Abstract
BackgroundEven though new therapies are available against melanoma, novel approaches are needed to overcome resistance and high-toxicity issues. In the present study the anti-melanoma activity of Nicotinamide (NAM), the amide form of Niacin, was assessed in vitro and in vivo.MethodsHuman (A375, SK-MEL-28) and mouse (B16-F10) melanoma cell lines were used for in vitro investigations. Viability, cell-death, cell-cycle distribution, apoptosis, Nicotinamide Adenine Dinucleotide+ (NAD+), Adenosine Triphosphate (ATP), and Reactive Oxygen Species (ROS) levels were measured after NAM treatment. NAM anti-SIRT2 activity was tested in vitro; SIRT2 expression level was investigated by in silico transcriptomic analyses. Melanoma growth in vivo was measured in thirty-five C57BL/6 mice injected subcutaneously with B16-F10 melanoma cells and treated intraperitoneally with NAM. Interferon (IFN)-γ-secreting murine cells were counted with ELISPOT assay. Cytokine/chemokine plasmatic levels were measured by xMAP technology. Niacin receptors expression in human melanoma samples was also investigated by in silico transcriptomic analyses.ResultsNAM reduced up to 90% melanoma cell number and induced: i) accumulation in G1-phase (40% increase), ii) reduction in S- and G2-phase (about 50% decrease), iii) a 10-fold increase of cell-death and 2.5-fold increase of apoptosis in sub-G1 phase, iv) a significant increase of NAD+, ATP, and ROS levels, v) a strong inhibition of SIRT2 activity in vitro. NAM significantly delayed tumor growth in vivo (p ≤ 0.0005) and improved survival of melanoma-bearing mice (p ≤ 0.0001). About 3-fold increase (p ≤ 0.05) of Interferon-gamma (IFN-γ) producing cells was observed in NAM treated mice. The plasmatic expression levels of 6 cytokines (namely: Interleukin 5 (IL-5), Eotaxin, Interleukin 12 (p40) (IL12(p40)), Interleukin 3 (IL-3), Interleukin 10 (IL-10) and Regulated on Activation Normal T Expressed and Secreted (RANTES) were significantly changed in the blood of NAM treated mice, suggesting a key role of the immune response. The observed inhibitory effect of NAM on SIRT2 enzymatic activity confirmed previous evidence; we show here that SIRT2 expression is significantly increased in melanoma and inversely related to melanoma-patients survival. Finally, we show for the first time that the expression levels of Niacin receptors HCAR2 and HCAR3 is almost abolished in human melanoma samples.ConclusionNAM shows a relevant anti-melanoma activity in vitro and in vivo and is a suitable candidate for further clinical investigations.
Highlights
Even though new therapies are available against melanoma, novel approaches are needed to overcome resistance and high-toxicity issues
We show that NAM induces a significant increase of NAD+, Adenosine Triphosphate (ATP), and Reactive Oxygen Species (ROS) levels, a strong effect on cell cycle phases distribution, and a significant antimelanoma effect both in vitro and in vivo
Effect of NAM on human melanoma cells number A375 and SK-MEL-28 cells were treated with increasing NAM doses (1, 20, and 50 mM), in the presence of 10% fetal bovine serum (FBS)
Summary
Even though new therapies are available against melanoma, novel approaches are needed to overcome resistance and high-toxicity issues. In the present study the anti-melanoma activity of Nicotinamide (NAM), the amide form of Niacin, was assessed in vitro and in vivo. Nicotinamide (NAM) is the amide form of niacin (vitamin B3 or vitamin PP) It is a precursor of nicotinamideadenine dinucleotide NAD+ [1] and is known to play an essential role in energy metabolism and to act in several tissues including skin [2], nervous system [3, 4], and muscles [5]. Given its central role in controlling energy production and the activity of many enzymes, NAM has been proposed in the treatment of several clinical conditions, including chemoprevention of non-melanoma skin cancer [12, 21, 33] and chemoprevention of lung cancer [34]. We show that NAM induces a significant increase of NAD+, ATP, and ROS levels, a strong effect on cell cycle phases distribution, and a significant antimelanoma effect both in vitro and in vivo
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