Article Title: 1,8-cineole prevents UVB-induced skin carcinogenesis by targeting the aryl hydrocarbon receptor
Figure Lengend Snippet: Effect of 1,8-cineole on UVB induction of the AhR/c-Src/EGFR signaling pathway and direct binding with AhR (a) 1,8-cineole inhibits UVB-induced phosphorylation of c-Src Tyr-416 and EGFR Tyr-845 residues in HaCaT cells. Cells were pre-treated with 1,8-cineole at the indicated concentrations for 1 hour, irradiated with UVB, and then harvested after 15 min. Phosphorylation levels of proteins were detected by Western blotting. (b) and (c) 1,8-cineole suppresses UVB-induced AhR nuclear translocation and dissociation from c-Src in HaCaT cells. Cells were pretreated with 1,8-cineole for 1 hours, before irradiation with UVB for 30 min. (b) AhR (green) and p-c-Src (Tyr 416) (red) were detected by fluorescence microscopy. Nuclei were counterstained with DAPI (blue). White arrows indicate inhibition of AhR nuclear translocation and c-Src phosphorylation by 1,8-cineole treatment in HaCaT cells. Scale bar, 50 μm. (c) Co-immunoprecipitation assay was performed asdescribedin the ‘Materials and Methods’ and levels of AhR and c-Src were detected by Western blotting. (d) 1,8-cineole inhibits UVB-induced CYP1A1 mRNA upregulation in HaCaT cells. CYP1A1 mRNA levels were measured by qRT-PCR. Cells were pre-treated with 1,8-cineole at the indicated concentrations for 1 hour, and irradiated with UVB before total RNA was extracted after 4 hours. (e) 1,8-cineole directly binds to AhR but not (f) EGFR or (g) c-Src in HaCaT cells. For the DARTS assay, cells were pre-treated with 1,8-cineole at the indicated concentrations for 1 hour and lysed. The lysates were digested with pronase (at the indicated pronase to protein ratio) and subjected to Western blotting.
Article Snippet: Reagents and antibodies 1,8-cineole (99%) and N -Acetyl-L-cysteine (NAC, 99%) were purchased from Sigma Aldrich (St. Louis, MO, USA).
Techniques: Binding Assay, Irradiation, Western Blot, Translocation Assay, Fluorescence, Microscopy, Inhibition, Co-Immunoprecipitation Assay, Quantitative RT-PCR