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, 11 (4), 962-973
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Pyruvate Dehydrogenase Kinase 1 Interferes With Glucose Metabolism Reprogramming and Mitochondrial Quality Control to Aggravate Stress Damage in Cancer

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Pyruvate Dehydrogenase Kinase 1 Interferes With Glucose Metabolism Reprogramming and Mitochondrial Quality Control to Aggravate Stress Damage in Cancer

Xinyue Deng et al. J Cancer.

Abstract

Pyruvate dehydrogenase kinase 1 (PDK1) is a key factor in the connection between glycolysis and the tricarboxylic acid cycle. Restoring the mitochondrial OXPHOS function by inhibiting glycolysis through targeting PDK1 has become a hot spot for tumor therapy. However, the specific mechanism by which metabolic changes affect mitochondrial function remains unclear. Recent studies have found that mitochondrial quality control such as mitochondrial protein homeostasis plays an important role in maintaining mitochondrial function. Here, we focused on PDK1 and explored the specific mechanism by which metabolic changes affect mitochondrial OXPHOS function. We showed that glucose metabolism in HepG2 and HepG3B cells switched from anaerobic glycolysis to the mitochondrial tricarboxylic acid cycle under different concentrations of dichloroacetate (DCA) or short hairpin PDK1. After DCA treatment or knockdown of PDK1, the mitochondrial morphology was gradually condensed and exhibited shorter and more fragmented filaments. Additionally, expression of the mitochondrial autophagy proteins parkin and PTEN-induced kinase was down-regulated, and the biosynthetic protein peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α) and its regulated complex I, III, IV, and V protein were down-regulated. This indicated that PDK1 inhibition affected the level of mitochondrial quality control. Analysis of mitochondrial function revealed significantly increased mitochondrial reactive oxygen species and decreased membrane potential. Therefore, glucose metabolism reprogramming by PDK1 inhibition could induce mitochondrial quality control disorders to aggravate mitochondrial stress damage.

Keywords: DCA; PDK1.; glucose metabolic reprogramming; mitochondiral quality control; oxidative phosphorylation.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
DCA inhibits cell proliferation, arrests the cell cycle, and induces apoptosis. (A-B) HepG2 and HepG3B cells were treated with dichloroacetate (DCA) at 0, 20, 40, 60, or 80 mM for 24 h and 48 h and then tested by MTT assay. Cells exposed to DCA at the indicated doses for 24 h were stained with propidium iodide (C) and Annexin V/PI labeling (D), then subjected to flow cytometry analysis. Total cell death and the cell cycle were quantified by flow cytometry (E-F). Data are the mean ±SD, n=3, *P<0.05, **P<0.01, ***P<0.001 compared with respective controls.
Figure 2
Figure 2
DCA treatment or shPDK1 induce a metabolic shift from glycolysis to oxidative phosphorylation. HepG2 and HepG3B cells were treated with DCA for 24 h, western blot analysis the levels of p-PDH and PDH (A and B), lactate concentrations were determined in the culture media and normalized to protein amounts (C) .The oxygen consumption rates of 6h were measured in HepG2 and HepG3B cells in the presence of DCA (D). (E-F) HepG2 cells were treated with DCA at the indicated doses for 24 h, equal amounts of proteins were used for western blotting to determine the levels of LDHA and HK2. HepG2 cells were transiently transfected for 48 h with the shPDK1 expression vector or empty vector, and lactate concentrations were measured after incubation for 6, 12, 24, or 48 h under basal conditions and normalized to protein amounts production (G) , the oxygen consumption rate were measured (H), western blot analysis the levels of PDK1 ,p-PDH, PDH and LDHA (I-K). Data are the mean ± SD, n=3, *P<0.05, **P<0.01 compared with respective controls.
Figure 3
Figure 3
DCA or shPDK1 reduces the mitochondrial membrane potential and increases mitochondrial ROS. (A-B) HepG2 cells were treated with 80mM DCA for 6 h and for 24 h with or without pretreated with 5mM NAC for 1h, then assayed for the mitochondrial membrane potential with JC-1 and mtROS with MitoSOX by flow cytometry:the green fluorescence represents depolarized mitochondrial (J-monomer), and the red fluoresence represents the hyperpolarized mitochondrial (J-aggregates). The depolarization of Δψm is indicated by the increase in the ratio of J monomer/J aggregate. Data are the mean ± SD, n=3, *P<0.05, **P<0.01 compared with respective controls, #P<0.05 compared with DCA 24 h. (C-E) The mitochondrial membrane potential and mtROS were measured in HepG3B cells in the presence of DCA for 24 h. HepG2 cells were transiently transfected for 48 h with the shPDK1 expression vector or empty vector, mtROS was measured in HepG2 cells treated as indicated (magnification ×200)(G), and the mitochondrial membrane potential with JC-1 by flow cytometry (F). (H) Cell viability was determined by MTT assay in the presence of DCA with or without NAC pretreated. Data are the mean ± SD, n=3, *P<0.05, **P<0.01 compared with respective controls, #P<0.01 compared with DCA 40mM, $P<0.01 compared with DCA 80mM.
Figure 4
Figure 4
The effect of DCA on mitochondrial quality control. HepG2 and HepG3B cells treated with DCA were incubated for 24h. The expression of PGC-1α, Tfam (A)and mitochondrial DNA-encoded major subunits in respiratory chain complexes: ND1, CYTB, COX1, and ATP6 (B) were measured by western blot analysis. (C) The mitochondrial network of HepG2 and HepG3B cells after treatment with DCA were determined by staining and flurescent microscopy (400×). The proportion of cells (n=100 cells for each sample) with tubulated, intermediate and fragmented mitochondrial was quantified. Western blot analysis for DRP1, FIS1, Mfn2, OPA1 (D), and PINK, parkin (E) and LC3B (F-G) in cells treated as indicated. (I-J) Quantitation of the ratio of the indicate proteins. Data are the mean ± SD, n=3, *P<0.05, **P<0.01 compared with respective controls. (H) Western blot analysis for HSP10 and HSP60 in HepG2 and HepG3B cells treated with DCA at different time points as indicated.
Figure 5
Figure 5
The effect of shPDK1 on mitochondrial quality control. HepG2 cells were transiently transfected with the shPDK1 expression vector or empty vector for 48 h. (A) The mitochondrial network were determined by staining and flurescent microscopy (400×). The proportion of cells (n=100 cells for each sample) with tubulated, intermediate and fragmented mitochondrial was quantified. Western blot analysis of PDK1, PGC-1α and Tfam (B) mitochondrial DNA-encoded major subunits in respiratory chain complexes: ND1, CYTB, COX1, and ATP6 (C), DRP1, FIS1and Mfn2 (D), PINK, parkin (E) and LC3B (F) after treatments. (G) Quantitation of the ratio of PDK1. **P<0.01, ***P<0.01compared with respective controls.
Figure 6
Figure 6
DCA suppresses tumor growth in a subcutaneous xenograft. (A-B) Images of xenograft tumors formed in nude mice injected with normal saline (control group) and the DCA-treated group. (C) Body weight gain profiles of vehicle- and DCA-treated nude mice. (D and E) Tumor volume and weights were calculated from measurements. (F-G) Lactic acid in the serum and citric acid in the tissues of nude mice were measured. Data are the mean ± SD, n=4, *P<0.05, **P<0.01 compared with controls. (H-J and L-M) PDH, p-PDH, HK2, OPA1, Mfn2, FIS1, DRP1, parkin, PINK, LC3B, PGC-1α, ND1, COX1, CYTB, and ATP6 levels in xenografts were detected by immunoblotting. Data are the mean ± SD, n=3, *P<0.05, **P<0.01 compared with controls. (K) Immunohistochemistry of TUNEL, p-PDH, PGC-1α, and LDHA in HepG2 cell xenografts. TUNEL staining revealed obvious apoptosis in DCA-treated nude mice (bar, 100 μm).

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References

    1. Vander Heiden MG, Cantley LC, Thompson CB. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science. 2009;324:1029. - PMC - PubMed
    1. Warburg O. On the origin of cancer cells. Science. 1956;123:309–14. - PubMed
    1. Xiang Z, Chen R, Yu Z, Rui L, Li J, Zhao X. et al. Dichloroacetate restores drug sensitivity in paclitaxel-resistant cells by inducing citric acid accumulation. Molecular Cancer,14,1(2015-03-19) 2015;14:63. - PMC - PubMed
    1. R P, A H, JP J, G S. Mitochondria and cancer. Virchows Archiv An International Journal of Pathology. 2016;2013:1–2.
    1. Tatsuta T, Langer T. Quality control of mitochondria: protection against neurodegeneration and ageing. The EMBO Journal. 2014;27:306–14. - PMC - PubMed
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