Differential induction of reactive oxygen species through Erk1/2 and Nox-1 by FK228 for selective apoptosis of oncogenic H-Ras-expressing human urinary bladder cancer J82 cells.
OBJECTIVE
This study sought to reveal mechanisms for differential regulation of reactive oxygen species (ROS) in histone deacetylase inhibitor FK228-induced selective apoptosis of oncogenic H-Ras-expressing human cancer cells.


METHODS
Human urinary bladder cancer J82 and oncogenic H-Ras-expressing J82 cells were used to reveal FK228-induced differential Erk1/2 activation, Nox-1 elevation, ROS production, glutathione (GSH) depletion, caspase activation, and apoptosis. Specific inhibitors were used to suppress Nox-1 activity and ROS production. Mek1/2 inhibitor was used to suppress Erk1/2 activation. Validated-specific siRNAs were used to knock down Nox-1. ROS levels, GSH levels, and caspase-3/7 activities were measured by GSH assay, flow cytometry and luminescence assays, respectively. Western blot analysis determined levels of Erk1/2 and Nox-1.


RESULTS
Erk1/2, Nox-1, ROS, caspase-3/7, and cell death were differentially induced, whereas GSH was differentially depleted by FK228 in oncogenic H-Ras-expressing J82 versus parental cells. Blockage of the ERK pathway resulted in suppressing oncogenic H-Ras- and FK228-induced Nox-1 elevation, ROS production, caspase activation, and cell death. Knockdown of Nox-1 by specific siRNAs reduced FK228-induced ROS production, caspase activation, and cell death.


CONCLUSIONS
Oncogenic H-Ras expression and FK228 treatment synergistically induced the ERK pathway, resulting in differentially increased Nox-1 elevation, ROS production, and GSH depletion, leading to differential caspase activation and cell death in oncogenic H-Ras-expressing J82 versus parental cells.
Choudhary
Shambhunath
S
Anticancer Molecular Oncology Laboratory, Department of Comparative Medicine, College of Veterinary Medicine, The University of Tennessee, Knoxville, TN 37996, USA. schoudha@utk.edu
Rathore
Kusum
K
Wang
Hwa-Chain Robert
HC
D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D047630:Depsipeptides / Q000494:pharmacology*; D004790:Enzyme Induction / Q000187:drug effects; D005978:Glutathione / Q000172:deficiency / Q000378:metabolism; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects; D008928:Mitochondria / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D000074624:NADPH Oxidase 1; D019255:NADPH Oxidases / Q000096:biosynthesis / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000096:biosynthesis*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D001749:Urinary Bladder Neoplasms / Q000188:drug therapy* / Q000201:enzymology* / Q000473:pathology
D000903:Antibiotics, Antineoplastic; D047630:Depsipeptides; D017382:Reactive Oxygen Species; C087123:romidepsin; D000074624:NADPH Oxidase 1; D019255:NADPH Oxidases; C402799:NOX1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020169:Caspases; D016283:Proto-Oncogene Proteins p21(ras); D005978:Glutathione
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