Specific inhibition by synthetic analogs of pyruvate reveals that the pyruvate dehydrogenase reaction is essential for metabolism and viability of glioblastoma cells

Abstract

The pyruvate dehydrogenase complex (PDHC) and its phosphorylation are considered essential for oncotransformation, but it is unclear whether cancer cells require PDHC to be functional or silenced. We used specific inhibition of PDHC by synthetic structural analogs of pyruvate to resolve this question. With isolated and intramitochondrial PDHC, acetyl phosphinate (AcPH, K i AcPH = 0.1 μM) was a much more potent competitive inhibitor than the methyl ester of acetyl phosphonate (AcPMe, K i AcPMe = 40 μM). When preincubated with the complex, AcPH also irreversibly inactivated PDHC. Pyruvate prevented, but did not reverse the inactivation. The pyruvate analogs did not significantly inhibit other 2-oxo acid dehydrogenases. Different cell lines were exposed to the inhibitors and a membrane-permeable precursor of AcPMe, dimethyl acetyl phosphonate, which did not inhibit isolated PDHC. Using an ATP-based assay, dependence of cellular viability on the concentration of the pyruvate analogs was followed. The highest toxicity of the membrane-permeable precursor suggested that the cellular action of charged AcPH and AcPMe requires monocarboxylate transporters. The relevant cell-specific transcripts extracted from Gene Expression Omnibus database indicated that cell lines with higher expression of monocarboxylate transporters and PDHC components were more sensitive to the PDHC inhibitors. Prior to a detectable antiproliferative action, AcPH significantly changed metabolic profiles of the investigated glioblastoma cell lines. We conclude that catalytic transformation of pyruvate by pyruvate dehydrogenase is essential for the metabolism and viability of glioblastoma cell lines, although metabolic heterogeneity causes different cellular sensitivities and/or abilities to cope with PDHC inhibition.

Synthetic analogues of 2-oxo acids discriminate metabolic contribution of the 2-oxoglutarate and 2-oxoadipate dehydrogenases in mammalian cells and tissues

“…To grow cells for the viability tests, the cell-optimized densities were used: C6 cells were seeded in 96-well plates in 100 µl DMEM at a density of 5*10 3 cells/well for 5 h incubation or 1*10 3 cells/well for 96 h incubation; MCF-7 cells were seeded in 100 µl DMEM/F12 at a density of 1.5*10 4 cells/well for 5 h incubation or 5*10 3 cells/well for 96 h incubation. After 24 h the media were exchanged for HBSS, supplemented with 1 g/L glucose, 1.3 mM CaCl 2 , 1.0 mM MgSO 4 , and phosphonate analogues (0-10 mM) for 5 h incubation, as previously described 91,92 . For 96 h incubation, the media were exchanged for the same ones including the phosphonates.…”

Known unknowns of cardiolipin signaling: The best is yet to come

“…2-oxoglutarate dehydrogenase (OGDH), branched-chain 2-oxoacid dehydrogenase (BCKDH), and pyruvate dehydrogenase (PDH) complexes are also capable of considerable superoxide/H 2 O 2 production. The 2-oxoacid dehydrogenase complexes in mitochondria can produce superoxide/hydrogen peroxide at much higher rates than complex I [141] and represent an additional source of ROS with the rates of their generation exceeding that of electron transport [142]. This redox-associated imbalance (also called oxidative stress) is believed to potentially turn into a completely uncontrolled pro-oxidant reactions catalyzed by high concentrations of a variety of catalysts containing transition metals.…”

How phosphorylation influences E1 subunit pyruvate dehydrogenase: A computational study

“…Given its central role in cellular metabolism 2 , 3 , PDC has been considered a promising target for the development of antibacterial 4 , 5 and anticancer drugs 6 , 7 . In fact, recent experimental investigations pointed out that an increase in PDC activity can sustain the progression of some cancer types (for example prostate cancer and glioblastoma) 8 as well as the survival of cancer stem cells 7 , 9 .…”