The metabolic bioactivation of caffeic acid phenethyl ester (CAPE) mediated by tyrosinase selectively inhibits glutathione S-transferase

Abstract

Glutathione S-transferase (GST) and multidrug resistance-associated proteins (MRPs) play major roles in drug resistance in melanoma. In this study, we investigated caffeic acid phenethyl ester (CAPE) as a selective GST inhibitor in the presence of tyrosinase, which is abundant in melanoma cells. Tyrosinase bioactivates CAPE to an o-quinone, which reacts with glutathione to form CAPE-SG conjugate. Our findings indicate that 90% CAPE was metabolized by tyrosinase after a 60-min incubation. LC-MS/MS analyses identified a CAPE-SG conjugate as a major metabolite. In the presence of tyrosinase, CAPE (10-25μM) showed 70-84% GST inhibition; whereas in the absence of tyrosinase, CAPE did not inhibit GST. CAPE-SG conjugate and CAPE-quinone (25μM) demonstrated ⩾85% GST inhibition via reversible and irreversible mechanisms, respectively. Comparing with CDNB and GSH, the non-substrate CAPE acted as a weak, reversible GST inhibitor at concentrations >50μM. Furthermore, MK-571, a selective MRP inhibitor, and probenecid, a non-selective MRP inhibitor, decrease the IC(50) of CAPE (15μM) by 13% and 21%, apoptotic cell death by 3% and 13%, and mitochondrial membrane potential in human SK-MEL-28 melanoma cells by 10% and 56%, respectively. Moreover, computational docking analyses suggest that CAPE binds to the GST catalytic active site. Caffeic acid, a hydrolyzed product of CAPE, showed a similar GST inhibition in the presence of tyrosinase. Although, as controls, 4-hydroxyanisole and L-tyrosine were metabolized by tyrosinase to form quinones and glutathione conjugates, they exhibited no GST inhibition in the absence and presence of tyrosinase. In conclusion, both CAPE and caffeic acid selectively inhibited GST in the presence of tyrosinase. Our results suggest that intracellularly formed quinones and glutathione conjugates of caffeic acid and CAPE may play major roles in the selective inhibition of GST in SK-MEL-28 melanoma cells. Moreover, the inhibition of MRP enhances CAPE-induced toxicity in the SK-MEL-28 melanoma cells.

Fig. 1

LC–MS/MS of CAPE-SG conjugate. Using selective/multiple reaction monitoring, both figures A and B represent two overlaid detection windows for m/z = 590 (CAPE-SG) peak and m/z = 285 (CAPE) peak on the LC/MS/MS detector. (A) After 5 min incubation of CAPE (62.5 µM) with glutathione (200 µM), the HPLC chromatogram revealed a peak at 7.8 min indicating that CAPE-SG conjugate was not formed in the absence of tyrosinase. (B) The glutathione conjugate of CAPE was determined by HPLC after 5 min incubation of reaction mixture containing CAPE (62.5 µM), glutathione (200 µM), and tyrosinase (20 U/mL). The peaks at 2.2 min and 7.8 min are CAPE-SG conjugate and CAPE, respectively. Note, the significant decrease in the intensity of CAPE peak at 7.8 min and the appearance of a new peak at 2.2 min. (C) The MS/MS analysis of the peak at 2.2 min showed CAPE-SG conjugate as the major metabolite (m/z 590 [M+H]⁺). The major daughter ions identified were 145, 264, 393, 461, 468, 515, and 590 m/z. The MS/MS analysis of the CAPE and glutathione reaction mixture detected only CAPE (m/z 285 [M+H]⁺), indicating that CAPE-SG conjugate was not formed in the absence of tyrosinase.

Fig. 2

The inhibition of GST. The inhibitory effects of CAPE and caffeic acid (a hydrolyzed product of CAPE) on human placenta GST with respect to CDNB. (A) CAPE-SG conjugate and CAPE-quinone at concentration of 10–50 µM demonstrated 68–96% and 70–93% GST inhibition, respectively. (B) CA-SG conjugate and caffeic acid-quinone at concentrations of 10–50 µM demonstrated 19–61% and 23–67% GST inhibition, respectively. At higher concentrations, CAPE alone showed negligible GST inhibition. 4-HA-SG conjugate, 4-HA-quinone, 4-HA, tyrosine-SG conjugate, tyrosine–quinone and tyrosine at concentrations of 10–50 µM did not show GST inhibition (data not shown).

Fig. 3

Reversible and irreversible nature of GST inhibition. (A) CAPE-SG conjugate, CAPE-quinone, CA-SG conjugate (caffeic acid glutathione conjugate), and CA-quinone showed significant GST inhibition. (B) Irreversible inhibition of human placenta GST. CAPE-quinone and CA-quinone showed significant irreversible inhibition of GST, whereas the GST activity was recovered after removing CAPE-SG and CA-SG conjugates by filtering the reaction mixture through Millipore centrifuge filter unit, indicating that both glutathione conjugates of CAPE and CA were reversible GST inhibitors.

Fig. 4

Lineweaver–Burk plots: competitive and non-competitive nature of GST inhibition. The experiments were performed on three different days using freshly prepared GST, CDNB, CAPE, GSH and tyrosinase stock solutions on a daily basis. The average of data points was used to graph the Lineweaver–Burk plot to determine the nature of competitive and non-competitive inhibition of GST. (A) Mixed GST inhibition by CAPE with respect to CDNB. (B) Mixed GST inhibition by CAPE-quinone with respect to CDNB. (C) Competitive GST inhibition by CAPE-SG conjugate with respect to CDNB. (D) Competitive GST inhibition by ethacrynic acid (EA) with respect to CDNB. (E) Mixed GST inhibition by CAPE with respect to GSH. (F) Competitive GST inhibition by CAPE-quinone with respect to GSH. (G) Mixed GST inhibition by CAPE-SG conjugate with respect to GSH. (H) Non-competitive GST inhibition by ethacrynic acid (EA) with respect to GSH. The unit of GST activity is µM/min (nmol/mL/min).

Fig. 5

The inhibition of GST in human SK-MEL-28 melanoma cell homogenate. CAPE (15–100 µM) led to a dose dependent GST inhibition ranging from 61 to 86% in human melanoma SK-MEL-28 cell homogenate. CA (15–100 µM) showed 21–53% GST inhibition whereas 4-HA (15–100 µM) and tyrosine (15–100 µM) did not show significant inhibition in comparison to CA (caffeic acid) and CAPE.

Fig. 6

Apoptosis assay. One hour of pretreatment of cells with MK-571, a selective MRP inhibitor, and probenecid, a non-selective MRP inhibitor, significantly increased CAPE induced apoptotic cell death. The effect of probenecid was significantly higher than MK-571.

Fig. 7

Mitochondrial membrane potential. One hour of cell pretreatment with MK-571, a selective MRP inhibitor, and probenecid, a non-selective MRP inhibitor, significantly decreased mitochondrial membrane potential. The effect of CAPE and probenecid combination was more significant than CAPE in combination with MK-571.

Fig. 8

Computational docking of CAPE and EA in the active sites of GST. The computational prediction of the interactions between GST and CAPE or EA was performed as described in Section 2. Sticks represent the ligands. Green surfaces represent the wild-type GST or C47S/Y108V double mutants GST. The GSH-binding site (G site) and the substrate-binding site (H site) are indicated. (A and B) X-ray crystal structures show EA-SG conjugate bound to the wild-type GST (PDB ID 11GS), and EA/GSH to C47S/Y108V mutant (PDB ID 3KM6) [36,37]. (C–F) The predicted bound conformation of EA (C and D) and CAPE (E and F), shown in black sticks, in the active sites of both wild type and C47S/Y108V GST mutant, respectively. The GSH, which was not included in the docking calculations, is present as red line to indicate the G site.

Scheme 1

Summary of selective GST inhibition by CAPE in the presence of tyrosinase with respect to CDNB. CAPE can cross biological cell membrane due to its high lipid solubility whereas caffeic acid, an organic anion, cannot cross cell membrane due to its ionization at physiological pH of 7.4 (more than 99.9% is ionized at pH 7.4). CAPE acts as an anti-melanoma agent through tyrosinase prodrug bioactivation to cytotoxic o-quinone, which further is conjugated with glutathione to form CAPE-SG conjugate. CAPE-quinone inhibits GST via an irreversible mixed mechanism with respect to CDNB whereas CAPE-SG conjugate inhibits GST via a reversible competitive mechanism with respect to CDNB. CAPE alone at high concentrations (>50 µM) marginally inhibits GST reversibly by a mixed mechanism of inhibition with respect to CDNB. In addition, CAPE can potentially be hydrolyzed to caffeic acid intracellularly, which can be metabolized by tyrosinase to form CA-quinone and CA-SG conjugate to inhibit GST irreversibly and reversibly, respectively.