Induction of apoptosis by flavopiridol in human neuroblastoma cells is enhanced under hypoxia and associated with N-myc proto-oncogene down-regulation.
OBJECTIVE
Neuroblastoma is the most common extracranial solid tumor of children that arises from the sympathetic nervous system. Survival rates for neuroblastoma patients is low despite intensive therapeutic intervention, and the identification of new effective drugs remains a primary goal. The cyclin-dependent kinase inhibitor, flavopiridol, has demonstrated growth-inhibitory and cytotoxic activity against various tumor types. Our aim was to investigate flavopiridol effects on advanced-stage, N-myc proto-oncogene (MYCN)-amplified human neuroblastomas and the modulation of its activity by hypoxia, a critical determinant of tumor progression and a major challenge of therapy.


METHODS
Cell viability was monitored by 3-(4,5 dimethyl-2 thiazolyl)-2,5 diphenyl-2H tetrazolium bromide (MTT) and trypan blue dye exclusion assays; DNA synthesis was assessed with the bromodeoxyuridine pulse-labeling technique; apoptosis was studied by Giemsa staining, DNA fragmentation, terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling reaction, flow cytometric determination of hypodiploid DNA content, and evaluation of caspase activity and cytochrome c (CytC) release; MYCN expression was determined by Northern and Western blotting.


RESULTS
Flavopiridol caused dose- and time-dependent decreases in neuroblastoma viability by inducing apoptosis, as confirmed by morphologic and biochemical criteria. Cell death was preceded by DNA synthesis inhibition and G1-G2 arrest, reversed by the pancaspase inhibitor, zVAD-fmk, and associated with caspase-3 and -2 activation and CytC increase. Moreover, flavopiridol strongly down-regulated MYCN mRNA and protein expression. Exposure to hypoxia enhanced both the extent of apoptosis and flavopiridol effects on CytC, caspase 3, and MYCN.


CONCLUSIONS
These results indicate that flavopiridol has growth-inhibitory and apoptotic activity against advanced-stage neuroblastomas in vitro and is worthy of further investigation for the treatment of this disease.
Puppo
Maura
M
Laboratory of Molecular Biology, Giannina Gaslini Institute, Genoa, Italy.
Pastorino
Sandra
S
Melillo
Giovanni
G
Pezzolo
Annalisa
A
Varesio
Luigi
L
Bosco
Maria Carla
MC
D017209:Apoptosis / Q000187:drug effects*; D001973:Bromodeoxyuridine; D020169:Caspases / Q000378:metabolism; D015687:Cell Hypoxia; D002470:Cell Survival / Q000187:drug effects; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors; D045304:Cytochromes c / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D015536:Down-Regulation; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology*; D016193:G1 Phase / Q000187:drug effects; D016195:G2 Phase / Q000187:drug effects; D016259:Genes, myc / Q000502:physiology*; D006131:Growth Inhibitors / Q000494:pharmacology*; D006801:Humans; D000860:Hypoxia; D020287:In Situ Nick-End Labeling; D009447:Neuroblastoma / Q000378:metabolism / Q000473:pathology*; D010880:Piperidines / Q000494:pharmacology*; D000090063:Proto-Oncogene Mas; D013778:Tetrazolium Salts; D013844:Thiazoles; D014407:Tumor Cells, Cultured
D004791:Enzyme Inhibitors; D005419:Flavonoids; D006131:Growth Inhibitors; C000715470:MAS1 protein, human; D010880:Piperidines; D000090063:Proto-Oncogene Mas; D013778:Tetrazolium Salts; D013844:Thiazoles; C077990:alvocidib; D045304:Cytochromes c; D004247:DNA; D018844:Cyclin-Dependent Kinases; D020169:Caspases; C022616:thiazolyl blue; D001973:Bromodeoxyuridine