RESUMO
The elevated expression of NQO1 in many human solid tumors along with its ability to activate quinone-based anticancer agents makes it an excellent target for enzyme-directed drug development. NQO1 plays an important role in melanogenesis and given its correlation with a poor patient outcome we propose this enzyme as an intriguing target for molecular-based therapeutic regimen against melanoma. Unfortunately, the natural product ß-Lapachone (ß-Lap), whose antitumor activity is based on NQO1, reported dose-limiting toxicity which hampered its pre-clinical and clinical use. Therefore, new effective and safe therapeutic NQO1-bioactivatable agents for melanoma treatment are desirable. Regarding NQO1, we demonstrated that halogenated ß-Lap derivative named PFB is an excellent substrate and effective tumor-selective anticancer compound. In addition, PFB resulted more attractive than the parent ß-Lap for treating metastatic-derived melanoma cells. In this context, it would be interesting to design strategies to induce NQO1 activity in cancer cells as a promising combinatorial approach with bioreductive drugs. In this sense, we had reported that photodynamic therapy (PDT) significantly upregulated NQO1 expression. Based on this event, here we demonstrated that the cytotoxic regimen consisting of PFB plus PDT improved synergistic therapeutic combination on melanoma cells. In conclusion, our contribution provides a strong rationale for using therapies that associate photo- and chemotherapy to effectively treat melanoma with modular NQO1 status.
Assuntos
Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/radioterapia , NAD(P)H Desidrogenase (Quinona)/biossíntese , Naftoquinonas/uso terapêutico , Fotoquimioterapia/métodos , Radiossensibilizantes/uso terapêutico , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Humanos , Camundongos , Naftoquinonas/farmacologia , Radiossensibilizantes/farmacologiaRESUMO
PURPOSE: To study the rhenium-188 labeling of polyamidoamine (PAMAM) generation 4 (G4) dendrimer and its evaluation on biodistribution and chromosomal aberrations in melanoma cells induced by ionizing radiation as potential treatment agent. MATERIALS AND METHODS: Dendrimers were first conjugated with Suc-HYNIC (succinimidyl 6-hydrazinopyridine-3-carboxylic acid hydrochloride). Dendrimer-HYNIC was then incubated with 188ReO4-. Biodistribution was performed administrating 188Re-dendrimer to normal (NM) or melanoma-bearing mice (MBM). Chromosome aberration test was conducted in order to measure treatment capacity of 188Re-dendrimer in melanoma cells. RESULTS: Radiolabeling yield of dendrimer was approx. 70%. Biodistribution studies in NM showed blood clearance with hepatic and renal depuration. MBM showed a similar pattern of biodistribution with tumor uptake of 6% of injected dose. Aberrant metaphases quantified in control cells were 7%, increasing to 29.5% in cells treated with 15µCi (0.555 MBq) of 188Re-dendrimer for 24 h. CONCLUSIONS: 188Re-dendrimer can produce double-stranded breaks in DNA induced by ionizing radiation in melanoma cells in vitro.
Assuntos
Aberrações Cromossômicas/efeitos da radiação , Dendrímeros/química , Melanoma Experimental/radioterapia , Radioisótopos/toxicidade , Rênio/toxicidade , Animais , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Marcação por Isótopo , Melanoma Experimental/genética , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Radioisótopos/farmacocinética , Rênio/farmacocinética , Distribuição TecidualRESUMO
Boron neutron capture therapy (BNCT) is a binary treatment involving selective accumulation of boron carriers in a tumor followed by irradiation with a thermal or epithermal neutron beam. The neutron capture reaction with a boron-10 nucleus yields high linear energy transfer (LET) particles, alpha and (7)Li, with a range of 5 to 9 µm. These particles can only travel very short distances and release their damaging energy directly into the cells containing the boron compound. We aimed to evaluate proliferation, apoptosis and extracellular matrix (ECM) modifications of B16F10 melanoma and normal human melanocytes after BNCT. The amounts of soluble collagen and Hsp47, indicating collagen synthesis in the ECM, as well as the cellular markers of apoptosis, were investigated. BNCT decreased proliferation, altered the ECM by decreasing collagen synthesis and induced apoptosis by regulating Bcl-2/Bax in melanoma. Additionally, BNCT also increased the levels of TNF receptor and the cleaved caspases 3, 7, 8 and 9 in melanoma. These results suggest that multiple pathways related to cell death and cell cycle arrest are involved in the treatment of melanoma by BNCT.
Assuntos
Apoptose/fisiologia , Terapia por Captura de Nêutron de Boro/métodos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Transferência Linear de Energia/fisiologia , Melanoma Experimental/radioterapia , Análise de Variância , Apoptose/efeitos da radiação , Western Blotting , Caspases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos da radiação , Colágeno/metabolismo , Primers do DNA/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/efeitos da radiação , Citometria de Fluxo , Proteínas de Choque Térmico HSP47/metabolismo , Humanos , Imuno-Histoquímica , Transferência Linear de Energia/efeitos da radiação , Melanócitos , Microscopia Eletrônica de Transmissão , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores do Fator de Necrose Tumoral/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
Information on (10)B distribution in normal tissues is crucial to any further development of boron neutron capture therapy (BNCT). The goal of this study was to investigate the in vitro and in vivo boron biodistribution in B16F10 murine melanoma and normal tissues as a model for human melanoma treatment by a simple and rapid colorimetric method, which was validated by HR-ICP-MS. The B16F10 melanoma cell line showed higher melanin content than human melanocytes, demonstrating a greater potential for boronophenylalanine uptake. The melanocytes showed a moderate viability decrease in the first few minutes after BNCT application, stabilizing after 75 min, whereas the B16F10 melanoma showed the greatest intracellular boron concentration at 150 min after application, indicating a different boron uptake of melanoma cells compared to normal melanocytes. Moreover, at this time, the increase in boron uptake in melanoma cells was approximately 1.6 times higher than that in normal melanocytes. The (10)B concentration in the blood of mice bearing B16F10 melanoma increased until 90 min after BNCT application and then decreased after 120 min, and remained low until the 240th minute. On the other hand, the (10)B concentration in tumors was increased from 90 min and maximal at 150 min after application, thus confirming the in vitro results. Therefore, the present in vitro and in vivo study of (10)B uptake in normal and tumor cells revealed important data that could enable BNCT to be possibly used as a treatment for melanoma, a chemoresistant cancer associated with high mortality.
Assuntos
Terapia por Captura de Nêutron de Boro , Boro/farmacocinética , Boro/uso terapêutico , Melanócitos/metabolismo , Melanócitos/efeitos da radiação , Melanoma Experimental/metabolismo , Melanoma Experimental/radioterapia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos da radiação , Feminino , Humanos , Melaninas/biossíntese , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Reprodutibilidade dos Testes , Distribuição TecidualRESUMO
The melanoma is a highly lethal skin tumor, with a high incidence. Boron Neutron Capture Therapy (BNCT) is a radiotherapy which combines Boron with thermal neutrons, constituting a binary system.B16F10 melanoma and L929 fibroblasts were treated with Boronophenylalanine and irradiated with thermal neutron flux. The electric potential of mitochondrial membrane, cyclin D1 and caspase-3 markers were analyzed.BNCT induced a cell death increase and cyclin D1 amount decreased only in B16F10 melanoma. Besides, there was not caspase-3 phosphorylation.
Assuntos
Ratos , Ciclo Celular/efeitos da radiação , Melanoma Experimental/induzido quimicamente , Melanoma Experimental/radioterapia , Terapia por Captura de Nêutron de Boro/métodos , /isolamento & purificação , Mitocôndrias/efeitos da radiação , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Técnicas de Cultura de CélulasRESUMO
Antiproliferative and oxidative damage effects occurring in Boron Neutron Capture Therapy (BNCT) in normal fibroblasts and melanoma cell lines were analyzed. Melanoma cells and normal fibroblasts were treated with different concentrations of Boronophenylalanine and irradiated with thermal neutron flux. The cellular viability and the oxidative stress were determined. BNCT induced free radicals production and proliferative potential inhibition in melanoma cells. Therefore, this therapeutic technique could be considered efficient to inhibit growth of melanoma with minimal effects on normal tissues.
Assuntos
Terapia por Captura de Nêutron de Boro , Radicais Livres/metabolismo , Melanoma Experimental/radioterapia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , CamundongosRESUMO
The melanoma is a highly lethal skin tumor, with a high incidence. Boron Neutron Capture Therapy (BNCT) is a radiotherapy which combines Boron with thermal neutrons, constituting a binary system. B16F10 melanoma and L929 fibroblasts were treated with Boronophenylalanine and irradiated with thermal neutron flux. The electric potential of mitochondrial membrane, cyclin D1 and caspase-3 markers were analyzed. BNCT induced a cell death increase and cyclin D1 amount decreased only in B16F10 melanoma. Besides, there was not caspase-3 phosphorylation.
Assuntos
Terapia por Captura de Nêutron de Boro , Pontos de Checagem do Ciclo Celular , Fragmentação do DNA , Melanoma Experimental/radioterapia , Animais , Caspase 3/metabolismo , Linhagem Celular Tumoral , Melanoma Experimental/patologia , CamundongosRESUMO
Erythropoietin (EPO) is a glycoprotein hormone responsible for regulating erythropoiesis. Expression of EPO and EPO receptors (EPOr) has recently been demonstrated in some neoplastic cell lines and tumours, suggesting a potential new target for therapy. In this work, EPO was labeled with iodine-125 using the lactoperoxidase method, known to prevent damage to protein during radioiodination, and labeling conditions were optimized. In vitro stability studies have shown that 125I-EPO is radiochemically stable for 20 days after radiolabeling. In vitro cell binding studies have demonstrated very low binding (<2 percent) of EPO to normal and neoplastic cell lines tested. As expected, the biodistribution in healthy mice exhibited comparatively high rates of fixation in the organs of the excretory system. Thyroid also proved to be a critical organ which may indicate in vivo dissociation of 125I-EPO. In mice with induced melanoma, only a residual fixation in the tumour was evident. Further studies are warranted on other tumoral cell lines to better understand the binding process and internalization into cells. Studies on EPO labeled with carbon-11 could be valuable, since there is a greater chance of preserving the biological activity of the protein using this method.
A eritropoetina (EPO) é um hormônio glicoprotéico responsável pela regulação da eritropoese. Recentemente foi demonstrado que os receptores de EPO (EPOr) estão expressos em algumas linhas celulares neoplásicas, o que sugere a sua potencialidade como um novo alvo terapêutico. Neste trabalho a EPO foi radiomarcada com iodo-125 através do método da lactoperoxidase, menos agressivo para a viabilidade biológica das proteínas. A 125I-EPO revelou ser radioquimicamente estável durante 20 dias após a síntese. Um estudo biológico in vitro em linhas celulares tumorais demonstrou que a 125I-EPO apresenta uma ligação muito fraca (<2 por cento), tanto em células normais como nas linhagens tumorais testadas. A biodistribuição em camundongos saudáveis apresentou taxas de fixação relativamente maiores nos órgãos excretores e a tireóide revelou ser o órgão crítico, o que pode indicar a dissociação in vivo da 125I-EPO. No estudo em camundongos com melanoma induzido a fixação no tumor foi residual. Serão, no entanto, necessários novos estudos em outras linhagens tumorais para entender o seu processo de internalização e ligação nas células. Estudos da EPO radiomarcada com carbono-11 poderão também revelar-se interessantes, já que neste método há maior probabilidade da atividade biológica ser preservada.
Assuntos
Animais , Feminino , Camundongos , Fenômenos Biológicos/análise , Compostos Radiofarmacêuticos/farmacocinética , Compostos Radiofarmacêuticos , Neoplasias/induzido quimicamente , Receptores da Eritropoetina , Eritropoese/efeitos da radiação , Glicoproteínas , Melanoma Experimental/induzido quimicamente , Melanoma Experimental/radioterapia , Radioisótopos do Iodo/análise , Radioisótopos do Iodo/uso terapêuticoRESUMO
PURPOSE: The aim of this study was to evaluate the induction and rejoining of DNA double strand breaks (DSBs) in melanoma cells exposed to low and high linear energy transfer (LET) radiation. METHODS AND MATERIALS: DSBs and survival were determined as a function of dose in melanoma cells (B16-F0) irradiated with monoenergetic proton and lithium beams and with a gamma source. Survival curves were obtained by clonogenic assay and fitted to the linear-quadratic model. DSBs were evaluated by the detection of phosphorylated histone H2AX (gammaH2AX) foci at 30 min and 6 h post-irradiation. RESULTS: Survival curves showed the increasing effectiveness of radiation as a function of LET. gammaH2AX labeling showed an increase in the number of foci vs. dose for all the radiations evaluated. A decrease in the number of foci was found at 6 h post-irradiation for low LET radiation, revealing the repair capacity of DSBs. An increase in the size of gammaH2AX foci in cells irradiated with lithium beams was found, as compared with gamma and proton irradiations, which could be attributed to the clusters of DSBs induced by high LET radiation. Foci size increased at 6 h post-irradiation for lithium and proton irradiations in relation with persistent DSBs, showing a correlation with surviving fraction. CONCLUSIONS: Our results showed the response of B16-F0 cells to charged particle beams evaluated by the detection of gammaH2AX foci. We conclude that gammaH2AX foci size is an accurate parameter to correlate the rejoining of DSBs induced by different LET radiations and radiosensitivity.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Histonas/metabolismo , Transferência Linear de Energia , Melanoma Experimental/radioterapia , Biomarcadores/análise , Biomarcadores/metabolismo , Sobrevivência Celular , Relação Dose-Resposta à Radiação , Histonas/análise , Humanos , Lítio/uso terapêutico , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Fosforilação , Terapia com Prótons , Tolerância a Radiação , Radioisótopos/uso terapêuticoRESUMO
El cáncer es una enfermedad del genoma, en el cual múltiples aberraciones genéticas conducen a la activación de oncogenes y a la inactivación de genes supresores tumorales, promoviendo un desequilibrio en el control de la proliferación, diferenciación y muertecelular. Las células tumorales pueden ser inducidas a un crecimiento controlado y a diferenciación terminal in vitro por tratamiento farmacológico que conduce a la restauración parcial del fenotipo celular normal. Sin embargo, el uso de inductores farmacológicos de diferenciarón, supresión del crecimiento y muerte celular como el ácido retinoíco, vitamina D, L-tirosina y bromodeoxiuridina, es limitado debido a que no se conocen los mecanismos moleculares y sus blancos de acción. La Bromodeoxiuridina, es un análogo de la timina, sensibilizador a la radiación ultravioleta e inductor de la supresión del crecimiento. Sin embargo, las bases moleculares de estas acciones no se conocen muy bien. La identificación de genes blancos moleculares (como cinasas y fosfatasas) de la Bromodeoxiuridina (BrdU) y su papel en el control de la proliferación celular y la radiosensibilización, conduciría al entendimiento y desarrollo de potenciales agentes terapéuticos para el control de la proliferación en células tumorales, como tratamiento alternativo a la quimioterapia y radioterapia convencionales, las cuales son altamente agresivas no sólo para las células tumorales, sino también para las células normales en proliferación. Los genes que codifican para proteínas fosfatasas se han implicado en la tumorigénesis. Recientemente en el laboratorio de fisiología molecular del INS se clonó y se secuenció el cADN que codifica para la tirosina fosfatasa PRL-1. Sin embargo, no se sabe si sus niveles de expresión cambian en células de melanoma proliferantes e inducidas a supresión del crecimiento y diferenciación celular. Por lo tanto, el objetivo de este trabajo fue determinar el nivel de expresión de ARN del gen que codifica para la tirosina-fosfatasa PRL-1, en células de melanoma murino B-16 inducidas a supresión del crecimiento y diferenciación celular. Por lo tanto, el objetivo de este trabajo fue determinar el nivel de expresión del ARN del gen que codifica para la tirosina-fosfatasa PRL-1, en células de melanoma murino B-16 inducidas a supresión del crecimiento por el tratamiento con Bromodeoxiuridina, radiación ultravioleta tipo C y sensibilización...