Comparative in vitro and in silico evaluation of the toxic effects of metformin and/or ascorbic acid, new treatment options in the treatment of Melasma

Abstract

Melasma is a chronic condition that leads to the buildup of melanin pigment in the epidermis and dermis due to active melanocytes. Even though it is considered a non-life-threatening condition, pigment disorders have a negative impact on quality of life. Since melasma treatment is not sufficient and complicated, new treatment options are sought. Research on metformin and ascorbic acid suggested that they might be used against melasma in the scope of "drug repositioning."The MNT-1 human melanoma cell line was used to assess the effects of metformin, ascorbic acid, and metformin+ascorbic acid combination on cytotoxicity and oxidative stress. Melanin, cAMP, L-3,4-dihydroxyphenylalanine (L-DOPA) and tyrosinase levels were determined by commercial ELISA kits and tyrosinase gene expression was analyzed with RT-qPCR. Cytopathological evaluations were performed by phase contrast microscopy. Tyrosinase expression was determined by immunofluorescence (IF) staining of MNT-1 cells. The online service TargetNet was used for biological target screening. The parameters were not significantly altered by ascorbic acid applied at non-cytotoxic concentrations. On the contrary, metformin dramatically raised tyrosinase and intracellular ROS levels. Moreover, intracellular ROS levels and tyrosinase levels were found to be considerably elevated with the combined treatment. Also, potential metformin and ascorbic acid interactions were determined. According to the results, it can be said that these parameters were not significantly altered by ascorbic acid. On the contrary, metformin dramatically raised tyrosinase and intracellular oxidative stress levels. Moreover, intracellular oxidative stress and tyrosinase levels were elevated with the combined treatment. In conclusion, individual treatments of ascorbic acid or metformin may only provide a limited effect when treating melasma and extensive in vitro and in vivo research are required.

Keywords: In silico; In vitro; MNT-1 cells; ascorbic acid; hyperpigmentation; metformin.

Fig. 1

A and B: Cell viability after ascorbic acid exposure in MNT-1 cells. C and 1D: Cell viability after metformin exposure in MNT-1 cells. E: ROS levels in study groups.

Fig. 2

Phase contrast microscopy images of the experimental groups. 1st panel: A: Control, B: Ascorbic acid (Asc), C: Metformin (met), D: Combined application (Asc + met) (phase contrast X100), 2nd panel: A: Control, B: Ascorbic acid (Asc), C: Metformin (met), D: Combined application (Asc + met) (phase contrast X400).

Fig. 3

Indirect immunofluorescence labeling with anti-tyrosinase in experimental groups. In the labeling with anti-tyrosinase, positive immunoreactivity with anti-tyrosinase (arrow) is observed in the cytoplasm of the cells. Mitotic cells (arrow in inset B) were observed in the ascorbic acid group. A: Control, B: Ascorbic acid (Asc), C: Metformin (met), D: Combined application (Asc + met). i: DAPI, ii: FITC, iii: Merged, X400.

Fig. 4

A Tyrosinase levels. A,bBars that do not share the same letters are significantly different from each other (P < 0.05). B: Tyrosinase gene expression. A,bBars that do not share the same letters are significantly different from each other (P < 0.05). C: Quantitative fluorescent image analysis of indirect immunofluorescent labeling with anti-tyrosinase in the experimental groups. Mean CTCF calculated from images at 40X magnification of experimental groups after anti-tyrosinase immunofluorescence labeling (CTCF in 5 fields for each group and at least 40 cells in each field). Data are given as mean ± standard error of mean (SEM). ^****P < 0.0001.