cytochrome-c-t and alvocidib

Although flavopiridol, a semisynthetic flavone, was initially thought to be a specific inhibitor of cyclin-dependent kinases, it has now been shown that flavopiridol mediates antitumor responses through mechanism(s) yet to be defined. We have shown previously that flavopiridol abrogates tumor necrosis factor (TNF)-induced nuclear factor-kappaB (NF-kappaB) activation. In this report, we examined whether this flavone affects other cellular responses activated by TNF. TNF is a potent inducer of activator protein-1 (AP-1), and flavopiridol abrogated this activation in a dose- and time-dependent manner. Flavopiridol also suppressed AP-1 activation induced by various carcinogens and inflammatory stimuli. When examined for its effect on other signaling pathways, flavopiridol inhibited TNF-induced activation of various mitogen-activated protein kinases, including c-Jun NH(2)-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and p44/p42 MAPK. It is noteworthy that this flavone also suppressed TNF-induced activation of Akt, a cell survival kinase, and expression of various antiapoptotic proteins, such as IAP-1, IAP-2, XIAP, Bcl-2, Bcl-xL, and TRAF-1. Flavopiridol also inhibited the TNF-induced induction of intercellular adhesion molecule-1, c-Myc, and c-Fos, all known to mediate tumorigenesis. Moreover, TNF-induced apoptosis was enhanced by flavopiridol through activation of the bid-cytochrome-caspase-9-caspase-3 pathway. Overall, our results clearly suggest that flavopiridol interferes with the TNF cell-signaling pathway, leading to suppression of antiapoptotic mechanisms and enhancement of apoptosis.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Caspases; Cell Line; Cell Nucleus; Cell Proliferation; Cytochromes c; Enzyme Activation; Flavonoids; Gene Expression Regulation; Genes, Reporter; Humans; Intercellular Adhesion Molecule-1; JNK Mitogen-Activated Protein Kinases; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Myeloid Cells; Neoplasms; p38 Mitogen-Activated Protein Kinases; Piperidines; Protein Transport; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-myc; Transcription Factor AP-1; Tumor Necrosis Factors

The mechanism and functional significance of XIAP and Mcl-1 down-regulation in human leukemia cells exposed to the histone deacetylase inhibitor vorinostat and the cyclin-dependent kinase inhibitor flavopiridol was investigated. Combined exposure of U937 leukemia cells to marginally toxic concentrations of vorinostat and flavopiridol resulted in a marked increase in mitochondrial damage and apoptosis accompanied by pronounced reductions in XIAP and Mcl-1 mRNA and protein. Down-regulation of Mcl-1 and XIAP expression by vorinostat/flavopiridol was associated with enhanced inhibition of phosphorylation of RNA polymerase II and was amplified by caspase-mediated protein degradation. Chromatin immunoprecipitation analysis revealed that XIAP and Mcl-1 down-regulation were also accompanied by both decreased association of nuclear factor-kappaB (XIAP) and increased E2F1 association (Mcl-1) with their promoter regions, respectively. Ectopic expression of Mcl-1 but not XIAP partially protected cells from flavopiridol/vorinostat-mediated mitochondrial injury at 48 h, but both did not significantly restored clonogenic potential. Flavopiridol/vorinostat-mediated transcriptional repression of XIAP, Mcl-1-enhanced apoptosis, and loss of clonogenic potential also occurred in primary acute myelogenous leukemia (AML) blasts. Together, these findings indicate that transcriptional repression of XIAP and Mcl-1 by flavopiridol/vorinostat contributes functionally to apoptosis induction at early exposure intervals and raise the possibility that expression levels may be a useful surrogate marker for activity in current trials.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Inducing Factor; Blast Crisis; Blotting, Western; Butyrates; Caspases; Chromatin Immunoprecipitation; Cyclin-Dependent Kinases; Cytochromes c; Down-Regulation; Drug Interactions; Flavonoids; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Leukemia, Myeloid, Acute; Membrane Potential, Mitochondrial; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Piperidines; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transcription, Genetic; Tumor Stem Cell Assay; U937 Cells; Vorinostat; X-Linked Inhibitor of Apoptosis Protein

Interactions between the cyclin-dependent kinase inhibitor flavopiridol (FP) and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/Apo2L), were examined in human leukemia cells (U937 and Jurkat). Coexposure of cells to marginally toxic concentrations of TRAIL and FP (24 h) synergistically increased mitochondrial injury (eg, cytochrome c, AIF, Smac/DIABLO release), cytoplasmic depletion of Bax, activation of Bid as well as caspase-8 and -3, PARP cleavage, and apoptosis. Coadministration of TRAIL markedly increased FP-induced apoptosis in leukemic cells ectopically expressing Bcl-2, Bcl-x(L), or a phosphorylation loop-deleted form of Bcl-2 (DeltaBcl-2), whereas lethality was substantially attenuated in cells ectopically expressing CrmA, dominant-negative-FADD, or dominant-negative-caspase-8. TRAIL/FP induced no discernible changes in FLIP, DR4, DR5, Mcl-1, or survivin expression, modest declines in levels of DcR2 and c-IAP, but resulted in the marked transcriptional downregulation of XIAP. Moreover, cells stably expressing an XIAP-antisense construct exhibited a pronounced increase in TRAIL sensitivity comparable to degrees of apoptosis achieved with TRAIL/FP. Conversely, enforced XIAP expression significantly attenuated caspase activation and TRAIL/FP lethality. Together, these findings suggest that simultaneous activation of the intrinsic and extrinsic apoptotic pathways by TRAIL and FP synergistically induces apoptosis in human leukemia cells through a mechanism that involves FP-mediated XIAP downregulation.

Topics: Apoptosis; Apoptosis Inducing Factor; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; bcl-X Protein; BH3 Interacting Domain Death Agonist Protein; Carrier Proteins; Caspases; Cell Cycle; Complement Membrane Attack Complex; Complement System Proteins; Cytochromes c; Down-Regulation; Drug Interactions; Drug Synergism; Flavonoids; Flavoproteins; Glycoproteins; HL-60 Cells; Humans; Intracellular Signaling Peptides and Proteins; Jurkat Cells; Leukemia; Membrane Glycoproteins; Membrane Proteins; Mitochondria; Mitochondrial Proteins; Multiple Myeloma; Phosphorylation; Piperidines; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Proteins; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; TNF-Related Apoptosis-Inducing Ligand; Transcription, Genetic; Tumor Necrosis Factor-alpha; U937 Cells; X-Linked Inhibitor of Apoptosis Protein

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

Interactions between the cyclin-dependent kinase inhibitor flavopiridol and the small-molecule Bcl-2 antagonist HA14-1 were examined in human multiple myeloma cells. Whereas individual treatment of U266 myeloma cells with 10 micromol/L HA14-1 or 100 nmol/L flavopiridol had little effect, exposure of cells to flavopiridol (6 hours) followed by HA14-1 (18 hours) resulted in a striking increase in mitochondrial dysfunction (cytochrome c and Smac/DIABLO release; loss of mitochondrial membrane potential), activation of the caspase cascade, apoptosis, and diminished clonogenic survival. Similar findings were noted in other myeloma cell lines (e.g., MM.1S, RPMI8226, and NCI-H929) as well as in those resistant to dexamethasone and cytotoxic agents (e.g., MM.1R, 8226/Dox40, and 8226/LR5). Combined exposure to flavopiridol and HA14-1 was associated with down-regulation of Mcl-1 and Bcl-xL, Bid cleavage, and mitochondrial translocation of Bax. Flavopiridol/HA14-1-treated cells also exhibited a pronounced activation of Jun NH2-terminal kinase, a modest activation of p38 mitogen-activated protein kinase, and down-regulation of cyclin D1. Flavopiridol/HA14-1-induced apoptosis was associated with a marked increase in reactive oxygen species generation; moreover,both events were attenuated by the antioxidant N-acetyl-l-cysteine. Finally, in contrast to dexamethasone, flavopiridol/HA14-1-induced lethality was unaffected by exogenous interleukin-6 or insulin-like growth factor-I. Together, these findings indicate that flavopiridol and the small-molecule Bcl-2 antagonist HA14-1 cooperate to trigger oxidant injury, mitochondrial dysfunction, caspase activation, and apoptosis in human multiple myeloma cells and suggest that this approach may warrant further evaluation as an antimyeloma strategy.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; bcl-X Protein; Benzopyrans; BH3 Interacting Domain Death Agonist Protein; Carrier Proteins; Cyclin D1; Cyclin-Dependent Kinases; Cytochromes c; Drug Synergism; Enzyme Activation; Enzyme Inhibitors; Flavonoids; Free Radicals; Humans; Intracellular Signaling Peptides and Proteins; JNK Mitogen-Activated Protein Kinases; Membrane Potentials; Mitochondria; Mitochondrial Proteins; Multiple Myeloma; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Nitriles; p38 Mitogen-Activated Protein Kinases; Piperidines; Protein Transport; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured

Flavopiridol was developed as a drug for cancer therapy due to its ability to inhibit cell cycle progression by targeting cyclin-dependent kinases (CDKs). In this study, we show that flavopiridol may also have a neuroprotective action. We show that at therapeutic dosage (or at micromolar range), flavopiridol almost completely prevents colchicine-induced apoptosis in cerebellar granule neurones. In agreement with this, flavopiridol inhibits both the release of cyt c and the activation of caspase-3 induced in response to colchicine treatment. We demonstrate that in this cellular model for neurotoxicity, neither re-entry in the cell cycle nor activation of stress-activated protein kinases, such as c-Jun N-terminal kinase (JNK) or p38 MAP kinase, is involved. In contrast, we show that colchicine-induced apoptosis correlates with a substantial increase in the expression of cdk5 and Par-4, which is efficiently prevented by flavopiridol. Accordingly, a cdk5 inhibitor such as roscovitine, but not a cdk4 inhibitor such as 3-ATA, was also able to protect neurons from apoptosis as well as prevent accumulation of cdk5 and Par-4 in response to colchicine. Our data suggest a potential therapeutic use of flavopiridol in disorders of the central nervous system in which cytoskeleton alteration mediated by cdk5 activation and Par-4 expression has been demonstrated, such as Alzheimer's disease.

Topics: Amino Acid Chloromethyl Ketones; Animals; Animals, Newborn; Anthracenes; Anti-Bacterial Agents; Apoptosis; Apoptosis Regulatory Proteins; Blotting, Western; Bromodeoxyuridine; Carrier Proteins; Caspase 3; Caspases; CDC2-CDC28 Kinases; Cell Count; Cell Survival; Cells, Cultured; Cerebellum; Chromatin; Colchicine; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 5; Cyclin-Dependent Kinases; Cytochromes c; Dose-Response Relationship, Drug; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Flavonoids; Flow Cytometry; Immunohistochemistry; Intracellular Signaling Peptides and Proteins; JNK Mitogen-Activated Protein Kinases; Kainic Acid; MAP Kinase Kinase 4; Microtubules; Minocycline; Mitogen-Activated Protein Kinase Kinases; Neurons; Neuroprotective Agents; Piperidines; Purines; Rats; Rats, Sprague-Dawley; Roscovitine; Time Factors; Tubulin

Flavopiridol is a cyclin-dependent kinase inhibitor currently under development by the National Cancer Institute both as a single agent and in combination with chemotherapy. There have been numerous reports that flavopiridol potently enhances the induction of apoptosis by chemotherapy. However, the effect of flavopiridol on radiotherapy (RT)-induced apoptosis has been largely untested. RT has become the cornerstone of adjuvant treatment of colorectal and gastric cancer. In view of this, we elected to evaluate the effect of flavopiridol on potentiating RT-induced apoptosis in the human colon cancer cell line HCT-116 and the gastric cancer cell line MKN-74.. The efficacy of combination of gamma-irradiation and flavopiridol was tested in vitro in MKN-74 and HCT-116 cells and correlated to changes in p21 expression. HCT-116 cells were also established as tumors in nude mice and treated with gamma-irradiation and flavopiridol either as single agents or in sequential combinations such that flavopiridol was either given 7 h before, concomitantly, or 3 and 7 h after gamma-irradiation.. Flavopiridol significantly enhanced the induction of apoptosis by gamma-irradiation in both cell lines as measured by quantitative fluorescent microscopy, caspase-3 activation, poly(ADP-ribose) polymerase cleavage, and cytochrome c release. To achieve the best effect, it was important to expose the tumor cells to gamma-irradiation before the flavopiridol. This sequence dependence was confirmed in vivo. When gamma-irradiation was administered 7 h before flavopiridol, 42% of the tumor-bearing animals were rendered disease free, compared with no animals treated with either gamma-irradiation or flavopiridol alone. Examination of the p21 status of HCT-116 and MKN-74 cells, after treatment with sequential gamma-irradiation and flavopiridol, indicated a loss of p21 protein expression. Loss of p21 was mainly due to cleavage by caspases. HCT-116 cells that lack p21 (p21(-/-)) also exhibited sensitization to gamma-irradiation and showed an even greater enhancement of gamma-irradiation-induced apoptosis by flavopiridol when compared with the parental HCT-116 cells.. These studies indicate that gamma-irradiation followed by flavopiridol enhances apoptosis and yields significantly increased tumor regressions and cures that are not achievable with radiation alone. These results indicate that flavopiridol can potently enhance the effect of gamma-radiation both in vitro and in vivo and may provide a new means to treat patients with locally advanced gastrointestinal cancers.

Topics: Animals; Antineoplastic Agents; Apoptosis; Caspase 3; Caspases; Cell Division; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; Cytochromes c; Enzyme Activation; Enzyme Inhibitors; Flavonoids; Gamma Rays; Male; Mice; Mice, Nude; Microscopy, Fluorescence; Piperidines; Poly(ADP-ribose) Polymerases; Stomach Neoplasms; Transplantation, Heterologous; Tumor Cells, Cultured

Topics: Cytochromes c; Flavonoids; NADH Dehydrogenase; Oxidoreductases; Piperidines; Saccharomyces cerevisiae; Yeasts