Abstract
We have further defined mechanism(s) by which 2-amino-N-{4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]-phenyl}acetamide [OSU-03012 (OSU)], a derivative of the cyclooxygenase-2 (COX2) inhibitor celecoxib but lacking COX2 inhibitory activity, kills transformed cells. In cells lacking expression of protein kinase R-like endoplasmic reticulum kinase (PERK-/-), the lethality of OSU was attenuated. OSU enhanced the expression of Beclin 1 and ATG5 and cleavage of pro-caspase 4 in a PERK-dependent fashion and promoted the Beclin 1- and ATG5-dependent formation of vacuoles containing LC3, followed by a subsequent caspase 4-dependent cleavage of cathepsin B and a cathepsin B-dependent formation of low pH intracellular vesicles; cathepsin B was activated and released into the cytosol and genetic suppression of caspase 4, cathepsin B, or apoptosis-inducing factor function significantly suppressed cell killing. In parallel, OSU caused PERK-dependent increases in 70-kDa heat shock protein (HSP70) expression and decreases in 90-kDa heat shock protein (HSP90) and Grp78/BiP expression. Changes in HSP70 expression were post-transcriptional. Knock-down or small-molecule inhibition of HSP70 expression enhanced OSU toxicity, and overexpression of HSP70 suppressed OSU-induced low pH vesicle formation and lethality. Our data demonstrate that OSU-03012 causes cell killing that is dependent on PERK-induced activation of multiple toxic proteases. OSU-03012 also increased expression of HSP70 in a PERK-dependent fashion, providing support for the contention that OSU-03012-induced PERK signaling promotes both cell survival and cell death processes.
Footnotes
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This work was funded by Public Health Service grants R01-DK52825, P01-CA104177, and R01-CA108520, Department of Defense Award DAMD17-03-1-0262 (all to P.D.) and by Public Health Service grants R01-CA63753 and R01-CA77141 and Leukemia Society of America Grant 6405-97 (all to S.G.). A portion of A.Y.'s funding is from the Department of Radiation Oncology, Virginia Commonwealth University, and P.D. is the holder of the Universal Inc. Professorship in Signal Transduction Research.
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ABBREVIATIONS: COX2, cyclooxygenase 2; OSU, OSU-03012 [2-amino-N-{4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]-phenyl}-acetamide]; ER, endoplasmic reticulum; zVAD, N-benzyloxycarbonyl-Val-Ala-Asp; AIF, apoptosis-inducing factor; PDK-1, pyruvate dehydrogenase kinase, isozyme 1; PI3K, phosphatidylinositol 3-kinase; AKT, protein kinase B; ERK1/2, extracellular signal-regulated kinase 1/2; PERK, PKR-like endoplasmic reticulum kinase; HSP70, 70-kDa heat shock protein; HSP90, 90-kDa heat shock protein; eIF2α, eukaryotic initiation factor-2α; HRP, horseradish peroxidase; GFP, green fluorescent protein; DMSO, dimethyl sulfoxide; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; 3MA, 3 methyl adenine; CMV, cytomegalovirus; siRNA, small-interfering RNA; CDTA, trans-1,2-diaminocyclohexane-N,N,N′,N′-tetra-acetic acid; MEF, mouse embryonic fibroblasts; GBM, glioblastoma multiforme; 17AAG, 17-(allylamino)-17-demethoxygeldanamycin; FITC, fluorescein isothiocyanate; SV40, simian virus 40; WT, wild type.
- Received October 15, 2007.
- Accepted December 20, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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