Pterostilbene, a Dimethyl Derivative of Resveratrol, Exerts Cytotoxic Effects on Melanin-Producing Cells through Metabolic Activation by Tyrosinase

Abstract

Pterostilbene (PTS), which is abundant in blueberries, is a dimethyl derivative of the natural polyphenol resveratrol (RES). Several plant species, including peanuts and grapes, also produce PTS. Although RES has a wide range of health benefits, including anti-cancer properties, PTS has a robust pharmacological profile that includes a better intestinal absorption and an increased hepatic stability compared to RES. Indeed, PTS has a higher bioavailability and a lower toxicity compared to other stilbenes, making it an attractive drug candidate for the treatment of various diseases, including diabetes, cancer, cardiovascular disease, neurodegenerative disorders, and aging. We previously reported that RES serves as a substrate for tyrosinase, producing an o-quinone metabolite that is highly cytotoxic to melanocytes. The present study investigated whether PTS may also be metabolized by tyrosinase, similarly to RES. PTS was oxidized as a substrate by tyrosinase to form an o-quinone, which reacted with thiols, such as N-acetyl-L-cysteine, to form di- and tri-adducts. We also confirmed that PTS was taken up and metabolized by human tyrosinase-expressing 293T cells in amounts several times greater than RES. In addition, PTS showed a tyrosinase-dependent cytotoxicity against B16BL6 melanoma cells that was stronger than RES and also inhibited the formation of melanin in B16BL6 melanoma cells and in the culture medium. These results suggest that the two methyl groups of PTS, which are lipophilic, increase its membrane permeability, making it easier to bind to intracellular proteins, and may therefore be more cytotoxic to melanin-producing cells.

Keywords: anti-aging; cytotoxic effects; melanin-producing cells; ortho-quinone; pterostilbene; resveratrol.

Figure 1

Structures of resveratrol (RES, 1) and pterostilbene (PTS, 2).

Figure 2

Scheme showing the tyrosinase-catalyzed oxidation of pterostilbene (PTS, 2) in the absence or presence of a thiol. The oxidation of PTS (2) gives PTS-quinone (3) as an immediate product, which rapidly decays. PTS-quinone (3) is reduced by ascorbic acid (AA) to form PTS-catechol (3′-hydroxyPTS, (4)). The tyrosinase-catalyzed oxidation of PTS (2) in the presence of the thiol N-acetyl-L-cysteine (NAC) affords the di-adduct DiNAC-PTS-catechol (5) and the tri-adduct TriNAC-PTS-catechol (6). These thiol adducts were isolated and identified as the NAC adducts; “+” means that DiNAC-PTS-catechol (5) and TriNAC-PTS-catechol (6) are produced together.

Figure 3

Time course of the tyrosinase-catalyzed oxidation of PTS (2) and PTS-catechol (4) and HPLC analyses of reaction products. (a) UV/visible spectral changes of PTS (2) at pH 6.8. (b) HPLC analysis following the tyrosinase-catalyzed oxidation of PTS (2) at pH 6.8, the reaction being stopped by the addition of NaBH₄, followed by HClO₄. The experiments were repeated once, and good reproducibility was obtained. The figure was from a single experiment and is representative.

Figure 4

Time course of the tyrosinase-catalyzed oxidation of PTS (2) in presence of ascorbic acid (AA). (a) UV/visible spectral changes of PTS (2) at pH 6.8 in presence of 10 mol eq. AA. (b) HPLC analysis following the tyrosinase-catalyzed oxidation of PTS (2) at pH 6.8 in presence of 10 mol eq. AA, the reaction being stopped by the addition of NaBH₄, followed by HClO₄. The experiments were repeated once, and good reproducibility was obtained. The figure was from a single experiment and is representative.

Figure 5

Time course of the tyrosinase-catalyzed oxidation of PTS (2) in presence of NAC. (a) UV/visible spectral changes of PTS (2) in presence of 2 mol eq. NAC at pH 6.8. (b) UV/visible spectral changes of PTS (2) in presence of 3 mol eq. NAC at pH 6.8. The experiments were repeated once, and good reproducibility was obtained. The figure was from a single experiment and is representative.

Figure 6

Time course of the tyrosinase-catalyzed oxidation of PTS (2) in presence of NAC. (a) HPLC chromatogram of the tyrosinase-catalyzed oxidation of PTS (2) for 5 min in presence of 3 mol eq. NAC at pH 6.8, the reaction being stopped by the addition of NaBH₄, followed by HClO₄. Peak: #1; tri-adduct (6), #2; di-adduct (5), #3; PTS-catechol (4), #4; PTS (2). HPLC analyses were performed at 45 °C at a flow rate of 0.7 mL/min. (b) HPLC analysis following the tyrosinase-catalyzed oxidation of PTS (2) in presence of 3 mol eq. NAC at pH 6.8, the reaction being stopped by the addition of NaBH₄, followed by HClO₄. The experiments were repeated once, and good reproducibility was obtained. The figure was from a single experiment and is representative.

Figure 7

Metabolism of RES (1) and PTS (2) in hTYR-293T cells yielding their adducts with CySH and GSH. (a) RES (1) and its metabolites in cells. (b) PTS (2) and its metabolites in cells. (c) RES (1) and its metabolites in the medium. (d) PTS (2) and its metabolites in the medium. Data represent means ± SD (n = 3 wells). Statistically significant differences: * p < 0.05, ** p < 0.01, *** p < 0.001 between each treatment concentration (in µM) at RES (1) and PTS (2). The statistically significance of the differences was determined by Student’s t-test (two-tailed).

Figure 8

(a) Level of Tyr mRNA in siRNA-transfected cells. (b) 4SCAP, (c) RES (1), and (d) PTS (2) induce tyrosinase-dependent reductions in the viability of B16BL6 melanoma cells. B16BL6 melanoma cells were transfected with a negative control siRNA or with a siRNA directed against Tyr for 24 h. The cells were then treated with the indicated concentrations of compounds for 24 h, and their viability and mRNA levels (in vehicle-treated cells) were measured. Data represent means ± SD (n = 3 wells). The experiments were repeated once, and good reproducibility was obtained. The figure was from a single experiment and representative. * p < 0.05 between siRNA control and siRNA Tyr at each treatment concentration (in µM). #1: p = 0.017 between RES (1) and PTS (2) in siRNA Ctrl. #2: p = 0.020 between RES (1) and PTS (2) in siRNA Tyr at 100 µM, respectively.

Figure 9

(a,b): Eumelanin and pheomelanin contents in B16BL6 melanoma cells. Data represent means ± SD (n = 3 wells). p value between control (0 μM) and each treatment concentration (µM) at eumelanin and pheomelanin in B16BL6 melanoma cells. Statistically significant differences: * p < 0.01, ** p < 0.001. (c,d): Eumelanin and pheomelanin contents in the medium. p value between control (0 μM) and each treatment concentration (µM) at eumelanin and pheomelanin in the medium. * p < 0.01, ** p < 0.001. #1: p = 0.026 between RES (1) and PTS (2) in eumelanin values at 3 µM. #2: p = 0.002 between RES (1) and PTS (2) in pheomelanin values at 3 µM.