Fig 1: Increased VDAC2 in NSTCs couples PFKP on mitochondrion to prevent its cytoplasmic release and inhibits PFKP-mediated glycolysis.a Western blot analyses of VDAC2 expression in GSCs relative to the matched NSTCs derived from human GBMs. COX IV is used as a mitochondrial marker for normalization. b qRT-PCR analysis of VDAC2 expression in GSCs and matched NSTCs (**p < 0.01). c Co-immunoprecipitation analysis showing the interactions between VDAC2 and PFKP. The anti-VDAC2 antibody (upper panel) and anti-PFKP antibody (lower panel) are used for immunoprecipitation, respectively. The input samples of NSTCs are used as positive controls. d Western blot analyses of VDAC2 and PFKP in mitochondrial and cytoplasmic fractions of NSTCs expressing shRNAs against VDAC2 (shVDAC2#1 and #2) or nontargeting shRNA (shNT). COX IV is used as a mitochondrial protein marker and ß-tubulin is used as a cytoplasmic protein marker for normalization. Silencing VDAC2 expression reduces the level of PFKP anchored on mitochondrion, but increases PFKP expression in cytoplasm. e PFK enzyme activity in NSTCs expressing shVDAC2 or shNT (***p < 0.001). f Analysis of the relative lactate production in NSTCs expressing shVDAC2 compared to those expressing shNT (***p < 0.001). g Co-immunoprecipitation assay showing the interactions between VDAC2 and PFKP in GSCs expressing VDAC2. The anti-VDAC2 antibody (upper panel) and anti-PFKP antibody (lower panel) are used for immunoprecipitation, respectively. The input samples of GSCs expressing VDAC2 are used as positive controls. h Analysis of PFK enzyme activity in GSCs expressing VDAC2 or control vector (***p < 0.001). i Analysis of the relative lactate production in GSCs expressing VDAC2 or control vector (***p < 0.001)
Fig 2: PFKP promotes NSCLC cell survival under GS via long-chain fatty acid oxidation.a Propidium iodide (PI) exclusion test of H1299 cells under control (Ctrl) or GS for 48 h. FSC: forward scatter. b Cell viability of H1299 (n = 9) or H1703 cells (n = 8) under control (Ctrl) or GS for 48 h. c Cell viability of H1299 with PFKP knock-down (KD) (n = 5) under control (Ctrl) or GS for 48 h. d Cell viability of H1299 KO cells with ACC2 KD (n = 9) under control (Ctrl) or GS for 48 h. e Cell viability of H1703 cells with AMPKα KD (n = 4) under control (Ctrl) or GS for 8 h. f Cell viability of H1703 cells treated with 1 mM PFKP inhibitor 2,5-Anhydro-D-glucitol-1,6-diphosphate (n = 4) under control (Ctrl) or GS for 24 h. g Cell viability of H1703 PFKP KO cells transfected with empty vector, Domain 1, or Flag-HA tagged PFKP (n = 4) under control (Ctrl) or GS for 24 h. h Cell viability of H1703 treated with 5 mM N-acetylcysteine (NAC) (n = 4) under control (Ctrl) or GS for 24 h. Data are shown as means ± SD with n indicating the number of biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed Student’s t-test.
Fig 3: Schematic model of the PFKP-AMPK-ACC2-mediated long-chain fatty acid oxidation which promotes cancer cell survival under GS.PFKP mediates the recruitment and activation of mitochondrial AMPK under GS, which results in enhanced mitochondrial ACC2 phosphorylation and facilitates long-chain fatty acid oxidation to maintain metabolic homeostasis, leading to the promotion of cancer cell survival.
Fig 4: A mechanistic illustration of meldonium amelioration of oxidative stress–induced mitochondrial damage. (A) Oxygen consumption rate (OCR) diagram, basal respiration, proton leak, and non-mitochondrial oxygen consumption in vitro after hypoxia for 24 h were detected using the Cell Mito Stress Test Kit (n > 3). (B) Protein expression of mitochondrial fission and fusion in vitro after hypoxia for 24 h were detected via western blot (n = 3–5). (C) A schematic diagram depicting a potential mechanism by which meldonium regulates glycolysis to alleviate hypoxia-induced lung injury. Mechanistically, meldonium can regulate and interact with PFKP to regulate glycolysis that is one of main energy metabolic pathway, while promoting Nrf2 transfer from the cytoplasm to the nucleus. Substantially, Nrf2 actives downstream pathways to prevent oxidative stress and alleviate mitochondrion damage and homeostasis imbalance, which protect the lung from hypoxia-induced injury. Black arrows denote hypoxia-induced changes. Red arrows denote meldonium-induced changes. ARE, antioxidant response elements; HRE, hypoxia response elements; HIF-1a, hypoxia inducible factor-1a; PFKP, platelet isoform of phosphofructokinase; PKM2, M2 type of pyruvate kinase; LDHA, lactate dehydrogenase A; PDH, pyruvate dehydrogenase; PDK1, pyruvate dehydrogenase kinase 1; Nrf2, nuclear factor E2-related factor 2; ROS, reactive oxygen species; ATP, adenosine triphosphate. Data are expressed as the mean ± SEM. Statistical analyses were performed using one-way ANOVA followed by Fisher’s LSD test. #p < 0.05, ##p < 0.01, ###p < 0.001 compared with the control group; *p < 0.05, **p < 0.01, ***p < 0.001 compared with the hypoxia group.
Fig 5: PFKP is an AMPK-interacting protein and highly expressed in lung adenocarcinoma.a Mass spectrometry analysis of interacting proteins using AMPKa or Flag-HA-PFKP as bait in NSCLC cells under GS for 4 h. b Diagram of glycolysis pathway and the step catalyzed by PFKP. G6P Glucose-6-Phosphate, F6P Fructose-6-Phosphate, FBP Fructose-1,6-bisphosphate, G3P Glyceraldehyde-3-Phosphate, DHAP Dihydroxyacetone-Phosphate, BPG 1,3-Bisphosphoglycerate, 3PG 3-Phosphoglycerate, 2PG 2-Phosphoglycerate, PEP Phosphoenolpyruvate. c Gene expression levels of PFKP in well, moderately, and poorly differentiated tumors from the NIH LUAD dataset. d Gene expression levels of PFKP, PFKL, and PFKM in tumor and adjacent normal tissue from the TCGA LUAD dataset. e Survival analysis of lung adenocarcinoma patients with high or low expression of PFKP, PFKL, and PFKM in the TCGA LUAD dataset (n = 501) and NIH LUAD dataset (n = 442). Data are shown as means ± SD with n indicating the number of patients in the datasets. P values by Mann–Whitney U test.
Supplier Page from Abcam for Anti-PFKP antibody [EPR17314]