Abstract
Purpose
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.
Conclusion
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.
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Acknowledgments
We thank Dr. KK Chan at the Ohio State University for providing FK228, Ms. DJ Trent for technique support in flow cytometric analysis of ROS contents and cell death, and Ms. M Bailey for textual editing of the manuscript. This work was supported by the University of Tennessee, College of Veterinary Medicine, Center of Excellence in Livestock Diseases and Human Health (HCR Wang).
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Choudhary, S., Rathore, K. & Wang, HC.R. 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. J Cancer Res Clin Oncol 137, 471–480 (2011). https://doi.org/10.1007/s00432-010-0910-z
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DOI: https://doi.org/10.1007/s00432-010-0910-z