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Molekularni mehanizmi citotoksičnog dejstva estara cikloheksil analoga etilendiamindipropanske kiseline na leukemijske ćelije in vitro

Molecular mechanisms of the in vitro antileukemic actionof cyclohexyl-functionalized ethylenediamine dipropanoic acid esters

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2012
Disertacija.pdf (1.707Mb)
Author
Misirlić-Denčić, Sonja T.
Mentor
Marković, Ivanka
Committee members
Sabo, Tibor
Isaković, Aleksandra
Trajković, Vladimir
Rakić, Ljubiša
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Abstract
Cilj ovog istraživanja je bio da ispita molekularne mehanizme u osnovi citotoksičnog dejstva estara (S,S)-etilendiamin-N,N’-di-2-(3- cikloheksil)propanske kiseline na leukemijskim ćelijama. Vijabilitet ćelija je procenjivan testom aktivnosti kisele fosfataze i laktat dehidrogenaze, parametri apoptotske smrti ćelija, kao i diferencijacije su analizirani protočnom citometrijom i elektronskom mikroskopijom. Intranukleusna lokalizacija faktora pokretača apoptoze (AIF) je utvrđena tehnikom imunoblota. Pokazano je da metil-, etil- i propil-estar deluju citotoksično na HL-60, REH, MOLT-4, KG-1, JVM-2 i K-562 leukemijske ćelijske linije, dok ishodno jedinjenje (kiselina) i butil-estar nisu pokazali toksičnost u in vitro uslovima. Citotoksičnost etil-estra je bila najveća (IC50: 10,7 – 45,4 μM), i uporediva sa cisplatinom, tipičnim proapoptotskim antitumorskim lekom. U osnovi antileukemijskog delovanja etil-estra na HL-60 ćelijsku liniju, najosetljiviju od ispitivanih, nalazila se hiperprodukci...ja superoksidnog anjona i depolarizacija mitohondrijalne membrane. Oštećenje mitohondrija je za posledicu imalo eksternalizaciju fosfatidil-serina, fragmentaciju DNK i sledstvenu apoptozu HL-60 ćelija. Fragmentacija DNK je bila posredovana translokacijom AIF-a iz mitohondrija u jedro, i prethodila je aktivaciji kaspaza, što je ukazalo na apoptozu nezavisnu od kaspaza. Ćelije tretirane subtoksičnim dozama etil-estra su pokazale morfološke znake granulocitne diferencijacije (segmentacija jedra i primarne granule u citoplazmi) kao i porast u ekspresiji markera diferencijacije CD11b i CD15. Sposobnost da uzrokuje apoptozu i diferencijaciju leukemijskih ćelija čini cikloheksil analoge etilendiamindipropanske kiseline po tipu estara potencijalnim kandidatima za dalja predklinička istraživanja sa ciljem ispitivanja mogućnosti ulaska u buduće kliničke studije...

The aim of the present study was to investigate antileukemic action of recently synthesized (S,S)-ethylenediamine-N,N’-di-2-(3-cyclohexyl)propanoic acid esters. Cell viability was assessed by the acid phosphatase and LDH release assay, apoptosis- and differentiation-related parameters were analyzed by flow cytometry/electron microscopy, while intracellular localization of apoptosis-inducing factor (AIF) was determined by immunoblotting. It was demonstrated that methyl, ethyl and propyl ester were toxic to HL-60, REH, MOLT-4, KG-1, JVM-2 and K-562 leukemic cell lines, while the non-esterified parental compound and butyl ester were devoid of in vitro antileukemic action. The cytotoxic activity of the ethyl ester was the highest (IC50: 10.7 - 45.4 μM) and comparable to that of the prototypical anticancer drug cisplatin. Its cytotoxic effect in HL-60 cells was associated with an increase in superoxide production and mitochondrial membrane depolarization, leading to apoptotic cell death cha...racterized by phosphatidylserine externalization and DNA fragmentation. DNA fragmentation preceded caspase activation and followed AIF translocation from mitochondria to nucleus, indicating that the observed apoptotic death was caspase-independent. Surviving cells displayed morphological signs of granulocytic differentiation (nuclear indentations and presence of cytoplasmic primary granules), as well as an increased expression of differentiation markers CD11b and CD15. The ability to induce both apoptosis and differentiation of leukemic cells makes the investigated cyclohexyl analogues plausible candidates for further preclinical examination and probably clinical trials...

Faculty:
Универзитет у Београду, Медицински факултет
Date:
11-07-2012
Projects:
  • Modulation of intracellular energy balance-controlling signalling pathways in therapy of cancer and neuro-immuno-endocrine disorders (RS-41025)
  • Fabrication and characterization of nano-photonic functional structrues in biomedicine and informatics (RS-45016)
Keywords:
leukemijske ćelije / leukemic cells / citotoksičnost / oksidativni stres / apoptoza nezavisna od kaspaza / diferencijacija / cytotoxicity / oxidative stress / caspase-independent apoptosis / differentiation

DOI: 10.2298/bg20120711misirlicdencic

[ Google Scholar ]
Handle
https://hdl.handle.net/21.15107/rcub_nardus_2352
URI
http://eteze.bg.ac.rs/application/showtheses?thesesId=202
https://nardus.mpn.gov.rs/handle/123456789/2352
https://fedorabg.bg.ac.rs/fedora/get/o:5453/bdef:Content/download
http://vbs.rs/scripts/cobiss?command=DISPLAY&base=70036&RID=41548047

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