Effect of LY294002 combined with dichloroacetate on apoptosis in human pulmonary arterial smooth muscle cells and AKT/GSK-3β/HK-2 signaling pathway

Abstract

Objective To evaluate the effect of phosphatidylinositol-3 kinase inhibitor LY294002 combined with dichloroacetate on apoptosis in human pulmonary arterial smooth muscle cells (SMCs) and AKT/GSK-3β/HK-2 signaling pathway.Methods Human pulmonary arterial SMCs were seeded into culture plates at a density of 2 x 104 cells/ml after 3-5 passages.After being incubated for 72 h,the SMCs were cultured in the medium supplemented with 0.2％ fetal bovine serum for 24 h to induce starvation prior to experiments.The cells were then randomly divided into 6 groups (n =6 each) using a random number table:control group (group C),positive control group (group F),LY294002 group (group L),different concentrations of dichloroacetate groups (D1 and D2 groups),and LY294002 combined with dichloroacetate group (group LD1).In group C,the cells were cultured in the medium supplemented with 0.2％ fetal bovine serum.In F,L,D1,D2 and LD1 groups,the cells were cultured in the medium supplemented with 10％ fetal bovine serum.LY294002 2 μmol/L was added to the medium in group L.Dichloroacetate l0 and 20 mmol/L were added to the medium in D1 and D2 groups,respectively.In group LD1,LY294002 (2 μmol/L) was added,and 30 min later dichloroacetate 10 mmol/L was added to the medium in LD1 group.The cells were incubated for 48 h.Flow cytometry was used to measure the cell apoptosis and mitochondrial membrane potential.The expression of phosphorylated AKT (p-Akt),phosphorylated glycogen synthase kinase 3β (p-GSK-3β),and hexokinase-2 (HK-2) was detected using Western blot.Apoptosis rate was calculated.Results Compared with group C,apoptosis rate was significantly increased,and mitochondrial membrane potential was decreased in D2 and LD1 groups,the expression of p-Akt,p-GSK-3β and HK-2 was up-regulated in group F,and no significant changes were found in apoptosis rate and mitochondrial membrane potential in F,L and D1 groups.Compared with group D2,apoptosis rate was significantly increased,mitochondrial membrane potential was decreased,and the expression of p-Akt,p-GSK-3β and HK-2 was down-regulated in LD1 group.The expression of p-Akt,p-GSK-3β and HK-2 was significantly lower in D2 and LD1 groups than in group F.Conclusion LY294002 combined with dichloroacetate can promote apoptosis in human pulmonary arterial SMCs possibly through blocking AKT/GSK-3β/HK-2 signaling pathway. Key words: 1-phosphatidylinositol 3-kinases; Dichloroacetate; Myocytes, smooth muscle; Apoptosis; Protein-serine-threonine kinases; Glycogen synthase kinases 3; Hexokinase