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
Author
Misirlić-Denčić, Sonja T.
Mentor
Marković, Ivanka
Committee members
Sabo, Tibor
Isaković, Aleksandra

Trajković, Vladimir

Rakić, Ljubiša
Metadata
Show full item recordAbstract
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 факултетDate:
11-07-2012Projects:
- 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)