-OH elevated with breast cancer risk reduced.The ratio of 4-OH estrogens to parent estrogen was elevated with an increased risk.In our study,4-OHE₁:4-Methoxy E₁ and total 4-OH estrogens:total 4-Methoxy estrogens were increased,which mean methyl group of 4pathway was not elevated as much as catechol estrogens of this pathway.These results meet the characteristics of EMs.Androgens and androgen metabolites have been hypothesized to increase breast cancer risk.In our study,Testosterone and Epi-Testosterone level were increased in breast cancer group.The level of Progesterone and 20α-OH-Progesterone came down in healthy women groupConclusions:This is the first time we evaluate associations between urinary EMs and breast cancer risk among premenopausal women from Mainland China.We also analyze the urinary progesterone and androgen metabolites levels in this population.In this comprehensive study of sexual hormone metabolism and risk of breast cancer in premenopausal women,we observed significant positive associations with urinary levels C-2 and C-4 pathway EMs,but not C-16αEMs.Women with lower urinary ratios of 2-OHE₁:16α-OHE₁ were at a significantly reduced risk of breast cancer.The data of C-2:C-16 ratio was inverse of previous studies.C-2:C-4 ratio was elevated with decreased risk,while C-4:C-4 methoxy ratio and C-4:parent estrogen ratio was elevated with an increased risk.Further research is necessary to confirm the associations we observed,investigate the role of sexual hormone metabolites in these results,and identify the determinants of sexual hormone metabolism patternsBackground:A female hormone,estrogen,is linked to breast cancer incidence.Estrogens undergo phase I and II metabolism by which they are biotransformed into genotoxic catechol estrogen metabolites and conjugate metabolites are produced for excretion or accumulation.The molecular mechanisms underlying estrogen-mediated mammary carcinogenesis remain unclear.Cell proliferation through activation of estrogen receptor（ER）by its agonist ligands and is clearly considered as one of carcinogenic mechanisms.Recent studies have proposed estrogen or estrogen metabolites are attributed to genotoxic effects and signal transduction through influencing redox sensitive transcription factors resulting in cell transformation,cell cycle,migration,and invasion of the breast cancer.In our study of Part I,we found the C-4 EMs increased in the breast cancer group.In order to know the molecular mechanism of 4-OH-E,We analyzed the effect of 4-OH-E2 and E2 on cell morphology in MCF10 A cellsMaterial and method: The cell were treated with 4-OH E2 for 8 week（MCF10A-H）or treated with E2 for 8 week（MCF10A-E）.The biological behavior changes of the cells were observed by colony formation assay,migration rate was measured by wound healing assay and cell migration assay,nude mouse tumorigenicity assay was used to observe the behavior in vivo.Result:MCF10A-H cells effectively induced cell-to-cell contact and led to a higher spreading with more formation of filo podia by comparison with the MCF10A-E and control groups.The MCF10 A cell colony formations were calculated.The MCF10A-H cells in the culture medium with 10-9M 4-OH E2 have much more colonies than other groups.4-OH E2 may inhibit the growth of the MCF10A and MCF10A-E,but no significant differences were found.4-OH E2 suppressed migratory potential.Determined by their migration in the wound gap after 24 h,the wound closure of MCF10A-H was 73.7% while the wound closure of MCF10 A was 53.3%.However,the wound closure of MCF10A-E was 44.5%.We investigated the 4-OH-E2 and E2 effects on the MCF10 A cell migration behavior via a transwell migration assay.Cell migration was significantly increased in MCF10A-H and MCF10A-E cell.The number of migrated MCF10A-H cells increased by nearly 3 folds when compared with MCF10 A,while MCF10A-E increased by 2 folds.To evaluate the tumor-suppressive functions of 4-OH-E2 in vivo,tumorigenicity of MCF10A-H and MCF10A-E cell were evaluated in nude mice.MCF10A-H cells and MCF10 A cells were injected into mammary fat pads of the mice.None of the nude mices injected MCF10 A cells formed a palpable mass when injected with Matrigel.90% of the MCF10A-H and 50% of MCF10A-E injected mice were discovered tumors after 2 weeks.MCF10A-E cells formed smaller tumor volume compared to the MCF10A-H cell.Conclusions: It was possible that 4-OH-E2 could induce malignant transformation of MCF-10 A cells,and 4-OH-E2 was more effective than E2.Background : The roles of estrogens and their metabolites in mammary carcinogenesis are under study.The direct or indirect modifications at the gene level is described as one of the prime mechanisms of tumor initiation by estrogen metabolites as well as estrogen-induced ROS.In the Part I and Part II,we found C-4 estrogen metabolisms were increased in breast cancer group and MCF-10 A could be turned malignant by 4-OH-E2.Material and method: In this part of study,gene expression profiles were generated by microarray（Affymetrix Gene Chip? Human Transcriptome Array 2.0.）to screen the changed genes in MCF10 A,MCF10A-E and MCF10A-H cells.Furthermore,differentially expressed genes were validated by quantitative reverse transcriptase-PCR（q RT-PCR）and analyzed compared with several databases.Mitotic index was get by immunofluorescence studiesResult:The genes change induced by 4-OH-E2 were focus on genes that are known to be involved in mitosis.CENPE was the gene changed most.This is a spindle assembly checkpoint gene.The mitotic index was lower in MCF10A-H than MCF 10 A when treated by docetaxel.That may reveal the the destruction of the spindle-assembly checkpoint.Conclusions: As the result,we hypothesis that genotoxic of C-4 hydroxylations is cause by abnormal chromosome segregation.