title: Cyclohexyl analogues of ethylenediamine dipropanoic acid induce caspase-independent mitochondrial apoptosis in human leukemic cells.
abstract: We investigated the cytotoxicity of recently synthesized (S,S)-ethylendiamine-N,N'-di-2-(3-cyclohexyl)propanoic acid esters toward human leukemic cell lines and healthy blood mononuclear cells. Cell viability was assessed by acid phosphatase assay, apoptosis, and differentiation were analyzed by flow cytometry and electron microscopy, while intracellular localization of apoptosis-inducing factor (AIF) was determined by immunoblotting. It was demonstrated that methyl, ethyl, and n-propyl esters were toxic to HL-60, REH, MOLT-4, KG-1, JVM-2, and K-562 leukemic cell lines, while the nonesterified parental compound and n-butyl ester were devoid of cytotoxic action. The ethyl ester exhibited the highest cytotoxic activity (IC₅₀ 10.7 μM-45.4 μM), which was comparable to that of the prototypical anticancer drug cisplatin. The observed 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 in the absence of autophagic response. DNA fragmentation preceded caspase activation and followed AIF translocation from mitochondria to nucleus, which was indicative of caspase-independent apoptotic cell death. HL-60 cells treated with subtoxic concentration of the compound 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 cyclohexyl analogues of ethylenediamine dipropanoic acid were also toxic to peripheral blood mononuclear cells of both healthy controls and leukemic patients, the latter being more sensitive. Our data demonstrate that the toxicity of the investigated cyclohexyl compounds against leukemic cell lines is mediated by caspase-independent apoptosis associated with oxidative stress, mitochondrial dysfunction, and AIF translocation.
authors.affiliation: Institute of Medical and Clinical Biochemistry, Faculty of Medicine, University of Belgrade, Belgrade, Serbia.
authors.forename: Sonja
authors.initials: S
authors.lastname: Misirlic Dencic
authors.forename: Jelena
authors.initials: J
authors.lastname: Poljarevic
authors.forename: Urosh
authors.initials: U
authors.lastname: Vilimanovich
authors.forename: Andrija
authors.initials: A
authors.lastname: Bogdanovic
authors.forename: Aleksandra J
authors.initials: AJ
authors.lastname: Isakovic
authors.forename: Tamara
authors.initials: T
authors.lastname: Kravic Stevovic
authors.forename: Marija
authors.initials: M
authors.lastname: Dulovic
authors.forename: Nevena
authors.initials: N
authors.lastname: Zogovic
authors.forename: Andjelka M
authors.initials: AM
authors.lastname: Isakovic
authors.forename: Sanja
authors.initials: S
authors.lastname: Grguric-Sipka
authors.forename: Vladimir
authors.initials: V
authors.lastname: Bumbasirevic
authors.forename: Tibor
authors.initials: T
authors.lastname: Sabo
authors.forename: Vladimir
authors.initials: V
authors.lastname: Trajkovic
authors.forename: Ivanka
authors.initials: I
authors.lastname: Markovic
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D051033:Apoptosis Inducing Factor / Q000378:metabolism; D039481:CD11b Antigen / Q000378:metabolism; D020169:Caspases / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D003510:Cyclohexanes / Q000737:chemistry / Q000633:toxicity*; D053938:DNA Fragmentation / Q000187:drug effects; D018922:HL-60 Cells; D006801:Humans; D020014:K562 Cells; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism; D016256:Lewis X Antigen / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D010718:Phosphatidylserines / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism
chemical_list: D051033:Apoptosis Inducing Factor; D039481:CD11b Antigen; D003510:Cyclohexanes; D016256:Lewis X Antigen; D010718:Phosphatidylserines; D013481:Superoxides; C506365:Cyclohexane; D020169:Caspases