title: Synergistic apoptosis of CML cells by buthionine sulfoximine and hydroxychavicol correlates with activation of AIF and GSH-ROS-JNK-ERK-iNOS pathway.
abstract: BACKGROUND
  Hydroxychavicol (HCH), a constituent of Piper betle leaf has been reported to exert anti-leukemic activity through induction of reactive oxygen species (ROS). The aim of the study is to optimize the oxidative stress -induced chronic myeloid leukemic (CML) cell death by combining glutathione synthesis inhibitor, buthionine sulfoximine (BSO) with HCH and studying the underlying mechanism.
  
  
  METHODS
  Anti-proliferative activity of BSO and HCH alone or in combination against a number of leukemic (K562, KCL22, KU812, U937, Molt4), non-leukemic (A549, MIA-PaCa2, PC-3, HepG2) cancer cell lines and normal cell lines (NIH3T3, Vero) was measured by MTT assay. Apoptotic activity in CML cell line K562 was detected by flow cytometry (FCM) after staining with annexin V-FITC/propidium iodide (PI), detection of reduced mitochondrial membrane potential after staining with JC-1, cleavage of caspase- 3 and poly (ADP)-ribose polymerase proteins by western blot analysis and translocation of apoptosis inducing factor (AIF) by confocal microscopy. Intracellular reduced glutathione (GSH) was measured by colorimetric assay using GSH assay kit. 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA) and 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) were used as probes to measure intracellular increase in ROS and nitric oxide (NO) levels respectively. Multiple techniques like siRNA transfection and pharmacological inhibition were used to understand the mechanisms of action.
  
  
  RESULTS
  Non-apoptotic concentrations of BSO significantly potentiated HCH-induced apoptosis in K562 cells. BSO potentiated apoptosis-inducing activity of HCH in CML cells by caspase-dependent as well as caspase-independent but apoptosis inducing factor (AIF)-dependent manner. Enhanced depletion of intracellular GSH induced by combined treatment correlated with induction of ROS. Activation of ROS- dependent JNK played a crucial role in ERK1/2 activation which subsequently induced the expression of inducible nitric oxide synthase (iNOS). iNOS- mediated production of NO was identified as an effector molecule causing apoptosis of CML cells.
  
  
  CONCLUSIONS
  BSO synergizes with HCH in inducing apoptosis of CML cells through the GSH-ROS-JNK-ERK-iNOS pathway.
authors.affiliation: Division of Cancer Biology and Inflammatory Disorder, Council of Scientific and Industrial Research (CSIR)-Indian Institute of Chemical Biology (IICB), Kolkata, India.
authors.forename: Avik Acharya
authors.initials: AA
authors.lastname: Chowdhury
authors.forename: Jaydeep
authors.initials: J
authors.lastname: Chaudhuri
authors.forename: Nabendu
authors.initials: N
authors.lastname: Biswas
authors.forename: Anirban
authors.initials: A
authors.lastname: Manna
authors.forename: Saurav
authors.initials: S
authors.lastname: Chatterjee
authors.forename: Sanjit K
authors.initials: SK
authors.lastname: Mahato
authors.forename: Utpal
authors.initials: U
authors.lastname: Chaudhuri
authors.forename: Parasuraman
authors.initials: P
authors.lastname: Jaisankar
authors.forename: Santu
authors.initials: S
authors.lastname: Bandyopadhyay
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D051033:Apoptosis Inducing Factor / Q000378:metabolism*; D015153:Blotting, Western; D019328:Buthionine Sulfoximine / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D004357:Drug Synergism; D005054:Eugenol / Q000031:analogs & derivatives* / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D056945:Hep G2 Cells; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D020014:K562 Cells; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D051379:Mice; D018613:Microscopy, Confocal; D041681:NIH 3T3 Cells; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D020298:U937 Cells; D014709:Vero Cells
chemical_list: C494829:AIFM1 protein, human; D051033:Apoptosis Inducing Factor; D017382:Reactive Oxygen Species; C051268:2-hydroxychavicol; D009569:Nitric Oxide; D005054:Eugenol; D019328:Buthionine Sulfoximine; D052247:Nitric Oxide Synthase Type II; D048049:Extracellular Signal-Regulated MAP Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D005978:Glutathione
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