For decades skin malignancy incidence has increased mainly due to oncogenic signaling pathways activated by solar ultraviolet (UV) irradiation (i. ethyl caffeate] isolated from var. (Wehrhahn) Grierson. HOEC strongly inhibited neoplastic transformation of JB6 C14l cells without toxicity. PI3-K ERK1/2 and p38 kinase activities were suppressed by direct binding with HOEC Our docking data showed that HOEC binds at the ATP-binding site of each kinase. The inhibition of solar UV-induced PI3-K ERK1/2 and p38 kinase activities resulted in suppression of their downstream signaling pathways and AP-1 and NF-κB transactivation in JB6 cells. SB-408124 Furthermore topical application of HOEC reduced skin cancer incidence and tumor volume in SKH-1 hairless mice chronically exposed to solar UV. In summary our results show that HOEC exerts inhibitory effects on multiple kinase targets and their downstream pathways activated by solar UV and var. (Wehrhahn) Grierson. Although the plants of the genus are widely produced for ornamental purposes especially in China spices recently received attention for its anti-nociceptive effects (25). In particular HOEC isolated from model of solar UV-induced skin carcinogenesis HOEC significantly reduced tumor volume and tumor number by inhibiting the PI3-K ERK1/2 and p38 signaling pathways. Materials and Methods Reagents HOEC (2-(1-hydroxyl-4-oxocyclohexyl)ethyl caffeate) is usually SB-408124 SB-408124 a natural compound isolated from the whole plants of and the compound used in this study was synthesized according to previous reports (27). The purity of HOEC was assessed by HPLC and found to be greater than 97%. Active p110 (PI3-K) active ERK1 active SB-408124 ERK2 active p38α inactive RSK2 and inactive ATF2 recombinant proteins for kinase assays were purchased from Millipore (Temecula CA). Antibodies to detect phosphorylated tyrosines (p-Tyr i.e. p-p110) phosphorylated Akt (p-Akt S473) phosphorylated ERK1/2 SB-408124 (p-ERK1/2 Thr202/Tyr204) phosphorylated p38 (p-p38 Thr180/Tyr182) phosphorylated RSK2 (p-RSK2 Ser380) phosphorylated Rabbit Polyclonal to Vimentin. MSK1 (p-MSK1 Ser376) phosphorylated ATF2 (p-ATF2 Thr69/71) phosphorylated S6 ribosomal protein (p-S6 ribosomal protein Ser235/236) phosphorylated c-Fos (p-c-Fos Ser32) phosphorylated c-Jun (p-c-Jun Ser63) total p110 total ERKs total RSK total Akt total ATF2 total MSK total S6 ribosomal protein total c-Fos and total c-Jun were purchased from Cell Signaling Technology (Beverly MA). The antibody against β-actin was from Santa Cruz Biotechnology (Santa Cruz CA). CNBr-sepharose 4B beads were obtained from Amersham Pharmacia Biotech (Piscataway NJ). The luciferase assay substrate and the Cell Titer 96 Aqueous One Answer Reagent [3-(4 5 inner salt (MTS)] kit for the cell proliferation assay were from Promega (Madison WI). Cell culture The JB6 Cl41 murine epidermal cell collection (a promotion-sensitive clone of the JB6 P+ cell collection) was cultured in Eagle’s Minimum Essential Medium (MEM) and HaCaT human keratinocytes were cultured in Dulbecco’s Modified Eagle Medium (DMEM)/high glucose made up of penicillin (100 models/mL) streptomycin (100 μg/mL) and 4% or 10% fetal bovine serum (FBS; Gemini Bio-Products Calabasas CA) respectively. Cells were maintained in a 5% CO2 37 humidified incubator. Cells were cytogenetically tested and authenticated before being frozen. Each vial of frozen cells was thawed and managed in culture for a maximum of 8 weeks. kinase assay The kinase assay was performed according to the instructions provided by Millipore. Briefly for ERK1 ERK2 or p38α activity analysis the relevant active protein SB-408124 (100 ng) was incubated with HOEC (0 10 or 20 μmol/L) for 30 min at 30°C. Then each reaction combination was mixed with isotope-unlabeled ATP and 10 μCi [γ-32P] ATP with each compound in 10 μL of reaction buffer made up of 20 mmol/L HEPES (pH 7.4) 10 mmol/L MgCl2 10 mmol/L MnCl2 and 1 mmol/L dithiothreitol (DTT). After incubation at 30°C for 30 min the reaction was stopped by adding 5 μL protein loading buffer and the combination was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). For measuring PI3-K activity an active p110α protein (100 ng) was incubated with HOEC (0 10 or 20 μmol/L) for 30 min at 30°C. Then 20 μl of 0.5 mg/mL phosphatidylinositol (Avanti Polar Lipids Alabaster AL) were added and the mixture was incubated for 5 min at room temperature. Reaction buffer (100 mmol/L.