nitrophenols and Neuroblastoma

Catechol-O-methyltransferase (COMT) is an enzyme that inactivates dopamine and other catecholamines by O-methylation. Tolcapone, a drug commonly used in the treatment of Parkinson's disease, is a potent inhibitor of COMT and previous studies indicate that Tolcapone increases the bioavailability of dopamine in cells. In this study, we demonstrate that Tolcapone kills neuroblastoma (NB) cells in preclinical models by inhibition of COMT. Treating four established NB cells lines (SMS-KCNR, SH-SY5Y, BE(2)-C, CHLA-90) and two primary NB cell lines with Tolcapone for 48 h decreased cell viability in a dose-dependent manner, with IncuCyte imaging and Western blotting indicating that cell death was due to caspase-3-mediated apoptosis. Tolcapone also increased ROS while simultaneously decreasing ATP-per-cell in NB cells. Additionally, COMT was inhibited by siRNA in NB cells and showed similar increases in apoptotic markers compared to Tolcapone. In vivo xenograft models displayed inhibition of tumor growth and a significant decrease in time-to-event in mice treated with Tolcapone compared to untreated mice. These results indicate that Tolcapone is cytotoxic to neuroblastoma cells and invite further studies into Tolcapone as a promising novel therapy for the treatment of neuroblastoma.

Topics: Animals; Antineoplastic Agents; Apoptosis; Benzophenones; Caspase 3; Catechol O-Methyltransferase; Catechol O-Methyltransferase Inhibitors; Cell Line, Tumor; Cell Proliferation; Cell Survival; Female; Humans; Mice, Nude; Neuroblastoma; Nitrophenols; Oxidative Stress; Reactive Oxygen Species; Tolcapone; Tumor Cells, Cultured

The pediatric solid tumor neuroblastoma (NB) often depends on the anti-apoptotic protein, Mcl(-)1, for survival through Mcl(-)1 sequestration of pro-apoptotic Bim. High affinity Mcl(-)1 inhibitors currently do not exist such that novel methods to inhibit Mcl(-)1 clinically are in high demand. Receptor tyrosine kinases (RTK) regulate Mcl(-)1 in many cancers and play a role in NB survival, yet how they regulate Bcl(-)2 family interactions in NB is unknown. We found that NB cell lines derived to resist the Bcl(-)2/-xl/-w antagonist, ABT-737, acquire a dependence on Mcl(-)1 and show increased expression and activation of the RTK, EGFR. Mcl(-)1 dependent NB cell lines derived at diagnosis and from the same tumor following relapse also have increased EGFR expression compared to those dependent on Bcl(-)2. Inhibition of EGFR by shRNA or erlotinib in Mcl(-)1 dependent NBs disrupts Bim binding to Mcl(-)1 and enhances its affinity for Bcl(-)2, restoring sensitivity to ABT-737 as well as cytotoxics in vitro. Mechanistically treatment of NBs with small molecule inhibitors of EGFR (erlotinib, cetuximab) and ERK (U0126) increases Noxa expression and dephosphorylates Bim to promote Bim binding to Bcl(-)2. Thus, EGFR regulates Mcl(-)1 dependence in high-risk NB via ERK-mediated phosphorylation of Bim such that EGFR/ERK inhibition renders Mcl(-)1 dependent tumors now reliant on Bcl(-)2. Clinically, EGFR inhibitors are ineffective as single agent compounds in patients with recurrent NB, likely due to this transferred survival dependence to Bcl(-)2. Likewise, EGFR or ERK inhibitors warrant further testing in combination with Bcl(-)2 antagonists in vivo as a novel future combination to overcome therapy resistance in the clinic.

Topics: Antineoplastic Agents; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Biphenyl Compounds; Cell Line, Tumor; Drug Resistance, Neoplasm; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Gene Expression; Humans; Membrane Proteins; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Recurrence, Local; Neuroblastoma; Nitrophenols; Phosphorylation; Piperazines; Protein Binding; Protein Processing, Post-Translational; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; RNA Interference; RNA, Small Interfering; Signal Transduction; Sulfonamides

Chemotherapeutic drug resistance is one of the major causes for treatment failure in high-risk neuroblastoma (NB), the most common extra cranial solid tumor in children. Poor prognosis is typically associated with MYCN amplification. Here, we utilized a loss-of-function kinome-wide RNA interference screen to identify genes that cause cisplatin sensitization. We identified fibroblast growth factor receptor 2 (FGFR2) as an important determinant of cisplatin resistance. Pharmacological inhibition of FGFR2 confirmed the importance of this kinase in NB chemoresistance. Silencing of FGFR2 sensitized NB cells to cisplatin-induced apoptosis, which was regulated by the downregulation of the anti-apoptotic proteins BCL2 and BCLXL. Mechanistically, FGFR2 was shown to activate protein kinase C-δ to induce BCL2 expression. FGFR2, as well as the ligand fibroblast growth factor-2, were consistently expressed in primary NB and NB cell lines, indicating the presence of an autocrine loop. Expression analysis revealed that FGFR2 correlates with MYCN amplification and with advanced stage disease, demonstrating the clinical relevance of FGFR2 in NB. These findings suggest a novel role for FGFR2 in chemoresistance and provide a rational to combine pharmacological inhibitors against FGFR2 with chemotherapeutic agents for the treatment of NB.

Topics: Antineoplastic Agents; Apoptosis; bcl-X Protein; Biphenyl Compounds; Blotting, Western; Cell Line, Tumor; Cell Survival; Cisplatin; Drug Resistance, Neoplasm; Enzyme Activation; Fibroblast Growth Factor 2; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Neuroblastoma; Nitrophenols; Oligonucleotide Array Sequence Analysis; Piperazines; Protein Kinase C-delta; Proto-Oncogene Proteins c-bcl-2; Pyrroles; Receptor, Fibroblast Growth Factor, Type 2; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Signal Transduction; Sulfonamides

Fenretinide (4-HPR) is a cytotoxic retinoid with minimal systemic toxicity that has shown clinical activity against recurrent high-risk neuroblastoma. To identify possible synergistic drug combinations for future clinical trials, we determined whether ABT-737, a small-molecule BH3-mimetic that inhibits most proteins of the antiapoptotic Bcl-2 family, could enhance 4-HPR activity in neuroblastoma.. Eleven neuroblastoma cell lines were tested for the cytotoxic activity of 4-HPR and ABT-737 as single agents and in combination using the DIMSCAN fluorescence digital imaging cytotoxicity assay. The effect of these agents alone and in combination on mitochondrial membrane depolarization and apoptosis (by flow cytometry), cytochrome c release, caspases, Bax-α, t-Bid, and Bak activation, and subcutaneous xenografts in nu/nu mice was also determined.. Multilog synergistic cytotoxicity was observed for the drug combination in all of the 11 neuroblastoma cell lines tested, including MDR lines and those insensitive to either drug as single agents. 4-HPR + ABT-737 induced greater mitochondrial membrane depolarization and mitochondrial cytochrome c release, greater activation of caspases, Bax-α, t-Bid, and Bak, and a higher level of apoptosis than either drug alone. In vivo, 4-HPR + ABT-737 increased the event-free survival of the MDR human neuroblastoma line CHLA-119 implanted subcutaneously in nu/nu mice (194.5 days for the combination vs. 68 days for ABT-737 and 99 days for 4-HPR).. Thus, the combination of 4-HPR with a BH3-mimetic drug warrants clinical trials in recurrent neuroblastoma.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Biphenyl Compounds; Caspases; Cell Line, Tumor; Cytochromes c; Disease-Free Survival; Female; Fenretinide; Humans; Membrane Potential, Mitochondrial; Mice; Mice, Nude; Neuroblastoma; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Sulfonamides; Xenograft Model Antitumor Assays

Neuroblastoma is a common solid tumor of childhood and advanced disease carries a poor prognosis despite intensive multimodality therapy. Hypoxia is a common feature of solid tumors because of poorly organized tumor-induced neovasculature. Hypoxia is associated with advanced stage and poor outcome in a range of tumor types, and leads to resistance to clinically relevant cytotoxic agents in neuroblastoma and other pediatric tumors in vitro. Resistance to apoptosis is a common feature of tumor cells and leads to pleiotropic drug resistance, mediated by Bcl-2 family proteins. ABT-737 is a novel small-molecule inhibitor of Bcl-2 and Bcl-x(L) that is able to induce apoptosis in a range of tumor types. Neuroblastoma cell lines are relatively resistant to ABT-737-induced apoptosis in normoxia, but in contrast to the situation with conventional cytotoxic agents are more sensitive in hypoxia. This sensitization is because of an increase in ABT-737-induced apoptosis and is variably dependent upon the presence of functional hypoxia-inducible factor 1 (HIF-1) α. In contrast to the situation in colon carcinoma and non-small cell lung cancer cells, hypoxia does not result in downregulation of the known ABT-737 resistance factor, Mcl-1, nor any other Bcl-2 family proteins. ABT-737 sensitizes neuroblastoma cells to clinically relevant cytotoxic agents under normal levels of oxygen, and importantly, this sensitization is maintained under hypoxia when neuroblastoma cells are resistant to these agents. Thus rational combinations of ABT-737 and conventional cytotoxics offer a novel approach to overcoming hypoxia-induced drug resistance in neuroblastoma.

Topics: Antineoplastic Agents; Apoptosis; Biphenyl Compounds; Cell Hypoxia; Cell Line, Tumor; Drug Evaluation, Preclinical; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; Myeloid Cell Leukemia Sequence 1 Protein; N-Myc Proto-Oncogene Protein; Neuroblastoma; Nitrophenols; Nuclear Proteins; Oncogene Proteins; Oxygen; Piperazines; Proto-Oncogene Proteins c-bcl-2; Sulfonamides; Treatment Outcome

Bcl-2 family proteins regulate mitochondrial apoptosis downstream of diverse stressors. Cancer cells frequently deregulate Bcl-2 proteins leading to chemoresistance. We have optimized a platform for solid tumors in which Bcl-2 family resistance patterns are inferred. Functional mitochondria were isolated from neuroblastoma (NB) cell lines, exposed to distinct BH3-domain peptides, and assayed for cytochrome c release. Such BH3 profiles revealed three patterns of cytochrome c response. A subset had a dominant NoxaBH3 response implying Mcl1 dependence. These cells were more sensitive to small molecules that antagonize Mcl1 (AT-101) than those that antagonize Bcl-2, Bcl-xL and Bcl-w (ABT-737). A second subset had a dominant BikBH3 response, implying a Bcl-xL/-w dependence, and was exquisitely sensitive to ABT-737 (IC(50) <200 nM). Finally, most NB cell lines derived at relapse were relatively resistant to pro-death BH3 peptides and Bcl-2 antagonists. Our findings define heterogeneity for apoptosis resistance in NB, help triage emerging Bcl-2 antagonists for clinical use, and provide a platform for studies to characterize post-therapy resistance mechanisms for NB and other solid tumors.

Topics: Apoptosis; Biphenyl Compounds; Cell Line; Cell Line, Tumor; Cytochromes c; Gossypol; Humans; Immunoblotting; Immunoprecipitation; Mitochondria; Neuroblastoma; Nitrophenols; Peptide Fragments; Piperazines; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Sulfonamides

13-cis-Retinoic acid (13-cis-RA) is given at completion of cytotoxic therapy to control minimal residual disease in neuroblastoma. We investigated the effect of combining 13-cis-RA with cytotoxic agents employed in neuroblastoma therapy using a panel of 6 neuroblastoma cell lines. The effect of 13-cis-RA on the mitochondrial apoptotic pathway was studied by flow cytometry, cytotoxicity by DIMSCAN, and protein expression by immunoblotting. Pretreatment and direct combination of 13-cis-RA with etoposide, topotecan, cisplatin, melphalan, or doxorubicin markedly antagonized the cytotoxicity of those agents in 4 out of 6 tested neuroblastoma cell lines, increasing fractional cell survival by 1 to 3 logs. The inhibitory concentration of drugs (IC(99)) increased from clinically achievable levels to nonachievable levels, greater than 5-fold (cisplatin) to greater than 7-fold (etoposide). In SMS-KNCR neuroblastoma cells, 13-cis-RA upregulated expression of Bcl-2 and Bcl-xL RNA and protein, and this was associated with protection from etoposide-mediated apoptosis at the mitochondrial level. A small molecule inhibitor of the Bcl-2 family of proteins (ABT-737) restored mitochondrial membrane potential loss and apoptosis in response to cytotoxic agents in 13-cis-RA treated cells. Prior selection for resistance to RA did not diminish the response to cytotoxic treatment. Thus, combining 13-cis-RA with cytotoxic chemotherapy significantly reduced the cytotoxicity for neuroblastoma in vitro, mediated at least in part via the antiapoptotic Bcl-2 family of proteins.

Topics: Alkylating Agents; Apoptosis; Biphenyl Compounds; Cell Line, Tumor; Cisplatin; Cytochromes c; Cytoprotection; Doxorubicin; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Etoposide; Gene Expression Regulation, Neoplastic; Humans; Isotretinoin; Melphalan; Membrane Potential, Mitochondrial; Mitochondria; Neuroblastoma; Nitrophenols; Piperazines; Protective Agents; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; Signal Transduction; Sulfonamides; Topoisomerase Inhibitors; Tumor Suppressor Protein p53

Neuroblastoma (NB) is a common, highly lethal pediatric cancer, with treatment failures largely attributable to the emergence of chemoresistance. The pro-survival Bcl2 homology (BH) proteins critically regulate apoptosis, and may represent important therapeutic targets for restoring drug sensitivity in NB. We used a human NB tumor tissue microarray to survey the expression of pro-survival BH proteins Mcl1 and Bcl2, and correlated expression to clinical prognostic factors and survival. Primary NB tumors heterogeneously expressed Mcl1 or Bcl2, with high expression correlating to high-risk phenotype. Co-expression is infrequent (11%), but correlates to reduced survival. Using RNA interference, we investigated the functional relevance of Mcl1 and Bcl2 in high-risk NB cell lines (SK-N-AS, IMR-5, NLF). Mcl1 knockdown induced apoptosis in all NB cell lines, while Bcl2 knockdown inhibited only NLF, suggesting functional heterogeneity. Finally, we determined the relevance of Mcl1 in resistance to conventional chemotherapy (etoposide, doxorubicin) and small molecule Bcl2-family antagonists (ABT-737 and AT-101). Mcl1 silencing augmented sensitivity to chemotherapeutics 2- to 300-fold, while Bcl2 silencing did not, even in Bcl2-sensitive NLF cells. Resistance to ABT-737, which targets Bcl2/-w/-x, was overcome by Mcl1 knockdown. AT-101, which also neutralizes Mcl1, had single-agent cytotoxicity, further augmented by Mcl1 knockdown. In conclusion, Mcl1 appears a predominant pro-survival protein contributing to chemoresistance in NB, and Mcl1 inactivation may represent a novel therapeutic strategy. Optimization of compounds with higher Mcl1 affinity, or combination with additional Mcl1 antagonists, may enhance the clinical utility of this approach.

Topics: Biphenyl Compounds; Cell Line, Tumor; Cell Proliferation; Combined Modality Therapy; Down-Regulation; Gene Silencing; Gossypol; Humans; Microscopy, Phase-Contrast; Myeloid Cell Leukemia Sequence 1 Protein; Neuroblastoma; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; RNA Interference; Sulfonamides; Transfection

Tolcapone and entacapone are catechol-O-methyltransferase (COMT) inhibitors used as adjuncts to levodopa in the treatment of Parkinson's disease (PD). The use of tolcapone has been limited by its hepatotoxicity, the cause of which remains uncertain. Tolcapone compound is an uncoupler of mitochondrial respiration in isolated mitochondria and this action may be relevant to its effect on liver function. We have examined the actions of COMT inhibitors on cultured cells, comparing them with those of the classical uncoupler carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), in order to provide insight into their mechanism of potential toxicity. Tolcapone and FCCP were shown to be toxic to human neuroblastoma SH-SY5Y cells and caused a profound reduction in ATP synthesis. Entacapone was not toxic to SH-SY5Y. Tolcapone and FCCP were shown to be equally toxic to cells depleted of mtDNA and thus devoid of a functional respiratory chain. This study demonstrates that tolcapone markedly inhibits ATP synthesis in cultured cells mirroring the effects of a classical uncoupler. However its toxicity may also involve a mechanism independent of its effects upon oxidative phosphorylation.

Topics: Benzophenones; Catechol O-Methyltransferase; Catechol O-Methyltransferase Inhibitors; Catechols; Cell Survival; DNA, Mitochondrial; Dose-Response Relationship, Drug; Enzyme Inhibitors; Humans; Neuroblastoma; Nitriles; Nitrophenols; Tolcapone; Tumor Cells, Cultured

Amifostine [WR-2721; H2N-(CH2)3-NH-(CH2)2-S-PO3H2] is used as a protecting agent in the chemotherapy of neuroblastoma. It is supposed that Amifostine will be transformed into its active form, the free thiol (WR-1065), easier by normal cells than by tumour cells. Analytical capillary isotachophoresis was used to determine the dephosphorylation of Amifostine in serum and on neuroblastoma cells and peripheral blood cells. Furthermore, the biological effects of Amifostine and its free thiol, on cell proliferation of neuroblastoma cells were measured in combination with Carboplatin. It was found that neuroblastoma cells did not split phosphate less efficiently than normal peripheral blood cells. Furthermore, neither Amifostine (as expected) nor the free thiol (not expected according to the theory) were able to inhibit the effects of Carboplatin. Therefore, the current hypothesis concerning the mode of action of Amifostine must be questioned.

Topics: Amifostine; Carboplatin; Cell Division; Electrophoresis; Humans; Neuroblastoma; Nitrophenols; Organophosphorus Compounds; Phosphates; Phosphorylation; Sulfhydryl Compounds; Tumor Cells, Cultured

Beta-Xylosides stimulate 2- to 6-fold the synthesis of glycosaminoglycans by three types of nonconnective tissue cells (RG-C6, NB41A, and rat hepatoma cells, and normal and simian virus 40 (SV40)-transformed normal human skin fibroblasts. The effect, which is specific for the anomeric linkage and the glycone, is observed in the presence and absence of puromycin. Beta-Xylosides may substitute for xylosylated core protein as initiators of synthesis of chondroitin sulfate chains. No stimulation of synthesis of heparan sulfate was observed. With the use of a fluorogenic xyloside, 4-methylumbelliferyl-beta-D-xyloside, it was demonstrated that the free chondroitin sulfate chains secreted into the medium bear the xyloside at the reducing end, and have an average molecular weight of 16,500.

Topics: Animals; Carcinoma, Hepatocellular; Cells, Cultured; Chondroitin; Chromatography, Gel; Dermatan Sulfate; Fibroblasts; Galactose; Glycosaminoglycans; Glycosides; Heparitin Sulfate; Humans; Hyaluronic Acid; Hymecromone; Liver Neoplasms; Mice; Neuroblastoma; Neuroglia; Nitrophenols; Rats; Simian virus 40; Skin; Spectrometry, Fluorescence; Sulfur Radioisotopes; Sulfuric Acids; Tritium; Xylose