Licochalcone A, a polyphenol present in licorice, suppresses UV-induced COX-2 expression by targeting PI3K, MEK1, and B-Raf

Abstract

Licorice is a traditional botanical medicine, and has historically been commonly prescribed in Asia to treat various diseases. Glycyrrhizin (Gc), a triterpene compound, is the most abundant phytochemical constituent of licorice. However, high intake or long-term consumption of Gc has been associated with a number of side effects, including hypertension. However, the presence of alternative bioactive compounds in licorice with anti-carcinogenic effects has long been suspected. Licochalcone A (LicoA) is a prominent member of the chalcone family and can be isolated from licorice root. To date, there have been no reported studies on the suppressive effect of LicoA against solar ultraviolet (sUV)-induced cyclooxygenase (COX)-2 expression and the potential molecular mechanisms involved. Here, we show that LicoA, a major chalcone compound of licorice, effectively inhibits sUV-induced COX-2 expression and prostaglandin E2 PGE2 generation through the inhibition of activator protein 1 AP-1 transcriptional activity, with an effect that is notably more potent than Gc. Western blotting analysis shows that LicoA suppresses sUV-induced phosphorylation of Akt/ mammalian target of rapamycin (mTOR) and extracellular signal-regulated kinases (ERK)1/2/p90 ribosomal protein S6 kinase (RSK) in HaCaT cells. Moreover, LicoA directly suppresses the activity of phosphoinositide 3-kinase (PI3K), mitogen-activated protein kinase kinase (MEK)1, and B-Raf, but not Raf-1 in cell-free assays, indicating that PI3K, MEK1, and B-Raf are direct molecular targets of LicoA. We also found that LicoA binds to PI3K and B-Raf in an ATP-competitive manner, although LicoA does not appear to compete with ATP for binding with MEK1. Collectively, these results provide insight into the biological action of LicoA, which may have potential for development as a skin cancer chemopreventive agent.

Figure 1

Treatment with licochalcone A (LicoA) but not Glycyrrhizin (Gc) inhibits solar ultraviolet (sUV)-induced prostaglandin E2 PGE₂ production by suppressing cyclooxygenase (COX)-2 expression in HaCaT cells. (A) Chemical structure of LicoA and Gc; (B) LicoA suppressed sUV-induced PGE₂ generation. HaCaT cells were treated with LicoA, Gc, and celecoxib at the indicated concentrations for 1 h before exposure to SUV, and harvested 24 h later. PGE₂ production was measured using a PGE₂ assay kit as described in the Experimental Section; (C) Treatment with LicoA or Gc did not suppress COX-2 activity. COX-2 activity was measured as described in the Experimental Section; (D) LicoA suppressed sUV-induced COX-2 expression in HaCaT cells. Cells were more potently inhibited by LicoA than Gc. Data is representative of 3 independent experiments that yielded similar results; (E) LicoA suppressed sUV-induced AP-1 transactivation in HaCaT cells. For the luciferase assay, HaCaT cells stably transfected with AP-1-luciferase reporter plasmids were cultured as described in the Materials and Methods. The cells were then starved in 0.1% fetal bovine serum (FBS)/minimal essential medium (MEM) in the presence or absence of LicoA or Gc at the indicated concentrations (5, 10 μM) for 1 h before exposure to sUV for 6 h. Luciferase activity was then assayed. activator protein 1 AP-1 activity is expressed relative to that of the control cells (without sUV irradiation). Data are presented as mean AP-1 luciferase activity ± SD calculated from three independent experiments; (F) The effect of LicoA was not attributable to any detectable effects on HaCaT cell viability. Cells were treated with 1.25, 2.5, 5, or 10 μM LicoA for 1 h before sUV radiation for 24 h. Cell viability was measured using (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay. Data are shown as mean ± S.D. and asterisks indicate significant inhibition by LicoA or Gc compared to the group treated with sUV alone (** p < 0.01).

Figure 2

Effects of LicoA on sUV-dependent phosphorylation of the Akt/ mammalian target of rapamycin (mTOR), mitogen-activated protein kinase kinase (MEK)1/ extracellular signal-regulated kinases (ERKs)/p90 ribosomal protein S6 kinase (RSK), c-Jun N-terminal kinases (JNK)/c-Jun, and p38/Elk pathways in HaCaT cells. Cells were treated with 1.25, 2.5, 5, or 10 μM LicoA for 1 h before sUV radiation, and harvested 30 min later. Phosphorylation levels as well as total mitogen-activated protein kinases (MAPKs) and Akt protein content were determined by Western blot analysis, as described in the Materials and Methods, using antibodies specific for the corresponding phosphorylated and total proteins.

Figure 2

Effects of LicoA on sUV-dependent phosphorylation of the Akt/ mammalian target of rapamycin (mTOR), mitogen-activated protein kinase kinase (MEK)1/ extracellular signal-regulated kinases (ERKs)/p90 ribosomal protein S6 kinase (RSK), c-Jun N-terminal kinases (JNK)/c-Jun, and p38/Elk pathways in HaCaT cells. Cells were treated with 1.25, 2.5, 5, or 10 μM LicoA for 1 h before sUV radiation, and harvested 30 min later. Phosphorylation levels as well as total mitogen-activated protein kinases (MAPKs) and Akt protein content were determined by Western blot analysis, as described in the Materials and Methods, using antibodies specific for the corresponding phosphorylated and total proteins.

Figure 3

Effect of LicoA on phosphoinositide 3-kinase PI3K, MEK1, B-Raf, and C-Raf kinase activity. (A) LicoA inhibits PI3K activity. Active PI3K (100 ng) was preincubated with LicoA or LY294002 at the indicated concentrations for 10 min at 30 °C, then incubated with phosphatidylinositol substrate and [γ-³²P]ATP for an additional 30 min at 30 °C. The resulting ³²P-labeled phosphatidylinositol-3-phosphate (PIP) was measured as described in the Experimental Section; (B–D) LicoA inhibits MEK1 (B), and B-Raf (C), but not C-Raf (D) kinase activity in vitro. The MEK1, B-Raf, and C-Raf in vitro kinase assays were performed as described in the Experimental Section, and kinase activity is expressed as percent inhibition relative to the activity of the untreated kinase control. The average ³²P count was determined from three separate experiments, and the data are presented as the mean values ± S.D. ** p < 0.01.

Figure 4

LicoA directly binds with PI3K, MEK1, and B-Raf. (A, C and E) LicoA specifically binds with PI3K (A), MEK1 (C), and B-Raf (E) in vitro. The PI3K (or MEK1, B-Raf)—LicoA binding was confirmed by immunoblotting using antibodies against PI3K(p110), MEK1 or B-Raf: Lane 1 (input control), PI3K, MEK1, or B-Raf protein standard; Lane 2 (control), Sepharose 4B was used to immunoprecipitate PI3K, MEK1, or B-Raf, as described in the Experimental Section; Lane 3, LicoA-Sepharose 4B affinity beads were used to immunoprecipitate PI3K, MEK1, or B-Raf. (B, D and F) LicoA competes with ATP to bind with PI3K and B-Raf but not MEK1. Active PI3K, MEK1, or B-Raf (200 ng) was incubated with ATP at different concentrations (0, 10, or 100 µM) and 100 µL of LicoA-Sepharose 4B or 100 µL of Sepharose 4B (negative control) in reaction buffer for a final volume of 500 µL. The mixtures were incubated at 4 °C overnight with shaking. After washing, the immunoprecipitated proteins were detected by Western blotting: Lane 1 (input control), PI3K, MEK1, or B-Raf protein standard; Lane 2 (negative control), PI3K, MEK1, or B-Raf bound with Sepharose 4B; Lane 3 (positive control), PI3K, MEK1, or B-Raf binding with LicoA—Sepharose 4B. Each experiment was performed a minimum of three times and representative blots are shown.

Figure 5

Hypothetical models of PI3K, B-RAF, and MEK1 in complex with LicoA. (A) and (B), Model structure of PI3K in complex with LicoA (A), and magnified view (B). The Ras-binding C2 domain, and the helical domain of PI3K are colored gray. LicoA (atomic structure) binds to the ATP binding site in the catalytic domain of PI3K and ATP (black) is overlaid for comparison; (C) and (D), Model structure of B-Raf in complex with LicoA (C) and magnified view (D). LicoA (atomic structure) binds to the ATP binding site of B-Raf and ATP (black) is overlaid for comparison. (E) and (F), Model structure of MEK1 (yellow) in complex with LicoA (E) and magnified view (F). LicoA (atomic structure) binds to the pocket adjacent to ATP’s (black) binding site. PD308088 (violet) has been overlaid on the model structure of MEK1-ATP-LicoA for comparison. In (A) and (B) the N-lobe, C-lobe, and hinge loop are colored violet, orange, and cyan, respectively. The residues involved in the interaction with LicoA are labeled and the hydrogen bonds are depicted as dotted lines.

Figure 6

Hypothetical model for the inhibitory mechanism of LicoA against sUV-induced COX-2 expression. Red line, ++, dotted cycle