Figure 1.
Chemical structure of 5-Hydroxy-3,6,7,8,3′,4′-hexamethoxyflavone (5-OH-HxMF) and nobiletin.
Figure 2.
Morphological characteristics of 5-OH-HxMF- and nobiletin-treated PC12 cells.
PC12 cells were seeded on poly-L-lysine-coated 6-well plates in low serum medium for 24 h prior to exposure to vehicle (0.1% DMSO) or indicated agents for additional 48 h. Cell morphology was observed using phase-contrast microscopy and photographed by the digital camera. Arrowheads indicate the neurite bearing cells in 5-OH-HxMF- or nobiletin-treated groups. Scale bar, 100 µm. (B) Neurite bearing cells were analyzed as described in Materials and Methods. Data represent the mean ± SD from three independent experiments. The maximal neurite length for each of the differentiated cells was analyzed and the average neurite length was calculated as described in Materials and Methods. Data represent the mean ± SD from three independent experiments. **p<0.01 represents significant differences compared with those of the vehicle-treated cells.
Figure 3.
Effects of 5-OH-HxMF on GAP-43 mRNA and protein expression in PC12 cells.
PC12 cells were seeded on poly-L-lysine-coated 6-well plates or 100 mm dishes in normal medium for 24 h and then shifted to low serum medium (1% HS and 0.5% FBS) for 24 h prior to exposure to indicated agents. (A) Cells were treated with vehicle (0.1% DMSO), NGF (50 ng/ml; 0.38 nM) and 5-OH-HxMF (10 and 20 µM) for 24 h, respectively. Cellular RNA was then prepared and GAP-43 mRNA level was detected by RT-Q-PCR as described in Materials and Methods. Data represent the mean ± SD of three independent experiments. (B) Cells were treated with vehicle (0.1% DMSO), NGF (50 ng/ml; 0.38 nM) and 5-OH-HxMF (10 and 20 µM) for 24 h. GAP-43 protein expression was detected by Western blotting as described in Materials and Methods. The immunoblot experiments were replicated three times and a representative blot was shown. Normalized intensity of GAP-43 versus β-actin is presented as the mean ± SD of three independent experiments. *p<0.05 and **p<0.01 represent significant differences compared with vehicle-treated cells.
Figure 4.
Effects of 5-OH-HxMF and nobiletin on the phosphorylation of CREB protein in PC12 cells.
PC12 cells were seeded on poly-L-lysine-coated 100 mm dishes in normal medium for 24 h and then shifted to low serum medium (1% HS and 0.5% FBS) for 24 h prior to exposure to indicated agents. Cells were treated with vehicle (0.1% DMSO), 5-OH-HxMF or nobiletin (20 µM) for 0 min, 30 min, 60 min or 120 min. Phosphor-CREB (p-CREB) and CREB proteins were analyzed by Western blotting as described in Materials and Methods. The immunoblot experiments were replicated at least three times and a representative blot was shown. Normalized intensity of p-CREB versus CREB is presented as the mean ± SD of three independent experiments. **p<0.01 represent significant differences compared with 0 min group.
Figure 5.
Effects of 5-OH-HxMF, nobiletin, and inhibitor KG-501 on CRE-dependent gene transcription and neurite outgrowth in PC12 cells.
(A) PC12 cells were transfected with a CRE-mediated luciferase reporter construct and Renilla luciferase control plasmid for 24 h. After transfection, PC12 cells were treated with vehicle (0.1% DMSO), 5-OH-HxMF (20 µM), or nobiletin (20 µM) for 8 h. For treatment of cells with inhibitor, the transfected cells were pre-incubated with KG-501 (10 µM) for 30 min, then exposure of cells with vehicle, 5-OH-HxMF, or nobiletin for additional 8 h. Cells were harvested and luciferase activities were determined as described in Materials and Methods. The intensity of the luciferase reactions measured in the lysates of the transfectants was normalized to their Renilla luciferase control activity. (B) PC12 cells were cultured on poly-L-lysine-coated 6-well plates in low serum medium and treated with vehicle (0.1% DMSO) and 5-OH-HxMF (20 µM) for indicated period. Cellular RNA was then prepared and c-fos mRNA level was detected by RT-Q-PCR as described in Materials and Methods. Data represent the mean ± SD of three independent experiments. (C) PC12 cells were seeded on poly-L-lysine-coated 6-well plates in normal medium for 24 h and then shifted to low serum medium (1% HS and 0.5% FBS) for 24 h prior to exposure to vehicle (0.1% DMSO), 5-OH-HxMF (20 µM) or nobiletin (20 µM) for additional 48 h. For treatment of cells with inhibitor, adherent cells were pre-incubated with KG-501 (10 µM) for 30 min and then exposure to indicated agents for additional 48 h. Neurite bearing cells were analyzed as described in Materials and Methods. Data represent the mean ± SD from three independent experiments. (D) The maximal neurite length for each of the differentiated cells was analyzed and the average neurite length was calculated as described in Materials and Methods. Data represent the mean ± SD from three independent experiments. *p<0.05 and **p<0.01 represent significant differences compared with vehicle-treated cells. #p<0.05 and ##p<0.01 represent significant differences compared with respective KG-501-untreated group.
Figure 6.
Effects of the molecular inhibitors on the 5-OH-HxMF -mediated CREB activation.
(A) PC12 cells were transfected with a CRE-mediated luciferase reporter construct and Renilla luciferase control plasmid for 24 h. Following transfection, cells were pre-treated for 30 min with inhibitors 10 µM U0126, 2.5 µM BIM, 20 µM LY294002, 10 µM KN-62, 500 µM SQ22536, 10 µM H-89 or vehicle (0.1% DMSO), followed by exposure to 5-OH-HxMF (20 µM) for 8 h. Forskolin (2 µM)-treated transfected cells as a positive control for CRE-luciferase reporter assay. The intensity of the luciferase reactions measured in the lysates of the transfectants was normalized to their Renilla luciferase control activity. (B) PC12 cells were seeded on poly-L-lysine-coated 100 mm dishes in normal medium for 24 h and then shifted to low serum medium (1% HS and 0.5% FBS) for further 24 h culture. Cells were treated with inhibitor SQ22536 or H-89 for 30 min prior to exposure of vehicle (0.1% DMSO) or 5-OH-HxMF (20 µM) for 60 min. Phosphor-CREB (p-CREB) and CREB were analyzed by Western blotting as described in Materials and Methods. The immunoblot experiments were replicated at least three times and a representative blot was shown. Normalized intensity of p-CREB versus CREB is presented as the mean ± SD of three independent experiments. ** p<0.01 represents significant differences compared with vehicle. ## p<0.01 represents significant differences compared with respective inhibitors-untreated group.
Figure 7.
The involvement of cAMP production and protein kinase A (PKA) activation in 5-OH-HxMF-induced neruite outgrowth.
PC12 cells were seeded on poly-L-lysine-coated 6-well and then shifted to low serum medium (1% HS and 0.5% FBS) for further 24 h culture. (A) Cells were then incubated with 5-OH-HxMF (20 µM) for indicated period and cAMP level was determined by using enzyme immunoassay kit as described in Materials and Methods. (B) Cells were then incubated with 5-OH-HxMF (20 µM) for indicated period and PKA activity was detected using ELISA kit as described in Materials and Methods. Data represent the mean ± SD of three independent experiments. *p<0.05 and ** p<0.01 represents significant differences compared with 0 min group. (C) Cells were then treated for 30 min with inhibitors 500 µM SQ22536 (adenylate cyclase), 10 µM H-89 (PKA) or vehicle (0.1% DMSO), prior to exposure to 5-OH-HxMF (20 µM) for 48 h. Percentage of neurite bearing cells and maximal neurite length for each of the differentiated cells was analyzed as described in Materials and Methods. (D) Cells were then treated for 30 min with TrkA antagonist K252a (100 nM) prior to exposure to NGF (100 ng/ml; 0.76 nM) or 5-OH-HxMF (20 µM) for 48 h. The neurite bearing cells were analyzed as described in Materials and Methods. Data represent the mean ± SD from three independent experiments. ** p<0.01 represents significant differences compared with vehicle-treated cells. ## p<0.01 represents significant differences compared with respective inhibitor-untreated group.
Figure 8.
Hypothetic mechanism of 5-OH-HxMF in promoting neurite outgrowth in PC12 cells.
5-OH-HxMF could induce neurite outgrowth associated with expression of neuronal differentiation marker (Figure 2 and 3). 5-OH-HxMF induced neurite outgrowth through stimulating CREB phosphorylation and activation of CREdependent transcription activity (Figure 4 and 5). 5-OH-HxMF stimulated CREB phosphorylation and neurite outgrowth mainly through activation of cAMP-dependent protein kinase A (PKA) and TrkAindependent pathway in PC12 cells (Figure 6 and 7). Another PKAindependent pathway, protein kinase C (PKC), was also partly involved in the 5-OH-HxMF-induced neurite outgrowth. In addition, the PKAindependent but CRE-dependent pathways may also partially contribute to the 5-OH-HxMF-mediated neurite outgrowth. As a result, 5-OHHxMF promotes neurite outgrowth mainly through activation of cAMP/PKA/CREB pathway in PC12 cells.