ABSTRACT
Objective To investigate the cross-talk between extracellular signal-regulated kinase (ERK) and nuclear factor κB (NF-κB) signal transduction pathways in A375 human melanoma cells.Methods Cultured A375 cells were randomly divided into 5 groups:control group receiving no treatment,two U0126 (a selective inhibitor of the ERK signaling pathway) groups treated with U0126 of 10 and 5 μmol/L,and two BMS-345541 groups treated with BMS-345541 of 10 and 5 μmol/L.After 24-hour treatment,Western blot and reverse transcription PCR were performed to measure the protein expressions of NF-κB P65,phosphorylated IκBα (p-IκBα),ERK1/2,as well as p-ERK1/2,and the mRNA expressions of NF-κB P65 and ERK1,respectively.One-way analysis of variance and least significant difference (LSD)-t test were carried out for statistical analysis.Results After 24 hours of treatment with U0126 of 10 and 5 μmol/L,a significant decrease was noted in the relative expression level of NF-κB p65 protein (0.60 ± 0.04 and 0.56 ± 0.06 vs.1.54 ± 0.15,both P＜ 0.01) and mRNA (0.79 ± 0.05 and 0.75 ± 0.04 vs.0.86 ± 0.05,both P ＜ 0.01),but a statistical increase in that of p-IκBα protein (0.90 ± 0.05 and 0.70 ± 0.02 vs.0.61 ± 0.03,both P ＜ 0.01) in the two U0126 groups compared with the control group; significant differences were observed in the expression level of p-IκBo protein (P ＜ 0.01) but not in that of NF-κB p65 protein (P ＞ 0.01) between the two U0126 groups.The relative expression levels of ERK1/2 and p-ERK1/2 proteins as well as ERK1 mRNA were significantly higher in the control A375 cells than those in the cells treated with BMS-345541 of 10 μmol/L (0.73 ± 0.07,0.75 ± 0.09,1.51 ± 0.02,all P ＜ 0.01),but similar to those treated with BMS-345541 of 5 μmol/L (0.94 ± 0.11,0.99 ± 0.04,1.62 ± 0.03,all P ＞ 0.05).Conclusion There is a cross-talk between ERK and NF-κB signal transduction pathways in A375 melanoma cells.
ABSTRACT
Objective To investigate the expression of Ptch-1 and Gli-1, hedgehog pathway-related genes in squamous cell carcinoma (SCC), and the effect of cyclopamine, a specific inhibitor of hedgehog signaling pathway, on the proliferation of a SCC cell line Tca. Methods Skin samples were resected from 42 patients with SCC and 10 normal human controls. Immunohistochemistry and in situ hybridization were employed to study the expression and distribution of Ptch-1 and Gli-1 in these specimens. Tca cells were incubated with cyclopamine (1, 2, 5, 10 μmol/mL) for 48 hours, or cyclopamine (5 μmol/mL) for 1-8 days. The same concentrations of lycopersicin served as the control treatment. Then, MTT assay was performed to detect the proliferation of Tca cells. A fraction of Tca cells were cultured in the presence of 5 μmol/mL cyclopamine for 72 hours followed by BrdU assay for the evaluation of cell growth and proliferation. Results A significant increment was shown in the expression of both Patch-1 and Gli-1 by immunohistochemistry (χ2= 5.656, 6.732, P<0.05, 0.01, respectively) and in situ hybridization (χ2=6.787, 9.600, respectively, both P<0.01) in SCC tissue compared with the control specimens. And both of them were predominantly distributed in the cytoplasm of SCC cells. As MTT assay revealed, cyclopamine notably inhibited the proliferation of Tea cells, and the effect increased with the concentration and action time of cyclopamine. Further more, the percentage of BrdU-positive cells was 26% in cyclopamine-treated Tca cells, significantly higher than that in the blank control cells (77%) and lycopersicin-treated cellls (72%). Conclusions Hedgehog signaling pathway is activated in the lesions of SCC, and inhibition of the pathway may facilitate the treatment of SCC.
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Objective To study the biological characteristics of dendritic cells (DCs) derived from peripheral blood mononuclear cells (PBMCs) of patients diagnosed with syphilis. Methods PBMCs were isolated from 16 patients clinically and serologically diagnosed with syphilis, and from 16 healthy human controls, then cultured with GM-CSF and IL-4. On day 10, the monocyte-derived dendritic cells (MoDCs)of the patients and controls were collected and subjected to the detection of surface molecules by flow cytometry; TpN17 was used to stimulate MoDCs from the controls, the expression of phosphorylated ERK was detected by Westem blotting 20 minutes following the stimulation. Results The positivity rate of CD80 was significantly increased in the patients with syphilis than that in the controls (51.90% vs 33.67,P ＜ 0.05), while no significant difference was observed in the expressions of CD83, CD86 or HLA-DR be tween the two groups (16.53% vs 15.99%, 66.13% vs 59.32%, 91.29% vs 90.51%, all P 0.05). The ex pressions of CD80 and CD83 on the surface of MoDCs were enhanced in a dose-dependent manner after ex-posure to TpN17. The expression of cytoplasmic phosphorylated ERK was observed in MoDC stimulated by TpN17, but not in those without the treatment. Conclusions Antigenic stimulation with Treponema pal-lidum may be a reason for phenotypic abnormality of MoDCs derived from patients with syphilis. TpN17 may stimulate the maturation of DCs through the ERK signal transduction pathway.