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Previous issue date: 2015-03-10 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Conselho Nacional de Pesquisa e Desenvolvimento Cient?fico e Tecnol?gico - CNPq / Financiadora de Estudos e Projetos - Finep / Glioblastoma (grade IV) is among the most prevalent primary intracranial tumors and is considered a challenge in oncology and neurosurgery due to the highly aggressive nature, and the elevated mortality rates. The location of the tumor and its invasive nature avoid the standard-of-care therapy, which includes surgical resection followed by radiotherapy and chemotherapy. Nevertheless, the current gold standard treatment has not been effective to prevent tumor evolution, as indicated by the poor
survival rates. In this context, we analyzed the GPCRs for kinin and the high-voltagegated calcium channels (VGCC) as feasible new therapeutic approaches in malignant gliomas. Thereby, the signaling triggered by bradykinin or the disruption of calcium signaling might contribute with pivotal mechanisms underlying glioma progression,
such as cell proliferation. Therefore, the aim of this study was to further evaluate the relevance of B1 (B1R) and B2 (B2R) kinin receptors as well as the P/Q- and N-type VGCC in glioma development, by using in vitro and in vivo glioma model.
Cell culture assay showed that the treatment with the selective B1R des-Arg9-BK (1-100 nM) and B2R BK (1-100 nM) agonists induced a marked enhancement of cell proliferation and viability through ERK1/2 and PI3K/Akt signaling, according to evaluation of U-138MG and U-251MG cell lines. Meanwhile, the incubation of either
B1R SSR240612 (1-30 ?M) or B2R HOE-140 (1-100 ?M) antagonists induced a marked cell death with mixed apoptosis/necrosis characteristics. The in vivo mouse model of
GL261-induced glioma of C57/BL6 or B1R and B2R knockout mice showed an uncontrolled tumor growing in KOB1R mice. Conversely, there was no significant change of the tumor development in KOB2R mice. Notably, the genetic ablation or the pharmacological combined antagonism of B1R and B2R (SSR240612; 25 nmol/site +
HOE-140; 50 pmol/site) diminished the tumor progression as well as the mitotic index of the GL261-induced glioma.
To understand the potential anti-tumor effects of the blockade of P/Q- and Ntype
VGCC, we used animal-derived inhibitors namely PhTx3-3 (P/Q-type blocker) and Ph?1? (N-type blocker) from P. nigriventer, or MVIIC (P/Q-type blocker) and MVIIA (N-type blocker) from C. magus. The PhTx3-3 (0.3 - 100 pM), Ph?1? (0.3 - 100 pM) and MVIIA (0.3 - 100 pM) displayed a significant inhibitory effect on proliferation and
viability of M059J, U-138MG and U-251MG glioma tested cell lines, and evoked cell death mainly with apoptosis characteristics. In the glioblastoma in vivo model, the Ntype VGCC blockade by either Ph?1? (50 pmol/site; i.c.v. and i.t.) or MVIIA (10pmol/site; i.c.v.) caused significant reductions of glioma growth and progression. Of
note, the N-type inhibition by Ph?1? and MVIIA led to a marked increase of GFAPactivated astrocytes and Iba-1-positive microglia in the peritumoral area, which might be related to the inhibitory effects of immune system in tumor development.
Using molecular and pharmacological approaches, our data provide clear evidence on the beneficial effects of the simultaneous inhibition of both B1R and B2R as well as the P/Q- and N-type blockade on glioma development. Thus, we propose that
the combined selective antagonism of B1R and B2R, such as the P/Q-, and especially NP type high-VGCC inhibition could markedly modify the tumor progression, which might represent an attractive alternative for the treatment of malignant gliomas in the future. / O glioblastoma apresenta a maior incid?ncia entre todos os gliomas e se
caracteriza como o mais agressivo e fatal (grau IV) dos tumores prim?rios do SNC.
Atualmente o glioblastoma ? considerado uns dos grandes desafios da oncologia e da
neurocirurgia, devido ao seu car?ter altamente agressivo. A sobrevida m?dia dos
pacientes ? bastante baixa e o progn?stico ? desfavor?vel, j? que a grande maioria destes tumores apresenta um padr?o difuso e infiltrativo de crescimento, o que dificulta as abordagens atuais para a terapia tumoral. Neste estudo foram analisados os efeitos dos receptores da fam?lia dos GPCRs de cininas e dos canais de c?lcio voltagem dependentes (CCVD) como base para poss?veis alvos no tratamento dos gliomas malignos, a fim de caracterizar novas abordagens terap?uticas. O efeito da sinaliza??o desencadeada pela BK e da sinaliza??o Ca2+-dependente pode estar envolvida na regula??o do crescimento e progress?o dos gliomas e na migra??o das c?lulas tumorais. Neste sentido, este trabalho visou explorar o papel dos receptores B1 (B1R) e B2 (B2R) de cininas e da sinaliza??o de Ca2+ via CCVD tipo-P/Q e -N em modelo de glioma in vitro e in vivo. Ensaios em cultura celular utilizando as linhagens de glioma humano U-138MG e U-251MG demonstraram que a ativa??o dos B1R e B2R pelo uso dos agonistas desarg9-BK (1-100 nM) e BK (1-100 nM) aumentou a prolifera??o das linhagens celulares testadas, atrav?s da ativa??o das vias ERK1/2 e PI3K/Akt. Enquanto que a exposi??o aos antagonistas seletivos para estes receptores, SSR240612 (1-30 ?M) e HOE-140 (1-100 ?M), provocou intensa morte celular com caracter?sticas de necrose/apoptose. A parte in vivo compreendeu a t?cnica de implante das c?lulas GL261 de glioma (grau IV) em animais C57/BL6 e knockout para os B1R e B2R. A dele??o apenas do B1R provocou um importante crescimento tumoral nos animais knockout para este receptor, enquanto que os animais com dele??o de B2R n?o tiveram o desenvolvimento tumoral alterado. Notavelmente, tanto a dele??o g?nica como o antagonismo farmacol?gico combinado dos receptores B1 e B2 (SSR240612; 25 nmol/s?tio + HOE-140; 50 pmol/s?tio) diminuiu o crescimento tumoral e o ?ndice mit?tico dos gliomas implantados. Para compreender o envolvimento dos CCVD tipo-P/Q e -N na fisiopatologia dos gliomas foram utilizadas fra??es da toxina da aranha Phoneutria nigriventer (PhTx3-3 bloqueadora de canais do tipo-P/Q; Ph?1? bloqueadora de canais do tipo-N) e ?-conotoxinas provenientes do Conus magus (MVIIC bloqueadora de canais do tipo-P/Q; MVIIA bloqueadora de canais do tipo-N). Os experimentos in vitro evidenciaram que o bloqueio dos canais de Ca2+ tipo-P/Q e -N pelas toxinas PhTx3-3 (0.3 - 100 pM), Ph?1? (0.3 - 100 pM) e MVIIA (0.3 - 100 pM) inibiram a prolifera??o e a viabilidade das linhagens celulares M059J, U-138MG e U-251MG de glioma humano, com intensa caracter?stica de morte celular por apoptose. Os resultados utilizando o modelo de glioblastoma in vivo, demonstraram que ambas as toxinas bloqueadoras dos canais do tipo-N, Ph?1? (50 pmol/s?tio) e MVIIA (10 pmol/s?tio), foram efetivas em diminuir o crescimento e a progress?o tumoral nos animais tratados, com intensa ativa??o de
astr?citos e micr?glia, destacando o poss?vel envolvimento do sistema imune na
inibi??o do crescimento tumoral. Atrav?s do uso de ferramentas moleculares e farmacol?gicas, nossos resultados demonstraram o envolvimento importante tanto dos B1R e B2R de cininas, como dos CCVD tipo-P/Q e -N no desenvolvimento dos gliomas malignos. Desta maneira, podemos propor que o bloqueio farmacol?gico combinado de antagonistas seletivos para os receptores B1 e B2, assim como a inibi??o dos CCVD tipo-P/Q e -N surgem como potenciais alvos terap?uticos no manejo dos tumores cerebrais e podem representar alternativas promissoras no tratamento dos gliomas.
| Identifer | oai:union.ndltd.org:IBICT/oai:tede2.pucrs.br:tede/6009 |
| Date | 10 March 2015 |
| Creators | Nicoletti, Nat?lia Fontana |
| Contributors | Morrone, Fernanda Bueno, Campos, Maria Martha |
| Publisher | Pontif?cia Universidade Cat?lica do Rio Grande do Sul, Programa de P?s-Gradua??o em Biologia Celular e Molecular, PUCRS, Brasil, Faculdade de Bioci?ncias |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Format | application/pdf |
| Source | reponame:Biblioteca Digital de Teses e Dissertações da PUC_RS, instname:Pontifícia Universidade Católica do Rio Grande do Sul, instacron:PUC_RS |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | 8198246930096637360, 600, 600, 600, 600, 600, 600, 36528317262667714, -1634559385931244697, 2075167498588264571, -2555911436985713659, -1790502019031048300 |
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