Search

Objective: To explore whether ERK1/2 and p38 pathways mediate P2Y receptor-induced prostate cancer invasion., Methods: The two subclones from the PC-3 human prostate carcinoma cell line: 1E8 (highly metastatic) and 2B4 (non-metastatic), were cultured and transfected with the plasmid pcDNA3-KA-MEK1 containing the dominant negative mutant of MEK1 (KA-MEK1) and wild type MKP-5 (a dual-specificity phosphatase of p38). P2Y receptor-activated ERK1/2 and p38 kinases were detected using phospho-specific antibodies directed against the dually phosphorylated active forms of these kinases by Western blotting. P2Y receptor agonists ATP and P2Y receptor antagonist suramin were used respectively to observe their effects on the activity of ERK1/2. The roles ERK1/2 and p38 pathways play in P2Y receptor-induced in vitro invasion were detected by in vitro invasion assay. The cells were pre-treated with ATP, SB203580, a p38 inhibitor, and PD98059, a blocker of ERK1/2 pathway, respectively., Results: ATP activated ERK1/2 and p38 kinase time-dependently. Suramin significantly inhibited the activation of ERK1/2 and p38 kinase by ATP. ATP stimulated prostate cancer cell invasion. The stimulated cancer cell invasion was significantly inhibited by pretreatment of the cells with PD98059 or SB203580. Transfected of 1E8 cells with KA-MEK1 or up-regulation of MKP-5 both, while inhibiting phosphorylation of ERK1/2 or p38, significantly reduced the invasion of prostate cancer cells in vitro., Conclusion: P2Y receptor-induced prostate cancer cell invasion is mainly regulated through ERK1/2 and p38 pathways.

Objective: To explore the mechanism of basic fibroblast growth factor (bFGF)-mediated hypoxia inducible factor (HIF-1) activation and the down-stream signaling pathways involved., Methods: Human breast cancer cells of the line T47D were cultured and lysed to extract the total protein in the supernatant. The amounts of extracellular signal kinase 1/2 (ERK1/2), Akt, p38, and beta-tubulin were measured. T47D cells were inoculated into a 24-well plate, co-transfected with luciferase vector OB-HRE containing HIF-1 functional sequence (HRE) and pRL-SV40 (as inner marker), pretreated with SU5402, inhibitor of FGFR1, SB203580, inhibitors of PI-3K, PD98059, inhibitors of MEK1, or LY294002, inhibitors of p38, treated with basic fibroblast growth factor (bFGF), and then lysed. The amounts of ERK1/2, Akt, p38, and beta-tubulin were measured. Western blotting was used to detect the HIF-1alpha level in total protein. Dual luciferase assay was used to analyze the transactivity of HIF-1. The firefly/renilla luciferase ratio was measured to access the transcription activity of HIF-1. Western blotting was used to detect the expression of HIF-1alpha protein and phosphorylated Akt, ERK1/2 and p38 in whole cell extract., Results: After the addition of bFGF Western blotting showed that the and phosphorylation of Akt, ERK1/2 and p38 in the T47D cells were increased time- and dose-dependent manner, and dual luciferase assay showed that the fluorescent intensity was increased, signifying the increase of expression of HIF-1alpha protein. Ten minutes after the addition of CHX the expression of HIF-1alpha protein began to be decreased and ceased 90 minutes after. SU5402 remarkably dose-dependently blocked the bFGF-induced phosphorylation of ERK1/2, Akt and p38. 15 micromol/L LY294002 completely blocked the bFGF-induced phosphorylation of Akt. 5 micromol/L PD98059 blocked 80% of the bFGF-induced phosphorylation of ERK1/2. 10 approximately 20 micromol/L SB203580 basically blocked the bFGF-induced phosphorylation of p38. SU5402 and LY294002 100% inhibited the bFGF-induced expression of HIF-1alpha protein. However, PD98059 and SB203580 did not significantly influence the expression of HIF-1alpha protein induced by bFGF. Luciferase assay showed that SU5402 and PD98059 inhibited the bFGF-induced transcription activity of HIF-1 by 94.8% and 81.7% respectively. LY294002 not only completely inhibited the bFGF-induced transcription activity of HIF-1 but also inhibited the basic transcription of HIF-1, and SB203580 did not significantly influence the transcription activity of HIF-1., Conclusion: bFGF activates HIF-1 via the PI-3K/Akt and MEK1/ERK pathways that co-operatively and differentially regulate this process with PI-3K/Akt pathway playing a more important role.

Objective: To investigate the effects of mitogen-activated protein kinase phosphatase-5 (MKP5) on the biological behaviors of cell and the activation of mitogen-activated protein kinase (MAPK) induced by P2Y purinergic receptor agonist ATP in the prostate cancer cell line 1E8., Methods: 1E8 cells were cultured. The recombinant plasmid pcDL-SRalpha296-MKP5 containing the human wild type MKP5 cDNA and blank vector pcDL-SRalpha were co-transfected with pcDNA3 vector plasmid into the 1E8 cells respectively to get strong expression clones. RT-PCR was used to detect the expression of MKP5 mRNA. ATP, MEK inhibitor PD 98059, and p38 inhibitor SB 203580 were added into the culture. Then the total protein was extracted, SDS-PAGE and Western blotting were used to detect the activation of p38 and ERK1/2 induced by ATP with phosphospecific antibodies directed against the dually phosphorylated, active forms of p38 and ERK1/2. The in vitro growth curve was drawn, soft agar colony formation test was made, and Matrigel membrane-penetrating experiment was made to test the invasion ability of 1E8 cells., Results: The activity of p38 and that of ERK1/2 of the 1E8 cells untreated by ATP were almost undetected. ATP time-dependently stimulated the activities of ERK1/2 and p38 kinases, inhibited the in vitro growth and colony formation on soft agar, and promoted the in vitro invasion of 1E8 cells. MKP5-transfection effectively inhibited the p38 activity induced by ATP and blocked the effects by ATP stimulation on 1E8 cells. The activity of p38 kinase was significantly decreased both in the control and MKP5 transfected cells that were pretreated with SB203580 and then with ATP with an inhibition rates of 83.14% and 58.00% (P < 0.001 and P = 0.003). The growth of cells transfected with MKP5 was significantly inhibited (P < 0.001) and further inhibited by addition of ATP. The inhibition of growth of MKP5-transfected cells by ATP could be partially reversed by pretreatment of PD98059 (P < 0.05). The number of cells transfected with MKP5 that penetrated the membrane was significantly less than that of the control cells (P = 0.000 9). ATP increased the penetrating ability of different cells (all P < 0.01), and this action was partially inhibited by MKP5 transfection and addition of SB203580 (P < 0.001), but not by the addition of PD98059. ATP treatment decreased the colony formation on soft agar of different cells, especially of the MKP5 transfected cells (P < 0.001). Such action was partially reversed by SB203580 and PD98059., Conclusion: The p38 and ERK1/2 pathways exert different effects on the in vitro growth, invasion and colony formation of 1E8 prostate cancer cells related to ATP. The p38 pathway may be more important in the invasive capability of 1E8 cells. MKP5 plays an important role in the regulation of p38 action.