Caffeic acid, a phenolic phytochemical in coffee, directly inhibits Fyn kinase activity and UVB-induced COX-2 expression

Abstract

Caffeic acid (3,4-dihydroxycinnamic acid) is a well-known phenolic phytochemical present in many foods, including coffee. Recent studies suggested that caffeic acid exerts anticarcinogenic effects, but little is known about the underlying molecular mechanisms and specific target proteins. In this study, we found that Fyn, one of the members of the non-receptor protein tyrosine kinase family, was required for ultraviolet (UV) B-induced cyclooxygenase-2 (COX-2) expression, and caffeic acid suppressed UVB-induced skin carcinogenesis by directly inhibiting Fyn kinase activity. Caffeic acid more effectively suppressed UVB-induced COX-2 expression and subsequent prostaglandin E(2) production in JB6 P+ mouse skin epidermal (JB6 P+) cells compared with chlorogenic acid (5-O-caffeoylquinic acid), an ester of caffeic acid with quinic acid. Data also revealed that caffeic acid more effectively induced the downregulation of COX-2 expression at the transcriptional level mediated through the inhibition of activator protein-1 (AP-1) and nuclear factor-kappaB transcription activity compared with chlorogenic acid. Fyn kinase activity was suppressed more effectively by caffeic acid than by chlorogenic acid, and downstream mitogen-activated protein kinases (MAPKs) were subsequently blocked. Pharmacological Fyn kinase inhibitor (3-(4-chlorophenyl)1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine and leflunomide) data also revealed that Fyn is involved in UVB-induced COX-2 expression mediated through the phosphorylation of MAPKs in JB6 P+ cells. Pull-down assays revealed that caffeic acid directly bound with Fyn and non-competitively with adenosine triphosphate. In vivo data from mouse skin also supported the idea that caffeic acid suppressed UVB-induced COX-2 expression by blocking Fyn kinase activity. These results suggested that this compound could act as a potent chemopreventive agent against skin cancer.

Fig. 1.

Effect of caffeic acid or chlorogenic acid on UVB-induced COX-2 expression and PGE₂ production in JB6 P+ cells. (A) Chemical structures of caffeic acid (left) and chlorogenic acid (right). (B and C) UVB-induced COX-2 expression in JB6 P+ cells is inhibited more strongly by caffeic acid than by chlorogenic acid. JB6 P+ cells were treated with caffeic acid or chlorogenic acid at the indicated concentrations (0, 10, 30, 50, 75 or 100 μM) for 1 h before being exposed to 0.5 kJ/m² UVB and harvested 4 h later. The cells were disrupted and COX-2 protein level was determined by western blot analysis as described in Materials and Methods. β-Actin was detected to verify equal loading of proteins. Data are representative of two independent experiments that gave similar results. (D) UVB-induced PGE₂ production in JB6 P+ cells is inhibited more strongly by caffeic acid than by chlorogenic acid. JB6 P+ cells were treated with caffeic acid or chlorogenic acid at the indicated concentration (0, 10, 50 or 100 μM) for 1 h before being exposed to 0.5 kJ/m² UVB and harvested 18 h later. PGE₂ production was measured using a PGE₂ assay kit as described in Materials and Methods. Asterisks indicate significant inhibition of PGE₂ production by caffeic acid or chlorogenic acid compared with the group treated with UVB alone (*P < 0.05 and **P < 0.01).

Fig. 2.

Effect of caffeic acid or chlorogenic acid on UVB-induced COX-2 promoter activity, and AP-1 or NF-κB transactivation in JB6 P+ cells. Caffeic acid is more effective than chlorogenic acid at suppressing UVB-induced COX-2 promoter activity (A), AP-1 (B) or NF-κB (C) transactivation. JB6 P+ cells, which were stably transfected with COX-2, AP-1 or NF-κB luciferase reporter plasmids, were treated with caffeic acid or chlorogenic acid at the indicated concentration (0, 10, 25, 50 or 100 μM) for 1 h before being exposed to 0.5 kJ/m² UVB and harvested 24 h later (i.e. when determining COX-2 activity) or 12 h later (i.e. when determining AP-1 or NF-κB activity). Relative activities were determined using a luciferase assay as described in Materials and Methods. Data are presented as means ± SDs values of the COX-2, AP-1 or NF-κB luciferase activity from three independent experiments. Asterisks indicate significant inhibition of luciferase activity by caffeic acid or chlorogenic acid compared with the group treated with UVB alone (*P < 0.05, **P < 0.01 and ***P < 0.001).

Fig. 3.

Effect of caffeic acid or chlorogenic acid on UVB-induced phosphorylation of MAPKs and on the activation of Fyn. (A) UVB-induced phosphorylation of JNK, p38 MAPK or ERK is inhibited more strongly by caffeic acid than by chlorogenic acid at the same relative concentration. JB6 P+ cells were treated with caffeic acid or chlorogenic acid at the indicated concentration (0, 10, 50 or 100 μM) for 1 h before being exposed to 0.5 kJ/m² UVB and harvested 15 min later. The cells were disrupted and the levels of phosphorylated and total JNK, p38 and ERK proteins were measured by western blot as described in Materials and Methods using specific antibodies against each protein. Data are representative of two independent experiments that gave similar results. (B) Caffeic acid is more effective than chlorogenic acid at directly suppressing Fyn kinase activity. A direct Fyn kinase assay was performed as described in Materials and Methods, and the effect of caffeic acid or chlorogenic acid is expressed as the percent inhibition relative to the activity of the Fyn protein alone. (C) Caffeic acid is more effective than chlorogenic acid at suppressing UVB-induced Fyn kinase activity. JB6 P+ cells were treated with caffeic acid or chlorogenic acid at the indicated concentration (0, 10, 50 or 100 μM) for 1 h before being exposed to 0.5 kJ/m² UVB and harvested 15 min later. The cells were disrupted, and Fyn kinase activity was measured as described in Materials and Methods. The effect of caffeic acid or chlorogenic acid is expressed as the percent inhibition relative to the group treated with UVB alone. Data in (B) and (C) are shown as means ± SDs values as determined from three independent experiments. Asterisks indicate significant inhibition of Fyn kinase activity by caffeic acid or chlorogenic acid compared with the active Fyn only group (A) or the group treated with UVB (B) only (**P < 0.01 and ***P < 0.001).

Fig. 4.

Involvement of Fyn as an upstream kinase of MAPKs in mediating UVB-induced COX-2 upregulation. (A) PP2 or leflunomide inhibits UVB-induced COX-2 expression in JB6 P+ cells. JB6 P+ cells were treated with PP2 (0, 5 or 10 μM) or leflunomide (0, 50 or 100 μM) for 1 h before being exposed to 0.5 kJ/m² UVB and harvested 4 h later. The cells were disrupted and the level of the COX-2 protein was determined by western blot analysis as described in Materials and Methods. (B) PP2 or leflunomide suppresses UVB-induced COX-2 promoter activity in JB6 P+ cells. JB6 P+ cells stably transfected with a COX-2 luciferase reporter plasmid were treated with PP2 (0, 5 or 10 μM) or leflunomide (0, 50 or 100 μM) for 1 h before being exposed to 0.5 kJ/m² UVB and harvested 24 h later. Relative activity was measured using a luciferase assay as described in Materials and Methods. Data were presented as means ± SDs values of the COX-2 luciferase activity from three independent experiments. Asterisks indicate significant inhibition of luciferase activity by PP2 or leflunomide compared with the group treated with UVB only (**P < 0.01 and ***P < 0.001). (C) PP2 or leflunomide suppresses UVB-induced phosphorylation of JNK, p38 and ERK in JB6 P+ cells. JB6 P+ cells were treated with PP2 (0, 5 or 10 μM) or leflunomide (0, 50 or 100 μM) for 1 h before being exposed to 0.5 kJ/m² UVB and harvested 15 min later. The cells were lysed and then the levels of phosphorylated and total JNK, p38 and ERK proteins were determined by western blot as described in Materials and Methods using specific antibodies against each protein. (D) U0126, SB203580 or SP600125 inhibits UVB-induced COX-2 expression in JB6 P+ cells. JB6 P+ cells were treated with U0126, SB203580 or SP600125 at the indicated concentrations (0, 10 or 20 μM) for 1 h before being exposed to 0.5 kJ/m² UVB and harvested 4 h later. The cells were disrupted, and the level of the COX-2 protein was determined by western blot analysis as described in Materials and Methods. Data in (A), (C) and (D) are representative of two independent experiments that gave similar results.

Fig. 5.

Direct binding of caffeic acid with Fyn. (A) Caffeic acid, but not chlorogenic acid, binds directly with the Fyn protein. The Fyn–caffeic acid and Fyn–chlorogenic acid binding were confirmed by immunoblotting using an antibody against Fyn: lane 1 (input control)—Fyn protein standard; lane 2 (control)—Sepharose 4B was used to pull down Fyn as described in Materials and Methods and lanes 3 and 4—Fyn was pulled down using caffeic acid–Sepharose 4B or chlorogenic acid–Sepharose 4B beads as described in Materials and Methods, respectively. (B) Caffeic acid, but not chlorogenic acid, specifically binds with the UVB-activated Fyn protein. The Fyn–caffeic acid or Fyn–chlorogenic acid binding in UVB-exposed JB6 P+ cells was confirmed by immunoblotting using an antibody against Fyn: lane 1 (input control)—whole-cell lysates from JB6 P+ cells; lane 2 (control)—lysates of JB6 P+ cells were precipitated with Sepharose 4B beads as described in Materials and Methods and lanes 3 and 4—whole-cell lysates from JB6 P+ cells were precipitated by caffeic acid–Sepharose 4B or chlorogenic acid–Sepharose 4B beads as described in Materials and Methods, respectively. (C) Caffeic acid binds to Fyn non-competitively with ATP. Active Fyn (0.2 μg) was incubated with ATP at the indicated concentration (0, 10 or 100 μM) and 100 μl of caffeic acid–Sepharose 4B or 100 μl of Sepharose 4B (as a negative control) in a reaction buffer to a final volume of 500 μl. The pulled-down proteins were detected by western blot analysis as described in Materials and Methods: lane 1 (input control)—Fyn protein standard; lane 2 is the negative control, which indicates that Fyn does not bind with Sepharose 4B and lane 3 is the positive control, which indicates that Fyn binds with caffeic acid–Sepharose 4B. Data presented in (A), (B) and (C) are representative of three independent experiments that gave similar results. (D) Hypothetical model of the Fyn kinase domain in complex with caffeic acid or chlorogenic acid. Putative caffeic acid-binding sites in the homology model structure of Fyn. Different parts of Fyn are presented in different colors: the N-terminal domain in red, SH3 in cyan, SH2 in violet and the kinase domain in yellow. Caffeic acid (atomic color) binds to both the ATP-binding site in the kinase domain of Fyn and the putative allosteric site between the SH2 domain and the kinase domain. In the close-up view of the inhibitor interaction in the allosteric site, the hydrogen bonds and salt bridge are depicted as dashed lines and the hydrophobic contacts by small curves.

Fig. 6.

Effects of caffeic acid on UVB-induced COX-2 expression and Fyn kinase activity in mouse dorsal skin. (A) Caffeic acid inhibits UVB-induced COX-2 expression in mouse skin extracts. The levels of COX-2 and β-actin were determined by western blot analysis using specific antibodies against the corresponding COX-2 and β-actin proteins. Each band was quantified by densitometry. Results are shown as means ± SD (n = 5). The pound symbols (##) indicate a significant difference (P < 0.01) between the control group and the group exposed to UVB only, and the asterisks (**) indicate a significant difference at P < 0.01 between groups treated with caffeic acid and irradiated with UVB and the group exposed to UVB alone. (B) Caffeic acid inhibits UVB-induced Fyn kinase activity in mouse skin extracts. For the Fyn 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. Results are shown as means ± standard errors (n = 5). The pound sign (#) indicates a significant difference (P < 0.05) between the control group and the group exposed to UVB (5 kJ/m²) only; the asterisk (*) indicates a significant difference (P < 0.05) between groups treated with UVB and caffeic acid and the group exposed to UVB alone. (C) Caffeic acid specifically binds with Fyn in mouse skin extracts. The Fyn–caffeic acid binding in vivo was confirmed by immunoblotting using an antibody against Fyn; lane 1 (input control)—mouse dorsal skin lysate; lane 2 (control)—a lysate of mouse dorsal skin precipitated with Sepharose 4B beads as described in Materials and Methods; lane 3—mouse dorsal skin lysate precipitated by caffeic acid–Sepharose 4B affinity beads. Each experiment was performed three times.