Induction of Apoptosis by Flavopiridol in Human Neuroblastoma Cells Is Enhanced under Hypoxia and Associated With N-myc Proto-oncogene Down-Regulation

Abstract

Purpose: 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.Experimental Design: 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 pulselabeling 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 G 1 -G 2 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 advancedstage neuroblastomas in vitro and is worthy of further investigation for the treatment of this disease.

Flavopiridol causes early mitochondrial damage in chronic lymphocytic leukemia cells with impaired oxygen consumption and mobilization of intracellular calcium

“…4 On the contrary, our experiments using primary CLL tumor cells suggest that since flavopiridol-induced loss of ⌬⌿ m is not rescued by either a pan-caspase inhibitor or specific inhibitors of caspase-8 and caspase-9, this process is either caspase-independent or lies upstream of caspase activation. Furthermore, Puppo et al 43 showed that treatment of human neuroblastoma cells with flavopiridol causes an early activation of caspase-3 prior to extensive cell death, similar to what we reported previously in CLL patient cells. 44 However, in this report, caspase-8 and caspase-9 were not activated, suggesting that in flavopiridol-mediated cytotoxicity caspase-3 may be acting upstream of these activator caspases.…”

Purging of the Neuroblastoma Stem Cell Compartment and Tumor Regression on Exposure to Hypoxia or Cytotoxic Treatment

“…Hypoxic conditions (0.1% O 2 ) were achieved by culturing the cells in an anaerobic workstation incubator (Concept 400, Jouan, Milan, Italy) flushed with a gas mixture containing 0.1% O 2 , 5% CO 2 , and balanced N 2 at 37jC in a humidified atmosphere (10). Oxygen tension in the medium was measured with a portable trace oxygen analyzer (Dräger PACIII).…”

Hypoxia inducible factor-2α: a critical mediator of aggressive tumor phenotypes

“…Accumulation of HIF-1α and/or HIF-2α proteins has been observed in vitro in various advanced-stage, metastatic human neuroblastoma cell lines exposed to hypoxia [28]. Hypoxia has a profound impact on aggressive neuroblastoma behavior: increased production of the vascular endothelial growth factor (VEGF), inhibitor of differentiation (ID), and Notch family proteins was observed in hypoxic neuroblastoma cells both in vitro and in vivo [29,30]. Interestingly, high levels of HIF-2α expression have been proposed to promote an aggressive neuroblastoma phenotype [21].…”