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Title:Molekularni in celični odziv na oksidativni stres, hipoksijo in naravne antioksidante, pridobljene s konvencionalno in superkritično ekstrakcijo, pri akutni in kronični mieloični levkemiji in vitro : doktorska disertacija
Authors:ID Jurgec, Staša (Author)
ID Potočnik, Uroš (Mentor) More about this mentor... New window
ID Knez, Željko (Comentor)
Files:.pdf DOK_Jurgec_Stasa_2023.pdf (3,23 MB)
MD5: 4890E3B6160FFE88ECFDEB1BA5FD892C
 
Language:Slovenian
Work type:Doctoral dissertation
Typology:2.08 - Doctoral Dissertation
Organization:FKKT - Faculty of Chemistry and Chemical Engineering
Abstract:Akutna mieloična levkemija (angl. Acute Myeloid Leukemia, AML) in kronična mieloična levkemija (angl. Chronic Myeloid Leukemia, CML) sta skupini hematoloških rakov, za kateri je značilna nekontrolirana delitev krvnih celic mieloične linije. Kljub napredku v poznavanju genetskih dejavnikov in molekularnih mehanizmov, ki lahko privedejo do AML in CML, so trenutno razpoložljivi načini zdravljenja nezadostni. Za boljše razumevanje patofiziologije nastanka in razvoja AML in CML smo v doktorski nalogi proučili uravnavanje celične redoks homeostaze pri tej skupini rakov. V prvem delu doktorske naloge izvedli meta-analizo transkriptomskih podatkov iz 4 študij CML in 1 študije AML. V drugem delu smo na celičnih linijah AML HL-60 in KASUMI1 in celičnih linijah CML K562 in Ku812F natančneje proučili vpliv oksidativnega stresa, povzročenega z vodikovim peroksidom, hipoksije in naravnih ekstraktov origana, konoplje in brusnice, pridobljenih s hladno maceracijo, ekstrakcijo z ultrazvokom in superkritično tekočinsko ekstrakcijo, na molekularnem in celičnem nivoju. Z meta-analizo transkriptomskih podatkov smo med prvimi 100 najbolj diferencialno izraženimi geni identificirali gen za nekodirajočo RNA LINC01554 in dva pseudogene RPS27AP20 in FAM133CP, ki predhodno še niso bili povezani z AML in CML, in potrdili, da se AML in CML razlikujeta v izražanju genov, poveznih z uravnavanjem redoks homeostaze. Izpostavitev oksidativnemu stresu je znižala celično rast/proliferacijo pri proučevanih celičnih linijah, pri tem pa je bil vpliv večji pri celičnih linijah AML kot CML. Oksidativni stres je še znižal metabolno aktivnost celice pri celičnih linijah AML in jo povišal pri celičnih linijah CML. Ugotovljene razlike v celični rasti/proliferaciji in metabolizmu med AML in CML v pogojih oksidativnega stresa so bile posledica zmanjšanega preživetja celičnih linij AML v primerjavi s celičnimi linijami CML in ne spremenjenega celičnega cikla. Na molekularnem nivoju so se proučevane celične linije razlikovale v izražanju AQP1, 3, 8, 9 in 11, SOD1, CAT in AATF. Tretiranje z ekstrakti je v pogojih oksidativnega stresa znižalo celično rast/proliferacijo pri celičnih linijah HL-60, KASUMI1 in K562 in povišalo količino reaktivnih kisikovih in dušikovih spojin (ROS/RNS) pri celični liniji HL-60 oziroma znižalo njihovo količino pri celični liniji KASUMI1. Ekstrakti niso vplivali na celično rast/proliferacijo pri celični linji Ku812F in na količino ROS/RNS pri celičnih linijah K562 in Ku812F. Znižana celična rast v prisotnosti oksidativnega stresa in ekstraktov je bila posledica zmanjšanega preživetja celic in ne spremenjenega celičnega cikla. Tretiranje z ekstrakti brusnice je povišalo izražanje SOD1 pri celični liniji Ku812F, v prisotnosti oksidativnega stresa in specifičnih ekstraktov pa smo ugotovili še razlike v izražanju SOD1, NQO1 in PRDX1 med proučevanimi celičnimi linijami. Hipoksija je znižala celično rast/proliferacijo pri vseh proučevanih celičnih linijah, pri tem pa so bile celične linije AML bolj dovzetne za vplive hipoksije kot celične linije CML. Še več, hipoksija je specifično znižala metabolizem celice pri celičnih linijah AML. Znižana rast/proliferacija in metabolizem celičnih linij AML v primerjavi s celičnmi linijami CML v hipoksiji sta bila posledica zmanjšanega preživetja in ne spremenjenega celičnega cikla. Rezultati doktorske naloge kažejo, da se AML in CML razlikujeta v molekularnem in celičnem odzivu na dejavnike, ki vplivajo na celično redoks homeostazo. Molekularni mehanizmi, ki omogočajo večjo dovzetnost AML za ROS/RNS, ki vodijo v znižanje celične rasti/proliferacije in preživetja celic, bi lahko v prihodnje predstavljali potencialne klinične tarče za nadzorovanje in zdravljenje te bolezni.
Keywords:AML, CML, oksidativni stres, hipoksija, naravni ekstrakt, RT-qPCR
Place of publishing:Maribor
Place of performance:Maribor
Publisher:[S. Jurgec]
Year of publishing:2023
Number of pages:XI, 124 str.
PID:20.500.12556/DKUM-83677 New window
UDC:616.155.392:543.382(043.3)
COBISS.SI-ID:159269891 New window
Publication date in DKUM:19.07.2023
Views:662
Downloads:72
Metadata:XML DC-XML DC-RDF
Categories:KTFMB - FKKT
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Licences

License:CC BY-NC-ND 4.0, Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International
Link:http://creativecommons.org/licenses/by-nc-nd/4.0/
Description:The most restrictive Creative Commons license. This only allows people to download and share the work for no commercial gain and for no other purposes.
Licensing start date:23.01.2023

Secondary language

Language:English
Title:Molecular and cellular response to oxidative stress, hypoxia and natural antioxidants obtained by conventional and supercritical fluid extraction in acute and chronic myeloid leukemia in vitro
Abstract:Acute myeloid leukemia (AML) and chronic myeloid leukemia (CML) are a group of haematological cancers characterised by the uncontrolled growth and proliferaton of blood cells of the myeloid lineage. Despite advances in our understanding of the genetic factors and molecular mechanisms that lead to AML and CML, currently available treatments are inadequate. To better understand the pathophysiology of AML and CML development and progression, in this PhD thesis we investigated the regulation of cellular redox homeostasis in this gruop of cancers. In the first part of the thesis, we performed a meta-analysis of transcriptomic data from 4 studies of CML and 1 study of AML. In the second part, we further investigated the effects of hydrogen peroxide-induced oxidative stress, hypoxia and natural extracts of oregano, cannabis and cranberry, obtained by cold maceration, ultrasound-assisted extraction and supercritical fluid extraction, at the molecular and cellular levels in the AML cell lines HL-60 and KASUMI1 and the CML cell lines K562 and Ku812F. A meta-analysis of the transcriptomic data identified among the first 100 differentially expressed genes the non-coding RNA gene LINC01554 and two pseudogenes RPS27AP20 and FAM133CP, which have not been previously associated with AML and CML, and confirmed that AML and CML differ in the expression of genes related to the regulation of redox homeostasis. Exposure to oxidative stress reduced cell growth/proliferation in the cell lines studied, with a greater effect in AML than in CML cell lines. Furthermore, oxidative stress decreased cell metabolic activity in AML cell lines and increased cell metabolic activity in CML cell lines. The observed differences in cell growth/proliferation and metabolism between AML and CML under oxidative stress conditions were due to the reduced survival of AML cell lines compared to CML cell lines, whereas oxidative stress had no effect on the cell cycle in the cell lines studied. At the molecular level, under oxidative stress, the cell lines studied differed in the expression of AQP1, 3, 8, 9 and 11, SOD1, CAT and AATF. Under oxidative stress, treatment with the extracts decreased cell growth/proliferation in HL-60, KASUMI1 and K562 cell lines and increased and decreased reactive oxygen nitrogen species (ROS/RNS) in HL-60 and KASUMI1 cell lines, respectively. The extracts had no effect on cell growth/proliferation in the Ku812F cell line and on the amount of ROS/RNS in the K562 and Ku812F cell lines. The reduced cell growth in the presence of oxidative stress and the extracts was due to reduced cell survival and not altered cell cycle. Treatment with cranberry extracts increased the expression of SOD1 in the Ku812F cell line, and in the presence of oxidative stress and specific extracts, we also found differences in the expression of SOD1, NQO1 and PRDX1 between the cell lines studied. Hypoxia reduced cell growth/proliferation in all cell lines studied, with AML cell lines being more susceptible to the effects of hypoxia than CML cell lines. In addition, hypoxia specifically reduced cell metabolism in AML cell lines. As with oxidative stress, reduced cell growth/proliferation was due to reduced survival rather than altered cell cycle. The results of the thesis show that AML and CML differ in the molecular and cellular response to factors affecting cellular redox homeostasis. The molecular mechanisms that render acute myeloid leukemia more susceptible to ROS/RNS, leading to a reduction in cell growth/proliferation and cell survival, may represent potential future clinical targets for controlling and treating the disease.
Keywords:AML, CML, oxidative stress, hypoxia, natural extract, RT-qPCR


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