RESUMEN
Two mononuclear copper(II) compounds, [Cu(isad)(H2O)Cl]Cl 1 and [Cu(isah)(H2O)Cl]Cl 2, and its corresponding heterobinuclear species containing also platinum(II), [CuCl(isad)Pt(NH3)Cl2] 3 and [CuCl(isah)Pt(NH3)Cl2] 4 (where isad and isah are oxindolimine ligands, (E)-3-(2-(3-aminopropylamino)ethylimino)indolin-2-one, and (E)-3-(3-amino-2-hydroxypropylimino)indolin-2-one, respectively), have been previously synthesized and characterized by different spectroscopic techniques in our laboratory. Cytotoxicity assays performed with B16F10 murine cancer cells, and MES-SA human uterine sarcoma cells, showed IC50 values lower or in the same order of cisplatin. Herein, in order to better elucidate their probable modes of action, possible interaction and damage to DNA, as well as their effect on the activity of crucial proteins were verified. Both mononuclear complexes and the binuclear compound 4 displayed a significant cleavage activity toward plasmid DNA, while compound 3 tends to protect DNA from oxidative damage, avoiding degradation. Complementary experiments indicated a significant inhibition activity toward cyclin-dependent kinase (CDK1/cyclinB) activity in the phosphorylation of histone H1, and only moderate inhibition concerning alkaline phosphatase. Results also revealed that the reactivity is reliant on the ligand structure and on the nature of the metal present, in a synergistic effect. Simulation studies complemented and supported our results, indicating different bindings of the binuclear compounds to DNA. Therefore, the verified cytotoxicity of these complexes comprises multiple modes of action, including modification of DNA conformation, scission of DNA strands by reactive oxygen species, and inhibition of selected proteins that are crucial to the cellular cycle.
Asunto(s)
Fosfatasa Alcalina/antagonistas & inhibidores , Complejos de Coordinación/farmacología , ADN/metabolismo , Iminas/farmacología , Oxindoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Animales , Línea Celular Tumoral , Complejos de Coordinación/metabolismo , Cobre/química , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , División del ADN/efectos de los fármacos , Humanos , Iminas/metabolismo , Ligandos , Ratones , Simulación del Acoplamiento Molecular , Oxindoles/metabolismo , Platino (Metal)/química , Inhibidores de Proteínas Quinasas/metabolismoRESUMEN
In vertebrate species, testosterone seems to inhibit spermatogonial differentiation and proliferation. However, this androgen can also be converted, via aromatase, into estrogen which stimulates spermatogonial differentiation and mitotic activity. During seasonal spermatogenesis of adult bullfrogs Lithobates catesbeianus, primordial germ cells (PGCs) show enhanced testosterone cytoplasm immunoexpression in winter; however, in summer, weak or no testosterone immunolabelling was observed. The aim of this study was to confirm if PGCs express stem cell markers - alkaline phosphatase (AP) activity and GFRα1 (glial-cell-line-derived neurotrophic factor) - and verify whether testosterone is maintained in these cells by androgen receptors (ARs) and/or sex hormone-binding globulin (SHBG) in winter. Furthermore, regarding the possibility that testosterone is converted into estrogen by PGCs in summer, the immunoexpression of estrogen receptor (ER)ß was investigated. Bullfrog testes were collected in winter and in summer and were embedded in glycol methacrylate for morphological analyses or in paraffin for the histochemical detection of AP activity. GFRα1, AR, SHBG and ERß expression were detected by Western blot and immunohistochemical analyses. The expression of AP activity and GFRα1 in the PGCs suggest that these cells are spermatogonial stem cells. In winter, the cytoplasmic immunoexpression of ARs and SHBG in the PGCs indicates that testosterone is maintained by these proteins in these cells. The cytoplasmic immunoexpression of ERß, in summer, also points to an ER-mediated action of estrogen in PGCs. The results indicate a participation of testosterone and estrogen in the control of the primordial spermatogonia during the seasonal spermatogenesis of L. catesbeianus.
Asunto(s)
Receptor beta de Estrógeno/metabolismo , Rana catesbeiana/fisiología , Receptores Androgénicos/metabolismo , Globulina de Unión a Hormona Sexual/biosíntesis , Espermatogonias/metabolismo , Células Madre/metabolismo , Animales , Diferenciación Celular/fisiología , Humanos , Inmunohistoquímica , Masculino , Globulina de Unión a Hormona Sexual/metabolismo , Espermatogénesis , Espermatogonias/citología , Células Madre/citologíaRESUMEN
Bullfrog stem spermatogonia, also named primordial germ cells (PGCs), show strong testosterone immunolabeling in winter, but no or weak testosterone immunoexpression in summer. Thus, the role of testosterone in these cells needs to be clarified. In this study, we proposed to evaluate whether PGCs express aromatase and estrogen receptors, and verify a possible role of estrogen in PGCs seasonal proliferation. Testes of male adult bullfrogs, collected in winter (WG) and summer (SG), were fixed and embedded in historesin, for quantitative analysis, or paraffin for immunohistochemistry (IHC). The number of haematoxylin/eosin stained PGCs/lobular area was obtained. Proliferating cell nuclear antigen (PCNA), aromatase, estrogen receptor ß (ERß) and PCNA/ERß double immunolabeling were detected by IHC. The number of PCNA-positive PGCs and the histological score (HSCORE) of aromatase and ERß immunolabeled PGCs were obtained. Although the number of PGCs increased significantly in WG, a high number of PCNA-positive PGCs was observed in summer. Moreover, aromatase and ERß HSCORE was higher in SG than WG. The results indicate that PGCs express a seasonal proliferative activity; the low mitotic activity in winter is related to the maximal limit of germ cells which can be supported in the large lobules. In SG, the increased ERß and aromatase HSCORE suggests that testosterone is converted into estrogen from winter to summer. Moreover, the parallelism between the high PGCs mitotic activity and ERß immunoexpression suggest a participation of estrogen in the control of the PGCs seasonal proliferative activity which guarantee the formation of new germ cysts from summer to next autumn.