RESUMEN
We have shown that antioxidant N-acetylcysteine (NAC, 2-10 mM) quickly (for 2 hours) and completely inactivates the activity of matrix metalloproteinases (gelatinases MMP-2 and MMP-9, and collagenases MMP-1 and MMP-8) secreted by transformed mouse fibroblasts 3T3-SV40 into the medium. The same MMP inhibition took place in the cell-free conditioned medium of HT-1080 fibroblasts, which suggests a direct chemical interaction between NAC and MMP resulting in the loss of MMP activity. Besides inhibitory effect, NAC decreased MMP-1 and MMP-9 (but not MMP-2) production in the cell medium. However, the level of MMP-1 and MMP-9 inhibitor (TIMP-1) remained normal, indicating a shift in the balance between the enzyme and inhibitor. The correlation between MMP-2 level and tissue enzyme inhibitor TIMP-2 was similar in control and NAC treated cells. At the same time, reorganization of type I collagen at the cell surface occurred. All together permits the conclusion that NAC action results in the extracellular matrix remodeling and changing in cellular functions.
Asunto(s)
Acetilcisteína/farmacología , Antioxidantes/farmacología , Matriz Extracelular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Animales , Línea Celular Transformada , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Matriz Extracelular/enzimología , Matriz Extracelular/ultraestructura , Metaloproteinasa 1 de la Matriz/genética , Metaloproteinasa 1 de la Matriz/metabolismo , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 8 de la Matriz/genética , Metaloproteinasa 8 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Células 3T3 NIH , Inhibidor Tisular de Metaloproteinasa-1/genética , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Inhibidor Tisular de Metaloproteinasa-2/genética , Inhibidor Tisular de Metaloproteinasa-2/metabolismoRESUMEN
We studied the effect of antioxidants such as N-acetylcysteine (NAC, 10 mM) and alpha-lipoic acid (ALA, 1.25 mM) and of the hormone melatonin (1 microM) on the ability of murine hepatoma cells MH22a to develop tumors in syngenic mice (C3HA) after subsutaneous injection. Tumor formation and development slowed down and mouse mortality decreased when the injected cells were pretreated by NAC, ALA or melatonin during 20 h. Melatonin had the most marked effect. Tumors appeared in 100 % cases after 10 days in control mice when untreated cells had been injected; injection of cells pretreated by NAC or ALA resulted in tumor formation only in 40 and 53 % of mice, respectively. When cells were pretreated with melatonin the tumors appeared only in 18-20 days after injection. Until the end of the observation (36 days) 67 % of control mice died, but when the cells were pretreated by NAC or ALA mouse death-rate was 20 and 53 %, respectively. In the case of melatonin we did not observed any dead mice at all. We showed that treatment by antioxidants delayed (NAC) or completely inhibited (ALA) cell cycle of hepatoma cells. Cell cycle was restored after removal of the antioxidants. Melatonin did not change cell cycle phase distribution. We conclude that there is no direct correlation between loss of tumorigenic properties and changing of proliferative activity of hepatoma cells. Different mechanisms of antioxidants and melatonin action resulting in transient tumor phenotype normalization are discussed.
Asunto(s)
Antioxidantes/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Neoplasias Hepáticas/tratamiento farmacológico , Melatonina/farmacología , Acetilcisteína/farmacología , Acetilcisteína/uso terapéutico , Animales , Antioxidantes/uso terapéutico , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/prevención & control , Ciclo Celular/efectos de los fármacos , Citometría de Flujo , Humanos , Inyecciones Subcutáneas , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/prevención & control , Melatonina/uso terapéutico , Ratones , Ratones Endogámicos C3H , Ácido Tióctico/farmacología , Ácido Tióctico/uso terapéutico , Trasplante Isogénico , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We studied N-acetylcysteine (NAC) ability to change the phenotype properties of several transformed and embryonic cells. We examined human epidermoid carcinoma A431 cells, murine hepatoma MH22a cells, and murine embryonic fibroblasts (MEFs) in terms of the sensitivity to natural killer (NK) recognition and abolishment. We have demonstrated that treatment with NAC (10 mM) results in a loss of susceptibility to NK cell activity by transformed A431 and MH22a cells similar to 3T3-SV40 transformed cells whose partial reversion caused by NAC was revealed by us before. We have shown that MEFs are also sensitive to NK activity and abolished by NK cells as well as transformed cells. MEFs pretreated with 10 mM NAC as well as transformed cells lose their susceptibility to NK cell activity. The loss of cell sensitivity to NK cytolytic activity was accompanied by a reorganization of the actin cytoskeleton and the appearance of well-pronounced stress-fibers.
Asunto(s)
Acetilcisteína/farmacología , Citotoxicidad Inmunológica , Embrión de Mamíferos/efectos de los fármacos , Células Asesinas Naturales/inmunología , Neoplasias/inmunología , Células 3T3 , Actinas/metabolismo , Animales , Línea Celular Transformada , Línea Celular Tumoral , Embrión de Mamíferos/inmunología , Embrión de Mamíferos/ultraestructura , Fibroblastos/efectos de los fármacos , Fibroblastos/inmunología , Humanos , Masculino , Ratones , Fibras de Estrés/metabolismoRESUMEN
The purpose of this study was to compare the effects of two antioxidants, alpha-lipoic acid (ALA) and N-acetylcysteine (NAC) on the sensitivity of 3T3-SV40 fibroblasts to lytic activity of natural killer (NK) cells. ALA (1.25 mM) reduced significantly the fibroblast sensitivity in several hours, whereas NAC (10 mM) did not change it. Subsequent removal of the antioxidants from the cultivation medium resulted in gradual recovery of the sensitivity in the case of ALA and in complete loss of it in the case of NAC. Inactivation of gelatinase MMP-2 (matrix metalloproteinase) using pretreatment of the cells with the inhibitor of MMP, G6001, or specific antibodies to MMP-2 or MMP-9 resulted in decrease of 3T3-SV40 sensitivity to NK cells activity. This effect was similar to that of ALA, not to the NAC one. Pretreatment of NK cells with G6001 did not influence their lytic activity. The results obtained demonstrate that the direct antioxidant, NAC (having reduced thiol groups), and the indirect one, ALA (reducing thiol groups and acting as a direct antioxidant only inside the cell) activate principally different intracellular signal pathways. However, both NAC and ALA pathway includes inactivation of MMP-2.
Asunto(s)
Antioxidantes/farmacología , Transformación Celular Viral/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Células Asesinas Naturales/inmunología , Ácido Tióctico/farmacología , Acetilcisteína/farmacología , Animales , Células 3T3 BALB , Línea Celular Transformada , Transformación Celular Viral/inmunología , Citotoxicidad Inmunológica , Fibroblastos/inmunología , Depuradores de Radicales Libres/farmacología , RatonesRESUMEN
In this study we investigated the effect of alpha-lipoic acid (ALA) in concentration range 0.7-5.0 mM on the intracellular level of reduced glutathione, the cell cycle phase distribution, the structure of microfilaments and microtubules of normal (3T3) and transformed (3T3-SV40) fibroblasts. We obtained that ALA increased the glutathione content in transformed cells, but did not change its level in normal cells, induced cell cycle arrest of 3T3 cells (but not 3T3-SV40 cells), and disrupted actin microfilaments in cells of both lines. The effect of ALA was compared with N-acetylcysteine (NAC) action. The whole complex of findings allows us to affirm that each of these antioxidants acts on its own target molecules in normal and transformed cells and activates different signal and metabolic pathways in these cells. But at the same time the intermediate steps of ALA and NAC action can be common (alteration of the intracellular level of glutathione, reorganization of actin cytoskeleton, etc.).