Nuclear factor-kappa B and apoptosis inducing factor activation by doxorubicin analog WP744 in SH-SY5Y neuroblastoma cells.
BACKGROUND
Neuroblastoma (NB) is one of the most common extracranial tumors in children. The chemotherapeutic doxorubicin (Dox) remains a mainstay for treatment. However, the emergence of drug resistance seriously limits treatment efficacy. Numerous Dox analogues have been designed to more potently kill drug naive as well as drug-resistant NB. We have shown that the Dox analogue WP744 has enhanced the killing activity of NB compared to Dox, but the mechanism(s) of action is unclear.


METHODS
MTT assays were used to characterize the relative potencies of Dox and WP744 against SH-SY5Y NB cells. Western blotting was used to assess activation of caspases-3, -9, anti-poly (ADP-ribose) polymerase, p53, p21, AIF, IkappaBalpha, Bcl-2, Bcl-X(L), and cyclin D1. Nuclear factor (NF-kappaB) activation was assessed by electrophoretic mobility shift assay.


RESULTS
After WP744 treatment, enhanced apoptosis and cell death were seen, associated with cleavage of caspases-3, -9, and anti-poly (ADP-ribose) polymerase, an increase in p53 protein levels, and the induction of p21. WP744 also induced translocation of apoptosis-inducing factor from mitochondria to nuclei. Most remarkably, WP744 was 50-fold more potent than Dox in activating NF-kappaB. WP744 treatment also resulted in the down-regulation of expression of downstream targets of NF-kappaB.


CONCLUSIONS
WP744 is a novel Dox analogue that triggers apoptosis and cell killing by activation of proapoptotic mediators in NB cells. Enhanced cytotoxicity of this novel drug compared with Dox may be related to more effective activation of NF-kappaB and apoptosis-inducing factor in tumor cells.
Wu
Jianqiang
J
Department of Pediatric Surgery and Children's Hospital Research Foundation, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio 45229, USA.
Harris
Nathaniel L
NL
Inge
Thomas H
TH
D018943:Anthracyclines / Q000494:pharmacology*; D017209:Apoptosis; D051033:Apoptosis Inducing Factor; D001692:Biological Transport / Q000187:drug effects; D020169:Caspases / Q000378:metabolism; D016923:Cell Death; D045744:Cell Line, Tumor; D004317:Doxorubicin / Q000031:analogs & derivatives*; D004789:Enzyme Activation; D005420:Flavoproteins / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D009447:Neuroblastoma / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
C494829:AIFM1 protein, human; D018943:Anthracyclines; D051033:Apoptosis Inducing Factor; D005420:Flavoproteins; D008565:Membrane Proteins; D016328:NF-kappa B; D016159:Tumor Suppressor Protein p53; C451735:WP 744; D004317:Doxorubicin; D020169:Caspases