RESUMO
PTEN is one of the most commonly inactivated tumour suppressor genes in sporadic cancer. Germline heterozygous PTEN gene alterations also underlie PTEN hamartoma tumour syndrome (PHTS), a rare human cancer-predisposition condition. A key feature of systemic PTEN deregulation is the inability to adequately dampen PI3-kinase (PI3K)/mTORC1 signalling. PI3K/mTORC1 pathway inhibitors such as rapamycin are therefore expected to neutralise the impact of PTEN loss, rendering this a more druggable context compared with those of other tumour suppressor pathways such as loss of TP53. However, this has not been explored in cancer prevention in a model of germline cancer predisposition, such as PHTS. Clinical trials of short-term treatment with rapamycin have recently been initiated for PHTS, focusing on cognition and colon polyposis. Here, we administered a low dose of rapamycin from the age of 6 weeks onwards to mice with heterozygous germline Pten loss, a mouse model that recapitulates most characteristics of human PHTS. Rapamycin was well tolerated and led to a highly significant improvement of survival in both male and female mice. This was accompanied by a delay in, but not full blockade of, the development of a range of proliferative lesions, including gastro-intestinal and thyroid tumours and endometrial hyperplasia, with no impact on mammary and prostate tumours, and no effect on brain overgrowth. Our data indicate that rapamycin may have cancer prevention potential in human PHTS. This might also be the case for sporadic cancers in which genetic PI3K pathway activation is an early event in tumour development, such as endometrial cancer and some breast cancers. To the best of our knowledge, this is the first report of a long-term treatment of a germline cancer predisposition model with a PI3K/mTOR pathway inhibitor. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
Assuntos
Síndrome do Hamartoma Múltiplo , Neoplasias da Glândula Tireoide , Camundongos , Animais , Masculino , Feminino , Humanos , Lactente , Sirolimo/farmacologia , Sirolimo/uso terapêutico , Fosfatidilinositol 3-Quinases/genética , Longevidade , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Síndrome do Hamartoma Múltiplo/tratamento farmacológico , Síndrome do Hamartoma Múltiplo/genética , Síndrome do Hamartoma Múltiplo/patologia , Fosfatidilinositol 3-Quinase/genética , Inibidores de Fosfoinositídeo-3 Quinase , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Células Germinativas/metabolismo , Mutação em Linhagem GerminativaRESUMO
The widespread interest in free radicals in biology extends far beyond the effects of ionizing radiation, with recent attention largely focusing on reactions of free radicals derived from peroxynitrite (i.e., hydroxyl, nitrogen dioxide, and carbonate radicals). These radicals can easily be generated individually by reactions of radiolytically-produced radicals in aqueous solutions and their reactions can be monitored either in real time or by analysis of products. This review first describes the general principles of selective radical generation by radiolysis, the yields of individual species, the advantages and limitations of either pulsed or continuous radiolysis, and the quantitation of oxidizing power of radicals by electrode potentials. Some key reactions of peroxynitrite-derived radicals with potential biological targets are then discussed, including the characterization of reactions of tyrosine with a model alkoxyl radical, reactions of tyrosyl radicals with nitric oxide, and routes to nitrotyrosine formation. This is followed by a brief outline of studies involving the reactions of peroxynitrite-derived radicals with lipoic acid/dihydrolipoic acid, hydrogen sulphide, and the metal chelator desferrioxamine. For biological diagnostic probes such as 'spin traps' to be used with confidence, their reactivities with radical species have to be characterized, and the application of radiolysis methods in this context is also illustrated.
Assuntos
Ácido Peroxinitroso , Tirosina , Radicais Livres , Radical Hidroxila , OxirreduçãoRESUMO
Replication stress is a common feature of cancer cells, and thus a potentially important therapeutic target. Here, we show that cyclin-dependent kinase (CDK)-induced replication stress, resulting from Wee1 inactivation, is synthetic lethal with mutations disrupting dNTP homeostasis in fission yeast. Wee1 inactivation leads to increased dNTP demand and replication stress through CDK-induced firing of dormant replication origins. Subsequent dNTP depletion leads to inefficient DNA replication, DNA damage and to genome instability. Cells respond to this replication stress by increasing dNTP supply through histone methyltransferase Set2-dependent MBF-induced expression of Cdc22, the catalytic subunit of ribonucleotide reductase (RNR). Disrupting dNTP synthesis following Wee1 inactivation, through abrogating Set2-dependent H3K36 tri-methylation or DNA integrity checkpoint inactivation results in critically low dNTP levels, replication collapse and cell death, which can be rescued by increasing dNTP levels. These findings support a 'dNTP supply and demand' model in which maintaining dNTP homeostasis is essential to prevent replication catastrophe in response to CDK-induced replication stress.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Nucleotídeos/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Pontos de Checagem do Ciclo Celular , Dano ao DNA , Replicação do DNA , Código das Histonas , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Homeostase , Metilação , Schizosaccharomyces/metabolismo , Mutações Sintéticas Letais , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Tumour cells with BRCA1/2 gene mutations demonstrate increased sensitivity to platinum and poly (ADP-ribose) polymerase (PARP) inhibitors. 6-mercaptopurine (6MP) was found to selectively kill BRCA-defective cells in a xenograft model as effectively as the PARP inhibitor AG014699, even after these cells acquired resistance to a PARP inhibitor or cisplatin. METHODS: This phase II single-arm trial investigated the activity of 6MP 55-75 mg/m2 per day, and methotrexate 15-20 mg/m2 per week in advanced breast or platinum-resistant ovarian cancer patients with a BRCA1/2 germline mutation, who had progressed after ≥1 previous line of chemotherapy. The primary outcome was objective response including stable disease (SD) as an assessment of clinical benefit rate (CBR), at 8 weeks, by RECIST v1.1. Secondary outcomes included overall survival (OS) and progression-free survival (PFS). RESULTS: In total, 67 evaluable patients were recruited; 55 ovarian and 11 breast cancer patients. In total, 21 patients had SD (31%), one had a partial response (1.5%); CBR was 33% at 8 weeks. In total, 12/67 patients (18%) had SD at 16 weeks. In total, five ovarian cancer patients had SD for over 200 days. Median OS was 10.3 months (95% CI 6.9-14.5), median PFS 1.9 months (1.7-2.8). CONCLUSIONS: The overall activity of 6MP and methotrexate in these patients was low; however, there was a small group of patients who appeared to derive longer-term clinical benefit. TRIAL REGISTRATION: NCT01432145 http://www.ClinicalTrials.gov.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Recidiva Local de Neoplasia/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , Adulto , Idoso , Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Mercaptopurina/administração & dosagem , Mercaptopurina/efeitos adversos , Metotrexato/administração & dosagem , Metotrexato/efeitos adversos , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/mortalidade , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/mortalidade , Intervalo Livre de Progressão , Terapia de Salvação/métodosRESUMO
BACKGROUND: In the current study, the authors sought to determine the maximum tolerated dose (MTD) of the novel class 1 selective histone deacetylase inhibitor CXD101 in a dose escalation study in patients with advanced solid tumors or recurrent/refractory lymphoma. METHODS: The authors escalated the dose of CXD101 from 1 mg twice daily orally for 5 days in a 21-day cycle (3+3 design). RESULTS: A total of 39 patients were enrolled, 36 of whom received CXD101. Of the 30 patients in the escalation cohort, 29 were evaluable for determination of the dose-limiting toxicity (DLT). DLTs were noted at doses of 16 mg twice daily (1 of 6 patients), 20 mg twice daily (1 of 6 patients), and 24/25 mg twice daily (2 of 5 patients, both of whom developed neutropenic fever). The MTD was 20 mg twice daily, which achieved maximal plasma concentrations (±standard deviation) of 231±76 nM to 342±126 nM, which was within the biologically active range. Six patients received 20 mg twice daily in an expansion cohort. The most frequent adverse events were fatigue, nausea, and reversible cytopenia. Key grade 3 to 4 adverse events (according to Common Terminology Criteria for Adverse Events criteria [version 4.03]) included thrombocytopenia (11%), neutropenia (17%), and neutropenic fever (2%) across the 133 CXD101 cycles given. The toxicity profile was similar to that of licensing studies with other histone deacetylase inhibitors. In 22 evaluable patients receiving a dose of ≥16 mg twice daily (17 of whom had lymphoma and 5 of whom had solid tumors), 3 partial responses (2 in patients with classic Hodgkin lymphoma after allogenic stem cell transplantation and 1 in a patient with angioimmunoblastic T-cell lymphoma) and 1 complete response (in a patient with follicular lymphoma) were noted (overall response rate of 18%) in addition to 9 patients who achieved durable stable disease. Responses were noted predominantly among patients with lymphoma (tumor reduction noted in 63% of patients on standard computed tomography). CONCLUSIONS: The MTD in the current study was found to be 20 mg twice daily. Encouraging and durable activity was observed in patients with Hodgkin lymphoma, T-cell lymphoma, and follicular lymphoma.
Assuntos
Inibidores de Histona Desacetilases/administração & dosagem , Linfoma Cutâneo de Células T/tratamento farmacológico , Linfoma de Células T Periférico/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Adulto , Idoso , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Esquema de Medicação , Feminino , Inibidores de Histona Desacetilases/efeitos adversos , Humanos , Linfoma Cutâneo de Células T/metabolismo , Linfoma de Células T Periférico/metabolismo , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Neoplasias Cutâneas/metabolismo , Análise de Sobrevida , Resultado do Tratamento , Adulto JovemRESUMO
Purpose To demonstrate the feasibility and safety of using focused ultrasound planning models to determine the treatment parameters needed to deliver volumetric mild hyperthermia for targeted drug delivery without real-time thermometry. Materials and Methods This study was part of the Targeted Doxorubicin, or TARDOX, phase I prospective trial of focused ultrasound-mediated, hyperthermia-triggered drug delivery to solid liver tumors ( ClinicalTrials.gov identifier NCT02181075). Ten participants (age range, 49-68 years; average age, 60 years; four women) were treated from March 2015 to March 2017 by using a clinically approved focused ultrasound system to release doxorubicin from lyso-thermosensitive liposomes. Ultrasonic heating of target tumors (treated volume: 11-73 cm3 [mean ± standard deviation, 50 cm3 ± 26]) was monitored in six participants by using a minimally invasive temperature sensor; four participants were treated without real-time thermometry. For all participants, CT images were used with a patient-specific hyperthermia model to define focused ultrasound treatment plans. Feasibility was assessed by comparing model-prescribed focused ultrasound powers to those implemented for treatment. Safety was assessed by evaluating MR images and biopsy specimens for evidence of thermal ablation and monitoring adverse events. Results The mean difference between predicted and implemented treatment powers was -0.1 W ± 17.7 (n = 10). No evidence of focused ultrasound-related adverse effects, including thermal ablation, was found. Conclusion In this 10-participant study, the authors confirmed the feasibility of using focused ultrasound-mediated hyperthermia planning models to define treatment parameters that safely enabled targeted, noninvasive drug delivery to liver tumors while monitored with B-mode guidance and without real-time thermometry. Published under a CC BY 4.0 license. Online supplemental material is available for this article. See also the editorial by Dickey and Levi-Polyachenko in this issue.
Assuntos
Antibióticos Antineoplásicos/administração & dosagem , Doxorrubicina/administração & dosagem , Hipertermia Induzida/métodos , Neoplasias Hepáticas/terapia , Terapia por Ultrassom/métodos , Idoso , Sistemas de Liberação de Medicamentos , Liberação Controlada de Fármacos , Estudos de Viabilidade , Feminino , Humanos , Lipossomos , Masculino , Pessoa de Meia-Idade , Veículos Farmacêuticos , Estudos ProspectivosRESUMO
BACKGROUND: Previous preclinical research has shown that extracorporeal devices can be used to enhance the delivery and distribution of systemically administered anticancer drugs, resulting in increased intratumoural concentrations. We aimed to assess the safety and feasibility of targeted release and enhanced delivery of doxorubicin to solid tumours from thermosensitive liposomes triggered by mild hyperthermia, induced non-invasively by focused ultrasound. METHODS: We did an open-label, single-centre, phase 1 trial in a single UK hospital. Adult patients (aged ≥18 years) with unresectable and non-ablatable primary or secondary liver tumours of any histological subtype were considered for the study. Patients received a single intravenous infusion (50 mg/m2) of lyso-thermosensitive liposomal doxorubicin (LTLD), followed by extracorporeal focused ultrasound exposure of a single target liver tumour. The trial had two parts: in part I, patients had a real-time thermometry device implanted intratumourally, whereas patients in part II proceeded without thermometry and we used a patient-specific model to predict optimal exposure parameters. We assessed tumour biopsies obtained before and after focused ultrasound exposure for doxorubicin concentration and distribution. The primary endpoint was at least a doubling of total intratumoural doxorubicin concentration in at least half of the patients treated, on an intention-to-treat basis. This study is registered with ClinicalTrials.gov, number NCT02181075, and is now closed to recruitment. FINDINGS: Between March 13, 2015, and March 27, 2017, ten patients were enrolled in the study (six patients in part I and four in part II), and received a dose of LTLD followed by focused ultrasound exposure. The treatment resulted in an average increase of 3·7 times in intratumoural biopsy doxorubicin concentrations, from an estimate of 2·34 µg/g (SD 0·93) immediately after drug infusion to 8·56 µg/g (5·69) after focused ultrasound. Increases of two to ten times were observed in seven (70%) of ten patients, satisfying the primary endpoint. Serious adverse events registered were expected grade 4 transient neutropenia in five patients and prolonged hospital stay due to unexpected grade 1 confusion in one patient. Grade 3-4 adverse events recorded were neutropenia (grade 3 in one patient and grade 4 in five patients), and grade 3 anaemia in one patient. No treatment-related deaths occurred. INTERPRETATION: The combined treatment of LTLD and non-invasive focused ultrasound hyperthermia in this study seemed to be clinically feasible, safe, and able to enhance intratumoural drug delivery, providing targeted chemo-ablative response in human liver tumours that were refractory to standard chemotherapy. FUNDING: Oxford Biomedical Research Centre, National Institute for Health Research.
Assuntos
Antibióticos Antineoplásicos/administração & dosagem , Doxorrubicina/análogos & derivados , Hipertermia Induzida , Neoplasias Hepáticas/tratamento farmacológico , Ultrassonografia , Idoso , Doxorrubicina/administração & dosagem , Sistemas de Liberação de Medicamentos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Polietilenoglicóis/administração & dosagemRESUMO
BACKGROUND: Src is involved in cancer invasion and metastasis. AZD0424, an oral inhibitor of Src and ABL1, has shown evidence of anti-tumour activity in pre-clinical studies. METHODS: A phase Ia, dose escalation study was performed to assess the safety of continuous oral dosing with AZD0424 in advanced solid tumours. Secondary objectives included investigation of AZD0424 pharmacokinetics, effect on Src activity using markers of bone turnover, and anti-tumour activity. RESULTS: 41 patients were treated; 34 received AZD0424 once-daily at doses ranging from 5 mg to 150 mg, and 7 received 40 mg bi-daily 41.5% of patients experienced at least one AZD0424-related adverse event that was Grade 3-5 in severity, with patients treated at doses above 60 mg per day experiencing multiple treatment-related toxicities. The most commonly observed AZD0424-related adverse events were nausea, fatigue, anorexia and alopecia. Cmax and AUC increased linearly with dose and the mean±standard deviation t1/2 was 8.4±2.8 h. Clear evidence of Src target inhibition was seen at doses ⩾20 mg per day. No responses were observed and 7 patients (17.1%) achieved stable disease lasting 6 weeks or more. CONCLUSIONS: AZD0424 displayed no evidence of efficacy as monotherapy despite a clear pharmacodynamic effect. Further evaluation of AZD0424 monotherapy in patients with solid tumours is not recommended.
Assuntos
Antineoplásicos/efeitos adversos , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/efeitos adversos , Administração Oral , Adulto , Idoso , Antineoplásicos/administração & dosagem , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Neoplasias/enzimologia , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Quinases da Família src/antagonistas & inibidoresRESUMO
Nucleotide synthesis is a universal response to DNA damage, but how this response facilitates DNA repair and cell survival is unclear. Here we establish a role for DNA damage-induced nucleotide synthesis in homologous recombination (HR) repair in fission yeast. Using a genetic screen, we found the Ddb1-Cul4(Cdt)² ubiquitin ligase complex and ribonucleotide reductase (RNR) to be required for HR repair of a DNA double-strand break (DSB). The Ddb1-Cul4(Cdt)² ubiquitin ligase complex is required for degradation of Spd1, an inhibitor of RNR in fission yeast. Accordingly, deleting spd1(+) suppressed the DNA damage sensitivity and the reduced HR efficiency associated with loss of ddb1(+) or cdt2(+). Furthermore, we demonstrate a role for nucleotide synthesis in postsynaptic gap filling of resected ssDNA ends during HR repair. Finally, we define a role for Rad3 (ATR) in nucleotide synthesis and HR through increasing Cdt2 nuclear levels in response to DNA damage. Our findings support a model in which break-induced Rad3 and Ddb1-Cul4(Cdt)² ubiquitin ligase-dependent Spd1 degradation and RNR activation promotes postsynaptic ssDNA gap filling during HR repair.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Quinases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Quinase do Ponto de Checagem 2 , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Deleção de Genes , Nucleotídeos/metabolismo , Recombinação Genética , Ribonucleotídeo Redutases/metabolismoRESUMO
Nitric oxide (NO) is a very effective radiosensitizer of hypoxic mammalian cells, at least as efficient as oxygen in enhancing cell death in vitro. NO may induce cell death through the formation of base lesions which are difficult to repair, and if they occur within complex clustered damage common to ionizing radiation, they may lead to replication-induced DNA strand breaks. It has previously been shown that 8-azaguanine and xanthine result from the reaction of guanine radicals with nitric oxide. We have now shown that adenine radicals also react with NO to form hypoxanthine and 8-azaadenine. Cells irradiated in exponential growth in the presence of NO are twice as radiosensitive compared to those irradiated in anoxia alone, whereas confluent cells are less radiosensitive to (â¢)NO. In addition, the numbers of DNA double strand breaks observed as γH2AX staining following radiosensitization by NO, are higher in exponential cells than in confluent cells. DNA damage, detected as 53BP1 foci, is also higher in HF-19 cells expressing Cyclin A, a marker for cells in S and G2 phases of the cell cycle, following radiosensitization by NO. RAD51 foci are highest in V79-4 cells irradiated in the presence of NO compared to in anoxia, 24h after radiolysis. This work presents evidence that radiosensitization of cells by NO is in part through the formation of specific DNA damage, difficult to repair, which in dividing cells may induce the formation of stalled replication forks and as a consequence replication-induced DNA strand breaks which may lead to cell death.
Assuntos
Dano ao DNA , Replicação do DNA/efeitos dos fármacos , DNA/efeitos dos fármacos , DNA/efeitos da radiação , Óxido Nítrico/toxicidade , Radiossensibilizantes/toxicidade , Adenina/metabolismo , Animais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Radiação IonizanteRESUMO
Modification of tyrosine (TyrOH) is used as a marker of oxidative and nitrosative stress. 3,3'-Dityrosine formation, in particular, reflects oxidative damage and results from the combination of two tyrosyl phenoxyl radicals (TyrO·). This reaction is in competition with reductive processes in the cell which 'repair' tyrosyl radicals: possible reductants include thiols and ascorbate. In this study, a rate constant of 2 x 106 M⻹ s⻹ was estimated for the reaction between tyrosyl radicals and glutathione (GSH) at pH 7.15, generating the radicals by pulse radiolysis and monitoring the tyrosyl radical by kinetic spectrophotometry. Earlier measurements have suggested that this 'repair' reaction could be an equilibrium, and to investigate this possibility the reduction (electrode) potential of the (TyrO·,H+/TyrOH) couple was reinvestigated by observing the fast redox equilibrium with the indicator 2,2'-azinobis(3-ethylbenzothiazoline-6-sulphonate). Extrapolation of the reduction potential of TyrO· measured at pH 9-11 indicated the mid-point reduction potential of the tyrosyl radical at pH 7, E(m7)(TyrO·,H+/TyrOH) = 0.93 ± 0.02 V. This is close to the reported reduction potential of the glutathione thiyl radical, E(m7) = 0.94 ± 0.03V, confirming the 'repair' equilibrium constant is of the order of unity and suggesting that efficient reduction of TyrO· by GSH might require removal of thiyl radicals to move the equilibrium in the direction of repair. Loss of thiyl radicals, facilitating repair of TyrO·, can arise either via conjugation of thiyl with thiol/thiolate or oxygen, or unimolecular transformation, the latter important at low concentrations of thiols and oxygen.
Assuntos
Radicais Livres/metabolismo , Glutationa/metabolismo , Tirosina/metabolismo , Radicais Livres/química , Glutationa/química , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Cinética , Oxirredução , Oxigênio/metabolismo , Fenóis/química , Fenóis/metabolismo , Radiólise de Impulso , Espectrofotometria , Tirosina/análogos & derivados , Tirosina/químicaRESUMO
Tyrosine is a critical component of many proteins and can be the subject of oxidative posttranslational modifications. Furthermore, the oxidation of tyrosine residues to phenoxyl radicals, sometimes quite stable, is essential for some enzymatic functions. The lifetime and fate of tyrosine phenoxyl radicals in biological systems are largely driven by the availability and proximity of oxidants and reductants. Tyrosine phenoxyl radicals have extremely low reactivity with molecular oxygen whereas reactions with nitric oxide are diffusion controlled. This is in contrast to equivalent reactions with tryptophanyl and cysteinyl radicals where reactions with oxygen are much faster. Despite, the quite disparate apparent reactivity of tyrosine phenoxyl radicals with oxygen and nitric oxide being known, the products of the reactions are not well established. Changes in the levels from expected basal concentrations of stable products resulting from tyrosine phenoxyl radicals, for example naturally occurring 3,3'-dityrosine, 3-nitrotyrosine, and 3-hydroxytyrosine, can be indicative of oxidative and/or nitrosative stress. Using the radiolytic generation of specific oxidizing radicals to form tyrosine phenoxyl radicals in an aqueous solution at a known rate, we have compared the products in the absence and presence of nitric oxide or oxygen. Possible reactions of the phenoxyl radicals with oxygen remain unclear although we show evidence for a small decrease in the yield of dityrosine and loss of tyrosine in the presence of 20% oxygen. Low concentrations of nitric oxide in anoxic conditions react with tyrosine phenoxyl radicals, by what is most probably through the formation of an unstable intermediate, regenerating tyrosine and forming nitrite.
Assuntos
Espectroscopia de Ressonância de Spin Eletrônica/métodos , Óxido Nítrico/metabolismo , Fenóis/metabolismo , HumanosRESUMO
Tumor hypoxia is associated with therapy resistance and poor patient prognosis. Hypoxia-activated prodrugs, designed to selectively target hypoxic cells while sparing normal tissue, represent a promising treatment strategy. We report the pre-clinical efficacy of 1-methyl-2-nitroimidazole panobinostat (NI-Pano, CH-03), a novel bioreductive version of the clinically used lysine deacetylase inhibitor, panobinostat. NI-Pano was stable in normoxic (21% O2) conditions and underwent NADPH-CYP-mediated enzymatic bioreduction to release panobinostat in hypoxia (<0.1% O2). Treatment of cells grown in both 2D and 3D with NI-Pano increased acetylation of histone H3 at lysine 9, induced apoptosis, and decreased clonogenic survival. Importantly, NI-Pano exhibited growth delay effects as a single agent in tumor xenografts. Pharmacokinetic analysis confirmed the presence of sub-micromolar concentrations of panobinostat in hypoxic mouse xenografts, but not in circulating plasma or kidneys. Together, our pre-clinical results provide a strong mechanistic rationale for the clinical development of NI-Pano for selective targeting of hypoxic tumors.
Assuntos
Antineoplásicos/farmacologia , Desenvolvimento de Medicamentos , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Hipóxia/tratamento farmacológico , Panobinostat/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/química , Hipóxia/metabolismo , Masculino , Camundongos , Camundongos Nus , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Panobinostat/síntese química , Panobinostat/química , Células Tumorais CultivadasRESUMO
In this study we compared three different microbubble-based approaches to the delivery of a widely used chemotherapy drug, gemcitabine: (i) co-administration of gemcitabine and microbubbles (Gem+MB); (ii) conjugates of microbubbles and gemcitabine-loaded liposomes (GemlipoMB); and (iii) microbubbles with gemcitabine directly bound to their surfaces (GembioMB). Both in vitro and in vivo investigations were carried out, respectively, in the RT112 bladder cancer cell line and in a murine orthotopic muscle-invasive bladder cancer model. The in vitro (in vivo) ultrasound exposure conditions were a 1 (1.1) MHz centre frequency, 0.07 (1.0) MPa peak negative pressure, 3000 (20,000) cycles and 100 (0.5) Hz pulse repetition frequency. Ultrasound exposure produced no significant increase in drug uptake either in vitro or in vivo compared with the drug-only control for co-administered gemcitabine and microbubbles. In vivo, GemlipoMB prolonged the plasma circulation time of gemcitabine, but only GembioMB produced a statistically significant increase in cleaved caspase 3 expression in the tumor, indicative of gemcitabine-induced apoptosis.
Assuntos
Antimetabólitos Antineoplásicos/administração & dosagem , Antimetabólitos Antineoplásicos/farmacocinética , Desoxicitidina/análogos & derivados , Sistemas de Liberação de Medicamentos/métodos , Microbolhas , Terapia por Ultrassom , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/terapia , Animais , Antimetabólitos Antineoplásicos/uso terapêutico , Desoxicitidina/administração & dosagem , Desoxicitidina/farmacocinética , Desoxicitidina/uso terapêutico , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Nus , Células Tumorais Cultivadas , GencitabinaRESUMO
Inhibition of IGF receptor (IGF1R) delays repair of radiation-induced DNA double-strand breaks (DSB), prompting us to investigate whether IGF1R influences endogenous DNA damage. Here we demonstrate that IGF1R inhibition generates endogenous DNA lesions protected by 53BP1 bodies, indicating under-replicated DNA. In cancer cells, inhibition or depletion of IGF1R delayed replication fork progression accompanied by activation of ATR-CHK1 signaling and the intra-S-phase checkpoint. This phenotype reflected unanticipated regulation of global replication by IGF1 mediated via AKT, MEK/ERK, and JUN to influence expression of ribonucleotide reductase (RNR) subunit RRM2. Consequently, inhibition or depletion of IGF1R downregulated RRM2, compromising RNR function and perturbing dNTP supply. The resulting delay in fork progression and hallmarks of replication stress were rescued by RRM2 overexpression, confirming RRM2 as the critical factor through which IGF1 regulates replication. Suspecting existence of a backup pathway protecting from toxic sequelae of replication stress, targeted compound screens in breast cancer cells identified synergy between IGF inhibition and ATM loss. Reciprocal screens of ATM-proficient/deficient fibroblasts identified an IGF1R inhibitor as the top hit. IGF inhibition selectively compromised growth of ATM-null cells and spheroids and caused regression of ATM-null xenografts. This synthetic-lethal effect reflected conversion of single-stranded lesions in IGF-inhibited cells into toxic DSBs upon ATM inhibition. Overall, these data implicate IGF1R in alleviating replication stress, and the reciprocal IGF:ATM codependence we identify provides an approach to exploit this effect in ATM-deficient cancers. SIGNIFICANCE: This study identifies regulation of ribonucleotide reductase function and dNTP supply by IGFs and demonstrates that IGF axis blockade induces replication stress and reciprocal codependence on ATM. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/8/2128/F1.large.jpg.
Assuntos
Quebras de DNA de Cadeia Dupla , Dano ao DNA , Replicação do DNA , Receptor IGF Tipo 1/antagonistas & inibidores , Ribonucleosídeo Difosfato Redutase/metabolismo , Ribonucleotídeo Redutases/metabolismo , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/metabolismo , Reparo do DNA , Desoxirribonucleosídeos/metabolismo , Regulação para Baixo , Fibroblastos , Xenoenxertos , Histonas/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases , Células MCF-7 , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Mutação , Receptores Nucleares Órfãos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Receptor IGF Tipo 1/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular , Esferoides CelularesRESUMO
PURPOSE: Tumor hypoxia fuels an aggressive tumor phenotype and confers resistance to anticancer treatments. We conducted a clinical trial to determine whether the antimalarial drug atovaquone, a known mitochondrial inhibitor, reduces hypoxia in non-small cell lung cancer (NSCLC). PATIENTS AND METHODS: Patients with NSCLC scheduled for surgery were recruited sequentially into two cohorts: cohort 1 received oral atovaquone at the standard clinical dose of 750 mg twice daily, while cohort 2 did not. Primary imaging endpoint was change in tumor hypoxic volume (HV) measured by hypoxia PET-CT. Intercohort comparison of hypoxia gene expression signatures using RNA sequencing from resected tumors was performed. RESULTS: Thirty patients were evaluable for hypoxia PET-CT analysis, 15 per cohort. Median treatment duration was 12 days. Eleven (73.3%) atovaquone-treated patients had meaningful HV reduction, with median change -28% [95% confidence interval (CI), -58.2 to -4.4]. In contrast, median change in untreated patients was +15.5% (95% CI, -6.5 to 35.5). Linear regression estimated the expected mean HV was 55% (95% CI, 24%-74%) lower in cohort 1 compared with cohort 2 (P = 0.004), adjusting for cohort, tumor volume, and baseline HV. A key pharmacodynamics endpoint was reduction in hypoxia-regulated genes, which were significantly downregulated in atovaquone-treated tumors. Data from multiple additional measures of tumor hypoxia and perfusion are presented. No atovaquone-related adverse events were reported. CONCLUSIONS: This is the first clinical evidence that targeting tumor mitochondrial metabolism can reduce hypoxia and produce relevant antitumor effects at the mRNA level. Repurposing atovaquone for this purpose may improve treatment outcomes for NSCLC.
Assuntos
Atovaquona/farmacologia , Regulação Neoplásica da Expressão Gênica , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Hipóxia Tumoral/efeitos dos fármacos , Hipóxia Tumoral/genética , Atovaquona/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Metabolismo Energético , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Imagem Molecular , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Fator de Transcrição STAT3/metabolismoRESUMO
Dihydrorhodamine 123 (RhH2) has been used to detect 'reactive nitrogen species', including peroxynitrite and its radical decomposition products, peroxynitrite probably oxidizing RhH2 to rhodamine (Rh) via radical products rather than directly. In this study, the radical intermediate (RhH(.)) was generated by pulse radiolysis, and shown to react with oxygen with a rate constant k approximately 7 x 10(8) M(-1) s(-1). This fast reaction was exploited in experiments observing Rh being formed slowly (k approximately 4-7 x 10(5) M(-1) s(-1)) from oxidation of RhH2 by nitrogen dioxide in a rate-limiting step, >1000-fold slower than the corresponding oxidation by carbonate radicals. The time-dependent uptake of RhH2 into mammalian cells was measured, with average intracellular levels reaching only approximately 10 microM with the protocol used. The combination of low loading and relatively low reactivity of oxidants towards RhH2 compared to competing cellular nucleophiles suggests rather a small fraction of peroxynitrite-derived radicals (mainly CO3(.-)) may be scavenged intracellularly by RhH2.
Assuntos
Fibroblastos/metabolismo , Dióxido de Nitrogênio/metabolismo , Ácido Peroxinitroso/metabolismo , Rodaminas/metabolismo , Animais , Linhagem Celular , Cricetinae , Radicais Livres/metabolismo , Cinética , Oxigênio/metabolismoRESUMO
Site-selective labelling of antibodies (Abs) can circumvent problems from heterogeneity of conventional conjugation. Here, we evaluate the industrially-applied chemoenzymatic 'Q-tag' strategy based on transglutaminase-mediated (TGase) amide-bond formation in the generation of 89Zr-radiolabelled antibody conjugates. We show that, despite previously suggested high regioselectivity of TGases, in the anti-Her2 Ab Herceptin™ more precise native MS indicates only 70-80% functionalization at the target site (Q298H), in competition with modification at other sites, such as Q3H critically close to the CDR1 region.
Assuntos
Anticorpos/química , Imunoconjugados/química , Radioisótopos/química , Zircônio/química , Amidas/química , Amidas/imunologia , Amidas/metabolismo , Anticorpos/imunologia , Imunoconjugados/imunologia , Estrutura Molecular , Transglutaminases/química , Transglutaminases/imunologia , Transglutaminases/metabolismo , Zircônio/imunologiaRESUMO
A possible route to S-nitrosothiols in biology is the reaction between thiyl radicals and nitric oxide. D. Hofstetter et al. (Biochem. Biophys. Res. Commun.360:146-148; 2007) claimed an upper limit of (2.8+/-0.6)x10(7) M(-1)s(-1) for the rate constant between thiyl radicals derived from glutathione and nitric oxide, and it was suggested that under physiological conditions S-nitrosation via this route is negligible. In the present study, thiyl radicals were generated by pulse radiolysis, and the rate constants of their reactions with nitric oxide were determined by kinetic competition with the oxidizable dyes 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) and a phenothiazine. The rate constants for the reaction of nitric oxide with thiyl radicals derived from glutathione, cysteine, and penicillamine were all in the range (2-3) x10(9) M(-1)s(-1), two orders of magnitude higher than the previously reported estimate in the case of glutathione. Absorbance changes on reaction of thiyl radicals with nitric oxide were consistent with such high reactivity and showed the formation of S-nitrosothiols, which was also confirmed in the case of glutathione by HPLC/MS. These rate constants imply that formation of S-nitrosothiols in biological systems from the combination of thiyl radicals with nitric oxide is much more likely than claimed by Hofstetter et al.
Assuntos
Glutationa/química , Óxido Nítrico/química , S-Nitrosotióis/química , Benzotiazóis , Radicais Livres/química , Raios gama , Cinética , Nitrosação , Fenotiazinas/química , Ácidos Sulfônicos/química , Tiazóis/químicaRESUMO
Carbonate radicals (CO3-) can be formed biologically by the reaction of OH with bicarbonate, the decomposition of the peroxynitrite-carbon dioxide adduct (ONOOCO2-), and enzymatic activities, i.e., peroxidase activity of CuZnSOD and xanthine oxidase turnover in the presence of bicarbonate. It has been reported that the spin-trap DMPO reacts with CO3(-) to yield transient species to yield finally the DMPO-OH spin adduct. In this study, the kinetics of reaction of CO3(-) with DMPO were studied by pulse radiolysis, yielding a second-order rate constant of 2.5 x 10(6) M(-1) s(-1). A Fenton system, composed of Fe(II)-DTPA plus H2O2, generated OH that was trapped by DMPO; the presence of 50-500 mM bicarbonate, expected to convert OH to CO3(-), markedly inhibited DMPO-OH formation. This was demonstrated to be due mainly to a fast reaction of CO3(-) with FeII-DTPA (k=6.1 x 10(8) M(-1) s(-1)), supported by kinetic analysis. Generation of CO3(-) by the Fenton system was further proved by analysis of tyrosine oxidation products: the presence of bicarbonate caused a dose-dependent inhibition of 3,4-dihydroxiphenylalanine with a concomitant increase of 3,3'-dityrosine yields, and the presence of DMPO inhibited tyrosine oxidation, in agreement with the rate constants with OH or CO3(-). Similarly, the formation of CO3(-) by CuZnSOD/H(2)O(2)/bicarbonate and peroxynitrite-carbon dioxide was supported by DMPO hydroxylation and kinetic competition data. Finally, the reaction of CO3(-) with DMPO to yield DMPO-OH was shown in peroxynitrite-forming macrophages. In conclusion, CO3(-) reacts quite rapidly with DMPO and may contribute to DMPO-OH yields in chemical and cellular systems; in turn, the extent of oxidation of other target molecules (such as tyrosine) by CO3(-) will be sensitive to the presence of DMPO.