Discovery of Orphan Olfactory Receptor 6M1 as a New Anticancer Target in MCF-7 Cells by a Combination of Surface Plasmon Resonance-Based and Cell-Based Systems

Abstract

Olfactory receptors (ORs) account for 49% of all G protein-coupled receptors (GPCRs), which are important targets for drug discovery, and hence ORs may also be potential drug targets. Various ORs are expressed in breast cancer cells; however, most of them are orphan receptors, and thus, their functions are unknown. Herein, we present an experimental strategy using a surface plasmon resonance (SPR) system and a cell-based assay that allowed the identification of orphan OR6M1 as a new anticancer target in the MCF-7 breast cancer cell line. After the construction of stable OR6M1-expressing cells, the SPR-based screening of 108 chemicals for ligand activity was performed against OR6M1-expressing whole cells (primary screening) or membrane fragments (secondary screening). As a result, anthraquinone (AQ) and rutin were discovered to be new OR6M1 ligands. Based on calcium imaging in OR6M1-expressing Hana3A cells, AQ and rutin were classified as an OR6M1 agonist and antagonist, respectively. Cell viability and live/dead assays showed that AQ induced the death of MCF-7 cells, which was inhibited by rutin. Therefore, OR6M1 may be considered an anticancer target, and AQ may be considered a chemotherapeutic agent. This combined method can be widely used to discover the ligands and functions of other orphan GPCRs.

Keywords: MCF-7 breast cancer cell line; OR6M1; anthraquinone; olfactory receptor; rutin; surface plasmon resonance.

Figure 1

Schematic illustration of a surface plasmon resonance (SPR) biosensor with immobilized stable cells or membrane fragments expressing the olfactory receptor 6M1 (OR6M1). EDC, 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; NHS, N-hydroxysuccinimide; CMD, carboxymethyl dextran; FC, flow channel; LED, light emitting diode; and 2D-CMOS, two dimensional-complementary metal oxide semiconductor.

Figure 2

Characterization of OR6M1 expression. (A) Detection of OR6M1 expression in MCF-7 cells by reverse transcription–polymerase chain reaction (left) and western blotting (right). (B) Plasmid construct containing the receptor-transporting protein 1 (RTP1) gene, glutathione S-transferase (GST)-tag, and human OR6M1 gene. (C) Immunocytochemical detection of OR6M1 in the constructed OR6M1 cell line. GST-tagged OR6M1, green; nuclei, blue. Live cells, green; dead cells, red; (D) OR6M1 expression in cells and membrane fragments.

Figure 3

Identification of anthraquinone (AQ) as a putative OR6M1 ligand using a cell-immobilized SPR biosensor. (A) Scanning electron microscopy (SEM) images and size distributions of cells immobilized on a CMD50M sensor chip. (B) Cell-based screening of 108 chemicals for ligand activity. (C,D) Sensorgrams of AQ at three different concentrations (8, 12, and 25 μM) using CMD50M-chip-immobilized OR6M1 cells (C) or HEK293T/17 cells (D). The mean signal values between 900 and 1100 s were used as the detected signal value.

Figure 4

Identification of AQ and rutin as OR6M1 ligands using membrane fragment-immobilized SPR biosensor. (A) SEM images and size distributions of membrane fragments immobilized on a CMD50M sensor chip. (B) Membrane-fragment-based screening of 108 chemicals for ligand activity. (C,D) Sensorgrams of AQ at three different concentrations (6.25, 12.5, and 25 μM) using CMD50M-chip-immobilized OR6M1-expressing cell membranes (C) or HEK293T/17 cell membranes (D). (E,F) Sensorgrams of rutin at three different concentrations (12.5, 25, and 50 μM) using CMD50M-chip-immobilized OR6M1-expressing cell membranes (E) or HEK293T/17 cell membranes (F).

Figure 5

Modulation of [Ca²⁺]_i by AQ and rutin in OR6M1-transfected Hana3A cells. (A) AQ treatment; (B) rutin treatment, and (C) co-treatment with AQ and rutin. Time response recording of the Fura-2AM fluorescence ratios (340/380 nm) (upper panel) and fluorescent images (lower panel).

Figure 6

Effects of AQ and rutin on the breast cancer cell line, MCF-7. (A–E) Time response recording of [Ca²⁺]_i in Fura-2AM-loaded MCF-7 cells after treatment with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), AQ (100 and 200 μM), rutin (100 μM), and AQ + rutin. (F) Concentration-response curves of the MCF-7 cell viability (%) after treatment with AQ or rutin for 48 h. (G) Live/dead cell imaging of MCF-7 cells treated with AQ (25–100 μM) for 48 h. Live cells, green; dead cells, red. (H) Concentration-response curves of the MCF-7 cell viability (%) after co-treatment with different concentrations of AQ and 100 μM rutin for 48 h. (I) Live/dead cell imaging of MCF-7 cells treated with AQ + rutin for 48 h. The data are expressed as the mean ± standard deviation. * p < 0.05 and *** p < 0.001 untreated vs. ligand-treated cells (one-way analysis of variance and Dunnett’s post-test).