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.

Antileukemic action of novel diamine Pt(II) halogenido complexes: Comparison of the representative novel Pt(II) with corresponding Pt(IV) complex

“…The antileukemic action of cyclohexyl analogues, esters ( L 2 – L 5 ) of ethylenediamine dipropanoic acid ( L 1 ),has been previously reported, with L 3 marked as the most potent ligand and the HL‐60 cell line as the most sensitive to its cytotoxic action . In the present study, human promyelocytic cell line (HL‐60) was also the most sensitive to antitumor action of all investigated substances.…”

“…The treatment with C3 complex (4 hr, 18 μ m ) caused more pronounced increase in DHR‐derived FL1 fluorescence compared with novel complexes (Figure a), suggesting that C3, despite the lack of noticeable increase in superoxide production (Figure b), induces rapid and prominent oxidative damage in HL‐60 cells, as observed previously in U251 glioma cells . On the other hand, it was shown that organic ligand induced mitochondria‐derived superoxide hyperproduction in HL‐60 cells, unlike cisplatin, indicating that cyclohexyl‐functionalized edda ligands significantly contributed to mitochondria‐derived oxidative damage.…”

“…Furthermore, 24‐hr treatment of HL‐60 cells with C3a (5 μ m ) and C3b (3 μ m ) caused massive disturbance in HL‐60 cell cycle distribution with cell accumulation in sub‐G 0 phase (>40%) indicating DNA fragmentation (data not shown). The intrinsic AIF‐mediated leukemic cell death has been reported as organic L 3 ligand mechanism of action . The obtained results demonstrated that Pt(II) complexes investigated herein induce apoptotic cell death of leukemic cells in the concentration several times lower compared with organic ligands.…”

“…Having in mind that oxidative stress was the initial event involved in antileukemic action of organic ligand L 3 , we first analyzed the ability of novel Pt(II) complexes to increase production of ROS and cause oxidative stress in HL‐60 cells.…”

Synthesis and biological evaluation of 99mTc tricarbonyl complex of O,O′‐diethylethylenediamine‐N,N′‐di‐3‐propanoate as potential tumour diagnostic agent

“…[6] In contrast to cisplatin, these complexes mostly cause rapid, caspase-independent, oxidative stressmediated, non-apoptotic cell death characterized by massive cytoplasmic vacuolization, cell membrane damage and the absence of protective autophagy. [7,8] Indeed, organic ligands themselves exert significant in vitro toxicity probably due to an incorporated ethylenediamine group, which is known for its positive contribution to the cytotoxicity effect. [9] Ethylenediamine-N,N′-di-3-propanoate (R 2 eddp)-type esters such as diisopentyl-, di-n-butyl-and di-n-pentyl-(S,S)-ethylenediamine-N,N′-di-2-propanoate show even higher cytotoxicity and better selectivity against cervix adenocarcinoma cell line (HeLa), human melanoma (Fem-x) and human chronic myelogenous leukaemia (K562) cells in comparison to cisplatin.…”