Quercetin-3-methyl ether suppresses proliferation of mouse epidermal JB6 P+ cells by targeting ERKs

Abstract

Chemoprevention has been acknowledged as an important and practical strategy for the management of skin cancer. Quercetin-3-methyl ether, a naturally occurring compound present in various plants, has potent anticancer-promoting activity. We identified this compound by in silico virtual screening of the Traditional Chinese Medicine Database using extracellular signal-regulated kinase 2 (ERK2) as the target protein. Here, we showed that quercetin-3-methyl ether inhibited proliferation of mouse skin epidermal JB6 P+ cells in a dose- and time-dependent manner by inducing cell cycle G(2)-M phase accumulation. It also suppressed 12-O-tetradecanoylphorbol-13-acetate-induced neoplastic cell transformation in a dose-dependent manner. Its inhibitory effect was greater than quercetin. The activation of activator protein-1 was dose-dependently suppressed by quercetin-3-methyl ether treatment. Western blot and kinase assay data revealed that quercetin-3-methyl ether inhibited ERKs kinase activity and attenuated phosphorylation of ERKs. Pull-down assays revealed that quercetin-3-methyl ether directly binds with ERKs. Furthermore, a loss-of-function ERK2 mutation inhibited the effectiveness of the quercetin-3-methyl ether. Overall, these results indicated that quercetin-3-methyl ether exerts potent chemopreventive activity by targeting ERKs.

Fig. 1.

Quercetin-3-methyl ether at 20 μM is cytotoxic to JB6 P+ cells. (A) Chemical structure of quercetin-3-methyl ether. (B) Cells were treated with quercetin-3-methyl ether (0–20 μM) or its vehicle, dimethyl sulfoxide, as a negative control in 5% FBS/EMEM for 24 or 48 h. Cell viability was determined by (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. Data are represented as means ± SE.

Fig. 2.

Quercetin-3-methyl ether inhibits cell growth by inducing G₂–M accumulation and suppresses transformation of JB6 P+ cells. (A) Quercetin-3-methyl ether inhibits cell growth. Cells were starved in serum-free medium for 24 h and then treated with quercetin-3-methyl ether (0–10 μM) or its vehicle dimethyl sulfoxide (control) in 5% FBS/EMEM for 24 or 48 h. At the end of each treatment time, cells were collected and processed for determination of total cell number. Data are shown as means ± SE. The asterisk (*) indicates a significant difference (P < 0.05) between groups treated with quercetin-3-methyl ether and the group treated with dimethyl sulfoxide. (B) Quercetin-3-methyl ether induces G₂/M accumulation and suppresses cyclin B1 protein expression in JB6 P+ cells. Cells were starved in serum-free medium for 24 h and then treated with quercetin-3-methyl ether (0–10 μM) or dimethyl sulfoxide (control) for 48 h. Cell cycle analysis was performed by flow cytometry. Data are shown as means ± SE. The asterisk (*) indicates a significant difference (P < 0.05) between groups treated with quercetin-3-methyl ether and the group treated with dimethyl sulfoxide. (C) For western blot analysis, cells were treated with quercetin-3-methyl ether at the indicated concentrations for 48 h. In vitro Cdk1/cyclin B kinase assay was performed as described in Materials and methods. Coomassie blue staining (lower) shows the histone H1 protein as a loading control. Lane 1, control, which indicates that active Cdk1/cyclin B phosphorylates the histone H1 protein; lanes 2 and 3, increasing amounts of quercetin-3-methyl ether suppress Cdk1/cyclin B kinase activity. (D) Quercetin-3-methyl ether inhibits TPA-induced transformation of JB6 P+ cells. Cells were treated as described under Materials and methods and colonies were counted under a microscope with the aid of Image-Pro Plus software (v.4). Data are shown as means ± SE. The asterisks (**) indicate a significant difference (P < 0.001) between groups treated with TPA and quercetin-3-methyl ether compared with the group treated with TPA alone.

Fig. 3.

Quercetin-3-methyl ether is more potent than quercetin in suppressing growth and transformation of JB6 P+ cells. (A) Quercetin-3-methyl ether inhibits cell growth more strongly compared with quercetin. Cells were treated with 10 μM quercetin-3-methyl ether or quercetin in 5% FBS/EMEM and cell growth was measured by (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay at the indicated times. (B) Compared with quercetin, quercetin-3-methyl ether more strongly inhibits TPA-induced transformation of JB6 P+ cells. Cells were treated as described in Materials and methods and colonies were counted 14 days later under a microscope with the aid of Image-Pro Plus software (v.4). Data are shown as means ± SE. The asterisk (**) indicates a significant difference (P < 0.001) between groups treated with TPA and quercetin-3-methyl ether compared with the group treated with TPA and quercetin.

Fig. 4.

Quercetin-3-methyl ether blocks UVB/TPA-induced AP-1 transactivation in JB6 cells by attenuating ERKs signaling. (A) For the luciferase assay, JB6 cells stably transfected with an AP-1 luciferase reporter plasmid were cultured in 5% FBS/EMEM. Cells were starved in serum-free medium for 24 h and then treated with quercetin-3-methyl ether (0–10 μM) or its vehicle dimethyl sulfoxide (control) in 5% FBS/EMEM for 2 h. Cells were then exposed to 4 kJ/m² UVB (upper panel) or 20 ng/ml TPA (lower panel) and harvested at 3 or 12 h, respectively. Luciferase activity was assessed and AP-1 activity is expressed relative to control cells without UVB or TPA treatment. Data are shown as means ± SE. The asterisk (*) indicates a significant difference (*P < 0.05; **P < 0.001) between groups treated with UVB/TPA and quercetin-3-methyl ether compared with the group treated with UVB/TPA alone. (B) Quercetin-3-methyl ether inhibits UVB/TPA-induced phosphorylation of ERKs but not JNKs or p38. Cells were treated with quercetin-3-methyl ether at the indicated concentrations (0–10 μM) for 2 or 6 h and then exposed to 4 kJ/0.5 m² UVB or 20 ng/ml TPA and harvested 30 min later. The levels of phosphorylated and total ERKs, mitogen-and stress activated protein kinase. JNKs and p38 proteins were determined by western blot analysis.

Fig. 5.

Quercetin-3-methyl ether inhibits ERK1 or ERK2 kinase activity and directly binds with ERK1 or ERK2. (A) Quercetin-3-methyl ether inhibits ERKs kinase activity. An in vitro ERK1 or ERK2 kinase assay was performed as described in Materials and methods. A glutathione S-transferase–ribosomal S6 kinase 2 fusion protein was used in the in vitro kinase assay with active ERK1 or ERK2 and results were visualized by autoradiography. Coomassie blue staining shows the glutathione S-transferase fusion protein as a loading control. Left: lane 1, ERK2 control; lane 2, ribosomal S6 kinase 2 substrate control; lane 3, positive control, which indicates that active ERK2 phosphorylates the glutathione S-transferase–ribosomal S6 kinase 2 fusion protein; lanes 4, 5, 6 and 7, increasing amounts of quercetin-3-methyl ether suppresses ERK2 kinase activity; lane 8, negative control, which indicates that CAY10561 (commercial ERK2 inhibitor) suppresses ERK2 kinase activity. Right: lane 1, ERK1 control; lane 2, substrate control; lane 3, positive control, which indicates that active ERK1 phosphorylates the glutathione S-transferase–ribosomal S6 kinase 2 fusion protein; lanes 4, 5 and 6, increasing amounts of quercetin-3-methyl ether suppressed ERK1 kinase activity. (B) Quercetin-3-methyl ether directly binds with ERK1 and ERK2. The ERK1- or ERK2-quercetin-3-methyl ether binding was confirmed by immunoblotting using an antibody against ERKs. Lane 1 (input control), ERK1 (left) or ERK2 (right) protein standard; lane 2 (control), Sepharose 4B was used to pull down ERK1 or ERK2 as described in Materials and methods and lane 3, ERK1 or ERK2 was pulled down using quercetin-3-methyl ether conjugated-Sepharose 4B beads as described in Materials and methods.

Fig. 6.

Quercetin-3-methyl ether inhibits cell proliferation by targeting ERKs. JB6 cells were transiently transfected with pCEP4-DN-ERK2-K52. Twenty-four hours after transfection, cells were treated with 10 μM quercetin-3-methyl ether or dimethyl sulfoxide vehicle and evaluated for cell growth at 48 h after drug treatment.