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Publication Details
AFRICAN RESEARCH NEXUS
SHINING A SPOTLIGHT ON AFRICAN RESEARCH
Synergistic Apoptosis of CML Cells by Buthionine Sulfoximine and Hydroxychavicol Correlates with Activation of AIF and GSH-ROS-JNK-ERK-iNOS Pathway
PLoS ONE, Volume 8, No. 9, Article e73672, Year 2013
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Description
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.Materials and 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 annexinV-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.Conclusion/Significance:BSO synergizes with HCH in inducing apoptosis of CML cells through the GSH-ROS-JNK-ERK-iNOS pathway. © 2013 Acharya Chowdhury et al.
Authors & Co-Authors
Chowdhury, Avik Acharya
India, New Delhi
Council of Scientific and Industrial Research India
Chaudhuri, Jaydeep
India, New Delhi
Council of Scientific and Industrial Research India
Biswas, Nabendu
India, New Delhi
Council of Scientific and Industrial Research India
India, Kolkata
Presidency University, Kolkata
Manna, Anirban
India, New Delhi
Council of Scientific and Industrial Research India
Chatterjee, Saurav
India, Kolkata
Csir-iicb
Mahato, Sanjit K.
India, Kolkata
Csir-iicb
South Africa, Johannesburg
University of Johannesburg
Chaudhuri, Utpal
India, Kolkata
Medical College and Hospital Kolkata
Jaisankar, Parasuraman
India, Kolkata
Csir-iicb
Bandyopadhyay, Santu
India, New Delhi
Council of Scientific and Industrial Research India
Statistics
Citations: 38
Authors: 9
Affiliations: 5
Identifiers
Doi:
10.1371/journal.pone.0073672
e-ISSN:
19326203
Research Areas
Cancer