title: The pyridone-annelated isoindigo (5'-Cl) induces apoptosis, dysregulation of mitochondria and formation of ROS in leukemic HL-60 cells.
abstract: OBJECTIVE
  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).
  
  
  CONCLUSIONS
  We demonstrated that the growth inhibitory effects of 5'-Cl in HL-60 cells involve multiple pathways of apoptosis and dysregulation of mitochondrial functions.
authors: Department of Basic Medical Sciences, College of Medicine, King Saud Bin Abdulaziz University for Health Sciences (KSAU-HS), National Guard Health Affairs, Riyadh, Kingdom of Saudi Arabia.
authors: Ayman M
authors: AM
authors: Saleh
authors: Ahmad
authors: A
authors: Aljada
authors: Mustafa M
authors: MM
authors: El-Abadelah
authors: Salim S
authors: SS
authors: Sabri
authors: Jalal A
authors: JA
authors: Zahra
authors: Amre
authors: A
authors: Nasr
authors: Mohammad A
authors: MA
authors: Aziz
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D045304:Cytochromes c / Q000378:metabolism; D018922:HL-60 Cells; D006801:Humans; D007211:Indoles / Q000737:chemistry / Q000494:pharmacology; D015473:Leukemia, Promyelocytic, Acute / Q000378:metabolism / Q000473:pathology; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D011728:Pyridones / Q000737:chemistry* / Q000494:pharmacology; D011759:Pyrrolidines / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013859:Thiocarbamates / Q000494:pharmacology; D051028:bcl-2-Associated X Protein / Q000378:metabolism
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D000970:Antineoplastic Agents; D007211:Indoles; D019253:Proto-Oncogene Proteins c-bcl-2; D011728:Pyridones; D011759:Pyrrolidines; D017382:Reactive Oxygen Species; D013859:Thiocarbamates; D051028:bcl-2-Associated X Protein; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C020972:pyrrolidine dithiocarbamic acid; C049010:isoindigotin; D045304:Cytochromes c; D020169:Caspases; D000111:Acetylcysteine