RESUMO
Glutathione (GSH) and its precursor cysteine (Cys) are both known to react within any cells with oxidative species and thus play an important role in cellular defense mechanisms against oxidative stress. In melanocytes, these are also important precursors of melanogenesis by reacting non-enzymatically with l-dopaquinone to form the sulfur-containing pheomelanin. Our aim was to assess pigment role in the cellular radioprotection mechanism using a human melanoma cell model of mixed-type melanin under GSH depletion to obtain a radiosensitizing effect. The latter has been achieved either by Cys deprivation or GSH specific depletion. We first compared cell survival of Cys-deprived and GSH-depleted cells vs. control cells. Cys deprivation was achieved by decreasing Cys concentration in the culture medium for 24 h. In this condition, no toxicity was observed, Cys and GSH levels decreased, melanogenesis switched to a higher eumelanin synthesis and cells were significantly more resistant to 10-Gy dose of ionizing radiations than untreated cells. Glutathione depletion was achieved with the gamma-glutamylcysteine synthetase inhibitor buthionine-S-sulfoximine (BSO) for 24 h at 50 microM, a concentration yielding no toxicity. In this condition, intracellular GSH level decreased but no change in pigmentation was observed and cells were slightly but significantly more sensitive to radiation than the control. We then compared DNA radio-induced damages by Comet assay in control cells, cells treated as above and cells with stimulated pigmentation by increasing Tyr concentration in the medium. Our results showed that, when intracellular eumelanin content increased, DNA damage decreased. By contrast, DNA damage increased in cells treated with BSO alone. It is concluded that increasing the intracellular eumelanin content by the melanin precursor Tyr or by favoring the Pheo- to Eumelanin switch, compensates for the loss of the two intracellular radioprotectors that are GSH and Cys.
Assuntos
Cisteína/fisiologia , Glutationa/fisiologia , Melanoma/radioterapia , Tolerância a Radiação/fisiologia , Butionina Sulfoximina/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Ensaio Cometa , Dano ao DNA , Relação Dose-Resposta à Radiação , Humanos , Melanoma/tratamento farmacológico , Melanoma/patologia , Pigmentação , Tolerância a Radiação/efeitos dos fármacos , Radiossensibilizantes/farmacologiaRESUMO
Exposure of MCF-7 cells to single and/or repeated low gamma-ray doses (0.5 to 8 Gy) resulted in a decrease in the capacity of these cells to concentrate tritiated estradiol ([3H]E2) (reduction of the number of binding sites). The decrease in the [3H]E2-binding capacity was higher than the survival rate, indicating that it could not be ascribed to cell death. Moreover, such low irradiation doses failed to similarly affect the specific incorporation of [3H]ORG 2058, even when the progesterone receptor was induced by E2, a finding that rejects the hypothesis of a nonspecific effect on all steroid hormone receptors. This loss of [3H]E2 binding was reflected by the elimination of the estrogen receptor alpha (ER) when the latter was assessed by immunocytochemistry. However, additional immunochemical studies (Western blot data) performed on cell extracts under denaturing conditions failed to show any similar elimination of the ER peptide, suggesting that the loss of E2-binding capacity would be relevant to subtle changes in the ER structure and/or ER-associated proteins. The loss of binding capacity, produced by a 3-Gy irradiation, failed to decrease the sensitivity of the cells to E2, since progesterone receptor induction and growth stimulation were maintained. Insufficient ER diminution may explain this observation.
Assuntos
Neoplasias da Mama/radioterapia , Sobrevivência Celular/efeitos da radiação , Estradiol/metabolismo , Receptores de Estrogênio/metabolismo , Sítios de Ligação , Western Blotting , Divisão Celular/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Corantes/farmacologia , Relação Dose-Resposta à Radiação , Eletroforese em Gel de Poliacrilamida , Humanos , Imuno-Histoquímica , Pregnenodionas/farmacologia , Ligação Proteica/efeitos da radiação , Receptores de Progesterona/metabolismo , Sais de Tetrazólio/farmacologia , Tiazóis/farmacologia , Fatores de Tempo , Células Tumorais CultivadasRESUMO
The relationship between cell pigmentation and radiosensitivity was investigated in two selected human melanoma cell lines with different melanin content (mixed type: eumelanin and pheomelanin, and pheomelanotic phenotypes). The same study was also done after stimulation of melanogenesis (1) by addition of the melanin precursor l-tyrosine to each of the cell lines separately and (2) by irradiation alone with doses ranging from 0 to 10 Gy. We found that a decrease in cell radiosensitivity was correlated with the type of melanin, with a clear involvement of eumelanin rather than pheomelanin. Increasing the intracellular content of both melanins promoted the growth of irradiated cells. Moreover, at a dose of 10 Gy, both tyrosinase activity and melanin cell content were significantly increased in the absence of any other melanogenesis promoter. Our data suggest that the amount of intracellular melanin is inversely related to the radiosensitivity of melanoma cells and may explain at least in part the controversial responses to ionizing radiations reported for melanoma.
Assuntos
Melaninas/fisiologia , Melanoma/radioterapia , Tolerância a Radiação/fisiologia , Sobrevivência Celular/fisiologia , Sobrevivência Celular/efeitos da radiação , Relação Dose-Resposta à Radiação , Humanos , Melaninas/metabolismo , Melanoma/patologia , Pigmentação , Células Tumorais CultivadasRESUMO
It is well known that ICAM-1 expression can be stimulated by TNF and by oxidative stress, via the activation of specific transcription factors. Two of these--NFkappaB and AP-1--can also be activated by reactive oxygen species, including the superoxide anion (also produced under TNF challenge). The latter is inactivated by superoxide dismutase of which two forms exist: Cu/Zn-SOD (cytoplasmic) and Mn-SOD (mitochondrial). We investigated whether superoxide anion direct generation or accumulation through specific SOD inhibition, may affect ICAM-1 expression in human melanoma and endothelial cells. Our results show a 20-50% increase in both SOD activities when cells were exposed to TNF or to an oxidative stress produced by Paraquat (a generator of superoxide anion radicals), both in terms of enzymes activity (zymogram) and protein levels (Western blotting and ELISA). Either with TNF or Paraquat, we could measure a significant increase of ICAM-1 expression with maxima ranging from 140 to 200%, depending on the cell line. Specific inhibition of Cu/Zn-SOD activity by DTIC (diethyldithiocarbamic acid), in presence of Paraquat or TNF, was followed by an upregulation of ICAM-1 expression (60 and 20%, respectively). In contrast, the addition of a SOD mimetic (MnTMPyP) completely inhibited Paraquat-stimulated ICAM-1 expression in melanoma cells and significantly decreased it in HUVEC (50%). In presence of TNF however, the same SOD mimetic inhibited TNF-stimulated ICAM-1 expression by 25% in melanoma and 17% in endothelial cells. In conclusion, these data provide evidence that melanoma and endothelial cells exposure to TNF or oxidative stress results in a significant increase of both Mn- and Cu/Zn-SOD activities. This increase seems to be associated with a reduction in the stimulation of ICAM-1 expression by TNF or oxidative stress.