Signal transduction pathways involved in protective effects of melatonin in C6 glioma cells

IRCCS Centro Neurolesi Bonino-Pulejo, Messina, Italy.
Journal of Pineal Research (Impact Factor: 9.6). 02/2008; 44(1):78-87. DOI: 10.1111/j.1600-079X.2007.00492.x
Source: PubMed


Melatonin (N-acetyl-5-methoxytryptamine), an indole hormone, is the chief secretory product of the pineal gland and is an efficient free radical scavenger and antioxidant, both in vitro and in vivo. The role of melatonin as an immunomodulator is, in some cases, contradictory. Although melatonin is reported to influence a variety of inflammatory and immune responses, evidence supporting its effects on important glioma cells-derived mediators is incomplete. We studied, in rat glioma cell line (C6), the role of melatonin (100 microm-1 mm) in the regulation of the expression of nitric oxide synthase (NOS) caused by incubation with lipopolysaccharide (LPS)/interferon (IFN)-gamma (1 microg/mL and 100 U/mL, respectively) and defined the mode of melatonin's action. Treatment with LPS/IFN-gamma for 24 hr elicited the induction of inducible (iNOS) activity as determined by nitrite and nitrate (NO(x)) accumulation in the culture medium. Preincubation with melatonin abrogated the mixed cytokines-mediated induction of iNOS. The effect of melatonin was concentration-dependent. Moreover, Western blot analysis showed that melatonin inhibited LPS/IFN-gamma-induced expression of COX-2 protein, but not that of constitutive cyclooxygenase. Inhibition of iNOS and COX-2 expression was associated with inhibition of activation of the transcription factor nuclear factor kappa B (NF-kappaB). The ability of melatonin to inhibit NF-kappaB activation was further confirmed by studies on the degradation of the inhibitor of NF-kappaB, IkappaB-alpha. Increased production of lipid peroxidation products using thiobarbituric acid assay were found in cellular contents from activated cultures. Lipid peroxidation was decreased by melatonin treatment in a concentration-dependent manner. Moreover, several genes having roles in heat-shock response were downregulated in melatonin-treated cells, such as 70 proteins, reflecting the reduced oxidative stress in these cells. The mechanisms underlying in vitro the neuroprotective properties of melatonin involve modulation of transcription factors and consequent altered gene expression, resulting in downregulation of inflammation.

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    • "However, melatonin has also been demonstrated to inhibit apoptotic pathways in a number of cell types (14–16), as well as having a role in stem cell proliferation and the epigenetic regulation of neural cell growth in vitro (17). These findings suggest that the modulation of melatonin may not be limited to treating cancer or exerting an apoptotic effect, and that numerous intracellular mechanisms may be involved in promoting cancer cell survival. "
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    ABSTRACT: A number of studies have suggested that melatonin possesses anticancer properties. However, conflicting data exists with regard to the role of melatonin in the treatment of cancer. In the present study, the effects of melatonin on the transcriptional regulation of three genes associated with cell proliferation (Nestin, Bmi-1 and Sox2), and on C6 glioma cell survival and viability, were investigated in vitro to evaluate the use of melatonin in cancer therapy. Melatonin was shown to increase the mRNA levels of Nestin, Bmi-1 and Sox2 in a similar pattern, with the highest mRNA levels noted at a concentration of 3 mM. At higher concentrations of melatonin (5 mM), the mRNA levels of Nestin, Bmi-1 and Sox2 were reduced from their peak levels, and were correlated with changes observed in immunofluorescence morphology studies, cell viability and survival assays. Immunofluorescence studies of Nestin-stained cells demonstrated that treatment with a higher concentration of melatonin (3 and 5 mM) led to the Nestin filaments condensing and rearranging around the cell nuclei, and an alteration in the cell morphology. C6 cell viability was also significantly decreased at 3 mM melatonin, and cell death was observed at 5 and 10 mM melatonin. These results suggested that Nestin, Bmi-1 and Sox2 were strongly correlated with the survival of C6 cells following treatment with melatonin, and that high therapeutic concentrations of melatonin (>5 mM) were required to induce cell death. These findings suggested that the implementation of melatonin in the treatment of glioma and other types of cancer may be inhibited by conflicting cell growth signals in cells. Therefore, adjunct therapy is required to improve the efficacy of melatonin in the treatment of cancer.
    Oncology letters 08/2013; 6(2):347-352. DOI:10.3892/ol.2013.1413 · 1.55 Impact Factor
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    • "The effects of melatonin on the viability of cultured tumor cells are documented in an extensive body of literature. Some examples are hepatocellular carcinomas [16,17], lymphomas [18], mammary tumors [19–21], pituitary prolactin-secreting tumors [22], gliomas [23], pheochromocytomas [24], and melanomas [12,25–30]. In the majority of cases, melatonin was capable of reducing tumor cell viability. "
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    ABSTRACT: Melatonin is a pleiotropic molecule with many cellular and systemic actions, including chronobiotic effects. Beneficial effects are widely documented concerning the treatment of neoplastic diseases in vivo as well as reductions in viability of cultured cells from melanoma, one of the most aggressive cancers in humans. However, studies of its effects on non-tumor cells in vitro have not focused on viability, except for experiments aiming to protect against oxidotoxicity or other toxicological insults. Furthermore, there is no agreement on the range of effective melatonin concentrations in vitro, and the mechanisms that reduce cell viability have remained unclear. Tumor cell-specific increases in the production of reactive oxygen and nitrogen species (ROS/RNS) may provide a possible explanation. Our aim was to analyze the potential inhibition of tumor (B16 melanoma 4A5) and non-tumor cell (3T3 Swiss albino) viability using a wide range of melatonin concentrations (10-11-10-2 M), and to determine whether intracellular ROS enhancement was involved in this process. In the absence of fetal bovine serum (FBS), low melatonin concentrations (10-9-10-5 M) reduced the proliferation of melanoma cells with no effect in fibroblasts, whereas, in the presence of FBS, they had no effect or even increased the proliferation of both fibroblast and melanoma cells. Melatonin concentrations in the upper millimolar range increased ROS levels and reduced the viability of both cell types, but more markedly so in non-tumor cells. Thus, low melatonin concentrations reduce proliferation in this specific melanoma cell line, whereas high concentrations affect the viability of both tumor (B16 4A5 melanoma) and non-tumor (3T3 fibroblasts) cells. Increased ROS levels in both lines indicate a role for ROS production in the reduction of cell viability at high-but not low-melatonin concentrations, although the mechanism of action still remains to be elucidated.
    International Journal of Molecular Sciences 02/2013; 14(2):3901-20. DOI:10.3390/ijms14023901 · 2.86 Impact Factor
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    • "Nitrite estimation was done in the culture medium according to Esposito et al. (2008). Briefly, the nitrite concentration in the samples was measured by the Griess reaction, by adding 100 ll of Griess reagent [0.1 % (w/v) napthylethylenediamine HCl and 1 % (w/v) sulfanilamide in 5 % (v/v) phosphoric acid (vol. "
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    ABSTRACT: Rotenone causes cytotoxicity in astrocytic cell culture by glial activation, which is linked to free radical generation. The present study is an investigation to explore whether rotenone could also cause cellular toxicity in mouse neuroblastoma cells (Neuro-2a) under treatment similar to astroglial cells. The effect of rotenone (0.1, 1, and 10 μM) on mitochondrial dehydrogenase enzyme activity by MTT reduction assay, PI uptake, total reactive oxygen species (ROS)/superoxide levels, nitrite levels, extent of DNA damage (by comet assay), and nuclear morphological alteration by Hoechst staining was studied. Caspase-3 and Ca(2+)/calmodulin-dependent protein kinase II (CaMKIIα) gene expression was determined to evaluate the apoptotic cell death and calcium kinase, respectively. Calcium level was estimated fluorometrically using fura-2A stain. Rotenone decreased mitochondrial dehydrogenase enzyme activity and generated ROS, superoxide, and nitrite. Rotenone treatment impaired cell intactness and nuclear morphology as depicted by PI uptake and chromosomal condensation of Neuro-2a cells, respectively. In addition, rotenone resulted in increased intracellular Ca(+2) level, caspase-3, and CaMKIIα expression. Furthermore, co-exposure of melatonin (300 μM), an antioxidant to cell culture, significantly suppressed the rotenone-induced decreased mitochondrial dehydrogenase enzyme activity, elevated ROS and RNS. However, melatonin was found ineffective to counteract rotenone-induced increased PI uptake, altered morphological changes, DNA damage, elevated Ca(+2), and increased expression of caspase-3 and CaMKIIα. The study indicates that intracellular calcium rather than oxidative stress is a major factor for rotenone-induced apoptosis in neuronal cells.
    Archives of Toxicology 04/2012; 86(9):1387-97. DOI:10.1007/s00204-012-0853-z · 5.98 Impact Factor
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