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

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 hiperprodukcija 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 characterized 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...