Bioenergetics profile revealed that platelets pretreated with DCA exhibited decreased aerobic glycolysis in response to convulxin only. Targeting metabolic plasticity with DCA may be explored as a novel strategy to inhibit platelet function. For several years mitochondrial oxidative phosphorylation OXPHOS was considered the sole energy pathway for quiescent platelets.
Mitochondrial pyruvate dehydrogenase PDH complex converts pyruvate the end product of glycolysis in the cytosol to acetyl coenzyme A, which is further oxidized to tricarboxylic acid cycle to produce energy adenosine triphosphates [ATPs] in the OXPHOS pathway. The role of PDKs in platelet function and thrombosis remains unclear. Herein, we determined whether diverting metabolism in platelets from aerobic glycolysis back to OXPHOS would inhibit platelet aggregation and arterial thrombosis.
Platelets for infusion were isolated from 4- to 6-month-old donor wild-type WT mice. Human venous blood was obtained from healthy donors by the Declaration of Helsinki and approved by the Institutional Review Board, University of Iowa. All participants gave written informed consent. Platelet-rich plasma PRP or washed platelets 0. Aggregation was induced with different agonists convulxin, collagen, thrombin, and adenosine diphosphate [ADP].
Platelet-dense granule secretion was determined by measuring the release of ATP by adding luciferin-luciferase reagent. To examine the effect of DCA on platelet aggregation and secretion, washed platelets or PRP was pretreated with vehicle or DCA at different doses 10 and 25 mM for 10 minutes before treatment with various agonists.
Experiments were performed using a BioFlux Fluxion Biosciences microfluidics flow chamber. Experiments were performed in triplicate using whole blood from human and mice. FeCl 3 injury-induced carotid thrombosis was assessed by intravital microscopy as described.
Platelets labeled with calcein green 2. Whatman paper 0. Thrombus formation in real time was recorded using a high-speed electron-multiplying camera for 30 minutes or until occlusion occurred.
Videos were evaluated off-line using a Nikon computer-assisted image analysis program. The Micropoint laser ablation system Andor Technology was used to make an injury in the mesenteric arterioles as described. Fluorescent platelets labeled with calcein green 1. Infused platelets were isolated from adult months WT donor mice. The specific illumination of the area of interest was carried out through the microscope eyepiece. The power and frequency of pulses were regulated by software and empirically defined.
Videos were evaluated, and mean fluorescence intensity was calculated using a Nikon computer-assisted image analysis program. The TXB2 assay was done as described.
The supernatants were diluted with assay buffer. When 2-DG is added to cells, it is transported across the membrane and rapidly phosphorylated to 2-DGphosphate 2DG6P in the same manner as glucose and accumulates in the cell.
Luminescence produced by recombinant luciferase that is proportional to the concentration of 2DG6P was measured using a luminometer Spectramax i3X within 10 minutes after addition of detection reagent. For statistical analysis, GraphPad Prism software, version 7.
To determine the role of aerobic glycolysis in platelet function, PDKs activity in human and mouse platelets was inhibited with DCA, a potent inhibitor of all 4 forms of PDK The dose of DCA was chosen from published studies. At higher doses, DCA is known to induce apoptosis in cells. To rule out the possibility that observed impaired platelet aggregation could be related to apoptosis, we determined phosphatidylserine PS by Annexin V assay.
One of the earlier events of apoptosis is the translocation of PS from the inner side of the membrane to outside surface.
Annexin V, which has a high affinity for PS, is routinely used for detection of apoptosis using flow cytometry. We found that DCA up to a dose of mM did not induce apoptosis in human and mouse platelets supplemental Figure 1.
DCA inhibits agonist-induced platelet aggregation. A recent study suggested that glucose uptake and metabolism contribute to platelet function. We found that DCA did not inhibit glucose uptake in quiescent washed platelets Figure 2. Glucose uptake was increased by approximately twofold in response to suboptimal doses of agonists, including collagen, convulxin, thrombin, and ADP Figure 2. Both human and mouse platelets pretreated with DCA showed a significant decrease in glucose uptake in response to suboptimal doses of various agonists, suggesting an association between decreased glucose uptake and reduced platelet aggregation.
DCA inhibits glucose uptake in agonist-induced platelet activation. Measurement of 2-DG uptake in washed human A and mouse B platelets with a luminescence-based assay kit.
Luminescence produced by 2DG6P was monitored in resting and platelets response to suboptimal dose of collagen, convulxin, thrombin, and ADP. Recently, dichloroacetate DCA was proven to produce significant cytotoxic effects in certain tumor cells through this distinct mechanism. In this study, the effect of DCA on the metabolism of cervical cancer HeLa cells was explored and its synergistic growth inhibition with cisplatin was also evaluated.
For the evaluation of combination chemotherapy, HeLa cells were treated with a combination of DCA and cisplatin at various concentrations for 48 h. A modification of an antibody-mediated complement-dependent cytotoxicity CDC assay protocol developed for assessing antigen-specific antibodies in serum was used in order to assess alloantibody production 25 , At the end of the 7-day period of one-way MLRs, resting PBMCs similar to those used as stimulator cells in MLRs, albeit untreated, were counted and seeded in well plates at a number of 0.
The plates were incubated for 30 min on ice. Since cell-mediated cytotoxicity occurs in the course of an MLR 27 , and inevitably LDH is released in the supernatants and the used compounds affect the intensity of the MLRs, we avoided assessing antibody-mediated CDC with LDH release assay as it would provide us with erroneous data. Instead, we assessed cell survival by measuring the reduction of sodium 2,3-bis 2-methoxynitrosulfophenyl [ phenylamino -carbonyl]-2H-tetrazolium XTT , a yellow tetrazolium salt, to the orange formazan by metabolically viable active target cells.
Target cells were incubated with the XTT reagent for 1 h. Twelve such experiments were performed, each in triplicates and the results refer to the mean of the three measurements. The normality of the evaluated variables was confirmed by the one-sample Kolmogorov-Smirnov test. For comparison of means, paired t-test or one-way repeated measures analysis of variance ANOVA followed by Bonferronis correction test were used. The SPSS Cytotoxicity was None of the used compounds were cytotoxic.
Error bars correspond to 2 SD. The proliferation index was Two-way MLRs lasted 7 days. Six different two-way MLRs were performed. In this case, the proliferation index was Undiluted or diluted supernatants from these MLRs decreased target cell survival significantly to the One-way MLRs lasted 7 days.
Twelve different one-way MLRs were performed. CDC assay revealed that alloantibodies were present in the supernatants. Undiluted or diluted supernatants from LW6-treated one way MLRs increased target cell survival significantly from For this purpose, non-toxic concentrations of DCA and LW6 were used, which, however, are effective in inhibiting the aerobic glycolysis and Krebs cycle, respectively 20 , Their effect on T-cell clonal expansion was also assessed.
In consensus with a previous study 20 , DCA increased T-cell clonal expansion but only slightly. This is in contrast with another study, which has shown that DCA inhibits T-cell clonal expansion We used MLRs, which is a model of alloimmunity 27 , and DCA at a concentration of 1 mM, which is close to the blood concentrations of this drug when used for the treatment of hereditary lactic acidosis However, compared to the role of aerobic glycolysis, the role of Krebs cycle in T-cell activation has not been emphasized as Krebs cycle activity is required for an effective T-cell response 6 — Thus, regarding the T-cell clonal expansion part of cellular alloimmunity, the inhibitor of aerobic glycolysis DCA had a minor effect, whereas the inhibitor of the Krebs cycle LW6 decreased it significantly.
A number of studies using different murine experimental models have shown varying results regarding the effect of DCA on antibody production. In a previous study it was shown that DCA administration in lupus prone or normal mice enhances serum immunoglobulin levels 29 , whereas another study has shown that in lupus prone mice DCA had no effect on the levels of anti-dsDNA IgG 9.
On the other hand, in a murine model of collagen type II-induced arthritis, DCA alleviated arthritis only in female mice, in which it decreased anti-collagen type II antibodies without affecting the T cell response In addition, although upon activation murine B-cells exhibit a balanced increase in lactate production and oxygen consumption, the induction of glycolysis is critical for B-cell proliferation and antibody production since DCA abrogated both of these processes However, in that study, the human B cells, when used, were isolated and stimulated with a TLR9 activator This may be attributed to the direct effect of DCA on B cells.
Since plasma cells are mainly dependent on the Krebs cycle for covering their high energy requirements 12 , 16 — 18 , DCA-induced increased pyruvate entry into the mitochondria may enhance their ability to produce antibodies.
However, an indirect effect cannot be excluded. The combination of PLX a vemurafenib analogue with either of the two anti-diabetic biguanides, metformin and phenformin, showed synergistic inhibition of melanoma cell viability [ 35 ],[ 36 ]. Unlike DCA, metformin and phenformin both stimulate glycolysis and lactic acid production [ 37 ],[ 38 ], which could explain the growth-stimulating effects of metformin on some melanoma cell lines when used as a single agent.
In addition, the concentrations at which metformin was effective were above a therapeutically relevant level [ 35 ]. Phenformin was significantly more potent than metformin [ 36 ], but has been associated with a high risk of lactic acidosis [ 39 ], and was taken off the market for treatment of type 2 diabetes in many countries.
DCA, on the other hand, was here demonstrated to potentiate the effect of vemurafenib at concentrations down to 1 mM, and was previously shown to have few adverse effects when administered to patients [ 17 ],[ 19 ],[ 40 ]. This was reinforced by the demonstration that sensitivity to DCA was retained in melanoma cell lines with acquired resistance to vemurafenib. Although resistant cells showed an altered metabolic profile with significantly increased maximal mitochondrial respiration, as also shown by Corazao-Rozas et al.
Therefore, DCA could possibly provide a strategy to prevent the appearance of vemurafenib-tolerant subpopulations during initial treatment and thereby postpone or prevent the development of resistance.
We here provide a more elaborate understanding of the effects of DCA on the metabolism and growth of melanoma cells. Importantly, melanoma cells with acquired resistance to vemurafenib retained their sensitivity to DCA. These findings should encourage further investigation of this drug combination and the in vivo application of DCA.
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Cancer Lett. Br Med J. Invest New Drugs. Download references. You can also search for this author in PubMed Google Scholar. Correspondence to Per Guldberg. PG and CA planned and organized the study. CA performed the majority of the experiments and the processing of data. CD planned and performed the cell proliferation assay and helped interpret the results.
The final manuscript was read and approved by all authors. Additional file 1: Table S1. Forward, reverse and sequencing primers are denoted F, R and S, respectively. DOC 33 KB.
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