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2.
Sci Rep ; 10(1): 14174, 2020 08 25.
Article in English | MEDLINE | ID: mdl-32843660

ABSTRACT

Mitochondrial dysfunction and significant changes in metabolic pathways accompany cancer development and are responsible for maintaining the tumor microenvironment. Normal mitochondria can trigger intrinsic apoptosis by releasing cytochrome c into the cytosol. The survival of malignant cells highly depends on the suppression of this function. We validated that A250, a highly purified fraction of fermented wheat germ extract (FWGE), increases the carbon flux into the mitochondria, the expression of key elements of the Krebs cycle and oxidative phosphorylation (OXPHOS). The increased respiratory chain activity is related to the mitochondria's ability to release cytochrome c into the cytosol, which triggers the apoptotic cascade. The 68% tumor growth inhibitory effect observed in the murine melanoma study is related to this effect, as proteomic analysis validated similar changes in mitochondrial protein levels in the isolated tumor tissue samples. Blood count data indicated that this effect was not accompanied by general toxicity. This study is significant, as it shows that a highly concentrated form of FWGE is an effective agent that increases normal mitochondrial functionality. The lack of hepatotoxic and general toxic effects makes A250 an excellent candidate targeting mitochondria function in cancer therapy.


Subject(s)
Mitochondria/drug effects , Plant Extracts/pharmacology , Triticum/chemistry , Warburg Effect, Oncologic/drug effects , Animals , Apoptosis/drug effects , Carbon/metabolism , Cell Line, Tumor , Citric Acid Cycle/drug effects , Cytochromes c/metabolism , Drug Screening Assays, Antitumor , Fermentation , Gene Expression Regulation, Neoplastic/drug effects , HEK293 Cells , Humans , Liver/drug effects , Liver/pathology , Melanoma, Experimental/drug therapy , Methanol , Mice , Mitochondria/metabolism , Oxidative Phosphorylation/drug effects , Plant Extracts/isolation & purification , Plant Extracts/toxicity , Random Allocation , Solvents
3.
Mol Cell ; 58(2): 255-68, 2015 Apr 16.
Article in English | MEDLINE | ID: mdl-25866245

ABSTRACT

PTEN is proposed to function at the plasma membrane, where receptor tyrosine kinases are activated. However, the majority of PTEN is located throughout the cytoplasm. Here, we show that cytoplasmic PTEN is distributed along microtubules, tethered to vesicles via phosphatidylinositol 3-phosphate (PI(3)P), the signature lipid of endosomes. We demonstrate that the non-catalytic C2 domain of PTEN specifically binds PI(3)P through the CBR3 loop. Mutations render this loop incapable of PI(3)P binding and abrogate PTEN-mediated inhibition of PI 3-kinase/AKT signaling. This loss of function is rescued by fusion of the loop mutant PTEN to FYVE, the canonical PI(3)P binding domain, demonstrating the functional importance of targeting PTEN to endosomal membranes. Beyond revealing an upstream activation mechanism of PTEN, our data introduce the concept of PI 3-kinase signal activation on the vast plasma membrane that is contrasted by PTEN-mediated signal termination on the small, discrete surfaces of internalized vesicles.


Subject(s)
PTEN Phosphohydrolase/chemistry , PTEN Phosphohydrolase/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phosphatidylinositol Phosphates/metabolism , Transport Vesicles/metabolism , Animals , Binding Sites , Mice , Microtubules/enzymology , Models, Molecular , NIH 3T3 Cells , Protein Structure, Secondary , Signal Transduction
4.
Cell Rep ; 10(1): 8-19, 2015 Jan 06.
Article in English | MEDLINE | ID: mdl-25543136

ABSTRACT

Phosphatidylinositol phosphate (PIP) second messengers relay extracellular growth cues through the phosphorylation status of the inositol sugar, a signal transduction system that is deregulated in cancer. In stark contrast to PIP inositol head-group phosphorylation, changes in phosphatidylinositol (PI) lipid acyl chains in cancer have remained ill-defined. Here, we apply a mass-spectrometry-based method capable of unbiased high-throughput identification and quantification of cellular PI acyl chain composition. Using this approach, we find that PI lipid chains represent a cell-specific fingerprint and are unperturbed by serum-mediated signaling in contrast to the inositol head group. We find that mutation of Trp53 results in PIs containing reduced-length fatty acid moieties. Our results suggest that the anchoring tails of lipid second messengers form an additional layer of PIP signaling in cancer that operates independently of PTEN/PI3-kinase activity but is instead linked to p53.


Subject(s)
Fatty Acids/metabolism , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol Phosphates/metabolism , Tumor Suppressor Protein p53/genetics , Cell Line , High-Throughput Screening Assays , Humans , Lipid Metabolism/genetics , Mutation , PTEN Phosphohydrolase/genetics , PTEN Phosphohydrolase/metabolism , Phosphatidylinositol Phosphates/genetics , Phosphatidylinositol Phosphates/isolation & purification , Phosphorylation , Second Messenger Systems/genetics , Signal Transduction/genetics , Tumor Suppressor Protein p53/metabolism
5.
J Chromatogr Sci ; 52(9): 985-91, 2014 Oct.
Article in English | MEDLINE | ID: mdl-24046161

ABSTRACT

This study was conducted to analyze carotenoids from tomatoes by high-performance liquid chromatography using reversed-phase C18 silica having cross-linked end-capping with diode array and mass spectrometric detection. An efficient gradient elution system was developed to achieve good and reliable separation of both major and minor carotenoids as well as their isomers. Resolution of lycopene, ß-carotene and their isomers was 0.91-3.97 and 1.02-2.86 with cross-linked and conventional C18 column, respectively. The % recovery for zeaxanthin, lycopene and ß-carotene was found to be in the range of 89-97%. Limits of detection and quantification of 19.44 and 64.79 ng/mL for zeaxanthin, 15.6 and 52.4 ng/mL for lycopene and 8.28 and 27.61 ng/mL for ß-carotene were determined. More carotenoid compounds could be separated and detected with the new method as compared with conventional C18 column. Hyphenation of HPLC with photodiode array and mass spectrometry detectors assisted in detection of tetra-dehydrocarotenoid and fatty acid diesters of xanthophylls in tomato products. Content of all-trans-lycopene, ß-carotene and total carotenoid in different industrial tomatoes tested was found to range between 41.87 and 84.65, 0.89 and 1.50 and 53.22 and 112.60 µg/g fresh weight, respectively.


Subject(s)
Carotenoids/analysis , Chromatography, High Pressure Liquid/instrumentation , Chromatography, High Pressure Liquid/methods , Solanum lycopersicum/chemistry , Carotenoids/chemistry , Equipment Design , Isomerism , Limit of Detection , Lycopene , Mass Spectrometry/instrumentation , Mass Spectrometry/methods , Reproducibility of Results , Zeaxanthins/analysis , Zeaxanthins/chemistry , beta Carotene/analysis , beta Carotene/chemistry
6.
FEBS J ; 280(12): 2830-41, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23578302

ABSTRACT

The families of protein tyrosine phosphatases (PTPs) and protein tyrosine kinases (PTKs) function in a coordinated manner to regulate signal transduction events that are critical for cellular homeostasis. Aberrant tyrosine phosphorylation, resulting from disruption of either PTP or PTK function, has been shown to be the cause of major human diseases, including cancer and diabetes. Consequently, the characterization of small-molecule inhibitors of these kinases and phosphatases may not only provide molecular probes with which to define the significance of particular signaling events, but also may have therapeutic implications. BAY-11-7082 is an anti-inflammatory compound that has been reported to inhibit IκB kinase activity. The compound has an α,ß-unsaturated electrophilic center, which confers the property of being a Michael acceptor; this suggests that it may react with nucleophilic cysteine-containing proteins, such as PTPs. In this study, we demonstrated that BAY-11-7082 was a potent, irreversible inhibitor of PTPs. Using mass spectrometry, we have shown that BAY-11-7082 inactivated PTPs by forming a covalent adduct with the active-site cysteine. Administration of the compound caused an increase in protein tyrosine phosphorylation in RAW 264 macrophages, similar to the effects of the generic PTP inhibitor sodium orthovanadate. These data illustrate that BAY-11-7082 is an effective pan-PTP inhibitor with cell permeability, revealing its potential as a new probe for chemical biology approaches to the study of PTP function. Furthermore, the data suggest that inhibition of PTP function may contribute to the many biological effects of BAY-11-7082 that have been reported to date.


Subject(s)
Anti-Inflammatory Agents/chemistry , Nitriles/chemistry , Protein Processing, Post-Translational/drug effects , Protein Tyrosine Phosphatase, Non-Receptor Type 1/antagonists & inhibitors , Sulfones/chemistry , Animals , Anti-Inflammatory Agents/pharmacology , Catalytic Domain , Cell Line , Enzyme Inhibitors/pharmacology , Humans , Kinetics , Lipopolysaccharides/pharmacology , Macrophages/drug effects , Macrophages/enzymology , Macrophages/immunology , Mice , Models, Molecular , Nitriles/pharmacology , Phosphorylation , Protein Binding , Protein Tyrosine Phosphatase, Non-Receptor Type 1/chemistry , Protein Tyrosine Phosphatase, Non-Receptor Type 1/metabolism , Sulfones/pharmacology , Surface Properties , Tyrosine/metabolism , Vanadates/pharmacology
7.
Curr Med Chem ; 16(16): 1964-77, 2009.
Article in English | MEDLINE | ID: mdl-19519376

ABSTRACT

There are about 380 protein kinase inhibitors in drug development as of today and 15 drugs have been marketed already for the treatment of cancer. This time 139 validated kinase targets are in the focus of drug research of pharmaceutical companies and big efforts are made for the development of new, druglike kinase inhibitors. Plasma protein binding is an important factor of the ADME profiling of a drug compound. Human serum albumin (HSA) and alpha(1)-acid glycoprotein (AAG) are the most relevant drug carriers in blood plasma. Since previous literature data indicated that AAG is the principal plasma binding component of some kinase inhibitors the present work focuses on the comprehensive evaluation of AAG binding of a series of marketed and experimental kinase inhibitors by using circular dichroism (CD) spectroscopy approach. HSA binding was also evaluated by affinity chromatography. Protein binding interactions of twenty-six kinase inhibitors are characterized. The contribution of AAG and HSA binding data to the pharmacokinetic profiles of the investigated therapeutic agents is discussed. Structural, biological and drug binding properties of AAG as well as the applicability of the CD method in studying drug-protein binding interactions are also briefly reviewed.


Subject(s)
Marketing , Orosomucoid/metabolism , Protein Kinase Inhibitors/metabolism , Serum Albumin/metabolism , Animals , Humans , Orosomucoid/chemistry , Protein Binding , Protein Kinase Inhibitors/chemistry , Serum Albumin/chemistry
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