RESUMEN
In this study, we have investigated the genotoxic, cytostatic, antineoplastic and apoptotic effects of three newly synthesized modified steroidal esters, having as alkylating agent p-N,N-bis(2-chloroethyl) aminophenyl butyrate (CHL) or p-N,N-bis(2-chloroethyl) aminophenyl acetate (PHE) esterified with the steroidal nucleus modified in the B- and D-ring. The genotoxic and cytotoxic effects of the compounds were investigated both in vitro, in lymphocyte cultures obtained from blood samples of healthy donors and in vivo, in ascites cells of P388 leukemia obtained from the peritoneal cavity of DBA/2 mice. Preparations were scored for sister-chromatid exchange (SCE) and proliferation-rate indices (PRI). The newly synthesized compounds were also studied for antineoplastic activity against lymphocytic P388 and lymphoid L1210 leukemias in mice, by calculating the mean of the median survival of the drug-treated animals (T) versus the untreated control (C) (T/C%). The activity of caspase-2 and caspase-3, indicators of apoptosis, was assessed biochemically in primary cultures of human lymphocytes. Our results show that the newly synthesized compounds caused severe genotoxic effects by significantly increasing the frequency of SCE and decreasing the PRI values in cultures of peripheral lymphocytes in vitro and in ascites cells of lymphocytic P388 leukemia in vivo. A significant correlation was also observed in both the in vitro and in vivo experiments: the higher the SCE frequency the lower the PRI value (r=-0.65, P<0.001 and r=-0.99, P<0.01, respectively). The measured antileukemic potency was statistically increased by all test compounds in both types of tumours, while the activity of caspase-2 and caspase-3 showed a statistically significant increase after two periods of exposure. The genotoxic (increase of SCE), cytostatic/cytotoxic (decrease of PRI) and antileukemic effects (increase of T/C%) in combination with the induction of apoptosis (activation of caspase-2 and caspase-3) caused by the newly synthesized compounds, lead us to propose them as agents with potentially antineoplastic properties.
Asunto(s)
Androsterona/análogos & derivados , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Azaesteroides/farmacología , Citostáticos/farmacología , Compuestos de Mostaza Nitrogenada/farmacología , Esteroides/farmacología , Androsterona/síntesis química , Androsterona/química , Androsterona/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Ascitis/genética , Ascitis/metabolismo , Ascitis/patología , Azaesteroides/síntesis química , Azaesteroides/química , Caspasa 2/metabolismo , Caspasa 3/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Citostáticos/síntesis química , Citostáticos/química , Ensayos de Selección de Medicamentos Antitumorales , Ésteres , Femenino , Humanos , Leucemia L1210/patología , Leucemia L1210/prevención & control , Leucemia P388/patología , Leucemia P388/prevención & control , Linfocitos/citología , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Estructura Molecular , Pruebas de Mutagenicidad , Compuestos de Mostaza Nitrogenada/síntesis química , Compuestos de Mostaza Nitrogenada/química , Intercambio de Cromátides Hermanas/efectos de los fármacos , Esteroides/síntesis química , Esteroides/química , Análisis de SupervivenciaRESUMEN
Nitrosourea is decomposed under physiological conditions to react with biological macromolecules by two mechanisms: alkylation (with proteins and nucleic acids) and carbamoylation (with proteins but not nucleic acids). It has been suggested that the alkylating action is responsible for the therapeutic effects of nitrosoureas, and that the carbamoylation activity leads to toxicity effects. In order to reduce systemic toxicity and improve specificity and distribution for cancer therapy, 2-haloethyl nitrosourea has been esterified with modified steroids, which are used as biological platforms for transporting the alkylating agent to the tumor site in a specific manner. The cytogenetic and antineoplastic effect were studied of seven newly synthesized esters of N,N-bis(2-chloroethyl)alanyl carboxyl derivatives with a modified steroidal nucleus (compounds 1-7). As a very sensitive indicator of genotoxicity the Sister Chromatid Exchange (SCE) assay was used and as a valuable marker of cytostatic activity the cell Proliferation Rate Index (PRI) in cultures of normal human lymphocytes was used. The order of magnitude of the cytogenetic activity on a molar basis (15, 30, 120 microM) of the compounds was 7>>6>3>5>2>4>1. The most active compound 7 has an enlarged (seven carbon atoms) A ring modified with a lactam group (-NHCO-) with the nitrosourea moiety esterified at position 17 In the group of seven substances a correlation was observed between the magnitude of SCE response and the depression in PRI (r=-O, 65, p<0.001). According to the criterion of activity of National Cancer Institute (NCI), the order of antineoplastic activity of compounds on lymphoid L1210 leukemia is 7>6>2>5>4>3>1 and on lympocytic P388 leukemia cells is 7>2>6>5>4>3>1. The present results are in agreement with previous suggestions that the effectiveness in cytogenetic activity may well be correlated with antitumor effects [T/C: 248% for the compound 7 in 250 mg/kg b.w.; T/C: mean survival time of drug-treated animals (T) (excluding long term survivals) vs. corn-oil-treated controls (C)].