The impact of reactive oxygen species on the mTOR signaling pathway activity in patients with myeloproliferative neoplasms

Abstract

common characteristic of malignancies is an increase of reactive oxygen species (ROS) and reactive nitrogen species (RNS). In such circumstances macromolecules are damaged and the cell structure and function are disrupted. The oxidative DNA damage is considered to be the initial event in the developement of carcinogenesis. ROS affects the function of hematopoietic stem cells, changes the cell cycle progression, accelerаtes aging and induces hematological malignancies. The BCR/ABL oncogene and JAK2V617F mutation induces the formation of ROS in hematopoietic stem cells that leads to genomic instability and DNA damage. In the myeloproliferative neoplasm (MPN) patients, JAK2V617F mutation and BCR/ABL oncogene are associated with the constitutive activation of the PI3K/AKT/mTOR signaling pathway, which stimulates the growth and proliferation of cells. As ROS and RNS may affect the activity of mTOR, we have examined the parameters of antioxidant protection and oxidative damage of lipids and proteins in circulation as well as the AKT/mTOR signaling pathway activity in the MPN granulocytes and human erythroleukemia (HEL) cell line with JAK2V617F mutation. Methods: The study includes 110 de novo MPN patients: 30 polycythemia vera (PV), 24 essential thrombocythemia (ET), 34 primary myelofibrosis (PMF) as well as 22 patients with the diagnosis of chronic myeloid leukemia (CML) and 10 healthy subjects. Markers of oxidative and nitrosative stress in plasma and erythrocyte lysates were determined by the colorimetric method. The intracellular production of ROS was determined by fluorogenic reagent H2DCF-DA. The levels of nitrotyrosine, inducible NO synthase (iNOS) and the activation rate of AKT/mTOR/p70S6K kinases were determined in granulocytes of MPN patients using immunocytochemistry and immunoblotting methods. The influence of hydrogen peroxide (H2O2) and 2, 2'-Azobis (2-amidinopropane) dihydrochloride (AAPH) oxidants on AKT/mTOR signal pathway, cell cycle and survival were tested by Western blot, FACS and MTT test in HEL cells...

Zajednička karakteristika maligniteta je povećana koncentracija reaktivnih vrsta kiseonika (ROS) i reaktivnih vrsta azota (RNS) koje mogu da prouzrokuju oštećenja makromolekula u ćeliji i dovedu do narušavanja njene strukture i funkcije. Oksidativno oštećenje DNK smatra se inicijalnim događajem u razvoju kancerogeneze. ROS utiču na funkciju hematopoetskih matičnih ćelija, dovode do promena ćelijskog ciklusa, ubrzanog starenja ćelija ili do nastanka hematoloških maligniteta. BCR/ABL onkogen i JAK2V617F mutacija indukuju stvaranje ROS u hematopoetskim matičnim ćelijama što vodi genomskoj nestabilnosti i oštećenju DNK. Pod uticajem JAK2V617F mutacije i BCR/ABL onkogena kod hroničnih mijeloproliferativnih neoplazmi (HMN) dolazi do konstitutivne aktivacije PI3K/AKT/mTOR signalnog puta, koji reguliše metabolizam i stimuliše rast i proliferaciju ćelija. S obzirom da ROS i RNS mogu uticati na aktivnost mTOR kinaze, ispitani su parametri antioksidativne zaštite i oksidativno izmenjenih lipida i proteina u cirkulaciji, kao i stepen aktivacije AKT/mTOR signalnog puta u granulocitima bolesnika sa HMN i humanoj eritroleukemijskoj ćelijskoj liniji (eng. Human erythroleukemia cell line, HEL) sa JAK2V617F mutacijom. Metode: U studiju je uključeno 110 de novo HMN bolesnika: 30 sa policitemijom verom (PV), 24 sa esencijalnom trombocitemijom (ET), 34 sa primarnom mijelofibrozom (PMF), 22 bolesnika sa dijagnozom hronične mijeloidne leukemije (HML) i 10 zdravih ispitanika. Markeri oksidativnog i nitrozativnog stresa u plazmi i lizatima eritrocita određeni su kolorimetrijskom metodom. Količina intracelularne produkcije ROS određena je upotrebom fluorogenog reagensa H2DCF-DA. Nivo nitrotirozina, inducibilne NO sintaze (iNOS) i stepen aktivacije AKT/mTOR/p70S6K kinaza određeni su u granulocitima bolesnika sa HMN imunocitohemijskom metodom i metodom imunoblota. U in vitro uslovima ispitan je uticaj vodonik peroksida (H2O2) i N- acetil cisteina na aktivaciju mTOR i ekspresiju iNOS i nitrotirozina. Uticaj dva različita oksidansa, H2O2 i 2,2'-azobis(2-amidinopropan) dihidrohlorida (AAPH), na aktivaciju AKT/mTOR signalnog puta, preživljavanje i ćelijski ciklus HEL ćelija, u in vitro uslovima, ispitan je metodom imunoblota, FACS metodom i MTT testom...