RESUMEN
Anticancer therapy based on recombinant arginine-degrading enzymes has been proposed for the treatment of several types of malignant cells deficient in arginine biosynthesis. One of the predicted side effects of such therapy is restricted bioavailability of nitric oxide as arginine catabolic product. Prolonged NO limitation may lead to unwanted disturbances in NO-dependent vasodilation, cardiovascular and immune systems. This problem can be overcome by co-supplementation with exogenous NO donor. However, NO may potentially counteract anticancer effects of therapy based on arginine deprivation. In this study, we evaluate for the first time the effects of an exogenous NO donor, sodium nitroprusside, on viability and metastatic properties of two human melanoma cell lines SK-MEL-28 and WM793 under arginine-deprived conditions. It was revealed that NO did not rescue melanoma cells from specific effects evoked by arginine deprivation, namely decreased viability and induction of apoptosis, dramatically reduced motility, invasiveness and clonogenic potential. Moreover, sodium nitroprusside co-treatment augmented several of these antineoplastic effects. We report that a combination of NO-donor and arginine deprivation strongly and specifically impaired metastatic behavior of melanoma cells. Thus, sodium nitroprusside can be considered as an adjuvant for the more efficient treatment of malignant melanoma and possibly other tumors with arginine-degrading enzymes.
Asunto(s)
Antineoplásicos/farmacología , Arginina/deficiencia , Arginina/metabolismo , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , Donantes de Óxido Nítrico/farmacología , Nitroprusiato/farmacología , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Quimioterapia Combinada , Humanos , Melanoma/patología , Óxido Nítrico/biosíntesis , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Autophagy is a process of cytosol-to-lysosome vesicle trafficking of cellular constituents for degradation and recycling of their building blocks. Autophagy becomes very important for cell viability under different stress conditions, in particular under amino acid limitation. In this report we demonstrate that single amino acid arginine deprivation triggers profound prosurvival autophagic response in cultured human ovarian cancer SKOV3 cells. In fact, a significant drop in viability of arginine-starved SKOV3 cells was observed when autophagy was inhibited by either coadministration of chloroquine or transcriptional silencing of the essential autophagy protein BECLIN 1. Enzymatic arginine deprivation is a novel anticancer therapy undergoing clinical trials. This therapy is considered nontoxic and selective, as it allows controlling the growth of malignant tumours deficient in arginine biosynthesis. We propose that arginine deprivation-based combinational treatments that include autophagy inhibitors (e.g., chloroquine) may produce a stronger anticancer effect as a second line therapy for a subset of chemoresistant ovarian cancers.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Arginina , Autofagia , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Ováricas/metabolismo , Beclina-1 , Línea Celular Tumoral , Supervivencia Celular , Femenino , Humanos , Neoplasias Ováricas/patología , Neoplasias Ováricas/terapiaRESUMEN
AIM: To study the dynamics of Ras-dependent signalling in the course of Herbimycin A induced erythroid differentiation of human erythroleukemia K562 cells. METHODS: p21Ras functional activity was analyzed by direct measurement of GTP/GDP ratio in anti-p21Ras immunoprecipitates of K562 cells previously incubated with H3(32)PO4. Dynamics of protein tyrosine phosphorylation was studied using Western blotting. Electrophoretic mobility shift assay was used to monitor Erk2 activation. Phosphotyrosine (pY)-containing proteins bound to recombinant glutathione-S-tranferase (GST)-fused form of adaptor protein Grb2 were identified using GST in vitro binding assay. RESULTS: It was shown that the relative quantity of GTP associated with Ras protein in non-induced cells varied from 27% to 37% upon 72 h of cell culturing. Herbimycin A caused 15% increase of GTP/GDP ratio at 3rd h. This index decreased during further investigated periods, although it did not reach control values even at 72nd h. Transient rise of Ras-GTP level at 3rd h of incubation in the presence of Herbimycin A correlated with the increase in tyrosine phosphorylation of proteins with apparent molecular weight of 210, 160, 140, 116 and 42 kDa, as well as with the activation of Erk2 and increase of binding of a set of pY-containing proteins with recombinant GST-fusion form of Ras activator, adaptor protein Grb2. Dramatic inhibition of interaction between docking protein Shc and GST-Grb2 was observed at late stages of cell induction (48-72 h) while binding of pY-containing proteins during this period did not differ significantly in control and differentiated cells. CONCLUSION: The obtained results suggest that time-dependent changes in Grb2-mediated network of protein-protein interaction events might define implication of Ras-dependent signalling in Herbimycin A-induced erythroid differentiation of K562 cells.