Tolcapone induces oxidative stress leading to apoptosis and inhibition of tumor growth in Neuroblastoma

Abstract

Catechol-O-methyltransferase (COMT) is an enzyme that inactivates dopamine and other catecholamines by O-methylation. Tolcapone, a drug commonly used in the treatment of Parkinson's disease, is a potent inhibitor of COMT and previous studies indicate that Tolcapone increases the bioavailability of dopamine in cells. In this study, we demonstrate that Tolcapone kills neuroblastoma (NB) cells in preclinical models by inhibition of COMT. Treating four established NB cells lines (SMS-KCNR, SH-SY5Y, BE(2)-C, CHLA-90) and two primary NB cell lines with Tolcapone for 48 h decreased cell viability in a dose-dependent manner, with IncuCyte imaging and Western blotting indicating that cell death was due to caspase-3-mediated apoptosis. Tolcapone also increased ROS while simultaneously decreasing ATP-per-cell in NB cells. Additionally, COMT was inhibited by siRNA in NB cells and showed similar increases in apoptotic markers compared to Tolcapone. In vivo xenograft models displayed inhibition of tumor growth and a significant decrease in time-to-event in mice treated with Tolcapone compared to untreated mice. These results indicate that Tolcapone is cytotoxic to neuroblastoma cells and invite further studies into Tolcapone as a promising novel therapy for the treatment of neuroblastoma.

Keywords: COMT; ROS; Apoptosis; neuroblastoma; tolcapone.

Figure 1

Predicted downstream dopamine pathway of COMT. Inhibition of COMT by Tolcapone results in overexpression of dopamine, leading to apoptosis in NB which characteristically overexpress dopamine.

Figure 2

Tolcapone exhibits cytotoxicity as a single agent in four NB cell lines and two primary tumors. (A) IC₅₀ values were calculated 48 h postintroduction of Tolcapone drug treatment using Calcein AM fluorescent assays. (B) GI₅₀ values were calculated 48 h postinduction of Tolcapone drug treatment using BrdU assays. (C) RT‐qPCR was run on lysates from all cell lines used to determine basal mRNA levels of COMT. ∆CT represents the change in cycle threshold of COMT compared to β‐Actin. (D) Western blot was run on lysates from all cells to determine basal protein levels of MB‐COMT and S‐COMT. (E) Bands were normalized to β‐Actin control and quantified as a percent compared to β‐Actin expression.

Figure 3

Tolcapone treatment corresponds to a dose‐dependent increase in cell death in NB. (A) Images of established and primary NB cells treated with 200 nmol/L SYTOX and varying concentrations of Tolcapone (0–400 μmol/L) taken with the IncuCyte ZOOM. (B) Quantification of cleaved caspase‐3 expressed in images taken of NB cells treated with 5 μmol/L kinetic caspase‐3 reagent and varying concentrations of Tolcapone (0–400 μmol/L).

Figure 4

Inhibition of COMT through Tolcapone treatment or siRNA silencing induces apoptosis in NB cell lines. (A) Western blot analyses indicated a dose‐dependent increase in cleaved caspase‐3 and cleaved PARP and a reduction in whole caspase‐3 and whole PARP protein expression as NB cells were introduced to increasing concentrations of Tolcapone drug therapy. (B) Blots were normalized to actin control and quantified as a percent compared to β‐Actin expression. (C) COMT, whole and cleaved caspase‐3 protein expression for both SMS‐KCNR and BE(2)‐C cells treated with siRNA and 50 μmol/L Tolcapone. (D) SMS‐KCNR bands were normalized to β‐Actin control. (E) BE(2)‐C bands were normalized to β‐Actin control. (F) RT‐qPCR analysis confirmed a knockdown of COMT mRNA in SMS‐KCNR and BE(2)‐C cells.

Figure 5

Tolcapone treatment corresponds to a dose‐dependent decrease in cellular metabolic activity and increase in reactive oxygen species (ROS) in NB cell lines. (A) Cells were treated with Tolcapone (0–200 μmol/L) for 48 h. Cell Titer GLO was used to measure total cellular ATP and CyQuant was used to measure total cellular DNA (n = 3, graph is representative of one replicate). Values are mean ± SD. (B) Cells were stained with 20 μmol/L DCFDA to measure ROS for 45 min and then treated with varying concentrations of Tolcapone (0–100 μmol/L) with or without NAC for either 1 or 3 h (n = 3, graph is representative of one replicate). Values are mean ± SD (*P < 0.05, *^* P < 0.01, *^** P < 0.001).

Figure 6

Treatment with Tolcapone significantly reduced tumor volume compared to control in vivo. Mice were injected with 2 million SMS‐KCNR NB cells and treated orally with 125 mg/kg Tolcapone every 24 h. A) Mean tumor volume of both treatment groups over time (one replicate shown, P < 0.05). B) Growth pattern of mouse tumors over time with each line representing a specific mouse in the experiment (one replicate shown, P < 0.05).