Discovery of (-)-epigallocatechin gallate, a novel olfactory receptor 2AT4 agonist that regulates proliferation and apoptosis in leukemia cells

Abstract

Olfactory receptors (ORs), the largest family of G protein-coupled receptors (GPCRs), are ectopically expressed in cancer cells and are involved in cellular physiological processes, but their function as anticancer targets is still potential. OR2AT4 is expressed in leukemia cells, influencing the proliferation and apoptosis, yet the limited number of known OR2AT4 agonists makes it challenging to fully generalize the receptor's function. In this study, we aimed to identify new ligands for OR2AT4 and to investigate their functions and mechanisms in K562 leukemia cells. After producing the recombinant OR2AT4 protein, immobilizing it on a surface plasmon resonance chip, and conducting screening to confirm binding activity using 258 chemicals, five novel OR2AT4 ligands were discovered. As a result of examining changes in intracellular calcium by five ligands in OR2AT4-expressing cells and K562 cells, (-)-epigallocatechin gallate (EGCG) was identified as an OR2AT4 agonist in both cells. EGCG reduced the viability of K562 cells and induced apoptosis in K562 cells. EGCG increased the expression of cleaved caspase 3/8 and had no effect on the expression of Bax and Bcl-2, indicating that it induced apoptosis through the extrinsic pathway. Additionally, the initiation of the extrinsic apoptosis pathway in EGCG-induced K562 cells was due to the activation of OR2AT4, using an OR2AT4 antagonist. This study highlights the potential of EGCG as an anti-cancer agent against leukemia and OR2AT4 as a target, making it a new anti-cancer drug.

Keywords: (−)-Epigallocatechin gallate; Apoptosis; Myelogenous leukemia; OR2AT4; Olfactory receptor; Surface plasmon resonance.

Fig. 1

Optimization of expression and purification of OR2AT4-6xHis. (A) Schematic representation of pET-DEST42-Rho-OR2AT4-6xHis plasmid. (B) Expression of OR2AT4-6xHis induced with 1 mM IPTG by Coomassie staining of SDS-PAGE gel. (C) Separation of soluble (supernatant) and insoluble (pellet) fractions of OR2AT4-6xHis protein. (D) Comparison of OR2AT4-6xHis protein purification using cobalt and Ni-NTA resin. Detection of OR2AT4 protein by Coomassie staining (upper panel) and western blotting (lower panel) with anti-His. M, protein marker; (−), without IPTG; (+), induced protein; S, soluble fraction; IS, insoluble fraction; E, elution fraction.

Fig. 2

Discovery of novel OR2AT4 ligands using SPR and calcium imaging. (A) Sensorgram of OR2AT4 agonist, sandalore (100, 200, and 500 μM). (B–E) Sensorgram of berbamine (B), celastrol (C), EGCG (D), and rosmarinic acid (E). (F) Fluorescence image of intracellular calcium response of sandalore (200 μM), and dose-response curve of 0–1 mM sandalore in OR2AT4-expressing cells. (G) Fluorescence image of calcium response of celastrol and rosmarinic acid in OR2AT4-expressing cells. (H,I) Intracellular calcium response of concentration at 200 μM of berbamine (H) and EGCG (I). (J,K) Dose-response curve of calcium response of berbamine (J) and EGCG (K) in OR2AT4-expressing cells. EGCG, (−)-Epigallocatechin gallate.

Fig. 3

Effect of OR2AT4 agonists on K562 cells. (A) mRNA expression of OR2AT4 in K562 cells (143 bp). (B) Protein expression of OR2AT4 in membrane protein of K562 cells. (C) Intracellular calcium response of OR2AT4 agonist sandalore in K562 cells. (D) Intracellular calcium response of berbamine and EGCG in K562 cells. (E) Intracellular calcium levels following co-treatment with EGCG (200 μM) and OR2AT4 antagonist phenirat (200 μM). Mock, no RNA; Cyto, cytosolic protein; Mem, membrane protein.

Fig. 4

Effect of EGCG on cell proliferation and apoptosis in K562 cells (A) Cell viability of EGCG at different concentrations for 24, 48 (IC₅₀ = 146.5 μM), and 72 (IC₅₀ = 157.7 μM) hours. (B) Flow cytometric analysis of cell apoptosis with Annexin V & Dead cell assay in the absence or presence of EGCG (200 μM) for 24, 48, and 72 h. (C) Quantification of total apoptotic cells (%) in time-dependent. (D) Protein expression of p-Akt, Akt, p-p38 MAPK, p-38 MAPK, p-STAT5, and STAT5 following treatment with EGCG (200 μM). *p < 0.05, **p < 0.01, ***p < 0.001 when compared to 0 min. (E) Protein expression of apoptosis-related markers, cleaved caspase-3, caspase-3, Bax, Bcl-2, cleaved caspase-8 and capase-8 following treatment with EGCG (200 μM) for 24 and 48 h *p < 0.05, **p < 0.01 when compared to 0 h. (F) Protein expression of apoptosis-related markers following treated with EGCG dose-dependent for 48 h **p < 0.01, ***p < 0.001 when compared to 0 μM. Data are expressed as the mean ± SD (n ≥ 3). C-Cas3, cleaved caspase-3; Cas-3, caspase-3; C-Cas8, cleaved caspase-8; Cas-8, caspase-8.

Fig. 5

EGCG efficacy mediated by OR2AT4 in K562 cells. (A, B) Cell viability of EGCG (200 μM) co-treated with OR2AT4 antagonist, phenirat (200 μM) for 48 h. (B) Protein expression of cleaved caspase-3 and caspase-3 after simultaneous treatment with phenirat (200 μM) and EGCG (200 μM) for 48 h. (C) Silencing of OR2AT4 by siRNA in K562 cells. (D) Effect of OR2AT4 knockdown on the apoptosis of K562 cells. *p < 0.05, **p < 0.01, ***p < 0.001 when compared to control.

Fig. 6

Elimination of EGCG effect by Akt and p38 AMPK inhibitors in K562 cells. (A,B) Protein expression of p-Akt, Akt, cleaved caspase-3, and caspase-3 after treatment with perifosine, Akt inhibitor. (C,D) Protein expression of p-p38, p38 MAPK, cleaved caspase-3, and caspase-3 after treatment with SB203580, p38 MAPK inhibitor. *p < 0.05, **p < 0.01, ***p < 0.001 when compared to control. Data are expressed as the mean ± SD (n ≥ 3).

Fig. 7

Effect of EGCG on Molt-4 cells. (A) mRNA expression of OR2AT4 in Molt-4 cells. (B) Intracellular calcium response of sandalore (2 mM) and EGCG (200 μM) in Molt-4 cells. (C) Cell viability of EGCG at different concentrations for 24, 48, and 72 h. Data are expressed as the mean ± SD (n ≥ 3).