RESUMO
We analyzed the effect of nafoxidine on the earlier biological processes of angiogenesis and explored the role of different signaling pathways involved in the in vitro response of endothelial cells (HUVEC). Nafoxidine significantly inhibited adhesion, spreading, migration and invasion of HUVEC at concentrations ranging from 1 to 2.5 microM. Endothelial cord formation on Matrigel was inhibited by nafoxidine and cotreatment with phorbol-12-myristate-13-acetate (PMA) clearly prevented the antiangiogenic effect of the antiestrogen. On the contrary, cotreatment with the PKC inhibitor bisindolylmaleimide potentiated inhibition of cord formation. PMA also inhibited the nafoxidine-induced secretion of metalloproteinase-2 and tissue inhibitor of metalloproteinases-1 in HUVEC monolayers. Cotreatment with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine and the cAMP analog N6,2'-o-dibutyryladenosine 3',5'-cyclic monophosphate prevented the inhibition of endothelial cord formation induced by nafoxidine. Our work presents evidence about the signaling pathways involved in the antiangiogenic effect of nafoxidine, suggesting that PKC-dependent signaling pathways are essential in angiogenesis during endothelial cord formation.
Assuntos
Inibidores da Angiogênese/farmacologia , Endotélio Vascular/efeitos dos fármacos , Nafoxidina/farmacologia , Proteína Quinase C/fisiologia , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Endotélio Vascular/citologia , Endotélio Vascular/enzimologia , Endotélio Vascular/metabolismo , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Veias Umbilicais/citologiaRESUMO
During angiogenesis, proteases and their inhibitors interact in the remodelling of the basement membrane. It has been demonstrated that nafoxidine has antiangiogenic activity in the chick egg chorioallantoic membrane assay, but the precise mechanism of action is unknown. We have analyzed the effect of the partial estrogen antagonist nafoxidine on human umbilical vein endothelial cells (HUVEC). Our data indicated that in nafoxidine-treated endothelial cells MMP-2 was activated. Nafoxidine upregulated, in a dose-dependent manner, the secretion of a 66 kDa TIMP-1 dimer, that lacks anti-MMP activity and inhibited angiogenesis in the endothelial cord formation assay. We can postulate that nafoxidine induces an increase in TIMP-1, which has antiangiogenic activity in the late stages of tube formation, independent of its capacity to inhibit MMPs.
Assuntos
Inibidores da Angiogênese/farmacologia , Endotélio Vascular/efeitos dos fármacos , Moduladores de Receptor Estrogênico/farmacologia , Metaloproteinase 2 da Matriz/biossíntese , Nafoxidina/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-1/biossíntese , Células Cultivadas/efeitos dos fármacos , Meios de Cultivo Condicionados/química , Dimerização , Relação Dose-Resposta a Droga , Endotélio Vascular/enzimologia , Endotélio Vascular/metabolismo , Ativação Enzimática/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Substâncias de Crescimento/farmacologia , Humanos , Metaloproteinase 2 da Matriz/genética , Inibidor Tecidual de Metaloproteinase-1/genética , Veias UmbilicaisRESUMO
In human breast cancer the proliferating cells appear to differ from those containing estrogen receptors (ER) as shown by studies on isolated cellular subpopulations. In this paper the in vitro effect of 17-beta-estradiol on cell proliferation in 30 primary breast tumors was studied. The effect of several estradiol concentrations was assayed, and the influence of diethylstilbestrol, tamoxifen, and nafoxidine was also tested. The response to these compounds was measured through the thymidine labeling index (TLI). When exposed to 10(-9) mol/l and 10(-8) mol/l estradiol, 14 of 19 ER-positive tumors and six of 11 ER-negative tumors were induced to further proliferate. The TLI increase over the control was 219% (P less than 0.05) at 10(-9) mol/l E2 and 258% (P less than 0.05) at 10(-8) mol/l E2 for ER-positive tumors, and 233% (0.1 less than P less than 0.2) at 10(-9) mol/l E2 and 321% (0.1 less than P less than 0.2) at 10(-8) mol/l E2 for ER-negative tumors. The addition of diethylstilbestrol and antiestrogens in vitro inhibited, to varying degrees, the estradiol-induced increase in the TLI irrespective of the ER-status. The response to E2 was correlated with the expression of the ras p21 protein and carcinoembryonic antigen. It was found that the ras p21 protein is preferentially expressed in ER-negative tumors, the opposite being true for carcinoembryonic antigen. The ras p21 protein is preferentially expressed in those ER-positive tumors that do not respond to estradiol with an increase in the TLI.