alvocidib and Hypoxia

Angiogenesis is a critical step required for sustained tumor growth and tumor progression. The stimulation of endothelial cells by cytokines secreted by tumor cells such as vascular endothelial growth factor (VEGF) induces their proliferation and migration. This is a prominent feature of high-grade gliomas. The secretion of VEGF is greatly upregulated under conditions of hypoxia because of the transcription factor hypoxiainducible factor (HIF)-1alpha, which controls the expression of many genes, allowing rapid adaptation of cells to their hypoxic microenvironment. Flavopiridol, a novel cyclin-dependent kinase inhibitor, has been attributed with antiangiogenic properties in some cancer cell lines by its ability to inhibit VEGF production. Here, we show that flavopiridol treatment of human U87MG and T98G glioma cell lines decreases hypoxia-mediated HIF-1alpha expression, VEGF secretion, and tumor cell migration. These in vitro results correlate with reduced vascularity of intracranial syngeneic GL261 gliomas from animals treated with flavopiridol. In addition, we show that flavopiridol downregulates HIF-1alpha expression in the presence of a proteasome inhibitor, an agent that normally results in the accumulation and overexpression of HIF-1alpha. The potential to downregulate HIF-1alpha expression with flavopiridol treatment in combination with a proteasome inhibitor makes this an extremely attractive anticancer treatment strategy for tumors with high angiogenic activity, such as gliomas.

Topics: Animals; Blotting, Western; Boronic Acids; Brain Neoplasms; Cell Line, Tumor; Cell Movement; Down-Regulation; Female; Flavonoids; Glioma; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Matrix Metalloproteinase 2; Mice; Neovascularization, Pathologic; Piperidines; Polymerase Chain Reaction; Protease Inhibitors; Proteasome Endopeptidase Complex; Protein Kinase Inhibitors; RNA, Messenger; Transcription Factors; Transcription, Genetic; Vascular Endothelial Growth Factor A

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.. 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.. 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.. 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.

Topics: Apoptosis; Bromodeoxyuridine; Caspases; Cell Hypoxia; Cell Survival; Cyclin-Dependent Kinases; Cytochromes c; DNA; Down-Regulation; Enzyme Activation; Enzyme Inhibitors; Flavonoids; G1 Phase; G2 Phase; Genes, myc; Growth Inhibitors; Humans; Hypoxia; In Situ Nick-End Labeling; Neuroblastoma; Piperidines; Proto-Oncogene Mas; Tetrazolium Salts; Thiazoles; Tumor Cells, Cultured

We have investigated the effects of flavopiridol, a novel protein kinase inhibitor that is selective for cyclin-dependent kinases, on hypoxia-induced vascular endothelial growth factor (VEGF) expression in human monocytes. We found that hypoxia induces a time-dependent increase of VEGF mRNA expression and protein levels in human monocytes. Flavopiridol showed a minimal effect on the constitutive levels of VEGF mRNA but completely blocked hypoxia-induced VEGF mRNA and protein expression. The inhibitory effects of flavopiridol on VEGF mRNA induction also occurred in the presence of cycloheximide. The transcriptional activation of either a VEGF promoter-luciferase construct or a hypoxia-inducible factor 1 reporter plasmid was not affected by addition of flavopiridol in transient transfection experiments. In contrast, actinomycin D experiments demonstrated that flavopiridol dramatically decreased VEGF mRNA stability. These data provide the first evidence that flavopiridol can affect gene expression by altering mRNA stability. We propose that flavopiridol may interfere with one or more signaling events, leading to hypoxia-induced, protein kinase-modulated, RNA protein binding activity. An important clinical implication of our results is that flavopiridol, presently under investigation in clinical trials, might have antiangiogenic as well as direct antiproliferative effects.

Topics: Blotting, Northern; Dose-Response Relationship, Drug; Down-Regulation; Endothelial Growth Factors; Enzyme Inhibitors; Flavonoids; Humans; Hypoxia; Luciferases; Lymphokines; Monocytes; Piperidines; Protein Kinase Inhibitors; RNA, Messenger; Time Factors; Transcriptional Activation; Transfection; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors