Kaempferol inhibits UVB-induced COX-2 expression by suppressing Src kinase activity

Abstract

Ultraviolet (UV) radiation is the primary environmental risk factor in the development of nonmelanoma skin cancer, and UVB in particular promotes tumor growth through various signaling pathways. Kaempferol, a flavonoid with anti-inflammatory and anti-oxidative properties, has been studied as a chemopreventive agent; however, little is known regarding its effects on UVB-induced photo-carcinogenesis. Here, we examined the effect of kaempferol on UVB-induced skin inflammation. We found that kaempferol suppressed UVB-induced cyclooxygenase-2 (COX-2) protein expression in mouse skin epidermal JB6 P+ cells and attenuated the UVB-induced transcriptional activities of cox-2 and activator protein-1 (AP-1). Kaempferol attenuated the UVB-induced phosphorylation of several mitogen-activated protein kinases (MAPKs), including ERKs, p38, and JNKs, but had no effect on the phosphorylation of the upstream MAPK regulator Src. However, in vitro and ex vivo kinase assays demonstrated that kaempferol suppressed Src kinase activity. Furthermore, in vivo data from mouse skin support the idea that kaempferol suppresses UVB-induced COX-2 expression by blocking Src kinase activity. A pull-down assay revealed that kaempferol competes with ATP for direct binding to Src. Docking data suggest that kaempferol docks easily into the ATP-binding site of Src, which is located between the N and the C lobes of the kinase domain. Taken together, these results suggest that kaempferol is a potent chemopreventive agent against skin cancer through its inhibitory interaction with Src.

Fig. 1

Kaempferol attenuates UVB-induced COX-2 protein expression. (A) Chemical structure of kaempferol with its numbering scheme. (B) Kaempferol inhibits UVB-induced COX-2 protein expression in JB6 P+ cells. Cells were treated with various doses of kaempferol (0, 10, 20, or 40 μM) for 30 min, then stimulated with UVB (0.05 J/cm²) and harvested 4 h later. COX-2 and β-actin protein expression were determined by Western blotting using specific antibodies. Three separate experiments were performed with similar results and representative blots are shown. The asterisks (**) indicate a significant decrease in COX2 activity in groups treated with UVB and kaempferol compared with the group treated with UVB alone (p < 0.01).

Fig. 2

Kaempferol inhibits UVB-induced cox-2 and AP-1 promoter activity in JB6 P+ cells. (A and B) Kaempferol suppresses UVB-induced cox-2 and AP-1 promoter activity. For the luciferase assay, JB6 P+ cells stably transfected with a luciferase reporter plasmid bearing either cox-2 or AP-1 were cultured as described in Materials and Methods. Cells were then starved in 0.1% FBS-MEM and treated with various doses of kaempferol (0, 10, 20, or 40 μM) for 30 min. The cells were then subjected to UVB irradiation (0.05 J/cm²) and harvested 4 h later. Luciferase activity was assayed and cox-2 and AP-1 activities are expressed relative to control cells without UVB treatment. The values indicate means ± S.D. of cox-2- or AP-1-associated luciferase activity calculated from triplicate samples. The asterisk (**) indicates a significant decrease in luciferase activity in kaempferol treated cells compared to untreated control (p < 0.01).

Fig. 3

Kaempferol has differential effects on UVB-induced phosphorylation of Src, ERKs, p38, and JNKs in JB6 P+ cells. Cells were starved in 0.1% FBS-MEM and then treated with various doses of kaempferol (0, 10, 20, or 40 μM) for 30 min. The cells were then stimulated with UVB (0.05 J/cm²) and harvested 15 min later. Lysates were prepared and Western blotting was carried out to assess the phosphorylated and total protein levels of Src, ERKs, p38, and JNKs. Three separate experiments were performed with similar results and representative blots are shown. The asterisks (**) indicate a significant difference between groups treated with UVB and kaempferol and the group treated with UVB alone (p < 0.01).

Fig. 4

Kaempferol suppresses UVB-induced Src kinase and binding activities. (A) In vitro and (B) ex vivo Src kinase assays were performed as described in Materials and Methods. Active Src was incubated with kaempferol (A) or UVB-irradiated cells were treated with kaempferol (B) and Src kinase activity assessed. The mean ³²P count was determined from triplicate samples and the data are expressed as means ± S.D. The asterisks ** indicate a significant decrease in samples treated with kaempferol compared to untreated control samples (p < 0.01).

Fig. 5

Kaempferol inhibits UVB-induced COX-2 protein expression and Src kinase activity in mouse dorsal skin. (A) In a COX-2 expression assay, COX-2 and β-actin protein expression were analyzed by Western blotting using specific antibodies. Each band was quantified by densitometry. The data are expressed as means ± S.D. (n = 4). The asterisk (*) indicates a significant difference (p < 0.05) between the groups treated with kaempferol and UVB irradiation and the group exposed to UVB alone. (B) In the Src kinase activity assay, dorsal skin protein lysates were prepared from the epidermis and the assays were carried out as described in Materials and Methods. Each band was quantified by densitometry. The data are expressed as means ± S.D. (n = 3). The asterisks (**) indicate a significant difference at p < 0.01 between the groups treated with kaempferol and UVB irradiation and the group exposed to UVB alone.

Fig. 6

Kaempferol directly binds with Src. (A) Src-kaempferol binding was confirmed by immunoblotting with anti-Src: lane 1 (input control), Src protein standard; lane 2 (control), Sepharose 4B beads; lane 3, kaempferol-Sepharose 4B beads. (B) Kaempferol binds specifically to UVB-activated Src. Src-kaempferol binding in UVB-exposed JB6 P+ cells was confirmed by immunoblotting using anti-Src: lane 1 (input control), whole-cell lysates from JB6 P+ cells; lane 2 (control), lysates from JB6 P+ cells precipitated with Sepharose 4B beads; lane 3, whole-cell lysates from JB6 P+ cells precipitated with kaempferol-Sepharose 4B beads. (C) Kaempferol binds with Src in an ATP-competitive manner. Active Src (2 μg) was incubated with ATP (10 or 100 μM) and kaempferol-Sepharose 4B beads (50 μl) or Sepharose 4B beads (50 μl; as a negative control) in reaction buffer at a final volume of 500 μl. The mixtures were incubated at 4°C overnight with shaking. After washing, the pulled-down proteins were detected by Western blotting: lane 2, negative control, Src kinase cannot bind Sepharose 4B; lane 3, positive control, Src kinase binding with kaempferol-Sepharose 4B; lanes 4 and 5, increasing amounts of ATP decreased kaempferol binding with Src kinase. (D) Hypothetical computational models of Src in complex with kaempferol. In the close-up view, hydrogen bonds are depicted as white lines and hydrophobic contacts as white ellipses.