Metabolic reorganization in winter: Regulation of pyruvate dehydrogenase (PDH) during long-term freezing and anoxia

Wood frogs, Rana sylvatica, can undergo prolonged periods of whole body freezing during winter, locking as much as 65–70% of total body water into extracellular ice and imposing both anoxia and dehydration on their cells. Metabolic rate depression (MRD) is an adaptation used by R. sylvatica to survive these environmental stresses, where a finite amount of ATP generated through anaerobic metabolism is directed towards maintaining pro-survival functions, while most ATP-expensive cellular processes are temporarily reduced in function. Pyruvate dehydrogenase (PDH) is a vital metabolic enzyme that links anaerobic glycolysis to the aerobic TCA cycle and is an important regulatory site in MRD. PDH enzymatic activity is regulated via reversible protein phosphorylation in response to energetic demands of cells. This study explored the posttranslational regulation of PDH at three serine sites (S232, S293, S300) on the catalytic E1α subunit along with protein expression of four pyruvate dehydrogenase kinases (PDHK1-4) in response to 24 h Freezing, 8 h Thaw, 24 h Anoxia, and 4 h Recovery in the liver and skeletal muscle of R. sylvatica using Luminex multiplex technology and western immunoblotting. Overall, inhibitory regulation of PDH was evident during 24 h Freezing and 24 h Anoxia, which could indicate a notable reduction in glycoytic flux and carbon entry into the tricarboxylic acid cycle as part of MRD. Furthermore, the expression of PDHK1-4 and phosphorylation of PDH at S232, S293, and S300 were highly tissue and stress-specific, indicative of how different tissues respond differently to stress within the same organism.     

Mitochondrial free fatty acid β-oxidation supports oxidative phosphorylation and proliferation in cancer cells

Oxidative phosphorylation (OxPhos) is functional and sustains tumor proliferation in several cancer cell types. To establish whether mitochondrial β-oxidation of free fatty acids (FFAs) contributes to cancer OxPhos functioning, its protein contents and enzyme activities, as well as respiratory rates and electrical membrane potential (ΔΨm) driven by FFA oxidation were assessed in rat AS-30D hepatoma and liver (RLM) mitochondria. Higher protein contents (1.4–3 times) of β-oxidation (CPT1, SCAD) as well as proteins and enzyme activities (1.7–13-times) of Krebs cycle (KC: ICD, 2OGDH, PDH, ME, GA), and respiratory chain (RC: COX) were determined in hepatoma mitochondria vs. RLM. Although increased cholesterol content (9-times vs. RLM) was determined in the hepatoma mitochondrial membranes, FFAs and other NAD-linked substrates were oxidized faster (1.6–6.6 times) by hepatoma mitochondria than RLM, maintaining similar ΔΨm values. The contents of β-oxidation, KC and RC enzymes were also assessed in cells. The mitochondrial enzyme levels in human cervix cancer HeLa and AS-30D cells were higher than those observed in rat hepatocytes whereas in human breast cancer biopsies, CPT1 and SCAD contents were lower than in human breast normal tissue. The presence of CPT1 and SCAD in AS-30D mitochondria and HeLa cells correlated with an active FFA utilization in HeLa cells. Furthermore, the β-oxidation inhibitor perhexiline blocked FFA utilization, OxPhos and proliferation in HeLa and other cancer cells. In conclusion, functional mitochondria supported by FFA β-oxidation are essential for the accelerated cancer cell proliferation and hence anti-β-oxidation therapeutics appears as an alternative promising approach to deter malignant tumor growth.     

Glycolysis-mediated control of blood-brain barrier development and function

The blood-brain barrier (BBB) consists of differentiated cells integrating in one ensemble to control transport processes between the central nervous system (CNS) and peripheral blood. Molecular organization of BBB affects the extracellular content and cell metabolism in the CNS. Developmental aspects of BBB attract much attention in recent years, and barriergenesis is currently recognized as a very important and complex mechanism of CNS development and maturation. Metabolic control of angiogenesis/barriergenesis may be provided by glucose utilization within the neurovascular unit (NVU). The role of glycolysis in the brain has been reconsidered recently, and it is recognized now not only as a process active in hypoxic conditions, but also as a mechanism affecting signal transduction, synaptic activity, and brain development. There is growing evidence that glycolysis-derived metabolites, particularly, lactate, affect barriergenesis and functioning of BBB. In the brain, lactate produced in astrocytes or endothelial cells can be transported to the extracellular space via monocarboxylate transporters (MCTs), and may act on the adjoining cells via specific lactate receptors. Astrocytes are one of the major sources of lactate production in the brain and significantly contribute to the regulation of BBB development and functioning. Active glycolysis in astrocytes is required for effective support of neuronal activity and angiogenesis, while endothelial cells regulate bioavailability of lactate for brain cells adjusting its bidirectional transport through the BBB. In this article, we review the current knowledge with regard to energy production in endothelial and astroglial cells within the NVU. In addition, we describe lactate-driven mechanisms and action of alternative products of glucose metabolism affecting BBB structural and functional integrity in developing and mature brain.     

Short and prolonged exposure to hyperglycaemia in human fibroblasts and endothelial cells: Metabolic and osmotic effects

High blood glucose levels are the main feature of diabetes. However, the underlying mechanism linking high glucose concentration to diabetic complications is still not fully elucidated, particularly with regard to human physiology. Excess of glucose is likely to trigger a metabolic response depending on the cell features, activating deleterious pathways involved in the complications of diabetes. In this study, we aim to elucidate how acute and prolonged hyperglycaemia alters the biology and metabolism in human fibroblasts and endothelial cells.We found that hyperglycaemia triggers a metabolic switch from oxidative phosphorylation to glycolysis that is maintained over prolonged time. Moreover, osmotic pressure is a major factor in the early metabolic response, decreasing both mitochondrial transmembrane potential and cellular proliferation. After prolonged exposure to hyperglycaemia we observed decreased mitochondrial steady-state and uncoupled respiration, together with a reduced ATP/ADP ratio. At the same time, we could not detect major changes in mitochondrial transmembrane potential and reactive oxygen species.We suggest that the physiological and metabolic alterations observed in healthy human primary fibroblasts and endothelial cells are an adaptive response to hyperglycaemia. The severity of metabolic and bioenergetics impairment associated with diabetic complications may occur after longer glucose exposure or due to interactions with cell types more sensitive to hyperglycaemia.     

Interaction between APC and Fen1 during breast carcinogenesis

Aberrant DNA base excision repair (BER) contributes to malignant transformation. However, inter-individual variations in DNA repair capacity plays a key role in modifying breast cancer risk. We review here emerging evidence that two proteins involved in BER – adenomatous polyposis coli (APC) and flap endonuclease 1 (Fen1) – promote the development of breast cancer through novel mechanisms. APC and Fen1 expression and interaction is increased in breast tumors versus normal cells, APC interacts with and blocks Fen1 activity in Pol-β-directed LP-BER, and abrogation of LP-BER is linked with cigarette smoke condensate-induced transformation of normal breast epithelial cells. Carcinogens increase expression of APC and Fen1 in spontaneously immortalized human breast epithelial cells, human colon cancer cells, and mouse embryonic fibroblasts. Since APC and Fen1 are tumor suppressors, an increase in their levels could protect against carcinogenesis; however, this does not seem to be the case. Elevated Fen1 levels in breast and lung cancer cells may reflect the enhanced proliferation of cancer cells or increased DNA damage in cancer cells compared to normal cells. Inactivation of the tumor suppressor functions of APC and Fen1 is due to their interaction, which may act as a susceptibility factor for breast cancer. The increased interaction of APC and Fen1 may occur due to polypmorphic and/or mutational variation in these genes. Screening of APC and Fen1 polymorphic and/or mutational variations and APC/Fen1 interaction may permit assessment of individual DNA repair capability and the risk for breast cancer development. Such individuals might lower their breast cancer risk by reducing exposure to carcinogens. Stratifying individuals according to susceptibility would greatly assist epidemiologic studies of the impact of suspected environmental carcinogens. Additionally, a mechanistic understanding of the interaction of APC and Fen1 may provide the basis for developing new and effective targeted chemopreventive and chemotherapeutic agents.     

Acute fructose intake suppresses fasting-induced hepatic gluconeogenesis through the AKT-FoxO1 pathway

Excessive intake of fructose increases lipogenesis in the liver, leading to hepatic lipid accumulation and development of fatty liver disease. Metabolic alterations in the liver due to fructose intake have been reported in many studies, but the effect of fructose administration on hepatic gluconeogenesis is not fully understood. The aim of this study was to evaluate the acute effects of fructose administration on fasting-induced hepatic gluconeogenesis. C57BL/6J mice were administered fructose solution after 14 h of fasting and plasma insulin, glucose, free fatty acids, and ketone bodies were analysed. We also measured phosphorylated AKT and forkhead box O (FoxO) 1 protein levels and gene expression related to gluconeogenesis in the liver. Furthermore, we measured glucose production from pyruvate after fructose administration. Glucose-administered mice were used as controls. Fructose administration enhanced phosphorylation of AKT in the liver, without increase of blood insulin levels. Blood free fatty acids and ketone bodies concentrations were as high as those in the fasting group after fructose administration, suggesting that insulin-induced inhibition of lipolysis did not occur in mice administered with fructose. Fructose also enhanced phosphorylation of FoxO1 and suppressed gluconeogenic gene expression, glucose-6-phosphatase activity, and glucose production from pyruvate. The present study suggests that acute fructose administration suppresses fasting-induced hepatic gluconeogenesis in an insulin-independent manner.     

Bongkrekic acid facilitates glycolysis in cultured cells and induces cell death under low glucose conditions

Bongkrekic acid (BKA) inhibits adenine nucleotide translocator (ANT) and suppresses ADP/ATP exchange in the mitochondrial inner membrane. Previously, we demonstrated that BKA exhibited cytotoxic effects on 4T1 tumor cells, depending on the cell number in the culture, but not on NIH3T3 cells. However, the cause of this differential sensitivity was unelucidated. Here we demonstrate that BKA reduced the O₂ consumption in both cell lines and increased the mitochondrial membrane potential, thereby facilitating glucose consumption. BKA reduced cellular ATP in 4T1 cells in a dose-dependent manner but not in NIH3T3 cells. The cellular ATP of 4T1 cells was decreased with a reduced glucose concentration in the media, but that of NIH3T3 cells remained constant. We also demonstrated that BKA-induced cell death in both cell lines in low glucose media; however, the susceptibility to the reduced glucose concentration was slightly higher in 4T1 cells, which may be attributed to the difference in the dependency on glycolysis as their energy source. These results indicate that 4T1 tumor cells rely heavily on glucose for energy production. Our data demonstrate that BKA disturbs ATP production in mitochondria and increases the susceptibility to a low glucose condition.     

Mitochondria: A crossroads for lipid metabolism defect in neurodegeneration with brain iron accumulation diseases

Neurodegeneration with brain iron accumulation (NBIA) comprises a group of brain iron deposition syndromes that lead to mixed extrapyramidal features and progressive dementia. Exact pathologic mechanism of iron deposition in NBIA remains unknown. However, it is becoming increasingly evident that many neurodegenerative diseases are hallmarked by metabolic dysfunction that often involves altered lipid profile. Among the identified disease genes, four encode for proteins localized in mitochondria, which are directly or indirectly implicated in lipid metabolism: PANK2, CoASY, PLA2G6 and C19orf12. Mutations in PANK2 and CoASY, both implicated in CoA biosynthesis that acts as a fatty acyl carrier, lead, respectively, to PKAN and CoPAN forms of NBIA. Mutations in PLA2G6, which plays a key role in the biosynthesis and remodeling of membrane phospholipids including cardiolipin, lead to PLAN. Mutations in C19orf12 lead to MPAN, a syndrome similar to that caused by mutations in PANK2 and PLA2G6. Although the function of C19orf12 is largely unknown, experimental data suggest its implication in mitochondrial homeostasis and lipid metabolism. Altogether, the identified mutated proteins localized in mitochondria and associated with different NBIA forms support the concept that dysfunctions in mitochondria and lipid metabolism play a crucial role in the pathogenesis of NBIA.This article is part of a Directed Issue entitled: Energy Metabolism Disorders and Therapies.     

Resveratrol and curcumin synergistically induces apoptosis in cigarette smoke condensate transformed breast epithelial cells through a p21Waf1/Cip1 mediated inhibition of Hh-Gli signaling

Combination therapy using two or more small molecule inhibitors of aberrant signaling cascade in aggressive breast cancers is a promising therapeutic strategy over traditional monotherapeutic approaches. Here, we have studied the synergistic mechanism of resveratrol and curcumin induced apoptosis using in vitro (cigarette smoke condensate mediated transformed breast epithelial cell, MCF-10A-Tr) and in vivo (tumor xenograft mice) model system. Resveratrol exposure increased the intracellular uptake of curcumin in a dose dependent manner and caused apoptosis in MCF-10A-Tr cells. Approximately, ten fold lower IC₅₀ value was noted in cells treated with the combination of resveratrol (3 μM) and curcumin (3 μM) in comparison to 30 μM of resveratrol or curcumin alone. Resveratrol + curcumin combination caused apoptosis by increasing Bax/Bcl-xL ratio, Cytochrome C release, cleaved product of PARP and caspase 3 in cells. Interestingly, this combination unaltered the protein expressions of WNT-TCF and Notch signaling components, β-catenin and cleaved notch-1 val1744, respectively. Furthermore, the combination also significantly decreased the intermediates of Hedgehog-Gli cascade including SMO, SHH, Gli-1, c-MYC, Cyclin-D1, etc. and increased the level of p21^Waf/Cip1 in vitro and in vivo. A significant reduction of Gli- promoter activity was noted in combinational drug treated cells in comparison to individual drug treatment. Un-alteration of the expressions of the above proteins and Gli1 promoter activity in p21^Waf/Cip1 knockout cells suggests this combination caused apoptosis through p21^Waf/Cip1. Thus, our findings revealed resveratrol and curcumin synergistically caused apoptosis in cigarette smoke induced breast cancer cells through p2  ^Waf/Cip1 mediated inhibition of Hedgehog-Gli cascade.     

Exemestane metabolites suppress growth of estrogen receptor-positive breast cancer cells by inducing apoptosis and autophagy: A comparative study with Exemestane

Around 60–80% of all breast tumors are estrogen receptor-positive. One of the several therapeutic approaches used for this type of cancers is the use of aromatase inhibitors. Exemestane is a third-generation steroidal aromatase inhibitor that undergoes a complex and extensive metabolism, being catalytically converted into chemically active metabolites. Recently, our group showed that the major exemestane metabolites, 17β-hydroxy-6-methylenandrosta-1,4-dien-3-one and 6-(hydroxymethyl)androsta-1,4,6-triene-3,17-dione, as well as, the intermediary metabolite 6β-Spirooxiranandrosta-1,4-diene-3,17-dione, are potent aromatase inhibitors in breast cancer cells. In this work, in order to better understand the biological mechanisms of exemestane in breast cancer and the effectiveness of its metabolites, it was investigated their effects in sensitive and acquired-resistant estrogen receptor-positive breast cancer cells. Our results indicate that metabolites induced, in sensitive breast cancer cells, cell cycle arrest and apoptosis via mitochondrial pathway, involving caspase-8 activation. Moreover, metabolites also induced autophagy as a promoter mechanism of apoptosis. In addition, it was demonstrated that metabolites can sensitize aromatase inhibitors-resistant cancer cells, by inducing apoptosis. Therefore, this study indicates that exemestane after metabolization originates active metabolites that suppress the growth of sensitive and resistant breast cancer cells. It was also concluded that, in both cell lines, the biological effects of metabolites are different from the ones of exemestane, which suggests that exemestane efficacy in breast cancer treatment may also be dependent on its metabolites.     

An in situ study of bioenergetic properties of human colorectal cancer: The regulation of mitochondrial respiration and distribution of flux control among the components of ATP synthasome

The aim of this study is to characterize the function of mitochondria and main energy fluxes in human colorectal cancer (HCC) cells. We have performed quantitative analysis of cellular respiration in post-operative tissue samples collected from 42 cancer patients. Permeabilized tumor tissue in combination with high resolution respirometry was used.Our results indicate that HCC is not a pure glycolytic tumor and the oxidative phosphorylation (OXPHOS) system may be the main provider of ATP in these tumor cells. The apparent Michaelis–Menten constant (K_m) for ADP and maximal respiratory rate (V_m) values were calculated for the characterization of the affinity of mitochondria for exogenous ADP: normal colon tissue displayed low affinity (K_m = 260 ± 55 μM) whereas the affinity of tumor mitochondria was significantly higher (K_m = 126 ± 17 μM). But concurrently the V_m value of the tumor samples was 60–80% higher than that in control tissue. The reason for this change is related to the increased number of mitochondria. Our data suggest that in both HCC and normal intestinal cells tubulin β-II isoform probably does not play a role in the regulation of permeability of the MOM for adenine nucleotides.The mitochondrial creatine kinase energy transfer system is not functional in HCC and our experiments showed that adenylate kinase reactions could play an important role in the maintenance of energy homeostasis in colorectal carcinomas instead of creatine kinase.Immunofluorescent studies showed that hexokinase 2 (HK-2) was associated with mitochondria in HCC cells, but during carcinogenesis the total activity of HK did not change. Furthermore, only minor alterations in the expression of HK-1 and HK-2 isoforms have been observed.Metabolic Control analysis showed that the distribution of the control over electron transport chain and ATP synthasome complexes seemed to be similar in both tumor and control tissues. High flux control coefficients point to the possibility that the mitochondrial respiratory chain is reorganized in some way or assembled into large supercomplexes in both tissues.     

DNA methylation and histone deacetylation regulating insulin sensitivity due to chronic cold exposure

In this study, we investigated the causal relationship between chronic cold exposure and insulin resistance and the mechanisms of how DNA methylation and histone deacetylation regulate cold-reduced insulin resistance. 46 adult male mice from postnatal day 90–180 were randomly assigned to control group and cold-exposure group. Mice in cold-exposure group were placed at temperature from -1 to 4 °C for 30 days to mimic chronic cold environment. Then, fasting blood glucose, blood insulin level and insulin resistance index were measured with enzymatic methods. Immunofluorescent labeling was carried out to visualize the insulin receptor substrate 2 (IRS2), Obese receptor (Ob-R, a leptin receptor), voltage-dependent anion channel protein 1 (VDAC1), cytochrome C (cytC), 5-methylcytosine (5-mC) positive cells in hippocampal CA1 area. Furthermore, the expressions of some proteins mentioned above were detected with Western blot. The results showed: ① Chronic cold exposure could reduce the insulin resistance index (P < 0.01) and increase the number of IRS2 positive cells and Ob-R positive cells in hippocampus (P < 0.01). ② The expressions of mitochondrial energy-relative proteins, VDAC1 and cytC, were higher in cold-exposure group than in control group with both immunohistochemical staining and Western blot (P < 0.01). ③ Chronic cold exposure increased DNA methylation and histone deacetylation in the pyramidal cells of CA1 area and led to an increase in the expression of histone deacetylase 1 (HDAC1) and DNA methylation relative enzymes (P < 0.01). In conclusion, chronic cold exposure can improve insulin sensitivity, with the involvement of DNA methylation, histone deacetylation and the regulation of mitochondrial energy metabolism. These epigenetic modifications probably form the basic mechanism of cold-reduced insulin resistance.     

Energy metabolism disorders in rare and common diseases. Toward bioenergetic modulation therapy and the training of a new generation of European scientists

Energy metabolism alterations are found in a large number of rare and common diseases of genetic or environmental origin. The number of patients that could benefit from bioenergetic modulation therapy (BIOMET) is therefore very important and includes individuals with pathologies as diverse as mitochondrial diseases, acute coronary syndrome, chronic kidney disease, asthma or even cancer. Although, the alteration of energy metabolism is disease specific and sometimes patient specific, the strategies for BIOMET could be common and target a series of bioenergetic regulatory mechanisms discussed in this article. An excellent training of scientists in the field of energy metabolism, related human diseases and drug discovery is also crucial to form a young generation of MDs, PHDs and Pharma or CRO-group leaders who will discover novel personalized bioenergetic medicines, through pharmacology, genetics, nutrition or adapted exercise training. The Mitochondrial European Educational Training (MEET) consortium was created to pursue this goal, and we dedicated here a special issue of Organelle in Focus (OiF) to highlight their objectives. A total of 10 OiFs articles constitute this Directed Issue on Mitochondrial Medicine. As part of this editorial article, we asked timely questions to the PR. Jan W. Smeitink, professor of Mitochondrial Medicine and CEO of Khondrion, a mitochondrial medicine company. He shared with us his objectives and strategies for the study of mitochondrial diseases and the identification of future treatments.This article is part of a Directed Issue entitled: Energy Metabolism Disorders and Therapies.     

In vitro and ex vivo vanadium antitumor activity in (TGF-β)-induced EMT. Synergistic activity with carboplatin and correlation with tumor metastasis in cancer patients

Epithelial to mesenchymal transition (EMT) plays a key role in tumor progression and metastasis as a crucial event for cancer cells to trigger the metastatic niche. Transforming growth factor-β (TGF-β) has been shown to play an important role as an EMT inducer in various stages of carcinogenesis. Previous reports had shown that antitumor vanadium inhibits the metastatic potential of tumor cells by reducing MMP-2 expression and inducing ROS-dependent apoptosis. However, the role of vanadium in (TGF-β)-induced EMT remains unclear. In the present study, we report for the first time on the inhibitory effects of vanadium on (TGF-β)-mediated EMT followed by down-regulation of ex vivo cancer stem cell markers. The results demonstrate blockage of (TGF-β)-mediated EMT by vanadium and reduction in the mitochondrial potential of tumor cells linked to EMT and cancer metabolism. Furthermore, combination of vanadium and carboplatin (a) resulted in synergistic antitumor activity in ex vivo cell cultures, and (b) prompted G₀/G₁ cell cycle arrest and sensitization of tumor cells to carboplatin-induced apoptosis. Overall, the findings highlight the multifaceted antitumor action of vanadium and its synergistic antitumor efficacy with current chemotherapy drugs, knowledge that could be valuable for targeting cancer cell metabolism and cancer stem cell-mediated metastasis in aggressive chemoresistant tumors.     

Preliminary studies on the involvement of glutathione metabolism and redox status against zinc toxicity in radish seedlings by 28-Homobrassinolide

The effect of exogenous application of 28-Homobrassinolide (HBR) on radish (Raphanus sativus L.) seedlings under zinc (Zn²⁺) stress on glutathione (GSH) production, consumption and changes in redox status was investigated. Zinc toxicity resulted in oxidative burst as evidenced by increased accumulation of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) content. These stress indices were significantly decreased by HBR supplementation. Under Zn²⁺ stress, GSH pool was decreased, while the contribution of oxidized glutathione (GSSG) to total GSH increased (GSSH/GSH ratio), this translated into significant reduction of GSH redox homeostasis. In addition, an increase of phytochelatins (PCs) was observed. In radish seedlings under Zn²⁺ stress, the activities of gamma-glutamylcysteine synthetase (γ-ECS), glutathione synthetase (GS), glutathione peroxidase (GPX), glutathione-S-transferase (GST) and cysteine (Cys) levels increased but the activity of glutathione reductase (GR) decreased. However, application of HBR increased the GSH pool and maintained their redox ratio by increasing the enzyme activities of GSH biosynthesis (γ-ECS and GS) and GSH metabolism (GR, GPX and GST). The results of present study are novel in being the first to demonstrate that exogenous application of HBR modulates the GSH synthesis, metabolism and redox homeostasis to confer resistance against Zn²⁺ induced oxidative stress.     

Synthesis and antiproliferative activity of two diastereomeric lignan amides serving as dimeric caffeic acid-l-DOPA hybrids

Two new diastereomeric lignan amides (4 and 5) serving as dimeric caffeic acid-l-DOPA hybrids were synthesized. The synthesis involved the FeCl₃-mediated phenol oxidative coupling of methyl caffeate to afford trans-diester 1a as a mixture of enantiomers, protection of the catechol units, regioselective saponification, coupling with a suitably protected l-DOPA derivative, separation of the two diastereomers thus obtained by flash column chromatography and finally global chemoselective deprotection of the catechol units. The effect of hybrids 4 and 5 and related compounds on the proliferation of two breast cancer cell lines with different metastatic potential and estrogen receptor status (MDA-MB-231 and MCF-7) and of one epithelial lung cancer cell line, namely A-549, was evaluated for concentrations ranging from 1 to 256 μM and periods of treatment of 24, 48 and 72 h. Both hybrids showed interesting and almost equipotent antiproliferative activities (IC₅₀ 64–70 μM) for the MDA-MB-231 cell line after 24–48 h of treatment, but they were more selective and much more potent (IC₅₀ 4–16 μM) for the MCF-7 cells after 48 h of treatment. The highest activity for both hybrids and both breast cancer lines was observed after 72 h of treatment (IC₅₀ 1–2 μM), probably as the result of slow hydrolysis of their methyl ester functions.     

A cell-based high-throughput screen identifies tyrphostin AG 879 as an inhibitor of animal cell phospholipid and fatty acid biosynthesis

Inhibition of animal cell phospholipid biosynthesis has been proposed for anticancer and antiviral therapies. Using CHOK1 derived cell lines, we have developed and used a cell-based high-throughput procedure to screen a 1280 compound, small molecule library for inhibitors of phospholipid biosynthesis. We identified tyrphostin AG 879 (AG879), which inhibited phospholipid biosynthesis by 85–90% at a concentration of 10 μM, displaying an IC₅₀ of 1–3 μM. The synthesis of all phospholipid head group classes was heavily affected. Fatty acid biosynthesis was also dramatically inhibited (90%). AG879 inhibited phospholipid biosynthesis in all additional cell lines tested, including MDCK, HUH7, Vero, and HeLa cell lines. In CHO cells, AG879 was cytostatic; cells survived for at least four days during exposure and were able to divide following its removal. AG879 is an inhibitor of receptor tyrosine kinases (RTK) and inhibitors of signaling pathways known to be activated by RTK's also inhibited phospholipid biosynthesis. We speculate that inhibition of RTK by AG879 results in an inhibition of fatty acid biosynthesis with a resulting decrease in phospholipid biosynthesis and that AG879's effect on fatty acid synthesis and/or phospholipid biosynthesis may contribute to its known capacity as an effective antiviral/anticancer agent.     

Role of labile iron in the toxicity of pharmacological ascorbate

Pharmacological ascorbate has been shown to induce toxicity in a wide range of cancer cell lines. Pharmacological ascorbate in animal models has shown promise for use in cancer treatment. At pharmacological concentrations the oxidation of ascorbate produces a high flux of H₂O₂ via the formation of ascorbate radical (Asc^•-). The rate of oxidation of ascorbate is principally a function of the level of catalytically active metals. Iron in cell culture media contributes significantly to the rate of H₂O₂ generation. We hypothesized that increasing intracellular iron would enhance ascorbate-induced cytotoxicity and that iron chelators could modulate the catalytic efficiency with respect to ascorbate oxidation. Treatment of cells with the iron-chelators deferoxamine (DFO) or dipyridyl (DPD) in the presence of 2 mM ascorbate decreased the flux of H₂O₂ generated by pharmacological ascorbate and reversed ascorbate-induced toxicity. Conversely, increasing the level of intracellular iron by preincubating cells with Fe-hydroxyquinoline (HQ) increased ascorbate toxicity and decreased clonogenic survival. These findings indicate that redox metal metals, e.g., Fe³⁺/Fe²⁺, have an important role in ascorbate-induced cytotoxicity. Approaches that increase catalytic iron could potentially enhance the cytotoxicity of pharmacological ascorbate in vivo.