The Pyridone-Annelated Isoindigo (5‘-Cl) Induces Apoptosis, Dysregulation of Mitochondria and Formation of ROS in Leukemic HL-60 Cells

Abstract

Background/Aims: In our quest to develop an isoindigo with improved efficacy and bioavailability, we recently synthesized a series of novel substituted pyridone-annelated isoindigo and evaluated their antiproliferative effects. We identified the compound [(E)-1-(5'-Chloro-2'-oxoindolin-3'-ylidene)-6-ethyl-2,3,6,9-tetrahydro-2,9-dioxo-1H-pyrrolo[3,2-f] quinoline-8-carboxylic acid], abbreviated as 5'-Cl, which shows selective antiproliferative activities against various cancer cell lines mediated through apoptosis. Here we have investigated the molecular mechanisms underlying the apoptotic activity of 5'-Cl in the human promyelocytic leukemia HL-60 cells. Methods: We employed different methods to determine the apoptotic pathways triggered by 5'-Cl in HL-60 cells, using flow cytometry, nuclear staining, caspases activation, mitochondria functioning, generation of reactive oxygen species (ROS) and Western blotting techniques. Results: Low concentrations (1-8 µM) of 5'-Cl inhibited the growth of HL-60 cells in a dose and time-dependent manner. Cytotoxicity of this compound is found to be mediated by a caspase-dependent apoptosis. Also, there were indications of caspase independent apoptosis as z-VAD-FMK failed to fully rescue the cells from 5‘-Cl-induced apoptosis. In addition, the compound triggered generation of Reactive Oxygen Species (ROS), caused depolarization of the mitochondrial inner membrane, decreased the level of cellular ATP, modulated the expression and phosphorylation of Bcl-2 leading to loss of its association with Bax and increased the release of cytochrome c to the cytosol of treated cells. The effects of 5‘-Cl on mitochondria and apoptosis were substantially blocked in the presence of a combination between z-VAD-FMK and either of the ROS scavenger N-acetyl-L-cysteine (NAC) or pyrrolidine dithiocarbamate (PDTC). Conclusion: We demonstrated that the growth inhibitory effects of 5'-Cl in HL-60 cells involve multiple pathways of apoptosis and dysregulation of mitochondrial functions.

Comprehensive Analysis of the Chemical Composition and In Vitro Cytotoxic Mechanisms of Pallines Spinosa Flower and Leaf Essential Oils Against Breast Cancer Cells

“…The quantitative determination of viable cells after various treatments with F-PSEO and L-PSEO was performed after staining with the dual DNA intercalating fluorescent dyes kit from EMD Millipore Bioscience (Muse TM Cell Count & Viability Assay) as described in our recent publications [26, 27]. Briefly, 4 ×10 4 MCF-7, MDA-MB-231 and MCF-10-2A cells/mL, or 3 ×10 5 cells/mL of each of the hematological cell lines and 1x10 5 cells/mL of PB-MNCs, were treated with increasing concentrations of F-PSEO or L-PSEO (0.1 to 10.0 µg/mL) for 48 h. Subsequently, samples were incubated with the Cell Count & Viability reagent for 5 min, and the viability of treated cells were analyzed using the flow cytometry-based Muse TM Cell Analyzer (EMD Millipore Bioscience, Darmstadt, Germany) according to the manufacture's protocol.…”

“…7-AAD is excluded from living healthy cells, as well as early apoptotic cells. Percentages of cells in early (annexin-V +ve /7-AAD -ve ) and late stages of apoptosis (annexin-V +ve /7-AAD +ve ) were determined by a flow cytometer-based instrument (Muse TM Cell Analyzer) as previously described [26, 27]. In all experiments, the solvent DMSO concentration was ≤ 0.1%.…”

“…The assays were performed using the caspase-specific colorimetric tetrapeptide substrate Ac-DEVD-pNA as previously described in details [26-28]. …”

“…Sample preparation after treatment with the oils, primary antibodies used in this study and Western blot analysis were performed according to manufacturer's protocols and as previously described in detail [26, 29]. The density of visualized bands was quantitated by UN-SCAN-IT 7.0 gel and graph digitizing software and expressed as a relative-fold to untreated control in two independent experiments.…”

High Expression of AHSP, EPB42, GYPC and HEMGN Predicts Favorable Prognosis in FLT3-ITD-Negative Acute Myeloid Leukemia

“…Numerous somatic mutations have been discerned in AML. Somatic mutations in gene encoding nucleophosmin ( NPM1 ), CCAAT/enhancer binding protein alpha ( CEBPA ) and fms-related tyrosine kinase 3 ( FLT3 ), DNA (cytosine-5-)-methyltransferase 3 alpha ( DNMT3A ) [9], tet methylcytosine dioxygenase 2 ( TET2 ) [10], isocitrate dehydrogenase 1/2 ( IDH1/2 ) [11, 12], additional sex combs like 1 ( ASXL1 ) [13] and PHD finger protein 6 ( PHF6 ) [14, 15] have been implicated as well-established genetic marker in AML [16, 17]. …”