Structure-toxicity relationship of phenolic analogs as anti-melanoma agents: an enzyme directed prodrug approach

Abstract

The aim of this study was to identify a phenolic prodrug compound that is minimally metabolized by rat liver microsomes, but yet could form quinone reactive intermediates in melanoma cells as a result of its bioactivation by tyrosinase. In current work, we investigated 24 phenolic compounds for their metabolism by tyrosinase, rat liver microsomes and their toxicity towards murine B16-F0 and human SK-MEL-28 melanoma cells. A linear correlation was found between toxicities of phenolic analogs towards SK-MEL-28 and B16-F0 melanoma cells, suggesting similar mechanisms of toxicity in both cell lines. 4-HEB was identified as the lead compound. 4-HEB (IC(50) 48h, 75muM) showed selective toxicity towards five melanocytic melanoma cell lines SK-MEL-28, SK-MEL-5, MeWo, B16-F0 and B16-F10, which express functional tyrosinase, compared to four non-melanoma cells lines SW-620, Saos-2, PC3 and BJ cells and two amelanotic SK-MEL-24, C32 cells, which do not express functional tyrosinase. 4-HEB caused significant intracellular GSH depletion, ROS formation, and showed significantly less toxicity to tyrosinase specific shRNA transfected SK-MEL-28 cells. Our findings suggest that presence of a phenolic group in 4-HEB is critical for its selective toxicity towards melanoma cells.

Fig. 1

Postulated biochemical mechanism of action for phenolic prodrug toxicity in melanoma cells.

Fig. 2

Chemical structures of phenolic analogs.

Fig. 3

Enzymatic oxidation of 4-HEB and E4MB at pH 7.4 by tyrosinase. AA and NADH oxidations were monitored at 266 nm and 340 nm, respectively. Ethyl 4-hydroxybenzoate (4-HEB); Ethyl 4-methoxybenzoate (E4MB); n=3.

Fig. 4

Toxicity correlation between IC₅₀ of phenolic agents in murine B16-F0 and human SK-MEL-28 melanoma cell lines. 2-Hydroxyanisole, 3-hydroxyanisole, 4-hydroxybenzoic acid, 4-hydroxyacetophenone were considered as outliers, n=3.

Fig. 5

Intracellular GSH depletion in human melanocytic SK-MEL-28 and amelanotic C32 melanoma cells. (A) 4-HEB (75 μM-300 μM) showed dose and time dependent intracellular GSH depletion in human melanocytic SK-MEL-28 melanoma cells. (B) 4-HEB (75 μM-300 μM) caused significantly less GSH depletion in amelanotic C32 melanoma cells. (C-D) E4MB (0.75, 1.0, and 1.5 mM) caused significantly less intracellular GSH depletion in SK-MEL-28 and C32 melanoma cells. The IC₅₀ (48 h) for 4-HEB and E4MB in SK-MEL-28 cells was 75 μM and 750 μM, respectively. Ethyl 4-hydroxybenzoate (4-HEB); Ethyl 4-methoxybenzoate (E4MB), n=3.

Fig. 6

ROS formation in human melanocytic SK-MEL-28 and amelanotic C32 melanoma cells. (A) 4-HEB (75-1500 μM) led to a time- and dose dependent escalation in ROS formation in human melanocytic SK-MEL-28 cells. (B) 4-HEB (75-1500 μM) led to significantly less ROS formation in amelanotic C32 melanoma cells. (C-D) E4MB (750 μM -5 mM) lead to significantly less ROS formation in human melanocytic SK-MEL-28 and amelanotic C32 melanoma cells, n=6.

Fig. 7

4-HEB toxicity in melanocytic melanoma cells (black bars) and non-melanoma (open bars) cell lines. * significantly different, n=3.

Fig. 8

4-HEB toxicity in melanocytic melanoma and amelanotic melanoma cell lines. * significantly different, n=3.

Fig. 9

4-HEB toxicity in the presence and absence of tyrosinase shRNA silencing plasmid. (A) Tyrosinase protein levels were detected by western blotting with a specific anti-tyrosinase monoclonal antibody. Transfection with shRNA3 clone curtailed tyrosinase expression. (B) 4-HEB (75 μM) caused significantly less toxicity in SK-MEL-28 cells transfected with shRNA plasmid directed against tyrosinase. * significantly different when compared to control, n=3.