Biochemical mechanism of caffeic acid phenylethyl ester (CAPE) selective toxicity towards melanoma cell lines

Abstract

In the current work, we investigated the in vitro biochemical mechanism of Caffeic Acid Phenylethyl Ester (CAPE) toxicity and eight hydroxycinnamic/caffeic acid derivatives in vitro, using tyrosinase enzyme as a molecular target in human SK-MEL-28 melanoma cells. Enzymatic reaction models using tyrosinase/O(2) and HRP/H(2)O(2) were used to delineate the role of one- and two-electron oxidation. Ascorbic acid (AA), NADH and GSH depletion were used as markers of quinone formation and oxidative stress in CAPE induced toxicity in melanoma cells. Ethylenediamine, an o-quinone trap, prevented the formation of o-quinone and oxidations of AA and NADH mediated by tyrosinase bioactivation of CAPE. The IC(50) of CAPE towards SK-MEL-28 melanoma cells was 15muM. Dicoumarol, a diaphorase inhibitor, and 1-bromoheptane, a GSH depleting agent, increased CAPE's toxicity towards SK-MEL-28 cells indicating quinone formation played an important role in CAPE induced cell toxicity. Cyclosporin-A and trifluoperazine, inhibitors of the mitochondrial membrane permeability transition pore (PTP), prevented CAPE toxicity towards melanoma cells. We further investigated the role of tyrosinase in CAPE toxicity in the presence of a shRNA plasmid, targeting tyrosinase mRNA. Results from tyrosinase shRNA experiments showed that CAPE led to negligible anti-proliferative effect, apoptotic cell death and ROS formation in shRNA plasmid treated cells. Furthermore, it was also found that CAPE selectively caused escalation in the ROS formation and intracellular GSH (ICG) depletion in melanocytic human SK-MEL-28 cells which express functional tyrosinase. In contrast, CAPE did not lead to ROS formation and ICG depletion in amelanotic C32 melanoma cells, which do not express functional tyrosinase. These findings suggest that tyrosinase plays a major role in CAPE's selective toxicity towards melanocytic melanoma cell lines. Our findings suggest that the mechanisms of CAPE toxicity in SK-MEL-28 melanoma cells mediated by tyrosinase bioactivation of CAPE included quinone formation, ROS formation, intracellular GSH depletion and induced mitochondrial toxicity.

Figure 1. UV-Vis overlay scans for CAPE oxidation by tyrosinase/O₂ at pH 7.4

(A) Addition of tyrosinase enzyme to the reaction mixture containing CAPE resulted in the formation of the characteristic spectra at 320-400 nm. (B) Addition of GSH prior to tyrosinase did not result in the formation of the characteristic peaks. Ascorbic Acid (AA) and NADH demonstrate distinctive peaks at 266 nm and 340 nm, respectively. (C-E) Upon the addition of tyrosinase to the reaction mixture containing CAPE, the absorbance of 266 nm and 340 nm peaks significantly diminished, indicating oxidation of AA and NADH, respectively.

Figure 2. UV-Vis overlay scans for CAPE oxidation by HRP/H₂O₂ at pH 7.4

(A) Addition of HRP/H₂O₂ to the reaction mixture containing CAPE resulted in the formation of the characteristic spectra at 240-350 nm. (B) Addition of GSH prior to HRP/H₂O₂ addition did not result in the formation of the characteristic peaks. Ascorbic Acid (AA) and NADH demonstrate distinctive peaks at 266 nm and 340 nm, respectively. (C-E) Upon the addition of HRP/H₂O₂ to the reaction mixture containing CAPE, the absorbance of 266 nm and 340 nm peaks diminished significantly, indicating oxidation of AA and NADH, respectively.

Figure 3. Kinetic scans for CAPE oxidation by tyrosinase/O₂ and HRP/H₂O₂

AA and NADH oxidations were monitored at 266 and 340 nm, respectively. (A-B) The rate and extent of AA and NADH oxidation as a result of CAPE metabolism by tyrosinase/O₂ at pH 7.4. (C-D) The rate and extent of AA and NADH oxidation as a result of CAPE metabolism by HRP/H₂O₂ at pH 7.4. Addition of GSH prior to tyrosinase or HRP/H₂O₂ completely prevented AA and NADH oxidation. GSH diminished the rate of AA and NADH oxidations by CAPE/tyrosinase/O₂ or HRP/H₂O₂ oxidizing system.

Figure 4. CAPE toxicity in melanoma and non melanoma cell lines

(A) Tyrosinase expression in respective melanoma and non-melanoma cell lines. Tyrosinase protein levels were detected by western blotting with a specific anti-tyrosinase monoclonal antibody. The anti-tyrosinase monoclonal antibody recognizes tyrosinase (80 kDa). (B) CAPE toxicity in melanocytic melanoma SK-MEL-28, SK-MEL-5, Me Wo, B16-F0, B16-F10, non melanoma SW-620, Saos-2, PC 3 and amelanotic melanoma SK-MEL-24 and C32 cell lines. Note that the controls for all the cell lines were performed but not included in this figure. The control in the figure refers to untreated SK-MEL-28 control cells. The viability for the other respective cell lines treated with CAPE is indicated with reference to their respective control cell lines.

Figure 5. Toxicity of CAPE in the presence of various biochemical modulators (48 h)

(A) BH and DC significantly increased CAPE toxicity (*p<0.05); (B) CS and TF significantly reduced CAPE toxicity (*p<0.05) in SK-MEL-28 cells. Cyclosporin A (CS), Trifluoperazine (TF), inhibitors of PTP in mitochondria.

Figure 6. CAPE 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. The anti-tyrosinase monoclonal antibody recognizes tyrosinase (80 kDa). Transfection with shRNA3 clone curtailed tyrosinase expression for 50 %. (B) CAPE (15 μM) did not cause significant toxicity (*p<0.05) in SK-MEL-28 cells silenced with shRNA plasmid directed against tyrosinase.

Figure 7. ROS formation in human melanocytic SK-MEL-28 and amelanotic C32 melanoma cells

(A) CAPE (15-300 μM) led to a time- and concentration-dependent escalation in ROS formation in human melanocytic SK-MEL-28 cells. (B) CAPE (15-300 μM) led to significantly less (*p<0.05) ROS formation in amelanotic C32 melanoma cells. (C) CAPE caused significantly less ROS formation (*p<0.05) in SK-MEL-28 cells transfected with tyrosinase shRNA plasmid. (D) ROS formation in the presence of modulators in SK-MEL-28 cells (*significantly different p<0.05).

Figure 8. Mitochondrial membrane potential

(A) Concentration- and time-dependent decrease in mitochondrial membrane potential. (B) The protective effect of cyclosporin-A (CS), a PTP inhibitor, and ethylenediamine, an o-quinone trap, on mitochondrial membrane potential.

Figure 9. CAPE induced apoptotic cell death in presence and absence of tyrosinase shRNA

Transfection of SK-MEL-28 cells with tyrosinase shRNA significantly prevented CAPE induced apoptotic cell death.

Figure 10. Selective intracellular GSH depletion in human melanocytic SK-MEL-28 and amelanotic C32 melanoma cells

(A) Intracellular GSH depletion by CAPE in human melanocytic SK-MEL-28 melanoma cells, and (B) Intracellular GSH level in human amelanotic C32 melanoma cells. CAPE (15 μM-200 μM) showed concentration- and time-dependent intracellular GSH depletion in melanocytic human SK-MEL-28 cells but not in amelanotic C32 melanoma cells.

Figure 11. Proposed biochemical mechanism of CAPE induced toxicity in melanoma cells

Caffeic Acid Phenylethyl Ester (CAPE) underwent oxidation by tyrosinase through two-electron oxidation to quinone [37]. CAPE was oxidized by HRP/H₂O₂ through one-electron oxidation to a semiquinone [38]. CAPE toxicity towards SK-MEL-28 human melanoma cells was significantly enhanced by dicoumarol (DC), a diaphorase inhibitor, and 1-bromoheptane (BH), a GSH depleting agent. Ascorbic acid (AA), nicotinamide adenine dinucleotide (NADH), and glutathione (GSH) were depleted as a result of CAPE oxidation mediated by tyrosinase bioactivation. Ethylenediamine, an o-quinone trap [17, 18], reacts with CAPE o-quinone, preventing NADH and AA oxidation.