A Comparison of B16 Melanoma Cells and 3T3 Fibroblasts Concerning Cell Viability and ROS Production in the Presence of Melatonin Tested in a Wide Concentration Range

Int. J. Mol. Sci. 2013, 14, 3901-3920; doi:10.3390/ijms14023901 International Journal of Molecular Sciences ISSN 1422-0067 www.mdpi.com/journal/ijms Article A Comparison of B16 Melanoma Cells and 3T3 Fibroblasts Concerning Cell Viability and ROS Production in the Presence of Melatonin, Tested Over a Wide Range of Concentrations Maria Angeles Bonmati-Carrion 1, Nuria Álvarez-Sánchez 2,3, Rüdiger Hardeland 4, Juan Antonio Madrid 1,* and Maria Angeles Rol 1 1 Department of Physiology, College of Biology, University of Murcia, Murcia 30100, Spain; E-Mails: [email protected] (M.A.B.-C.); [email protected] (M.A.R.) 2 Institute of Biomedicine of Seville (IBiS), Virgen del Rocío University Hospital/CSIC/ University of Seville, Seville 41013, Spain; E-Mail: [email protected] 3 Department of Medical Biochemistry and Molecular Biology, University of Seville School of Medicine, Seville 41004, Spain 4 Johann Friedrich Blumenbach Institute of Zoology and Anthropology, University of Göttingen, Göttingen 37077, Germany; E-Mail: [email protected] * Author to whom correspondence should be addressed; E-Mail: jam[email protected]; Tel.: +34-868-88-4946 (ext. 123); Fax: +34-868-88-3963. Received: 22 January 2013; in revised form: 31 January 2013 / Accepted: 4 February 2013 / Published: 14 February 2013 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 OPEN ACCESS Int. J. Mol. Sci. 2013, 14 3902 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. Keywords: melanoma; fibroblast; melatonin; cell viability; intracellular ROS; tumor cell cultures; non-tumor cell cultures; in vitro 1. Introduction Melatonin (N-acetyl-5-methoxytryptamine) is a hormone that is synthesized and secreted during the dark phase of the day by the pineal gland. Moreover, an increasing body of evidence shows that it is also produced in various extrapineal organs, including the retina, gastrointestinal tract, bone marrow, lymphocytes [1,2], and heart [3], among others. In mammals, the nocturnal rise is controlled by the hypothalamic circadian pacemaker, the suprachiasmatic nuclei (SCN), which triggers sympathetic stimulation of the pineal gland via a specific neuronal pathway, whereas melatonin biosynthesis is inhibited by light. Photic information is transmitted to the SCN through the retinohypothalamic tract [4]. In addition to its properties of a chronobiotic agent, melatonin acts as a pleiotropic regulator that exerts a plethora of cellular and systemic effects [2,5]. Among other actions, it is now accepted that it has immune-enhancing [6], antioxidant [7] and oncostatic properties [8]. Antitumor effects of melatonin have been explored both in vivo and in vitro. In fact, melatonin has been widely documented to be beneficial in various cases of neoplastic diseases in vivo [9], including melanoma [8], one of the most invasive cancers in humans [10]. Although various suggestions concerning melatonergic signaling pathways and metabolic changes have been published for several tumors and respective cell lines [11–15], it remains to be clarified whether oncostatic and oncocidal effects, when present, are directly caused by the hormone via binding sites in the tumor cells, indirectly via modulation of the circadian and/or the immune systems or by several concomitant actions. A further problem with in vivo systems consists in the frequently observed interference of melatonin with other signals (e.g., by hormones, cytokines, cell-cell interactions or fatty acid uptake), making it difficult to interpret the results. For these reasons, among others, in vitro studies have been proposed as a useful tool to investigate the effects of melatonin and other agents. 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. However, there are some examples in which melatonin had no such effect [31], and rather Int. J. Mol. Sci. 2013, 14 3903 enhanced tumor cell proliferation or survival [24], in a certain concentration range. A moderate growth stimulation was even observed in melanoma B16 cell lines [28,32], at low doses of melatonin, whereas as inhibition was reported for another B16 subline [29]. These discrepancies are not entirely surprising, inasmuch as established cell lines, even if derived from the same carcinoma type or the same biopsy material, frequently differ substantially in their properties, because of multiple mutations and epigenetic effects by which tumor cells differently shut off certain genes. With regard to the action of a hormone such as melatonin, differences in the expression of receptors (cf. data by Helton et al. [28], Cos et al. [29] vs. Mengeaud et al. (1994) [33]) and other binding sites can lead to fundamental deviations in responsiveness between tumor cell sublines. In fact, comparisons of several human melanoma cell lines revealed considerable differences in their susceptibility to melatonin, also depending on stages and phenotypes [30]. Although in vitro studies are useful to avoid the above-mentioned problems of in vivo studies, variations among cell lines related to the state of differentiation or the number of mutations have to be taken into account, especially in the case of melanomas (cf. [30]). Corresponding assumptions are applicable to tumors in vivo and to comparisons of in vitro with in vivo studies [8,28]. With regard to this variability, there is no agreement on the concentrations of melatonin required for efficiently reducing cell viability in vitro. Some reports indicate that low concentrations are effective in melanoma cultures [29] and in other tumor cell lines [19], while other studies suggest that very high concentrations are required [17]. In addition, fetal bovine serum (FBS), a necessary component of culture media for most cell lines, has been reported to interfere with the action of melatonin, lowering its effectiveness in reducing cell viability [18,30]. The mechanisms involved in the action of melatonin on cell viability remain to be identified. Suggestions have been made concerning a role for melatonin receptors [17,21,34], the activation of several apoptotic pathways and cell cycle modifications [16,22]. Although melatonin is a potent radical scavenger and antioxidant at different levels, e.g., by increasing the activities of antioxidant enzymes [35,36] or by modulating mitochondrial activity (for a review, see ref. [37]), stimulations of ROS/RNS production have been also documented in some cell types, at both pharmacological and physiological concentrations. This is particularly evident in the immune system, in which melatonin directly activates monocytes [38–40]. However, most of the prooxidant effects are of indirect nature and related to the stimulation of proinflammatory cytokine secretion (IL-2, IL-6, IL-8, IL-12) [2,6,41] or suppression of the antiinflammatory IL-10 [42]. Although melatonin exhibits antiapoptotic properties in numerous models in which cells are toxicologically challenged [43], the contrary has been repeatedly described for cancer cells [44,45]. In the scientific literature of recent years, the hypothesis has gained increasing support that melatonin can also reduce cell viability and activate cell death pathways by enhancing intracellular ROS formation, particularly in cultured tumor cell lines [45–48]. Surprisingly, in non-tumor cell lines such as human leukocytes, it is the antioxidant effect of melatonin, at physiological concentrations, that are responsible for its protective actions [43]. Melatonin has been proposed as a potentially safe and effective oncostatic agent in cancer patients. However, and although many studies have investigated the effects of melatonin on non-tumor cells [49–52], they have not focused strictly on cell viability but rather on other aspects of melatonin actions, such as paracrine interactions [53,54]. Apart from controls in toxicological studies or Int. J. Mol. Sci. 2013, 14 3904 protection experiments against UVA and UVB radiation [55], only a few investigations have dealt with cell viability in non-tumor cell cultures. However, to the best of our knowledge, no single study has ever compared its effects on the viability of tumor and non-tumor cell lines in vitro using identical melatonin concentrations, culture media composition and exposure times. Therefore, the aim of this study was to investigate the possible differential effect of melatonin in a tumor and a non-tumor cell line, using a wide range of melatonin concentrations, to examine the interferential effect exerted by FBS on the action of melatonin, and to determine whether the enhancing effect of high melatonin concentrations on intracellular ROS constitutes a possible mechanism underlying the reduction of cell viability. Figure 1. Melanoma cell line (B16): Effects of melatonin on counts of viable cells (expressed as the percentage of MTT uptake with respect to the untreated control), in a low concentration range, in the absence (A and B) or the presence (C and D) of fetal bovine serum (FBS) after 24 (A and C) and 48 h (B and D) of treatment. DMSO vehicle controls were performed for each melatonin concentration, although for terms of clarity only the two highest concentrations are represented. Statistically significant differences compared to controls are marked with asterisks. (* indicates p < 0.05, ANOVA followed by Dunnett’s post hoc test). 2. Results and Discussion 2.1. Results The most relevant results concerning the effects of melatonin on cell viability are shown in Figures 1–4. In the absence of FBS, low melatonin concentrations ranging from 10−9 to 10−5 M reduced moderately, but significantly, counts of viable B16 melanoma cells (Figure 1A,B), as compared to untreated cells. However, when FBS was present (Figure 1C,D), low-range melatonin Int. J. Mol. Sci. 2013, 14 3905 concentrations (10−11–10−5) had no effect at 24 h, and significantly increased cell numbers after 48 h (with the exception of 10−7 M, which did not reach significance). In the case of fibroblast (non-tumor) 3T3 cells, no obvious tendency was seen for this same concentration range in terms of reducing cell numbers in the absence of FBS at either 24 h or 48 h (Figure 2A,B). After 48 h of treatment, a non-significant 30% reduction was found at the highest concentration (10−5 M) (Figure 2B), whereas 10−9 M melatonin caused a significant increase in cell number (p < 0.004). On the other hand, in the presence of FBS, and with this low range of melatonin concentration, practically no changes in cell viability were found after 24 or 48 h of exposure (Figure 2C,D). Thus, in the absence of FBS, low melatonin concentrations (10−11–10−5 M) significantly reduced the number of viable cells of the melanoma line, without affecting or even increasing that of the fibroblast cell line. Figure 2. Fibroblast cell line (3T3): Effects of melatonin on counts of viable cells (expressed as the percentage of MTT uptake with respect to the untreated control), in a low concentration range, in the absence (A and B) or the presence (C and D) of fetal bovine serum (FBS) after 24 (A and C) and 48 h (B and D) of treatment. DMSO vehicle controls were performed for each melatonin concentration, although for terms of clarity only the two highest concentrations are represented. Statistically significant differences compared to controls are marked with asterisks. (* indicates p < 0.05, ANOVA followed by Dunnett’s post hoc test). Figure 3 shows the effects by a range of high melatonin concentrations (1–10 mM), with and without FBS in the culture medium, on the melanoma cell line after 24 h and 48 h of exposure. In the absence of FBS and after 24 h of treatment, a 20% reduction was observed in the number of viable Int. J. Mol. Sci. 2013, 14 3906 cells, at a melatonin concentration of 5 mM (Figure 3A, p < 0.001). After 48 h, however, cell viability compared to untreated control cells was significantly lowered by melatonin concentrations above 2.5 mM, with reductions of 40% and 50% following the administration of 2.5 mM and 5 mM melatonin, respectively (Figure 3B, p < 0.001). At concentrations close to 8.75 mM, melatonin became strongly lethal. Cells exhibited a completely disorganized structure and their viability was reduced to 0% (Figure 3A,B, p < 0.001). In the presence of FBS (Figure 3C,D), a 5 mM melatonin concentration was needed to produce a 40% reduction in cell viability after 48 h of exposure (Figure 3D, p < 0.001), which is twice the concentration needed in the absence of FBS; at the same time, the lethal concentration decreased to 7.5 mM (Figure 3D, p < 0.001) (Figure 5A,B). Figure 3. Melanoma cell line (B16): Effects of melatonin on cell viability (expressed as the percentage of MTT uptake with respect to the untreated control), in a high concentration range, in the absence (A and B) or the presence (C and D) of fetal bovine serum (FBS) after 24 (A and C) and 48 h (B and D) of treatment. DMSO vehicle controls were performed for each melatonin concentration, although for terms of clarity only the two highest concentrations are represented. Statistically significant differences compared to controls are marked with asterisks. (* indicates p < 0.05, ANOVA followed by Dunnett’s post hoc test). When melatonin was applied in the millimolar range to 3T3 fibroblasts in the absence of FBS, cell viability declined with increasing melatonin concentrations. At 24 h, differences to controls became statistically significant at 1 mM (Figure 4A, 20%, p = 0.007). Similar results were obtained after 48 h of treatment (Figure 4B). At a concentration of 5 mM, melatonin became lethal after 24 h or 48 h of Int. J. Mol. Sci. 2013, 14 3907 treatment (p < 0.001). However, in the presence of FBS, 7.5 mM melatonin was needed in this cell line to elicit a reduction in cell viability (around 60%) after 24 h of exposure (Figure 4C, p < 0.001). After 48 h (Figure 4D), this inhibitory effect became statistically significant (p = 0.001) at lower melatonin concentrations (2.5 mM). At 8.75 mM, melatonin turned out to be completely lethal after 48 h (p < 0.001), losses in viability being also apparent in damaged cell structure (Figures 4D,5C,D). In summary, 3T3 fibroblasts were more sensitive to high concentrations of melatonin in the absence of FBS, but not in its presence. Figure 4. Fibroblast cell line (3T3): Effects of melatonin on cell viability (expressed as the percentage of MTT uptake with respect to the untreated control), in a high concentration range, in the absence (A and B) or the presence (C and D) of fetal bovine serum (FBS) after 24 (A and C) and 48 h (B and D) of treatment. DMSO vehicle controls were performed for each melatonin concentration, although for terms of clarity only the two highest concentrations are represented. Statistically significant differences compared to controls are marked with asterisks. (* indicates p < 0.05, ANOVA followed by Dunnett’s post hoc test). In order to ascertain that the effect of melatonin was not merely due to an increase in osmolarity or to a toxic effect by DMSO, controls for DMSO vehicle (Figures 1–4) and osmolarity (data not shown) (using sucrose to match the osmolarity of melatonin solutions, as detailed in Table 1) were also included. No differences were observed when comparing cells treated with DMSO or sucrose at different concentrations to non-treated controls in either tumor or non-tumor cells at 24 h or 48 h. In fact, the increase in osmolarity in melatonin solutions was due to DMSO, in such a way that the Int. J. Mol. Sci. 2013, 14 3908 vehicle-treated cells could easily serve as osmolarity controls (Table 1). This clearly demonstrates that the changes observed in cell viability result from melatonin treatment but not from an inappropriately high osmolarity or from DMSO toxicity. Figure 5. Lethal effect on melanoma (B16) (A and B) and fibroblast cell lines (3T3) (C and D) in the presence of FBS. Microphotographs in A and C represent untreated control cells. Microphotographs B and D are taken from cell cultures treated with 10 mM melatonin after 48 h. A loss of cell structure can be clearly observed. The images were obtained using a 20× objective. Table 1. Osmolality of solutions containing high melatonin concentrations. Treatment Osmolality (mOsm/Kg) Control 333 10 mM 425 8.75 mM 382 7.5 mM 364 5 mM 365 2.5 mM 346 1.25 mM 341 1 mM 342 To examine whether an intracellular increase in ROS induced by melatonin might be causative for a reduction in cell viability, the effect of three melatonin concentrations (10 pM, 0.1 µM and 5 mM) on intracellular dichlorofluorescin (DCFH)-reactive ROS levels was studied in both cell lines after 24 h and 48 h of treatment (Figure 6). Since flow cytometry requires cell integrity, tests were performed with non-lethal melatonin concentrations and in the presence of FBS (medium component that satisfies specific metabolic requirements for the culture of cells). The highest concentration tested (5 mM) Int. J. Mol. Sci. 2013, 14 3909 significantly increased intracellular DCFH-reactive ROS levels in both tumor and non-tumor cell lines. In the case of melanoma cells, intracellular DCFH-reactive ROS levels increased by 334% (Figure 6A, p < 0.001) and 545% (Figure 6B, p < 0.001) after 24 h and 48 h, respectively, as compared to untreated control cells. In the fibroblast cell line, the melatonin-induced increase in intracellular DCFH-reactive ROS levels after 24 h (Figure 6C) was similar (346%) to that seen for tumor cell lines (p < 0.001 vs. untreated control), but this level was maintained (373%) even after 48 h (Figure 6D, p < 0.001). Only in melanoma cells, a lower dose (10 pM) was able to produce an antioxidant effect after 48 h of exposure (Figure 6B). Catalase was used as a positive control for antioxidant activity, and as expected, it reduced intracellular DCFH-reactive ROS levels (p < 0.004). Figure 6. Intracellular DCFH-reactive ROS levels in melanoma (A and B) and fibroblast (C and D) cell lines treated with melatonin, in the presence of FBS, and their respective controls. DCFH-reactive ROS are expressed as the percentage of green fluorescence in the presence of melatonin at three different concentrations (10 pM, 0.1 µM, 5 mM) after 24 h (A and C) and 48 h (B and D). Catalase (1000 U/mL) was used as a positive control for antioxidant activity. Statistically significant differences compared to controls are marked with asterisks. (* indicates p < 0.05, ANOVA followed by Dunnett’s post hoc test). 2.2. 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