Figure 5
The anti-invasive effects of melatonin are mediated through the p38 MAPK signaling pathway. (a) The effect of melatonin on the phosphorylation of p38 MAPK. MCF-7/6 cells were serum-starved for 24 hours and treated with diluent (Control, 0.00001% ethanol) or melatonin (Mlt, 10-9 M) for 30 minutes in fresh medium supplemented with 10% fetal bovine serum. Expression of phospho-p38 (p-p38) and total p38 (p38) was analyzed by Western blot analysis. (b) The effect of melatonin on stromal-derived factor-1 (SDF-1)-induced p38 phosphorylation. MCF-7/CXCR4 cells were serum-starved for 48 hours and pre-treated with melatonin (10-9 M) or diluent (0.00001% ethanol) for 5 minutes followed by SDF-1 (100 ng/mL) stimulation for 2 minutes (Mlt + SDF-1). (c) The effect of H89 on melatonin regulation of p38 phosphorylation. MCF-7/6 cells were serum-starved for 24 hours and pre-treated with H89 (10 μM) for 45 minutes followed by a 30-minute treatment with melatonin (10-9 M). Figures in (a-c) are representative Western blots from three independent studies, respectively. In (a) and (c), the band intensity of phospho-p38 was normalized to that of total p38 and expressed in the graph as percentage of control (set as 100%). *P < 0.05 versus diluent-treated control (n = 3). (d) The effect of SB230580 on the invasive potential of MCF-7/6 breast cancer cells. MCF-7/6 cells were plated onto matrigel invasion chambers after 24-hour serum starvation and incubated in the medium containing diluent (ETOH, 0.00001% ethanol; dimethyl sulfoxide, or DMSO), melatonin (Mlt, 10-9 M), or SB230580 (20 μM). Data are presented as percentage of ethanol-treated control (100%). *P < 0.05 versus ethanol-treated control cells. **P < 0.05 versus DMSO-treated control cells. (e) Effect of CAMKK6b on melatonin's anti-invasive action. MCF-7/Her2.1 cells were transiently transfected with empty vector (V) or CAMKK6b plasmid for 24 hours, plated onto matrigel invasion chambers, and treated with diluent (Control, 0.00001% ethanol) or melatonin (Mlt, 10-9 M) for 2 days. Data are presented as percentage of vector-transfected diluent-treated control (100%). *P < 0.05 versus vehicle-treated control cells. Phosphorylation of p38 MAPK (p-p38) in vector- and CAMKK6b-transfected cells was analyzed by Western blot analysis. Expression of total p38 (p38) was used as loading control. MAPK, mitogen-activated protein kinase.