RESUMO
Calcitriol, a potent antineoplastic vitamin D metabolite, inhibits proliferation, induces apoptosis and slows the growth of tumors. Calcitriol also may exert either antiangiogenic or proangiogenic effects depending on the tissue. Vascular endothelial growth factor (VEGF) and thrombospondin-1 (Tsp-1) are key factors involved in promoting and inhibiting angiogenesis, respectively. The effects of calcitriol on Tsp-1 have not been studied in the mammary gland, while VEGF regulation is not clear, since opposite outcomes have been demonstrated. Therefore, the present study was undertaken to investigate the effects of calcitriol on VEGF and Tsp-1 expression in primary breast tumor-derived cells and a panel of established breast cancer cell lines. In vivo studies in athymic mice were also performed in order to gain further insight into the biological effects of calcitriol on angiogenesis. Real time-PCR and ELISA analyses showed that calcitriol stimulated VEGF mRNA expression and protein secretion while elicited the opposite effect on Tsp-1 in 7 out of 8 cell lines studied, independently of the cell phenotype (P<0.05 in n=5). In vivo, calcitriol significantly inhibited the relative tumoral volume after 4 weeks of treatment; however, serum VEGF was higher in calcitriol-treated animals compared to controls (P<0.05). The integrated fluorescence intensity analysis of CD31, a vessel marker, showed that xenografted breast cancer cells developed tumors with similar vascular density regardless of the treatment. Nevertheless, larger necrotic areas were observed in the tumors of calcitriol-treated mice compared to controls. Since the antineoplastic activity of calcitriol has been consistently demonstrated in several studies including this one, our results suggest that the antitumoral effect of calcitriol in vivo involve different mechanisms not necessarily related to the inhibition of tumor vascularization. Overall, our findings indicate that calcitriol can impact the angiogenic process in breast cancer by regulating VEGF and Tsp-1 expression. This article is part of a Special Issue entitled '16th Vitamin D Workshop'.
Assuntos
Inibidores da Angiogênese/farmacologia , Neoplasias da Mama/metabolismo , Calcitriol/farmacologia , Carcinoma Ductal de Mama/metabolismo , Neovascularização Patológica/tratamento farmacológico , Trombospondina 1/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Western Blotting , Conservadores da Densidade Óssea/farmacologia , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/tratamento farmacológico , Carcinoma Ductal de Mama/irrigação sanguínea , Carcinoma Ductal de Mama/tratamento farmacológico , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Trombospondina 1/genética , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Estradiol (E2) regulates several cellular functions through the interaction with estrogen receptor subtypes, ERα and ERß, which present different functional and regulation properties. ER subtypes have been identified in human astrocytomas, the most common and aggressive primary brain tumors. We studied the role of ER subtypes in cell growth of two human astrocytoma cell lines derived from tumors of different evolution grades: U373 and D54 (grades III and IV, respectively). E2 significantly increased the number of cells in both lines and the co-administration with an ER antagonist (ICI 182, 780) significantly blocked E2 effects. ERα was the predominant subtype in both cell lines. E2 and ICI 182, 780 down-regulated ERα expression. The number of U373 and D54 cells significantly increased after PPT (ERα agonist) treatment but not after DPN (ERß agonist) one. To determine the role of SRC-1 and SRC-3 coactivators in ERα induced cell growth, we silenced them with RNA interference. Coactivator silencing blocked the increase in cell number induced by PPT. The content of proteins involved in proliferation and metastasis was also determined after PPT treatment. Western blot analysis showed that in U373 cells the content of PR isoforms (PR-A and PR-B), EGFR, VEGF and cyclin D1 increased after PPT treatment while in D54 cells only the content of EGFR was increased. Our results demonstrate that E2 induces cell growth of human astrocytoma cell lines through ERα and its interaction with SRC-1 and SRC-3 and also suggest differential roles of ERα on cell growth depending on astrocytoma grade.
Assuntos
Astrocitoma/fisiopatologia , Neoplasias Encefálicas/fisiopatologia , Linhagem Celular Tumoral , Estradiol/farmacologia , Receptor alfa de Estrogênio/metabolismo , Coativador 1 de Receptor Nuclear/metabolismo , Coativador 3 de Receptor Nuclear/metabolismo , Astrocitoma/patologia , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/fisiologia , Ciclina D1/genética , Ciclina D1/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Receptor alfa de Estrogênio/genética , Humanos , Coativador 1 de Receptor Nuclear/genética , Coativador 3 de Receptor Nuclear/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferência de RNA , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Progesterone participates in the regulation of several reproductive functions in birds through its interaction with intracellular progesterone receptors (PR) which exhibit two isoforms (PR-A and PR-B) with different function and regulation. We determined the content of PR isoforms in several brain regions of immature (1.5 months-old), mature (12 months-old) and aged (48 months-old) male and female chickens by Western blot analysis. PR isoforms protein content changed in tissue-and age-specific manner in the brain of both male and female chickens. In female animals PR-B content was higher in the hypothalamus of mature animals than in other ages while PR-A content increased in the cerebellum of aged animals. No significant changes were observed in PR isoforms content in the cortex and the tectum of female animals. In contrast, in male animals PR-B content was higher in the hypothalamus of aged animals. The cerebellum, tectum and cortex of male animals did not present significant changes in PR isoforms content. In all studied regions of female animals PR-B was the predominant isoform, whereas, in males a significant predominance of PR-B was only found in the hypothalamus of aged animals. These results demonstrate that PR isoforms content presents a sexual dimorphism in the brain of chickens that changes in tissue- and age-specific manner, and suggest that these variations contribute to the regulation of progesterone actions in brain throughout the lifespan of male and female chickens.