Dichloroacetate and vitamin C synergistically suppress proliferation, migration and invasion of glioma U87 and U251 cells / 中国药理学通报

Aim To investigate the effects of dichloroacetate(DCA)combined with vitamin C(VC)on the malignant behavior of glioma U87 and U251 cells, and to explore the potential mechanism. Methods U87 and U251 cells were treated with different concentrations of DCA alone or in combination with 5 mmol·L-1 VC. The proliferation rate of each group was detected by CCK-8 method and the cooperative index was calculated. U87 and U251 cells were treated with DMSO, 15 mmol·L-1 DCA, 5 mmol·L-1 VC and their combination. The changes of clonal formation, reactive oxygen species content, apoptosis, cell cycle, migration and invasion were detected via in vitro experiments, while the proliferation of U251 cells in vivo in each group was detected by subcutaneous tumor-forming model. Western blot was used to detect the expression levels and degradation rates of BCL2A1 and CDC25A in each group of cells after network pharmacological analysis of DCA and VC targets and their value in glioma, and the expression levels of CDK4, CDK6, cytochrome C, caspase-7 and cleaved-caspase-7 were detected. Results The combined index of 15 mmol·L-1 DCA and 5 mmol·L-1 VC was the highest. Compared with the control and single drug groups, the clonal formation, migration and invasion ability of cells in combination group in vitro significantly decreased, the proliferation rate in vivo also decreased, and the content of reactive oxygen species, apoptosis rate and G1 phase arrest rate significantly increased. BCL2A1 and CDC25A proteins were important targets of DCA and VC in glioma. Compared with the control and single-drug groups, the expression levels of BCL2A1, CDC25A, CDK4, and CDK6 in the combination group were significantly reduced, and the expression levels of cytochrome C and cleaved-caspase-7 markedly increased, and the protein degradation rates of BCL2A1 and CDC25A significantly increased in the combination group. Conclusions VC can cooperate with DCA to promote the degradation of BCL2A1 and CDC25A, and inhibit the malignant behavior of glioma cells.

Medical research progresses on sodium dichloroacetate / 药学实践杂志

Sodium dichloroacetate (DCA) is a small molecule drug usually administered orally. It has therapeutic effects against several diseases, such as metabolic syndrome, cardiovascular and cerebrovascular diseases, and several solid tumors. In this review, the research progresses of DCA in mechanism of action, pharmacological action and toxicological studies were summarized from the recent literatures on the pharmacological actions of DCA.

Synergistic anti-tumor effect of metformin and dichloroacetate in MFCW-ampIified neuroblastoma BE-2C cells / 中国药理学通报

To investigate the effect of metformin production of combined group compared with alone combined with dichloroacetate on the proliferation of A/FC/V-amplified neuroblastoma BE-2C cells and its mechanisms. Methods The inhibitory effects of metformin and dichloroacetate alone or in combination on BE-2C cells was measured by CCK-8 assay; the concentrations of glucose and lactic acid in the medium were measured by a glucose test kit and L-lactic acid assay kit; cell apoptosis was determined by flow cytometry (FCM) with Annexin V-FITC conjugated propidium iodide (PI) staining; the expressions of apoptosis related protein were detected by Western blot. Results Metformin and dichloroacetate showed significant proliferation inhibition activity on BE-2C cells; there was obviously decreased glucose uptake and lactic acid production of combined group compared with alone group (P <0. 01) ; the apoptotic rate was significantly higher in combined group compared with that in alone group (P <0. 01) ; Bax and cleaved caspasce-3 protein expression markedly increased in combined group, but Bcl-2 protein expression significantly increased compared with alone group (P <0. 01). Conclusions Metformin combined with dichloroacetate shows a significant synergistic anti-tumor effect against BE-2C cells by reducing the accumulation of lactic acid caused by metformin and inducing apoptosis.

Effect of dichloroacetate on colony-forming, invasion and migration of the T24 bladder cancer cells line and its mechanism / 实用医学杂志

Objective To study the effects of dichloroacetate (DCA) on cell colony-forming,cell invasion and cell migration of the bladder cancer cells and to study the underlying mechanism.Methods The bldder cancer cells T24 were randomly divided into two groups:the observation group and the control group.Cells in the observation groups were treated with 5 mmol/L,10 mmol/L and 20 mmol/L dichloroacetate,and the control group was treated with the same amount of dimethyl sulfoxide.Colony formation assays were detected with Giemsa staining.Cell wound scratch assay and Transwell assay were applied to evaluate the ability of the T24 cell invasion and migration.Realtime PCR and Western blot were applied to detect the expression of the epithelial-mesenchymal transition (EMT)-related marker,including E-cadherin,N-cadherin,vimentin,Snail and Slug.Results Compared with the control group,the colony formation assays of T24 cells constantly decreased along with the increased doses in the observation group(P ＜ 0.01).The cell wound scratch assay showed that the scratch width of the observation groups were significantly higher along with the increased doses and prolonged time than that in the control group (P ＜ 0.01).The transwell assay showed that the invasion ability of the observation groups were significantly discreased along with the increased doses than that in the the control group (P ＜ 0.01).The expression levels of E-cadherin mRNA and protein in combination the control group were higher than those in the the observation groups (P ＜ 0.05).However,the expression levels of N-cadherin,vimentin,Snail and Slug mRNAs and proteins in combination the control group were lower than those in the the observation groups (P ＜ 0.05).Conclusion Dichloroacetate can inhibit the colony-forming,invasion and migration of bladder cancer T24 cells,and its mechanism may inhibit the expression of epithelial mesenchymal transition in T24 cells by down-regulating the expression of nuclear transcription factor Snail and Slug.

The influence of dichloroacetate combined with cisplatin on the apoptosis of colorectal carcinoma cells HCT116 / 中华普通外科杂志

Objective To explore the effects of dichloroacetate (DCA) combined with cisplatin on the apoptosis of HCT116 and possible mechanisms.Methods The inhibitory effects of DCA and cisplatin alone or in combination on colorectal carcinoma cell line HCT116 were examined by MTT and Hoechst 33342 staining,the mitochondrial membrane potential changes were measured by Rodanmine123 staining under fluorescent microscope.The expression of bcl-2 was checked by qPCR.The activity of caspase-3 was assayed.Results DCA or cisplatin alone could inhibit the growth of HCT116 in a time and dose dependent manner.Compared with single drug treatment,there was significantly synergistic effect after treatment of DCA combined with cisplatin for 48 hours.Compared with the single drug treatment,the nuclear morphological changes such as chromatin condensation and fragmentation were more severe,and the mitochondrial transmembrane potential declines were markedly apparent for DCA + cisplatin group.The expression of bcl-2 gene in combination group was inhibited (P ＜ 0.05),and the activity of caspase-3 significantly increased (P ＜ 0.01).Conclusions DCA could inhibit the proliferation and induce the apoptosis of HCT116 cells in a time and dose dependent manner.The combination use of DCA and cisplatin has a synergistic effect on the biological action of HCT116.This may be attributed to lowering of mitochondrial transmembrane potential and the suppressed expression of bcl-2 gene.

Effect of LY294002 combined with dichloroacetate on apoptosis in human pulmonary arterial smooth muscle cells and AKT/GSK-3β/HK-2 signaling pathway / 中华麻醉学杂志

Objective To evaluate the effect of phosphatidylinositol-3 kinase inhibitor LY294002 combined with dichloroacetate on apoptosis in human pulmonary arterial smooth muscle cells (SMCs) and AKT/GSK-3β/HK-2 signaling pathway.Methods Human pulmonary arterial SMCs were seeded into culture plates at a density of 2 x 104 cells/ml after 3-5 passages.After being incubated for 72 h,the SMCs were cultured in the medium supplemented with 0.2％ fetal bovine serum for 24 h to induce starvation prior to experiments.The cells were then randomly divided into 6 groups (n =6 each) using a random number table:control group (group C),positive control group (group F),LY294002 group (group L),different concentrations of dichloroacetate groups (D1 and D2 groups),and LY294002 combined with dichloroacetate group (group LD1).In group C,the cells were cultured in the medium supplemented with 0.2％ fetal bovine serum.In F,L,D1,D2 and LD1 groups,the cells were cultured in the medium supplemented with 10％ fetal bovine serum.LY294002 2 μmol/L was added to the medium in group L.Dichloroacetate l0 and 20 mmol/L were added to the medium in D1 and D2 groups,respectively.In group LD1,LY294002 (2 μmol/L) was added,and 30 min later dichloroacetate 10 mmol/L was added to the medium in LD1 group.The cells were incubated for 48 h.Flow cytometry was used to measure the cell apoptosis and mitochondrial membrane potential.The expression of phosphorylated AKT (p-Akt),phosphorylated glycogen synthase kinase 3β (p-GSK-3β),and hexokinase-2 (HK-2) was detected using Western blot.Apoptosis rate was calculated.Results Compared with group C,apoptosis rate was significantly increased,and mitochondrial membrane potential was decreased in D2 and LD1 groups,the expression of p-Akt,p-GSK-3β and HK-2 was up-regulated in group F,and no significant changes were found in apoptosis rate and mitochondrial membrane potential in F,L and D1 groups.Compared with group D2,apoptosis rate was significantly increased,mitochondrial membrane potential was decreased,and the expression of p-Akt,p-GSK-3β and HK-2 was down-regulated in LD1 group.The expression of p-Akt,p-GSK-3β and HK-2 was significantly lower in D2 and LD1 groups than in group F.Conclusion LY294002 combined with dichloroacetate can promote apoptosis in human pulmonary arterial SMCs possibly through blocking AKT/GSK-3β/HK-2 signaling pathway.

Low dose of dichloroacetate infusion reduces blood lactate after submaximal exercise in horses / Baixa dose de infusão de dicloroacetato reduz o lactato sanguíneo após exercício submáximo em cavalos

The acute administration of an indirect activator of the enzyme pyruvate dehydrogenase (PDH) in human athletes causes a reduction in blood lactate level during and after exercise. A single IV dose (2.5m.kg-1) of dichloroacetate (DCA) was administered before a submaximal incremental exercise test (IET) with five velocity steps, from 5.0 m.s-1 for 1 min to 6.0, 6.5, 7.0 and 7.5m.s-1 every 30s in four untrained mares. The blood collections were done in the period after exercise, at times 1, 3, 5, 10, 15 and 20 min. Blood lactate and glucose (mM) were determined electro-enzymatically utilizing a YSI 2300 automated analyzer. There was a 15.3% decrease in mean total blood lactate determined from the values obtained at all assessment times in both trials after the exercise. There was a decrease in blood lactate 1, 3, 5, 10, 15 and 20 min after exercise for the mares that received prior DCA treatment, with respective mean values of 6.31±0.90 vs 5.81±0.50, 6.45±1.19 vs 5.58±1.06, 6.07±1.56 vs 5.26±1.12, 4.88±1.61 vs 3.95±1.00, 3.66±1.41 vs 2.86±0.75 and 2.75±0.51 vs 2.04±0.30. There was no difference in glucose concentrations. By means of linear regression analysis, V140, V160, V180 and V200 were determined (velocity at which the rate heart is 140, 160, 180, and 200 beats/minute, respectively). The velocities related to heart rate did not differ, indicating that there was no ergogenic effect, but prior administration of a relatively low dose of DCA in mares reduced lactatemia after an IET.

A administração aguda de um ativador indireto da enzima piruvato desidrogenase (PD) em atletas da espécie humana provoca redução na concentração de lactato sanguíneo durante e após exercício. Uma dose única, intravenosa de 2.5m.kg-1 de dicloroacetato (DCA) foi administrada antes de um exercício teste incremental submáximo (ETI) com cinco etapas de velocidade sendo 5,0 ms-1 por 1 minuto e 6,0, 6,5, 7,0, e 7,5 ms-1 a cada 30 segundos em quatro éguas destreinadas. As coletas de sangue foram realizadas no período após o exercício, nos momentos de 1, 3, 5, 10, 15 e 20 min. Lactato e glicose (mM) foram determinados electro-enzimaticamente utilizando um analisador automático (YSI 2300). Houve redução de 15,3% no lactato sanguíneo total médio que foi determinado a partir dos valores obtidos em todos os momentos de avaliação em ambos os testes, após o exercício. Houve diminuição na lactatemia 1, 3, 5, 10, 15 e 20 minutos após exercício para as éguas que receberam infusão de DCA, com os respectivos valores médios de 6,31±0,90 versus 5,81±0,50, 6,45±1,19 versus 5,58±1,06, 6,07±1,56 versus 5,26±1,12, 4,88±1,61 versus 3,95±1,00, 3,66±1,41 versus 2,86±0,75 e 2,75±0,51 versus 2,04±0,30. Não houve diferença nas concentrações de glicose. Por meio de análise de regressão linear, V140, V160, V180 e V200 foram determinados (velocidades em que as taxas cardíacas alcançam 140, 160, 180 e 200 bpm, respectivamente). As velocidades relacionadas com a frequência cardíaca não diferiram, indicando que não houve efeito ergogênico, mas a administração prévia de uma dose relativamente baixa de DCA em éguas reduziu a lactatemia após um ETI.

Regulation of Muscle Pyruvate Dehydrogenase Complex in Insulin Resistance: Effects of Exercise and Dichloroacetate

Since the mitochondrial pyruvate dehydrogenase complex (PDC) controls the rate of carbohydrate oxidation, impairment of PDC activity mediated by high-fat intake has been advocated as a causative factor for the skeletal muscle insulin resistance, metabolic syndrome, and the onset of type 2 diabetes (T2D). There are also situations where muscle insulin resistance can occur independently from high-fat dietary intake such as sepsis, inflammation, or drug administration though they all may share the same underlying mechanism, i.e., via activation of forkhead box family of transcription factors, and to a lower extent via peroxisome proliferator-activated receptors. The main feature of T2D is a chronic elevation in blood glucose levels. Chronic systemic hyperglycaemia is toxic and can lead to cellular dysfunction that may become irreversible over time due to deterioration of the pericyte cell's ability to provide vascular stability and control to endothelial proliferation. Therefore, it may not be surprising that T2D's complications are mainly macrovascular and microvascular related, i.e., neuropathy, retinopathy, nephropathy, coronary artery, and peripheral vascular diseases. However, life style intervention such as exercise, which is the most potent physiological activator of muscle PDC, along with pharmacological intervention such as administration of dichloroacetate or L-carnitine can prove to be viable strategies for treating muscle insulin resistance in obesity and T2D as they can potentially restore whole body glucose disposal.

Pyruvate Dehydrogenase Kinase as a Potential Therapeutic Target for Malignant Gliomas

Metabolic aberrations in the form of altered flux through key metabolic pathways are the major hallmarks of several life-threatening malignancies including malignant gliomas. These adaptations play an important role in the enhancement of the survival and proliferation of gliomas at the expense of the surrounding normal/healthy tissues. Recent studies in the field of neurooncology have directly targeted the altered metabolic pathways of malignant tumor cells for the development of anti-cancer drugs. Aerobic glycolysis due to elevated production of lactate from pyruvate regardless of oxygen availability is a common metabolic alteration in most malignancies. Aerobic glycolysis offers survival advantages in addition to generating substrates such as fatty acids, amino acids and nucleotides required for the rapid proliferation of cells. This review outlines the role of pyruvate dehydrogenase kinase (PDK) in gliomas as an inhibitor of pyruvate dehydrogenase that catalyzes the oxidative decarboxylation of pyruvate. An in-depth investigation on the key metabolic enzyme PDK may provide a novel therapeutic approach for the treatment of malignant gliomas.

Glucose metabolism of lung adenocarcinoma A549 cells and its correlation with taxol-resistance / 肿瘤

Objective: To investigate the difference in glucose metabolism between lung adenocarcinoma A549 cell line and its taxol-resistant (A549/taxol) cell line, and to determine the effect of DCA (dichloroacetate) on glucose metabolism of these two cell lines. Methods: The taxol resistance of A549 and A549/taxol cell lines were firstly determined by CCK-8 (cell counting kit-8) assay. Then the productions of CO2 and lipid in A549 and A549/taxol cells were detected by scintillation counter after treatment with 14C-glucose. Furthermore, the uptake of 18F-FDG (18F-2-deoxy-β-D- glucose) and the production of lactate were detected by y-counter and lactate measurement kit, respectively. Results: All of CO2 production level, the 18F-FDG uptake rate and the lactate production level after treatment with 6-14C-glucose were lower in A549/taxol cells than those in A549 cells. The level of CO2 production after treatment with 6-14C-glucose was significantly higher in A549 cells treated with DCA than that in A549 cells without DCA treatment. However, DCA had no effect on the CO2 production after treatment with 6-14C-glucose in A549/taxol cells. Conclusion: There is a certain extent of mitochondrial oxidative breathing suppression in A549/taxol cells. DCA can promote mitochondrial oxidative respiration in A549 cells, but has no effect on that in A549/taxol cells. Copyright © 2013 by TUMOR.

Effect of dichloroacetate on expression of Kv1.5 in a rat model of pulmonary arterial hypertension / 中华麻醉学杂志

Objective To investigate the effect of dichloroacetate on the expression of Kv1.5 in a rat model of pulmonary arterial hypertension (PAH) .Methods Thirty-two male SD rats weighing 200-250 g were randomly divided into 4 groups ( n = 8 each): normal control group (group C), dichloroacetate control group (group D),PAH group, and PAH + dichloroacetate group (group PD). PAH was induced by left lung resection combined with subcutaneous injection of monocrotaline 60 mg/kg in PAH and PD groups. In group PD, dichloroacetate 80 mg/kg was given through a gastric tube into stomach once a day for 28 consecutive days after monocrotaline injection,while the equal volume of normal saline was given instead of dichloroacetate in group PAH. Group D only received dichloroacetate 80 mg/kg through a gastric tube into stomach once a day for 28 consecutive days. Pulmonary arterial pressure (PAP) was measured at day 28 after monocrotaline injection. The rats were then sacrificed and lung tissues were removed to calculate the percentage of thickness of the tunica media of pulmonary artery and right venicular hypertrophy index and to determine the proliferating cell nuclear antigen (PCNA) and Kv1.5 protein expression (by Western blot) and Kv1.5 mRNA expression (by RT-PCR).Results Compared with group C, the PAP,percentage of thickness of the tunica media, right ventricular hypertrophy index were significantly increased, Kv1.5 mRNA and protein expression was down-regulated and PCNA expression was up-regulated in groups PAH and PD ( P ＜ 0.05). Compared with group PAH, the PAP, percentage of thickness of the tunica media, right ventricular hypertrophy index were significantly decreased, Kv1.5 mRNA and protein expression was up-regulated and PCNA expression was down-regulated in group PD (P ＜ 0.05). There was no significant difference in the indexes mentioned above between group C and group D ( P ＞ 0.05). Conclusion Dichloroacetat alleviates PAH through upregulating Kv1.5 expression in lung tissues and inhibiting pulmonary vascular remodeling in rats.

Inhibition of Sarcoplasmic Reticulum Ca2+ Uptake by Pyruvate and Fatty Acid in H9c2 Cardiomyocytes: Implications for Diabetic Cardiomyopathy

High extracellular glucose concentration was reported to suppress intracellular Ca2+ clearing through altered sarcoplasmic reticulum (SR) function. In the present study, we attempted to elucidate the effects of pyruvate and fatty acid on SR function and reveal the mechanistic link with glucose-induced SR dysfunction. For this purpose, SR Ca2+-uptake rate was measured in digitonin-permeabilized H9c2 cardiomyocytes cultured in various conditions. Exposure of these cells to 5 mM pyruvate for 2 days induced a significant suppression of SR Ca2+-uptake, which was comparable to the effects of high glucose. These effects were accompanied with decreased glucose utilization. However, pyruvate could not further suppress SR Ca2+-uptake in cells cultured in high glucose condition. Enhanced entry of pyruvate into mitochondria by dichloroacetate, an activator of pyruvate dehydrogenase complex, also induced suppression of SR Ca2+-uptake, indicating that mitochondrial uptake of pyruvate is required in the SR dysfunction induced by pyruvate or glucose. On the other hand, augmentation of fatty acid supply by adding 0.2 to 0.8 mM oleic acid resulted in a dose-dependent suppression of SR Ca2+-uptake. However, these effects were attenuated in high glucose-cultured cells, with no significant changes by oleic acid concentrations lower than 0.4 mM. These results demonstrate that (1) increased pyruvate oxidation is the key mechanism in the SR dysfunction observed in high glucose-cultured cardiomyocytes; (2) exogenous fatty acid also suppresses SR Ca2+-uptake, presumably through a mechanism shared by glucose.

Antiapoptotic effects of rat hypertrophic cardiomyocytes induced with hypoxia/reoxygenation by dichloroacetate / 第三军医大学学报

Objective To investigate the effect of hypoxia/reoxygenation on apoptosis of rat hypertrophic cardiomyocytes and the metabolic pathway changes of hypertrophic cardiomyocytes by dichloroacetate.Methods The isolated ventricular cardiomyocytes from neonatal Wistar rat were cultured and purified with differential attachment,then BrdU was added to reduce the rate of non-myocytes.The cultured cells were identified by morphology,spontaneous contraction and specific immunocytochemical stain.Hypertrophic cardiomyocyte model induced by angiotensin Ⅱ was set up and assessed by -Leu incorporation.The cultured media were replaced by low glucose DMEM before hypoxia.The cardiomyocytes were incubated at 37 ℃ in an air-tight incubator containing 92% N_(2),5% CO_(2),3% O_(2) for 24 h to simulate hypoxia,then under the condition of 23% O_(2),5% CO_(2) for 4 h to simulate reoxygenation.Apoptotic cells was evaluated by a modified TUNEL assay(DeadEnd~(TM) Colorimetric TUNEL).Results The apoptotic rate of cardiomyocytes increased with time prolongation of hypoxia/reoxygenation(H/R).The apoptotic cell model induced by H/R in rat hypertropic cardiomyocytes was established successfully.When the hypertropic cardiomyocytes were pretreated with Dichloroacetate,the apoptotic rate decreased in a dose-dependent manner.The metabolic pathway changes had effect on the apoptosis of rat hypertrophic cardiomyocytes.Conclusion By collagense digestion,it is convenient to obtain primary neonatal Wistar rat ventricular cardiomyocytes. This method is one of vital research tools in the fields of cardiac hypertrophy.Angiotensin Ⅱ could accelerate cellular proliferation,structural protein biosynthesis and cardiac hypertrophy.Dichloroacetate has the effect of antiapoptosis.

Effects of Insulin, 2-Deoxyglucose and Dichloroacetate on Acute Focal Cerebral Ischemia in Rats

Hyperglycemia has been reported to worsen the tolerance of the brain to ischemia, and it has therefore been recommended that patient undergoing neurosurgical procedures not receive glucose-containing solutions. Since ischemic events lead to increased lactate production and accumulation and hence neuronal damage, the present study was designed to test the effect of insulin-induced hypoglycemia and decreased lactate by 2-Deoxyglucose and Dichloroacetate on focal cerebral ischemia in rats. Although the pre and post-ischemic blood glucose levels of control group and Dichloroacetate group showed no change, the blood glucose level of 2-Deoxyglucose group showed a significant increase(p=0.001), and insulim group a significant decrease(p=0.004). The reducing effects on the infarct zone in these three treated groups were found with statistical significance. As compared with control group, the tissue lactate levels of treated groups were decreased in both infarct zone and border zone but these data did not show statistical significance. From these observations, it is suggested that reduction of lactate production and accumulation could be beneficial by affording neuronal protection in ischemic tissues.

Determination of diisopropylamide dichloroacetate concentrations in urine by GC-HS / 重庆医科大学学报

Objective:To establish a method for determination of diisopropylamide dichloroacetate(DADA) concentrations in urine.Methods:GC-HS was applied to quantitative analysis.Using Agilent DB-5MS capillary column(30m?0.25mm),the column temperature was ascended from 40℃ to 200℃ according to program.Initial temperature was 40℃ and maintained 2 mins,then temperature rose to 80℃ at 5℃/min rate and maintained 2 mins,finally temperature rose to 200℃ at 20℃/min rate and maintained 8 mins.The injector temperature was 200℃.The detector temperature was 300℃.Nitrogen was used as carrier gas,and the flow rate was 1 ml/min.The flow rate of hydrogen was 35 ml/min.The flow rate of air was 350 ml/min.An internal standard was secondary butanol.Results:The calibration curve was linear within 8～800?g/ml.The recovery rate of the extraction were 88.75%～98.06%.The recovery rate were 97.36%～103.25%。Within-day and between-day RSD were 2.95%～6.62% and 2.45%～5.61% respectively.Detectable concentration limited was 2?g/mL.Conclusion:The GC-HS method is simple,reliable,and suitable for urine DADA concentration analysis.