RESUMO
Diarylheptanoids from Curcuma comosa, of the Zingiberaceae family, exhibit diverse estrogenic activities. In this study we investigated the estrogenic activity of a major hydroxyl diarylheptanoid, 7-(3,4 -dihydroxyphenyl)-5-hydroxy-1-phenyl-(1E)-1-heptene (compound 092) isolated from C. comosa. The compound elicited different transcriptional activities of estrogen agonist at low concentrations (0.1-1 µM) and antagonist at high concentrations (10-50 µM) using luciferase reporter gene assay in HEK-293T cells. In human breast cancer (MCF-7) cells, compound 092 showed an anti-estrogenic activity by down-regulating ERα-signaling and suppressing estrogen-responsive genes, whereas it attenuated the uterotrophic effect of estrogen in immature ovariectomized rats. Of note, compound 092 promoted mouse pre-osteoblastic (MC3T3-E1) cell differentiation and the related bone markers, indicating its positive osteogenic effect. Our findings highlight a new, nonsteroidal, estrogen agonist/antagonist of catechol diarylheptanoid from C. comosa, which is scientific evidence supporting its potential as a dietary supplement to prevent bone loss with low risk of breast and uterine cancers in postmenopausal women.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Curcuma/química , Diarileptanoides/administração & dosagem , Receptor alfa de Estrogênio/metabolismo , Osteoblastos/efeitos dos fármacos , Fitoestrógenos/administração & dosagem , Extratos Vegetais/administração & dosagem , Moduladores Seletivos de Receptor Estrogênico/administração & dosagem , Células 3T3 , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/fisiopatologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Receptor alfa de Estrogênio/genética , Feminino , Humanos , Camundongos , Osteoblastos/citologia , Osteoblastos/metabolismo , Ratos , Ratos Wistar , Útero/citologia , Útero/efeitos dos fármacos , Útero/crescimento & desenvolvimentoRESUMO
BACKGROUND: Concerns regarding potential endocrine-disrupting chemicals (EDCs) have led to a need for methods to evaluate candidate estrogenic chemicals. Our previous evaluations of two such EDCs revealed a response similar to that of estradiol (E2) at 2 hr, but a less robust response at 24 hr, similar to the short-acting estrogen estriol (E3). OBJECTIVES: Microarray analysis using tools to recognize patterns of response have been utilized in the cancer field to develop biomarker panels of transcripts for diagnosis and selection of treatments most likely to be effective. Biological effects elicited by long- versus short-acting estrogens greatly affect the risks associated with exposures; therefore, we sought to develop tools to predict the ability of chemicals to maintain estrogenic responses. METHODS: We used biological end points in uterine tissue and a signature pattern-recognizing tool that identified coexpressed transcripts to develop and test a panel of transcripts in order to classify potentially estrogenic compounds using an in vivo system. The end points used are relevant to uterine tissue, but the resulting classification of the compounds is important for other sensitive tissues and species. RESULTS: We evaluated biological and transcriptional end points with proven short- and long-acting estrogens and verified the use of our approach using a phytoestrogen. With our model, we were able to classify the diarylheptanoid D3 as a short-acting estrogen. CONCLUSIONS: We have developed a panel of transcripts as biomarkers which, together with biological end points, might be used to screen and evaluate potentially estrogenic chemicals and infer mode of activity.
Assuntos
Disruptores Endócrinos/toxicidade , Útero/efeitos dos fármacos , Animais , Biomarcadores/análise , Diarileptanoides/toxicidade , Estrogênios/toxicidade , Feminino , Perfilação da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Ovariectomia , Fitoestrógenos/toxicidade , Útero/metabolismoRESUMO
BACKGROUND: Diarylheptanoid (D3) isolated from the medicinal plant, Curcuma comosa, has estrogenic activity. OBJECTIVE: We aimed to elucidate the mechanism(s) of D3 action and compare it with that of 17ß-estradiol (E2) using both in vitro and in vivo uterine models. METHODS: We used human uterine (Ishikawa) cells to determine the estrogenic action of D3 on the activation and nuclear translocation of estrogen receptor α (ERα). In addition, we further characterized the uterine response to D3 treatment in vivo. RESULTS: D3 activated an estrogen responsive element (ERE) luciferase reporter through ERα, and molecular modeling suggested that D3 could be accommodated in the ERα binding pocket. Using modified ERα to assay ligand-dependent nuclear translocation, we observed D3-dependent ERα interaction and translocation. In mouse uteri, early- and late-phase estrogen-regulated gene responses were increased in D3-treated ovariectomized wild-type animals, in a manner similar to that of E2; no response was seen in ERα knockout animals. We observed a divergence in estrogen responses after D3 treatment: D3 induced robust DNA synthesis in uterine epithelial cells, linked to an increase in cell-cycle-related genes; however, no increase in uterine weight was observed 24 hr after treatment. D3 also affected uterine progesterone receptor expression patterns similar to E2. When D3 and E2 were administered together, we observed no additive or antagonistic effects of D3 on E2. Our findings suggest that D3 is a weak estrogenic agonist compound. CONCLUSION: D3 is a weakly acting phytoestrogen that mimics the mitogenic responses produced by E2 in an ERα-dependent manner, but it is unable to increase uterine weight or enhance or antagonize the effects of estrogen.
Assuntos
Curcuma/química , Diarileptanoides/metabolismo , Exposição Ambiental , Fitoestrógenos/metabolismo , Útero/efeitos dos fármacos , Animais , Linhagem Celular , Estradiol/metabolismo , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Camundongos , Microscopia Confocal , Ovariectomia , Extratos Vegetais/metabolismo , Reação em Cadeia da Polimerase , Receptores de Progesterona/metabolismo , Rizoma/químicaRESUMO
BACKGROUND: Diarylheptanoids isolated from Curcuma comosa Roxb. have been recently identified as phyto estrogens. However, the mechanism underlying their actions has not yet been identified. OBJECTIVES: We characterized the estrogenic activity of three active naturally occurring diarylheptanoids both in vitro and in vivo. METHODS: We characterized mechanisms of estrogenic action of the diarylheptanoids (3S)-1,7-diphenyl-(6E)-6-hepten-3-ol (D1), 1,7-diphenyl-(6E)-6-hepten-3-one (D2), and (3R)-1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol (D3) by using a real-time polymerase chain reaction assay, a mammalian transfection model, and a uterotrophic assay in mice. RESULTS: All diarylheptanoids up-regulated estrogen-responsive genes in estrogen-responsive breast cancer cells (MCF-7). In HepG2 cells transfected with estrogen receptor (ER) beta or different ERalpha functional receptor mutants and the Vit-ERE-TATA-Luc reporter gene, all diarylheptanoids induced transcription through a ligand-dependent human ERalpha-ERE-driven pathway, which was abolished with ICI 182,780 (ER antagonist), whereas only D2 was active with ERbeta. An ERalpha mutant lacking the functional AF2 (activation function 2) region was not responsive to 17beta-estradiol (E(2)) or to any of the diarylheptanoids, whereas ERalpha lacking the AF1 domain exhibited wild-type-like activity. D3 markedly increased uterine weight and proliferation of the uterine epithelium in ovariectomized mice, whereas D1 and D2 were inactive. D3, like E(2), up-regulated lactoferrin (Ltf) gene expression. The responses to D3 in the uterus were inhibited by ICI 182,780. In addition, D3 stimulated both classical (Aqp5) and nonclassical (Cdkn1a) ER-mediated gene regulation. CONCLUSIONS: The results suggest that the D3 diarylheptanoid is an agonist for ER both in vitro and in vivo, and its biological action is ERalpha selective, specifically requiring AF2 function, and involves direct binding via ER as well as ERE-independent gene regulation.
Assuntos
Curcuma/química , Diarileptanoides/farmacologia , Fitoestrógenos/farmacologia , Plantas Medicinais/química , Receptores de Estrogênio/fisiologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Diarileptanoides/química , Diarileptanoides/isolamento & purificação , Estradiol/análogos & derivados , Estradiol/farmacologia , Receptor alfa de Estrogênio/antagonistas & inibidores , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/fisiologia , Receptor beta de Estrogênio/antagonistas & inibidores , Receptor beta de Estrogênio/genética , Receptor beta de Estrogênio/fisiologia , Feminino , Fulvestranto , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Tamanho do Órgão/efeitos dos fármacos , Ovariectomia , Fitoestrógenos/química , Fitoestrógenos/isolamento & purificação , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Reação em Cadeia da Polimerase , Receptores de Estrogênio/antagonistas & inibidores , Receptores de Estrogênio/genética , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genética , Útero/citologia , Útero/efeitos dos fármacosRESUMO
Curcuma comosa Roxb. has traditionally been used as a dietary supplement for health promotion in peri- and postmenopausal women in Thailand. We investigated the estrogenic activity of 7 naturally occurring diarylheptanoids from the extracts of C. comosa both in vitro and in vivo. A yeast recombinant system containing human estrogen receptor alpha, coactivator TIF2 and a beta-galactosidase reporter gene was used to determine estrogenic activity of diarylheptanoids metabolically activated with rat liver S9-fraction prior to the assay. The most potent compound was (3R)-1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol, with a relative potency of 4% compared to 17beta-estradiol. The metabolic activation of diarylheptanoids markedly enhanced their efficiency. The chemical structure required for estrogenic activity of diarylheptanoids was the presence of a keto group at C3 and absence of hydroxyl moiety in ring B. Only diarylheptanoids showing full estrogenic efficiency in vitro were able to elicit uterotrophic activity of in immature ovariectomized rat. This is the first evidence for in vivo estrogenic activity of diarylheptanoids from C. comosa. This novel class of natural phytoestrogens has the potential to be developed for use as dietary supplement in the treatment of menopausal symptoms.