ABSTRACT
BACKGROUND: While Thioredoxin Reductase (TrxR) plays an important role in regulation of the intracellular redox balance and various signalling pathways, Glutathione S-Transferase (GSTs) enzymes belong to the detoxification family that catalyse the conjugation of glutathione with various endogenous and xenobiotic electrophiles. Since TrxR and GSTs are overexpressed in many cancer cells, they have been identified as potential targets to develop chemotherapeutic strategies. METHOD: The mitochondrial TrxR (TrxR2) enzyme and the cytosolic GST enzyme was purified from rat liver via affinity chromatography. After the purification, the in vitro inhibition effects of some anticancer drugs (cisplatin, calcium folinate, carboplatin, epirubicin hydrochloride, doxorubicin hydrochloride, paclitaxel, etoposide, fluorouracil, and methotrexate) were investigated on both enzymes. Since only methotrexate inhibits both enzymes among all the anticancer drugs, a molecular docking study was performed to determine the binding site and the binding affinity of methotrexate to the enzymes. RESULTS: Firstly, TrxR2 and GST were found to have a specific activity of 0.436, 1765 EU/mg proteins with a yield of 39.20%, 31.28% and 207.6, 3516.6 of purification fold, respectively. While TrxR2 was strongly inhibited by all of the anticancer drugs, GST was not inhibited by any of the anticancer drugs except methotrexate. CONCLUSION: Both enzymes were inhibited by only methotrexate in rat liver, and methotrexate was well placed in the active sites of both proteins. Therefore, it may be argued that methotrexate may be a more effective anticancer drug than all other drugs used in this study against the multi drug resistance that will occur during chemotherapy.
Subject(s)
Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Glutathione Transferase/antagonists & inhibitors , Liver/drug effects , Liver/enzymology , Thioredoxin-Disulfide Reductase/antagonists & inhibitors , Animals , Male , Molecular Docking Simulation , Rats , Rats, Sprague-DawleyABSTRACT
BACKGROUND: Thioredoxin reductase (E.C 1.6.4.5.; TrxR) is a widely distributed flavoprotein that catalyzes the NADPH-dependent reduction of thioredoxin (Trx) in many cellular events such as DNA synthesis, DNA repair, angiogenesis, antioxidative defense, and regulating apoptosis. Although TrxR is indispensible in protecting cells against oxidative stress, the overexpression of TrxR is seen in many aggressive tumors. Therefore, targeted inhibition of TrxR has been accepted as a new approach for chemotherapy. OBJECTIVE: In this study, in vitro inhibition effect of the lichen acids (diffractaic, evernic, lobaric, lecanoric, and vulpinic acid) on mitochondrial TrxR purified from rat lung was investigated. METHOD: It was the first time the enzyme was purified from rat lungs by using 2', 5'-ADP Sepharose 4B affinity chromatography. The purity of the enzyme was checked with SDS-PAGE. In vitro inhibition effect of the lichen acids was investigated spectrophotometrically. To emphasize the importance of the obtained data, the commercial anticancer drugs cisplatin and doxorubicin were used as positive controls. RESULTS: Molecular mass of the enzyme was calculated as approximately 52.4 kDa. The enzyme was purified with a 63.6% yield, 208.3 fold, and 0.5 EU/mg proteins specific activity. The IC50 values of five lichen acids were significantly lower than IC50 values of anticancer drugs. CONCLUSION: All of the lichen acids, especially lecanoric and vulpinic acid, exhibited much stronger inhibitory effect on TrxR than the anticancer drugs cisplatin and doxorubicin. These lichen acids have pharmacological potential as effective natural antioxidants, antimicrobials, and anticancer agents.
Subject(s)
Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Lichens/chemistry , Lung/enzymology , Thioredoxin-Disulfide Reductase/antagonists & inhibitors , Animals , Anisoles/chemical synthesis , Anisoles/chemistry , Anisoles/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cisplatin/chemistry , Cisplatin/pharmacology , Depsides/chemical synthesis , Depsides/chemistry , Depsides/pharmacology , Dose-Response Relationship, Drug , Doxorubicin/chemistry , Doxorubicin/pharmacology , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Furans/chemical synthesis , Furans/chemistry , Furans/pharmacology , Hydroxybenzoates/chemical synthesis , Hydroxybenzoates/chemistry , Hydroxybenzoates/pharmacology , Lactones/chemical synthesis , Lactones/chemistry , Lactones/pharmacology , Male , Mitochondria/drug effects , Mitochondria/metabolism , Molecular Structure , Phenylacetates/chemical synthesis , Phenylacetates/chemistry , Phenylacetates/pharmacology , Rats , Rats, Sprague-Dawley , Salicylates/chemical synthesis , Salicylates/chemistry , Salicylates/pharmacology , Structure-Activity Relationship , Thioredoxin-Disulfide Reductase/isolation & purification , Thioredoxin-Disulfide Reductase/metabolismABSTRACT
An extracellular thermostable alkaline serine protease enzyme from Aeribacillus pallidus C10 (GenBank No: KC333049), was purified 4.85 and 17. 32-fold with a yield of 26.9 and 19.56%, respectively, through DE52 anion exchange and Probond affinity chromatography. The molecular mass of the enzyme was determined through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with approximately 38.35 kDa. The enzyme exhibited optimum activity at pH 9 and at temperature 60 °C. It was determined that the enzyme had remained stable at the range of pH 7.0-10.0, and that it had preserved more than 80% of its activity at a broad temperature range (20-80 °C). The enzyme activity was found to retain more than 70% and 55% in the presence of organic solvents and commercial detergents, respectively. In addition, it was observed that the enzyme activity had increased in the presence of 5% SDS. KM and Vmax values were calculated as 0.197 mg/mL and 7.29 µmol.mL-1.min-1, respectively.
