Anti-Proliferative and Anti-Migratory Activity of Licorice Extract and Glycyrrhetinic Acid on Papillary Thyroid Cancer Cell Cultures

Abstract

Papillary thyroid cancer (PTC) is the 8th most common cancer among women overall. Licorice contains over 300 active compounds, many of them with anti-cancer properties. Glycyrrhetinic acid (GA) is a major component of licorice. The aim of this study was to investigate the potential anti-proliferative effects of licorice and GA on PTC cell cultures. Licorice extract (LE) was produced from the root and tested on BCPAP and K1 cell lines, as well as GA and aldosterone. We used the MTT test to investigate the anti-proliferative activity, the wound healing test for the migratory activity, and finally, we analyzed cell cycle distribution, apoptosis, and oxidative stress after LE, GA, or aldosterone incubation. Both LE and GA reduced cell viability at 48 h and cell migration at 24 h in both PTC cultures. Aldosterone reduced cell migration only in K1 cells. LE and GA induced cell cycle arrest in the G0/G1 phase in the BCPAP cell line, while LE and aldosterone induced it in the K1 culture. GA but not LE increased the apoptosis rate in both cell lines, whereas LE but not GA increased oxidative stress in both cultures. This study presents the first evidence of the in vitro anti-proliferative and anti-migratory activity of LE and GA on PTC.

Keywords: glycyrrhetinic acid; licorice; mineralocorticoid; papillary thyroid cancer; thyroid; thyroid cancer.

Figure 1

MTT assay for BCPAP and K1 cells treated for 48 h. (A) MTT assay in BCPAP cell line; (B) MTT assay in K1 cell line. Experiments were performed in triplicate and repeated three times. Aldo: Aldosterone; GA = Glycyrrhetinic acid; LE = Licorice extract; MTT assay: 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide assay.

Figure 2

Wound healing assay in papillary thyroid cancer cultures treated with licorice extract and glycyrrhetinic acid. (A) Representative photos of the migration of the BCPAP cell line at time zero and after 24 h; (B) BCPAP relative wound area. Control vs. LE after 24 h * = p < 0.0001; Control vs. GA after 24 h * = p < 0.0001 (C) Representative photos of the migration of the K1 cell line at time zero and after 24 h; (D) K1 relative wound area. Control vs. LE after 24 h * = p < 0.0001; Control vs. GA after 24 h * = p < 0.0001; Control vs. Aldo after 24 h * = p < 0.0001. GA = Glycyrrhetinic acid 0.01 mg/mL; LE = Licorice extract 0.13 mg/mL; Aldo = Aldosterone 1 µM.

Figure 3

Cell cycle distribution in papillary thyroid cancer cultures treated with licorice extract and glycyrrhetinic acid. (A) BCPAP Control; (B) BCPAP treated with licorice extract 0.13 mg/mL; (C) BCPAP treated with glycyrrhetinic acid 0.01 mg/mL; (D) BCPAP treated with Aldosterone 1 µM; (E) K1 Control; (F) K1 treated with licorice extract 0.13 mg/mL; (G) K1 treated with glycyrrhetinic acid 0.01 mg/mL; (H) K1 treated with Aldosterone 1 µM.

Figure 4

Apoptosis analysis in papillary thyroid cancer cultures treated with licorice extract and glycyrrhetinic acid. (A) Apoptosis analysis in BCPAP cell line; (B) Apoptosis analysis in K1 cell line. GA = glycyrrhetinic acid 0.01 mg/mL; LE = licorice extract 0.13 mg/mL; Aldo = Aldosterone 1 µM. Experiments were performed in triplicate. ** = p < 0.01; **** = p < 0.0001.

Figure 5

Oxidative stress levels in papillary thyroid cancer cultures treated with compounds. (A) Relative levels of ROS in BCPAP cell line; (B) Relative levels of ROS in K1 cell line. The cells were treated with the compounds for one hour. GA = glycyrrhetinic acid 0.01 mg/mL; LE = licorice extract 0.13 mg/mL; Aldo = Aldosterone 1 µM. Experiments were performed in triplicate. ** = p < 0.01; *** = p < 0.001.