Arrested proliferation and the activiations of Erk1/2 in MAPKs pathway of PC12 cells treated by manganese

Objective To employ a model in vitro for te st ing the concentration and time dependent relationships of Mn-treated Pheochromo cytoma (PC12) cells toxicity as well as the activiations of ERK in MAPKs pathway under the same conditions, in order to reveal the molecular mechanisms of dysfu nctions in basal ganglia caused by manganese. Methods Pheochrom ocytoma cells in logarithm period were incubated in culture media of manganese(M nCl 2) at 200,400,600,800 μmol/L for 1, 2, 3 and 4 days respectively. The cell viability was examined by MTT [3-(4,5- Dimethylthiazol-2-yl)-2,5-dipheny l tetrasoliumBromide]. Plate clone forming tests were performed to examine the cell growth,cell growth curve was made.Western-blot was used to test phosphryl ations of Erk1/2 (p-Erk2) of manganese-treated PC12 cell in different time c ourses and concentrations. Results The results of MTT and plate clone tests reve aled that manganese at different concentrations (MnCl 2 200,400,600,800 μmol/L ) could suppress the proliferation of PC12 cells in dose and time-dependent man ner after 1,2,3 or 4 days culturing respectively. The cell inhibited ratio on th e 4th day at 600 μmol/L MnCl 2 culture medium approached 50% or more. Wester n-blot tests showed that p-Erk2 of PC12 cells incubated at 600 μmol/L MnCl 2 culture medium was decreasing gradually on the 1st,2nd,3rd and 4th day. P-Erk2 of PC12 cells on the 2nd day decreased by 75%(n=3, P0.05) compared wi th t he controlled. P-Erk2 also gradually decreased at 200,400,600 μmol/L MnCl 2 treated PC12 cells on 4th day. P-Erk2 at 400 μmol/L MnCl 2 treated group on the 4th day decreased by 78%, compared with the control group(n=3, P0.05). Conclusion With specific inhibitor of MEK1/2 PD98059, it was s hown that manganese downregu lated p-Erk2 through MEK1/2 by phosphrylating its downstream Erk1/2. The decreased p-Erk2 probably induces proliferation arrest of PC12 cells.