Figure 4

Role of P38-MAPK cascade in MC3-mediated cellular apoptosis. (A) MC3 activated p38-MAPK. Panc1 cells were incubated with increasing concentrations of MC3 as indicated for 1 h. Whole cell lysates were subjected to immunoblot and probed with p38-MAPK and phospho-p38-MAPK antibodies. DMF was used as mock, ß-Actin as loading control and 40 μg proteins were loaded per lane. (B) MC3 activated p53 and induced PARP cleavage. Panc1 cells were treated with 5 μM MC3 as shown. Phospho-p53 and PARP cleavage were detected with respective antibodies in immunoblot. Phosphorylation of p38-MAPK, HSP27, Erk1/2, GSK3ß and Akt were determined by microarray analysis in concentration- (C) and time- (D) dependent studies. (E) MC3 activated expressions of p38-MAPK-related genes. Panc1 cells were incubated with MC3 5 μM for 6 h and the expression of ATF2, TP53 and Stat1 were analyzed by qRT-PCR. Data were normalized to the value of DMF-treated cells, shown is the mean ± SD of quadruplicates and representing three independent experiments. (F) p38-MAPK was activated by MC3 and MC4 treatment in ASPC1 and Panc1 cells. The cells were treated with gold(I) NHC complexes or ligand for 1 h. Phospho- and total-p38-MAPK were detected with corresponding antibodies. (G) p38 inhibitor (p38i), but not ROS scavengers, blocked MC3-activated p38-MAPK pathway within 1 h incubation. Panc1 cells were incubated with 5 μM MC3, 10 mM NAC, 10 μM p38i, 5 mM Glu or in combination as indicated for 1 h. Phosphorylation of p38-MAPK, ATF2 and p53 were detected by using specific antibodies. ROS scavenger did not influence on MC3-caused ROS formation (H) or mitochondrial membrane potential alternation (I). The densitometric analyses of corresponding immunoblots are provided in supporting information.