RESUMO
Respiratory system injury is the main cause of mortality for nitrogen mustard (NM)-induced damage. Previous studies indicate that reactive oxygen species (ROS) participates in NM-mediated respiratory injuries, but the detailed mechanism is not quite clear. Human bronchial epithelial cell lines 16HBE and BEAS-2B were treated with HN2, a type of NM. In detail, it was shown that HN2 treatment induced impaired cell viability, excessive mitochondrial ROS production and enhanced cellular apoptosis in bronchial epithelial cells. Moreover, impaired Sirt3/SOD2 axis was observed upon HN2 treatment, with decreased Sirt3 and increased acetylated SOD2 expression levels. Sirt3 overexpression partially ameliorated HN2-induced cell injury. Meanwhile, vitamin D3 treatment partially attenuated HN2-induced apoptosis and improved the mitochondrial functions upon HN2 intervention. In addition, HN2 exposure decreased VDR expression, thus inhibiting the Nrf2 phosphorylation and Sirt3 activation. Inhibition of Nrf2 or Sirt3 could decrease the protective effects of vitamin D3 and enhance mitochondrial ROS production via modulating mitochondrial redox balance. In conclusion, impaired VDR/Nrf2/Sirt3 axis contributed to NM-induced apoptosis, while vitamin D3 supplementation provides protective effects via the activation of VDR and the improvement of mitochondrial functions. This study provides novel mechanism and strategy for NM exposure-induced pulmonary injuries.
Assuntos
Apoptose/efeitos dos fármacos , Brônquios/efeitos dos fármacos , Colecalciferol/farmacologia , Células Epiteliais/efeitos dos fármacos , Compostos de Mostarda Nitrogenada/toxicidade , Substâncias Protetoras/farmacologia , Sistema Respiratório/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Humanos , Sistema Respiratório/fisiopatologiaAssuntos
Antineoplásicos/toxicidade , Autofagia/efeitos dos fármacos , Antineoplásicos/uso terapêutico , Humanos , Medicina Tradicional Chinesa , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Compostos de Mostarda Nitrogenada/química , Compostos de Mostarda Nitrogenada/uso terapêutico , Compostos de Mostarda Nitrogenada/toxicidadeRESUMO
Mustard agents are potent DNA alkylating agents with mutagenic, cytotoxic and vesicant properties. They include bi-functional agents, such as sulfur mustard (SM) or nitrogen mustard (mustine, HN2), as well as mono-functional agents, such as "half mustard" (CEES). Whereas SM has been used as a chemical warfare agent, several nitrogen mustard derivatives, such as chlorambucil and cyclophosphamide, are being used as established chemotherapeutics. Upon induction of specific forms of genotoxic stimuli, several poly(ADP-ribose) polymerases (PARPs) synthesize the nucleic acid-like biopolymer poly(ADP-ribose) (PAR) by using NAD(+) as a substrate. Previously, it was shown that SM triggers cellular poly(ADP-ribosyl) ation (PARylation), but so far this phenomenon is poorly characterized. In view of the protective effects of PARP inhibitors, the latter have been proposed as a treatment option of SM-exposed victims. In an accompanying article (Debiak et al., 2016), we have provided an optimized protocol for the analysis of the CEES-induced PARylation response in HaCaT keratinocytes, which forms an experimental basis to further analyze mustard-induced PARylation and its functional consequences, in general. Thus, in the present study, we performed a comprehensive characterization of the PARylation response in HaCaT cells after treatment with four different mustard agents, i.e., SM, CEES, HN2, and chlorambucil, on a qualitative, quantitative and functional level. In particular, we recorded substance-specific as well as dose- and time-dependent PARylation responses using independent bioanalytical methods based on single-cell immuno-fluorescence microscopy and quantitative isotope dilution mass spectrometry. Furthermore, we analyzed if and how PARylation contributes to mustard-induced toxicity by treating HaCaT cells with CEES, SM, and HN2 in combination with the clinically relevant PARP inhibitor ABT888. As evaluated by a novel immunofluorescence-based protocol for the detection of N7-ETE-guanine DNA adducts, the excision rate of CEES-induced DNA adducts was not affected by PARP inhibition. Furthermore, while CEES induced moderate changes in cellular NAD(+) levels, annexin V/PI flow cytometry analysis revealed that these changes did not affect CEES-induced short-term cytotoxicity 24h after treatment. In contrast, PARP inhibition impaired cell proliferation and clonogenic survival, and potentiated micronuclei formation of HaCaT cells upon CEES treatment. Similarly, PARP inhibition affected clonogenic survival of cells treated with bi-functional mustards such as SM and HN2. In conclusion, we demonstrate that PARylation plays a functional role in mustard-induced cellular stress response with substance-specific differences. Since PARP inhibitors exhibit therapeutic potential to treat SM-related pathologies and to sensitize cancer cells for mustard-based chemotherapy, potential long-term effects of PARP inhibition on genomic stability and carcinogenesis should be carefully considered when pursuing such a strategy.
Assuntos
Adenosina Difosfato Ribose/metabolismo , Antineoplásicos Alquilantes/toxicidade , Substâncias para a Guerra Química/toxicidade , Queratinócitos/efeitos dos fármacos , Gás de Mostarda/toxicidade , Compostos de Mostarda Nitrogenada/toxicidade , Poli(ADP-Ribose) Polimerases/metabolismo , Antídotos/toxicidade , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Clorambucila/toxicidade , Adutos de DNA/metabolismo , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Instabilidade Genômica/efeitos dos fármacos , Humanos , Queratinócitos/enzimologia , Queratinócitos/patologia , Mecloretamina/toxicidade , Micronúcleos com Defeito Cromossômico/induzido quimicamente , Gás de Mostarda/análogos & derivados , Inibidores de Poli(ADP-Ribose) Polimerases/toxicidade , Transdução de Sinais/efeitos dos fármacos , Fatores de TempoRESUMO
Exposure of humans and animals to vesicants, including sulfur mustard (SM) and nitrogen mustard (NM), causes severe and debilitating damage to the respiratory tract. Both acute and long term pathological consequences are observed in the lung following a single exposure to these vesicants. Evidence from our laboratories and others suggest that macrophages and the inflammatory mediators they release play an important role in mustard-induced lung injury. In this paper, the pathogenic effects of SM and NM on the lung are reviewed, along with the potential role of inflammatory macrophages and mediators they release in mustard-induced pulmonary toxicity.
Assuntos
Lesão Pulmonar/induzido quimicamente , Pulmão/efeitos dos fármacos , Compostos de Mostarda Nitrogenada/toxicidade , Pneumonia/induzido quimicamente , Animais , Antídotos/uso terapêutico , Modelos Animais de Doenças , Humanos , Mediadores da Inflamação/metabolismo , Exposição por Inalação , Pulmão/metabolismo , Pulmão/patologia , Pulmão/fisiopatologia , Lesão Pulmonar/tratamento farmacológico , Lesão Pulmonar/metabolismo , Lesão Pulmonar/patologia , Lesão Pulmonar/fisiopatologia , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/metabolismo , Pneumonia/tratamento farmacológico , Pneumonia/metabolismo , Pneumonia/patologia , Pneumonia/fisiopatologia , Transdução de Sinais/efeitos dos fármacosRESUMO
PURPOSE: PR-104, a bioreductive prodrug in clinical trial, is a phosphate ester which is rapidly metabolized to the corresponding alcohol PR-104A. This dinitrobenzamide mustard is activated by reduction to hydroxylamine (PR-104H) and amine (PR-104M) metabolites selectively in hypoxic cells, and also independently of hypoxia by aldo-keto reductase (AKR) 1C3 in some tumors. Here, we evaluate reductive metabolism of PR-104A in mice and its significance for host toxicity. METHODS: The pharmacokinetics of PR-104, PR-104A and its reduced metabolites were investigated in plasma and tissues of mice (with and without SiHa or H460 tumor xenografts) and effects of potential oxidoreductase inhibitors were evaluated. RESULTS: Pharmacokinetic studies identified extensive non-tumor reduction of PR-104A to the 5-amine PR-104H (identity of which was confirmed by chemical synthesis), especially in liver. However, high concentrations of PR-104H in tumors that suggested intra-tumor activation is also significant. The tissue distribution of PR-104M/H was broadly consistent with the target organ toxicities of PR-104 (bone marrow, intestines and liver). Surprisingly, hepatic nitroreduction was not enhanced when the liver was made more hypoxic by hepatic artery ligation or breathing of 10% oxygen. A screen of non-steroidal anti-inflammatory drugs identified naproxen as an effective AKR1C3 inhibitor in human tumor cell cultures and xenografts, suggesting its potential use to ameliorate PR-104 toxicity in patients. However, neither naproxen nor the pan-CYP inhibitor 1-aminobenzotriazole inhibited normal tissue reduction of PR-104A in mice. CONCLUSIONS: PR-104 is extensively reduced in mouse tissues, apparently via oxygen-independent two-electron reduction, with a tissue distribution that broadly reflects toxicity.
Assuntos
3-Hidroxiesteroide Desidrogenases/metabolismo , Antineoplásicos/farmacocinética , Hidroxiprostaglandina Desidrogenases/metabolismo , Compostos de Mostarda Nitrogenada/farmacocinética , Membro C3 da Família 1 de alfa-Ceto Redutase , Animais , Antineoplásicos/toxicidade , Linhagem Celular Tumoral , Elétrons , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Compostos de Mostarda Nitrogenada/toxicidade , Oxirredução , Pró-Fármacos , Distribuição Tecidual , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: PR-104 is a phosphate ester that is systemically converted to the corresponding alcohol PR-104A. The latter is activated by nitroreduction in tumours to cytotoxic DNA cross-linking metabolites. Here, we report a population pharmacokinetic (PK) model for PR-104 and PR-104A in non-human species and in humans. METHODS: A compartmental model was used to fit plasma PR-104 and PR-104A concentration-time data after intravenous (i.v.) dosing of humans, Beagle dogs, Sprague-Dawley rats and CD-1 nude mice. Intraperitoneal (i.p.) PR-104 and i.v. PR-104A dosing of mice was also investigated. Protein binding was measured in plasma from each species. Unbound drug clearances and volumes were scaled allometrically. RESULTS: A two-compartment model described the disposition of PR-104 and PR-104A in all four species. PR-104 was cleared rapidly by first-order (mice, rats, dogs) or mixed-order (humans) metabolism to PR-104A in the central compartment. The estimated unbound human clearance of PR104A was 211 L/h/70 kg, with a steady state unbound volume of 105 L/70 kg. The size equivalent unbound PR-104A clearance was 2.5 times faster in dogs, 0.78 times slower in rats and 0.63 times slower in mice, which may reflect reported species differences in PR-104A O-glucuronidation. CONCLUSIONS: The PK model demonstrates faster size equivalent clearance of PR-104A in dogs and humans than rodents. Dose-limiting myelotoxicity restricts the exposure of PR-104A in humans to approximately 25% of that achievable in mice.
Assuntos
Antineoplásicos/farmacocinética , Modelos Biológicos , Compostos de Mostarda Nitrogenada/farmacocinética , Pró-Fármacos/farmacocinética , Animais , Antineoplásicos/toxicidade , Proteínas Sanguíneas/metabolismo , Hipóxia Celular , Ensaios Clínicos Fase I como Assunto , Cães , Feminino , Humanos , Masculino , Camundongos , Camundongos Nus , Compostos de Mostarda Nitrogenada/toxicidade , Pró-Fármacos/toxicidade , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Especificidade da EspécieRESUMO
Primary treatment for classic Hodgkin lymphoma (HL) remains highly effective with chemotherapy alone or combined-modality therapy. The limitations of therapy have been related to toxicity and efficacy in subsets of patients. The introduction of a number of new and novel cytotoxic agents has provided opportunities for investigating their use in the treatment of HL. This article briefly reviews current primary treatment strategies for HL and examines the existing data for both new cytotoxic agents and other selected novel agents in the treatment of HL.
Assuntos
Antineoplásicos/uso terapêutico , Desoxicitidina/análogos & derivados , Doença de Hodgkin/terapia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais/toxicidade , Anticorpos Monoclonais Murinos , Antineoplásicos/toxicidade , Cloridrato de Bendamustina , Ensaios Clínicos como Assunto , Terapia Combinada , Desoxicitidina/uso terapêutico , Inibidores de Histona Desacetilases/uso terapêutico , Inibidores de Histona Desacetilases/toxicidade , Humanos , Lenalidomida , Compostos de Mostarda Nitrogenada/uso terapêutico , Compostos de Mostarda Nitrogenada/toxicidade , Rituximab , Talidomida/análogos & derivados , Talidomida/uso terapêutico , Talidomida/toxicidade , Vimblastina/análogos & derivados , Vimblastina/uso terapêutico , Vimblastina/toxicidade , Vinorelbina , GencitabinaRESUMO
OBJECTIVE: To investigate the influence factors of human peripheral blood B lymphocyte transformation by epstein-barr virus (EBV). METHODS: EB virus was obtained from B95-8 supernatant and quantitated by quantitative PCR amplification. The concentration of cyclosporine A (CSA) concentration of lymphocytes and different time of adding CSA to culture media were selected to examine the effects on the lymphocyte transformation, and the condition of lymphocyte transformation of worker exposed to trichloroethylene was further confirmed. RESULTS: The titer of two virus samples were 5.45 x 10(6) and 1.06 x 10(6) copies/ml. Peripheral blood mononuclear cells (PBMCs) was transformed by using CSA at different concentration and adding time to culture fluid. The concentration of PBMCs being transformed was 2 x 10(6)/ml for normal people, but 1 x 10(6)/ml for worker exposed to trichloroethylene. Cell transformation was confirmed by the gradual increase of cell size and cellular clusters. CONCLUSION: The results suggest the key issue is PBMCs being transformed at a concentration of 2 x 10(6)/ml, rather than using CSA at different concentration and adding time to culture fluid. The PBMCs at concentration of 1 x 10(6)/ml for worker exposed to trichloroethylene were transformed, which may be associated with sensitized status of lymphocytes.
Assuntos
Linfócitos B/virologia , Transformação Celular Viral/imunologia , Herpesvirus Humano 4/imunologia , Adulto , Linfócitos B/patologia , Células Cultivadas , Ciclosporina/farmacologia , Humanos , Ativação Linfocitária , Compostos de Mostarda Nitrogenada/toxicidade , Exposição OcupacionalRESUMO
PURPOSE: We investigated the in vitro toxicity of bendamustine and fludarabine to hematopoietic progenitors and stem cells from healthy donors. METHODS: Clonogenic agar colony assays, non-clonogenic long-term liquid cultures (LTC) and apoptosis assays were used to assess the cytotoxicity of both the agents. RESULTS: Total colony-forming units (CFU) were more sensitive to fludarabine than to bendamustine in agar colony assays (IC(50) 0.7 microM/L and 8.5 microM/L, respectively). Using the Bliss independence model and combining the two agents yielded additive inhibition of progenitors. Non-clonogenic assays, including LTC and an apoptosis assay detecting activated caspases showed that stem cells are characterized by low sensitivity to bendamustine. In contrast, fludarabine strongly inhibited the viability and growth of stem cells in LTC. CONCLUSIONS: Our data show that bendamustine is characterized by lower in vitro toxicity to hematopoietic progenitors and stem cells than fludarabine and might thus be preferable in regimens prior to stem cells apheresis.
Assuntos
Antineoplásicos/toxicidade , Células-Tronco Hematopoéticas/citologia , Compostos de Mostarda Nitrogenada/toxicidade , Células-Tronco/citologia , Vidarabina/análogos & derivados , Cloridrato de Bendamustina , Remoção de Componentes Sanguíneos/métodos , Células da Medula Óssea/citologia , Técnicas de Cultura de Células/métodos , Sobrevivência Celular/efeitos dos fármacos , Ensaio de Unidades Formadoras de Colônias , Fator Estimulador de Colônias de Granulócitos/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/patologia , Humanos , Modelos Biológicos , Valores de Referência , Células-Tronco/efeitos dos fármacos , Células-Tronco/patologia , Vidarabina/toxicidadeRESUMO
We recently reported the detection of mercapturic acid pathway metabolites of bendamustine, namely, cysteine S-conjugates in human bile, which are supposed to subsequently undergo further metabolism. In this study, we describe the identification and quantitation of consecutive bendamustine metabolites occurring in human bile using authentic reference standards and the synthesis and structural confirmation of these compounds. Mass spectrometry data along with high-performance liquid chromatography retention data (fluorescence detection) of the synthetic reference standards were consistent with those of the metabolites found in human bile after administration of bendamustine hydrochloride to cancer patients. Analysis of the purified synthetic reference compounds showed a purity of at least 95%. Structural confirmation was achieved by one- and two-dimensional proton as well as carbon-13 NMR spectroscopy and mass spectrometry. A total of 16 bendamustine-related compounds were detected in the bile of patients, 11 of them were recovered as conjugates. Eight conjugates have been structurally confirmed as novel mercapturic acids and sulfoxides. Biliary excretion of the sulfoxides was twice that of the mercapturate precursors. Glutathione S-conjugates of bendamustine have not been detected in bile samples, indicating rapid enzymatic cleavage in humans. Both the lack of glutathione (GSH) conjugates and occurrence of diastereomeric sulfoxides emphasize species-related differences in the GSH conjugation of bendamustine between humans and rats. The total amount recovered in the bile as the sum of all conjugates over the period of 24 h after dosing averaged 5.2% of the administered dose. The question of whether the novel metabolites contribute to urinary excretion should be a target of future investigations.
Assuntos
Bile/efeitos dos fármacos , Compostos de Mostarda Nitrogenada/toxicidade , Sulfóxidos/química , Animais , Cloridrato de Bendamustina , Bile/metabolismo , Ácido Gástrico/metabolismo , Humanos , Imunotoxinas/toxicidade , Compostos de Mostarda Nitrogenada/administração & dosagem , Compostos de Mostarda Nitrogenada/química , Ratos , Sulfóxidos/toxicidadeRESUMO
The biochemical sequelae to chloroethyl mustard exposure correspond very well to toxic processes initiated by free radicals. Additionally, mustard solutions contain spontaneously formed cyclic onium ions which produce carbon free radicals when reduced electrochemically. Therefore, we hypothesized that the onium ions of sulfur or nitrogen mustards might produce carbon free radicals upon being reduced enzymatically, and that these radicals might constitute a metabolic activation. We set out to document radical production using an in vitro metabolic system and electron paramagnetic resonance (EPR). Our system consisted of NADPH, one of several pyridine nucleotide-driven flavoprotein reductases, cytochrome c as a terminal electron acceptor, various sulfur or nitrogen mustards and the spin trap alpha-[4-pyridyl-1-oxide]-N-tert-butylnitrone in buffer. Reactions were started by adding the reductase to the other materials, vortexing and immediately transferring the mixture to a 10 mm EPR flat cell. Repeated scans on a Bruker ESP 300E EPR spectrometer produced a triplet of doublets with hyperfine splitting constants of a(N)=15.483 G and a(H)=2.512 G. The outcome supported our hypothesis that carbon-centered free radicals are produced when mustard-related onium ions are enzymatically reduced. The EPR results varied little with the chloroethyl compound used or with porcine or human cytochrome P450 reductase, the reductase domain of rat brain neuronal nitric oxide synthase or rat liver thioredoxin reductase. Our results offer new insight into the basis for mustard-induced vesication and the outcome of exposure to different mustards. The free radical model provides an explanation for similarities in the lesions arising from mustard exposure and energy-based lesions such as those from heat, ultraviolet and nuclear radiation as well as damage across tissue types such as skin, eyes or airway epithelium.
Assuntos
Substâncias para a Guerra Química/toxicidade , Radicais Livres/metabolismo , Gás de Mostarda/toxicidade , NADP/metabolismo , Compostos de Mostarda Nitrogenada/toxicidade , Animais , Encéfalo/metabolismo , Citocromos c/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Fígado/metabolismo , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Óxido Nítrico Sintase/metabolismo , Piridinas , Ratos , Detecção de Spin , Suínos , Tiorredoxina Dissulfeto Redutase/metabolismoRESUMO
Minor groove binding alkylating agents, which have potential as cancer drugs, generate cytotoxic DNA adducts that are relatively resistant to repair as a consequence of locating covalent attachment at purine N3 nitrogen atoms. Recently, we used electrospray and matrix-assisted laser desorption ionization mass spectrometry to study the binding of the minor groove-directed polybenzamide bis-half-mustard alkamin, and its monofunctional analogue alkamini, to the oligonucleotide d(CGCGAATTCGCG)(2), identifying a number of inter- and intrastrand alkamin cross-links involving the GAATTC sequence [ Abdul Majid , A. M. S. , Smythe , G. , Denny , W. A. , and Wakelin , L. P. G. ( 2007 ) Mol. Pharmacol. 71 , 1165 - 1178 ]. Here, we extend these studies to d(CGCAAATTTGCG)(2), A3T3, and d(CGCAAAAAAGCG).d(CGCTTTTTTGCG), A6/T6, in which the opportunity for both inter- and intrastrand cross-linking is enhanced. We find that both ligands alkylate all adenines in the longer AT-tracts, as well as the abutting guanines, whether they are in the same strand as the adenines or not, in a manner consistent with covalent attack on purine N3 atoms from the minor groove. Alkamin forms intrastrand cross-links involving A4 and A6 and A6 and G10 in A3T3 and all of the purines in the A6/T6 purine tract, including G10. In addition, it forms interstrand cross-links between A4, A5, A6 and A4', A5', A6', between G10 and the latter adenines in A3T3, and between G22 and adenines A5 and A6 in A6/T6. The reactivity of the abutting guanines provides unexpected opportunities for both inter- and intrastrand cross-linking by alkamin, such as the interstrand cross-link in the CAAAAAAG sequence. We conclude that positioning monofunctional mustard groups on either end of a minor groove-directed polybenzamide has the capacity to enhance interstrand cross-links at all manner of AT-tracts, including most in which the adenines are all in one strand.
Assuntos
Alquilantes/química , Anilidas/química , Compostos de Mostarda Nitrogenada/química , Oligonucleotídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Adenina/química , Alquilantes/toxicidade , Anilidas/toxicidade , Adutos de DNA/química , Compostos de Mostarda Nitrogenada/toxicidade , Timina/químicaRESUMO
Thirty-eight antileukemic steroidal esters possessing conformationally flexible nitrogen mustards were studied, and the 3D QSAR/CoMFA and CoMSIA methodologies were applied in order to derive the correlation between their structure and the in vivo antileukemic activity. These compounds show significantly reduced toxicity and possibly increased bioavailability compared to free nitrogen mustards and therefore constitute potent antileukemic drugs. Both the CoMFA and CoMSIA studies gave similar results indicating that the steric effect and the hydrophobic/hydrophilic balance especially in the steroidal part of the molecules probably determined their bioactivity. Of paramount interest is the observation that the orientation of the alkylating part of the SMEs toward the surface of ring B of the steroidal skeleton was related with increased activity. Concerning the steroidal part, the presence of hydrophobic groups in rings B and D was found to be important for enhanced activity. Enhancement of antileukemic potency is further observed if hydrophilic/H-bond acceptor groups are present at the positions 7 and 17 of the steroidal skeleton. Leapfrog simulations provided novel compounds which lead our future synthetic endeavor for obtaining SMEs with optimum bioactivity.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Leucemia/tratamento farmacológico , Compostos de Mostarda Nitrogenada/química , Compostos de Mostarda Nitrogenada/farmacologia , Esteroides/síntese química , Esteroides/farmacologia , Animais , Antineoplásicos/toxicidade , Desenho de Fármacos , Feminino , Humanos , Interações Hidrofóbicas e Hidrofílicas , Informática , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Compostos de Mostarda Nitrogenada/toxicidade , Relação Quantitativa Estrutura-Atividade , Ratos , Eletricidade Estática , Esteroides/toxicidade , TermodinâmicaRESUMO
BACKGROUND: Widely accepted somatic mutation theory of carcinogenesis states that mutations in oncogenes and tumor suppressor genes in genomes of somatic cells is the cause of neoplastic transformation. Identifying frequent mutations in cancer cells suggests the involvement of mutant genes in carcinogenesis. RESULTS: To develop an in vitro model for the analysis of genetic alterations associated with breast carcinogenesis, we used random mutagenesis and selection of human non-tumorigenic immortalized breast epithelial cells MCF-10A in tissue-culture conditions that mimic tumor environment. Random mutations were generated in MCF-10A cells by cultivating them in a tissue-culture medium containing the frameshift-inducing agent ICR191. The first selective condition we used to transform MCF1-10A cells was cultivation in a medium containing mutagen at a concentration that allowed cell replication despite p53 protein accumulation induced by mutagen treatment. The second step of selection was either cell cultivation in a medium with reduced growth-factor supply or in a medium that mimics a hypoxia condition or growing in soft agar. Using mutagenesis and selection, we have generated several independently derived cultures with various degrees of transformation. Gene Identification by Nonsense-mediated mRNA decay Inhibition (GINI) analysis has identified the ICR191-induced frameshift mutations in the TP53, smoothelin, Ras association (RalGDS/AF-6) domain family 6 (RASSF6) and other genes in the transformed MCF-10A cells. The TP53 gene mutations resulting in the loss of protein expression had been found in all independently transformed MCF-10A cultures, which form large progressively growing tumors with sustained angiogenesis in nude mice. CONCLUSION: Identifying genes containing bi-allelic ICR191-induced frameshift mutations in the transformed MCF-10A cells generated by random mutagenesis and selection indicates putative breast-tumor suppressors. This can provide a model for studying the role of mutant genes in breast carcinogenesis.
Assuntos
Aminacrina/análogos & derivados , Neoplasias da Mama/genética , Mutação da Fase de Leitura/genética , Genes Supressores de Tumor , Modelos Biológicos , Mutagênese/efeitos dos fármacos , Compostos de Mostarda Nitrogenada/toxicidade , Aminacrina/toxicidade , Animais , Linhagem Celular Transformada , Linhagem Celular Tumoral , Instabilidade Cromossômica/efeitos dos fármacos , Feminino , Mutação da Fase de Leitura/efeitos dos fármacos , Humanos , Neoplasias Mamárias Experimentais/genética , Camundongos , Transplante de Neoplasias , Hibridização de Ácido Nucleico , Estabilidade de RNA/efeitos dos fármacos , Cariotipagem EspectralRESUMO
To elucidate molecular and cellular mechanisms of peroxisome biogenesis, we have isolated Chinese hamster ovary (CHO) cell mutants defective in peroxisome biogenesis by making use of enhanced green fluorescent protein (EGFP) and a frameshift-inducing mutagen ICR191. CHO-TKa cells stably expressing Pex2p were transformed with a cDNA encoding EGFP fused with peroxisomal targeting signal type 2 (PTS2-EGFP), termed Tka/EG2. TKa/EG2 cells were mutagenized with ICR191 and cultured in the presence of P9OH (9-(1'-pyrene) nonanol) followed by an exposure to UV. P9OH/UV-resistant and morphologically peroxisome-deficient mutant cells were isolated by directly observing cytosolic localization of EGFP, without cell staining. By a combination of cell-fusion and PEX transfection, we determined complementation groups (CGs) of 16 cell mutants isolated here. The mutants were classified into five CGs, including pex2, pex3, pex5, pex6, and pex7 cell mutants. In contrast to typical pex6 mutants with the impaired import of both PTS1- and PTS2-proteins, two clones, ZPEG236 and ZPEG244, showed a distinct, novel phenotype where PTS1-protein import was normal despite the abrogated PTS2 import. Dysfunction of Pex3p in pex3 ZPEG 238 was due to one base (G) insertion in the codon for Asn7 resulting in a frameshift, thereby inducing a distinct 31 amino-acid sequence and a termination. pex2 ZPEG239 showed a mutation in codon GAG for Glu(201) to a nonsense mutation, TAG. Thus, the method developed here using ICR191 could be useful for isolation of further novel cell mutants impaired in peroxisome biogenesis.
Assuntos
Aminacrina/análogos & derivados , Mutagênicos/toxicidade , Mutação , Compostos de Mostarda Nitrogenada/toxicidade , Peroxissomos/efeitos dos fármacos , ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases/genética , Aminacrina/química , Aminacrina/toxicidade , Animais , Células CHO , Cricetinae , Cricetulus , Citosol/metabolismo , Análise Mutacional de DNA , Mutação da Fase de Leitura , Teste de Complementação Genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Estrutura Molecular , Mutagênicos/química , Compostos de Mostarda Nitrogenada/química , Receptor 2 de Sinal de Orientação para Peroxissomos , Receptor 1 de Sinal de Orientação para Peroxissomos , Peroxissomos/metabolismo , Mutação Puntual , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
We have investigated the ability of chlorophyllin (CHL) to interact with acridine mutagen ICR-191 (2-methoxy-6-chloro-9-(3-(2-chloroethyl)aminopropylamino)acridine) and also its ability to decrease binding of ICR-191 to DNA in a simple three-component competition system: CHL-ICR-DNA. Our data indicate a strong association of ICR-191 with CHL, stronger even than the association of ICR-191 with DNA. Calculations based on the measured affinity data show that a two- to three-fold excess of CHL reduces by about two-fold the concentration of the mutagen-DNA complex. We also exposed human leukemic HL-60 cells to ICR-191 in the absence and presence of CHL and measured the mutagen-induced DNA damage. The extent of DNA damage was assessed by analysis of histone H2AX phosphorylation. While ICR-191 induced significant increase in expression of phosphorylated H2AX (gammaH2AX), particularly in DNA replicating cells, this increase was totally abolished in the cells treated with ICR-191 in the presence of CHL.
Assuntos
Aminacrina/análogos & derivados , Clorofilídeos/farmacologia , Dano ao DNA , DNA/química , Mutagênicos/toxicidade , Compostos de Mostarda Nitrogenada/toxicidade , Aminacrina/química , Aminacrina/toxicidade , Antimutagênicos/química , Antimutagênicos/farmacologia , Ligação Competitiva/efeitos dos fármacos , Quimioprevenção , Clorofilídeos/química , DNA/efeitos dos fármacos , Células HL-60 , Humanos , Substâncias Macromoleculares/química , Mutagênicos/química , Compostos de Mostarda Nitrogenada/químicaRESUMO
PURPOSE: The efficacy and toxicity of bendamustine chemotherapy in relapsed small cell lung cancer (SCLC) was determined in this phase II trial. PATIENTS AND METHODS: Patients with cytologically or histologically proven SCLC, who had a sensitive relapse, which was defined as a relapse>or=2 months after completion of primary therapy, were eligible for this study. After informed consent patients received 120 mg/m2 of bendamustine on Days 1 and 2 every 3 weeks. A maximum of six cycles was administered. Primary endpoint was response rate, secondary endpoints included toxicity, progression free survival and overall survival (OS). RESULTS: Twenty-one patients with a median age of 59 years (range 47-76) were accrued to this trial. Six (29%) of 21 patients achieved a confirmed partial remission, 6 (29%) had stable disease and 9 (42%) patients progressed according to RECIST criteria. Median progression free survival was 4 months (95% CI 0-8, 3), median overall survival was 7 months (95% CI 5, 8-8, 2). One- and 2-year survival was 16% and 8%, respectively. Grade III/IV neutropenia occurred in 3 (15%) of 21 patients, 1 patient had a lethal Gram-negative sepsis in neutropenia. Two additional patients had pneumonia in the absence of neutropenia. Two patients (10%) had a grade III anemia, no grade III or IV thrombocytopenia was observed. CONCLUSION: This trial demonstrates efficacy of bendamustine in relapsed SCLC and a favourable toxicity profile. Therefore, single-agent bendamustine is a treatment option for patients with SCLC, who have responded to initial platinum containing chemotherapy and should further be investigated in randomized trials.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma de Células Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Compostos de Mostarda Nitrogenada/uso terapêutico , Idoso , Antineoplásicos/toxicidade , Cloridrato de Bendamustina , Carcinoma de Células Pequenas/mortalidade , Intervalo Livre de Doença , Feminino , Humanos , Neoplasias Pulmonares/mortalidade , Masculino , Pessoa de Meia-Idade , Compostos de Mostarda Nitrogenada/toxicidade , Recidiva , Análise de Sobrevida , Fatores de TempoRESUMO
The multifunctional molecule 11beta-dichloro consists of a ligand for the androgen receptor linked to a bifunctional alkylating group, permitting it to create DNA adducts that bind the androgen receptor. We propose that binding of the androgen receptor to 11beta-DNA adducts acts to both shield damaged sites from repair and disrupt the expression of genes essential for growth and survival. We investigated the formation 11beta-DNA adducts in tumor xenograft and nontumor tissues in mice. Using [14C]-11beta-dichloro, we show that the molecule remains intact in blood and is widely distributed in mouse tissues after i.p. injection. Covalent 11beta-guanine adducts identified in DNA that had been allowed to react with 11beta-dichloro in vitro were also found in DNA isolated from cells in culture treated with 11beta-dichloro as well as in DNA isolated from liver and tumor tissues of mice treated with the compound. We used accelerator mass spectrometry to determine the levels of [14C]-11beta-DNA adducts in LNCaP cells treated in culture as well as in liver tissue and LNCaP xenograft tumors in treated mice. The level of DNA adducts in tumor tissue was found to be similar to that found in LNCaP cells in culture treated with 2.5 micromol/L 11beta-dichloro. Our results indicate that 11beta-dichloro has sufficient stability to enter the circulation, penetrate tissues, and form DNA adducts that are capable of binding the androgen receptor in target tissues in vivo. These data suggest the involvement of our novel mechanisms in the antitumor effects of 11beta-dichloro.
Assuntos
Antineoplásicos/toxicidade , Antineoplásicos/uso terapêutico , Adutos de DNA/efeitos dos fármacos , Compostos de Mostarda Nitrogenada/toxicidade , Compostos de Mostarda Nitrogenada/uso terapêutico , Esteroides/toxicidade , Esteroides/uso terapêutico , Animais , Antineoplásicos/farmacocinética , Adutos de DNA/química , Humanos , Cinética , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Espectrometria de Massas , Camundongos , Neoplasias da Próstata/tratamento farmacológico , Espectrometria de Massas por Ionização por Electrospray , Distribuição Tecidual , Transplante HeterólogoRESUMO
Despite many years of research into chemical warfare agents, cytotoxic mechanisms induced by mustards are not well understood. Reactive oxygen and nitrogen species (ROS and RNS) are likely to be involved in chemical warfare agents induced toxicity. These species lead to lipid peroxidation, protein oxidation, and DNA injury, and trigger many pathophysiological processes that harm the organism. In this article, several steps of pathophysiological mechanisms and possible ways of protection against chemical warfare agents have been discussed. In summary, pathogenesis of mustard toxicity is explained by three steps: (1) mustard binds target cell surface receptor, (2) activates intracellular ROS and RNS leading to peroxynitrite (ONOO(-)) production, and (3) the increased ONOO(-) level damages organic molecules (lipids, proteins, and DNA) leading to poly(adenosine diphosphate-ribose) polymerase (PARP) activation. Therefore, protection against mustard toxicity could also be performed in these ways: (1) blocking of cell surface receptor, (2) inhibiting the ONOO(-) production or scavenging the ONOO(-) produced, and (3) inhibiting the PARP, activated by ONOO(-) and hydroxyl radical (OH(*)) induced DNA damage. As conclusion, to be really effective, treatment against mustards must take all molecular mechanisms of cytotoxicity into account. Combination of several individual potent agents, each blocking one of the toxic mechanisms induced by mustards, would be interesting. Therefore, variations of combination of cell membrane receptor blockers, antioxidants, nitric oxide synthase inhibitors, ONOO(-) scavengers, and PARP inhibitors should be investigated.