Role of reactive oxygen species in proapoptotic ability of oncogenic H-Ras to increase human bladder cancer cell susceptibility to histone deacetylase inhibitor for caspase induction.
OBJECTIVE
Reveal mechanisms for the novel proapoptotic ability of oncogenic H-Ras to increase cell susceptibility to a histone deacetylase inhibitor (HDACI) FR901228 for inducing caspase activation and selective apoptosis.


METHODS
Human urinary bladder cancer J82 and oncogenic H-Ras-expressing J82 cells were used to reveal differential induction of intracellular reactive oxygen species (ROS), caspase activation, and apoptosis by HDACI FR901228. ROS levels and caspase-8, -9, and -3/7 activities were measured by flow cytometry and luminescence assays, respectively. Specific inhibitors were used to suppress caspases and ROS. Western blot analysis determined modulators of caspase pathways.


RESULTS
ROS, caspase activity, and cell death was differentially increased by FR901228 in oncogenic H-Ras-expressing J82 versus parental cells. Blocking ROS resulted in reduced FR901228-induced cell death and caspase activation. Suppression of caspase-8 resulted in reduced FR901228-activated caspase-9 and -3/7. Suppression of caspase-9 resulted in reduced FR901228-activated caspase-3/7. Although FR901228 induced an ROS-dependent increase of FasL, FasL failed to induce caspase activation and cell death.


CONCLUSIONS
Increased ROS played an important role in the activation of the extrinsic and intrinsic caspase pathways to cooperatively induce executioner caspase-3/7 through a novel FasL-independent pathway in FR901228-induced selective apoptosis of oncogenic H-Ras-expressing J82 versus parental cells.
Choudhary
Shambhunath
S
Anticancer Molecular Oncology Laboratory, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996, USA.
Wang
Hwa-Chain Robert
HC
D017209:Apoptosis; D020169:Caspases / Q000096:biosynthesis*; D045744:Cell Line, Tumor; D047630:Depsipeptides / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D004790:Enzyme Induction / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D053222:Fas Ligand Protein / Q000502:physiology; D056572:Histone Deacetylase Inhibitors; D006801:Humans; D008928:Mitochondria / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D001749:Urinary Bladder Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology
D047630:Depsipeptides; D004791:Enzyme Inhibitors; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D056572:Histone Deacetylase Inhibitors; D017382:Reactive Oxygen Species; C087123:romidepsin; D020169:Caspases; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)