Reversal of the Warburg effect with DCA in PDGF-treated human PASMC is potentiated by pyruvate dehydrogenase kinase-1 inhibition mediated through blocking Akt/GSK-3 beta signalling

摘要

There is accumulating evidence indicating that the growth inhibitory effect of dichloroacetate (DCA) on pulmonary arterial smooth muscle cells (PASMCs) may be associated with the reversal of the Warburg effect and initiation of the mitochondria-dependent apoptotic pathway. Previous studies indicated that platelet-derived growth factor (PDGF) promoted the Warburg effect and resulted in apoptotic resistance of PASMCs, which was attributed to activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signalling pathway. However, the mechanism underlying the pro-apoptotic effect of DCA on PDGF-treated PASMCs has not been thoroughly elucidated, and the effect of the Akt/glycogen synthase kinase-3 beta (GSK-3) pathway inhibition concomitant with the effect of DCA on PASMC proliferation remains unclear. The growth of human PASMCs and the lactate concentration in extracellular medium of PASMCs were detected by Cell Counting Kit-8 assays and a Lactate Colorimetric Assay kit, respectively. Cell apoptosis was evaluated by fluorescence activated cell sorting. The mitochondrial membrane potential (Delta Psi m) was assessed with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazol-carbocy-anine iodide assays. The expression levels of phosphorylated Akt and GSK-3 beta, pyruvate dehydrogenase, cleaved caspase-3, pyruvate dehydrogenase kinase-1 (PDK-1), hypoxia inducible factor-1 alpha (HIF-1 alpha) and hexokinase-2 (HK-2) were measured with western blot analysis. Confocal analyses were employed to determine HK-2 co-localisation with the mitochondria. The results indicated that DCA inhibited human PASMC proliferation in a dose-dependent manner. DCA at 10 mM promoted apoptosis and the upregulation of activated caspase-3 in PASMCs pre-treated with 20 ng/ml PDGF-homeodimer BB (BB). Treatment with 5 mu M LY294002 produced minimal anti-proliferative effects on human PASMCs and barely induced cellular apoptosis and caspase-3 activation. However, co-administration of 10 mM DCA with LY294002 significantly decreased the cell proliferation index and induced cell apoptosis and caspase-3 activation. The combined administration of LY294002 with DCA significantly decreased lactate concentration, promoted the depolarisation of the Delta Psi m and repressed HIF-1 alpha upregulation and HK-2 activation in PASMCs treated with PDGF, which was attributed to the potentiation of DCA-induced PDK-1 inhibition by LY294002 via blockade of the Akt/GSK-3 beta/HIF-1 signalling pathway. In conclusion, inhibition of the Akt/GSK-3 beta pathway improved the pro-apoptotic effect of DCA on human PASMCs, which may be attributed to a reversal of the Warburg effect by blocking the mutual interaction between HIF-1 alpha and PDK-1, consequently downregulating HK-2. Therefore, combinatory treatment with DCA and PI3K inhibitors may represent a novel therapeutic strategy for the reversal of apoptosis resistance exhibited by PASMCs as a result of mitochondrial bioenergetic abnormalities, as well as the treatment of pulmonary vascular remodelling in pulmonary arterial hypertension.