Polypodium leucotomos targets multiple aspects of oral carcinogenesis and it is a potential antitumor phytotherapy against tongue cancer growth

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Polypodium leucotomos targets multiple aspects of oral carcinogenesis and it is a potential antitumor phytotherapy against tongue cancer growth

Introduction: Oral cancer refers to malignant tumors, of which 90% are squamous cell carcinomas (OSCCs). These malignancies exhibit rapid progression, poor prognosis, and often mutilating therapeutical approaches. The determination of a prophylactic and/or therapeutic antitumor role of the polyphenolic extract Polypodium leucotomos(PL) would be relevant in developing new tools for prevention and treatment.Methods: We aimed to determine the antitumor effect of PL by treating OSCC cell lines with PL metabolites and evaluating its action during OSCC progression in vivo.Results: PL treatment successfully impaired cell cycling and proliferation, migration, and invasion, enhanced apoptosis, and modulated macrophage polarization associated with the tumoral immune-inflammatory response of tongue cancer cell lines (TSCC). PL treatment significantly decreased the expression of MMP1 (p < 0.01) and MMP2 (p < 0.001), and increased the expression of TIMP1 (p < 0.001) and TIMP2 (p < 0.0001) in these cells. The mesenchymal-epithelial transition phenotype was promoted in cells treated with PL, through upregulation of E-CAD (p < 0.001) and reduction of N-CAD (p < 0.05). PL restrained OSCC progression in vivo by inhibiting tumor volume growth and decreasing the number of severe dysplasia lesions and squamous cell carcinomas. Ki-67 was significantly higher expressed in tongue tissues of animals not treated with PL(p < 0.05), and a notable reduction in Bcl2 (p < 0.05) and Pcna (p < 0.05) cell proliferation-associated genes was found in dysplastic lesions and TSCCs of PL-treated mice. Finally, N-cad(Cdh2), Vim, and Twist were significantly reduced in tongue tissues treated with PL.Conclusion: PL significantly decreased OSCC carcinogenic processes in vitro and inhibited tumor progression in vivo. PL also appears to contribute to the modulation of immune-inflammatory oral tumor-associated responses. Taken together, these results suggest that PL plays an important antitumor role in processes associated with oral carcinogenesis and may be a potential phytotherapeutic target for the prevention and/or adjuvant treatment of TSCC

Migração de neutrofilos induzida por enterotoxinas estafilococicas para a cavidade peritoneal de camundongos : participação de macrofagos

Orientador: Glaci Ribeiro da SilvaTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Este trabalho descreve a participação de macrófagos sobre o influxo de neutrófilos, induzido pelas enterotoxinas estafilocócicas A (SEA) e B (SEB) em cavidades peritoneais de camundongos. Nossos resultados demonstraram que estas toxinas induzem influxo de neutrófilos, dose e tempo-dependente, para a cavidade peritoneal de camundongos. Este fenômeno foi quantitativamente menor, quando estas SEs foram injetadas em uma cavidade artificial, desprovida de células residentes, tal como a bolsa de ar subcutânea. A migração de neutrófilos, induzi da por estas toxinas, foi potencializada em cavidades peritoneais com o número basal de macrófagos aumentado pelo prétratamento com Tg, e, reduzida em cavidades com número de macrófagos diminuído, através da lavagem prévia com salina estéril, confirmando a hipótese de que a habilidade destas toxinas em induzir migração de neutrófilos in vivo está correlacionada com o número de macrófagos. O influxo de neutrófilos induzido por estas SEs, na cavidade peritoneal, foi reduzido em animais pré-tratados com os seguintes inibidores e antagonistas farmacológicos: dexametasona; inibidor de lipoxigenase (BW A4C); antagonista de PAF (BN52021); antagonista H2 de histamina (cimetidina); depletor de neuropeptídeos de fibras C sensoriais (capsaicina), mas não foi reduzido com o uso de um inibidor da ciclooxigenase (indometacina). Estes dados sugerem que os mediadores inflamatórios envolvidos na infiltração de neutrófilos produzida por estas SEs incluem metabólitos da LO, PAF, histamina e neuropeptídeos de fibras C sensoriais. Os sobrenadantes resultantes da incubação in vitro de macrófagos peritoneais de camundongos com SEA (SM-SEA) ou com SEB (SM-SEB), induzem migração de neutrófilos in vivo. A liberação das substâncias quimiotáticas para neutrófilos nestes sobrenadantes depende de pH e temperatura próximos ao fisiológico e da presença de cálcio, magnésio e da glicose no meio de incubação, sugerindo que estas substâncias são secretadas por um processo ativo. A ciclohexemida, um inibidor de síntese proteíca, inibe a liberação destas substâncias quimiotáticas para neutrófilos. Além disto, a atividade quimiotática do SM-SEA ou do SM-SEB é reduzida após o aquecimento a 100°C. Estes resultados sugerem que as substâncias quimiotáticas para neutrófilos, descritas neste trabalho, são proteínas termolábeis. A liberação destas proteínas, foi dose e tempo-dependente e foi inibida pelo pré-tratamento dos macrófagos com dexametasona mas não com indometacina ou com BW755C. A proteína presente no SM-SEA possui peso molecular maior que 100.000, enquanto que a proteína presente no SM-SEB possui peso molecular entre 1.000-3.000. A migração de neutrófilos induzida pelo SM-SEB envolve mediadores tais como metabólitos da LO, PAF e histamina, enquanto que estes mediadores não participam da resposta migratória induzida pelo SM-SEA sugerindo que estas proteínas possuem características distintas e induzem acúmulo de neutrófilos por mecanismos distintos. Em ambos os casos, a migração de neutrófilos foi reduzida em animais prétratados com capsaicina ou com SR140333 (antagonista seletivo de receptores de taquicininas NK1), mas não em animais pré-tratados com SR48968 (antagonista seletivo de receptores de taquicininas NK2), sugerindo que estas proteínas induzem migração via liberação de substância P. Nossos resultados contribuem para o melhor esclarecimento do mecanismo de ação das SEs e sugerem que produtos de macrófagos modulam o influxo neutrófilos induzido por estas toxinasAbstract: In this study, the role role of macrophages in the neutrophil migration induced by staphylococcal enterotoxins A (SEA) and B (SEB) into the mouse peritoneal cavity was investigated. SEA and SEB induced dose- and timedependent neutrophil migration into the mouse peritoneal cavity. This migration was potentiated when the macrophage population was increased by pre-treating the mice with thioglycolate, and was reduced when the peritoneal cavities were washed with sterile saline. These results indicated that the neutrophil recruitment induced by these toxins was dependent on the number of resident macrophages. Dexamethasone inhibited the neutrophil migration induced by SEA and SEB. A similar response was observed with the P AF receptor antagonist (BN52021), the histamine H2 receptor (cimetidine), the lipoxygenase inhibitor (BW A4C) and a sensory C-fiber neuropeptide depletor (capsaicin). In contrast, indomethacin had no effect on the neutrophil chemotaxis. Thus, lipoxygenase metabolites, P AF, histamine and neuropeptides from sensory neurons play an important role in the neutrophil migration induced by SEA and SEB. When stimulated with SEA or SEB in vitro, mouse peritoneal macrophages secreted thermolabile proteins which induced neutrophil migration into the peritoneal cavities of naive mice. The release of these proteins was dose- and time-dependent and was inhibited by dexamethasone but not by indomethacin or BW755C The molecular mass of the neutrophil chemotactic protein secreted by SEB-stimulated macrophages was 1-3 kDa while that of the protein secreted by SEA was > 100 kDa. The neutrophil migration induced by supematants from SEB-stimulated macrophages involved inflammatory mediators such as lipoxygenase metabolites, P AF and histamine. In contrast, these same mediators had no role in the neutrophil migration induced by supematants from SEA-stimulated macrophages, suggesting that the neutrophil chemotactic proteins described above, induce neutrophil accumulation by different mechanisms. Capsaicin and neurokinin1 (NKl) receptor antagonist SR140333 reduced the neutrophil recruitment induced by supematants from macrophage monolayers stimulated with SEA or SEB. In contrast, the neurokinin2 (NK2) receptor antagonist SR48968 had no effect on the neutrophil migratory response of the supematants. These results suggest that the neutrophil migration induced by these chemotactic proteins may involve the release of neuropeptides such as SP from sensory neurons. lu conc1usion, macrophage products may play an important role in the neurogenic inflammation .induced by SEA and SEB by releasing specific chemotactic proteinsDoutoradoFisiologiaDoutor em Biologia Funcional e Molecula

Uso do camundongo como modelo animal para o estudo das enterotoxinas estafilococicas

Orientador : Glaci Ribeiro da SilvaDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências MédicasResumo: 1. A SEB produziu na pata de camundongos um edema de longa duração, dose e tempo-dependente. 2. Este edema foi bifásico; a primeira fase (edema precoce) durou aproximadamente 24 h , enquanto que a segunda fase (edema tardio), iniciou-se nas 24 h e se prolongou por mais 72 h. 3. Houve um paralelismo entre o edema e a exsudação de azul de Evans produzidos pela SEB, sugerindo ser a exsudação o componente principal deste edema. 4. Os metabólitos do ácido araquidônico parecem ser mediadores do edema de pata induzido pela SEB em camundongos; os produtos da lipoxigenase estão envolvidos no edema precoce e os da ciclooxigenase, no edema tardio. 5. O edema precoce produzido pela SEB, foi parcialmente bloqueado em camundongos previamente tratados com metissergida, ou com WEB 2086, um antagonista de PAF. 6. A depleção dos neuropeptídeos de fibras C sensoriais com capsaicina, inibiu o edema precoce produzido pela SEB. 7. O pré-tratamento dos animais com antagonistas histamínicos, tanto H1 como H2, bloqueou o edema e a exsudação induzidos por SEB. 8. Devido a vários paralelismos observados entre o edema de pata induzido pela SEB no camundongo, o vômito e a "pseudo-alergia" cutânea que a toxina produz no macaco, nossos resultados sugerem que, o edema de pata produzido pela SEB no camundongo, poderia ser usado como um modelo experimental para o estudo in vivo das enterotoxemias por SEsAbstract: 1. Staphylococcal enterotoxin B (SEB) induces a long-Iasting, dose- and timedependent edema in the mouse hind paw. 2. The edematogenic response was biphasic and consisted of a primary (early) phase which lasted approximately 24 h and a secondary (Iate) phase which started after 24 h and continued for up to 72 h. 3. There was a positive correlation between the measured edema and the amount of Evans blue extravasation produced by SEB, suggesting that this exudate is the principal component of the edema. 4. Arachidonic acid metabolites appear to be involved in the SEB-induced " edema: lipoxygenase products are involved in the early phase while cyclooxygenase products are involved in the late phase. 5. The early phase edema was partially prevented by pretreatment of the mice with either methysergide or with the PAF antagonist WEB 2086. 6. Pretreatment of mice with capsaicin to deplete the sensory C-fibers of their neuropeptides inhibited the early edema induced by SEB. 7. Pretreatment with H1 and H2 histamine receptor antagonists also inhibited the early phase edema and the accompanying Evans blue extravasation induced by SEB. 8. Based on the similarities between the SEB-induced edema in mice and the vomiting and cutaneous 'pseudoallergy' produced by this toxin in monkeys, our results suggest that mice may prove to be a useful model for studying SEmediated enterotoxemia.MestradoFarmacologiaMestre em Farmacologi

Polypodium leucotomos targets multiple aspects of oral carcinogenesis and it is a potential antitumor phytotherapy against tongue cancer growth

Abstract Introduction: Oral cancer refers to malignant tumors, of which 90% are squamous cell carcinomas (OSCCs). These malignancies exhibit rapid progression, poor prognosis, and often mutilating therapeutical approaches. The determination of a prophylactic and/or therapeutic antitumor role of the polyphenolic extract Polypodium leucotomos(PL) would be relevant in developing new tools for prevention and treatment. Methods: We aimed to determine the antitumor effect of PL by treating OSCC cell lines with PL metabolites and evaluating its action during OSCC progression in vivo. Results: PL treatment successfully impaired cell cycling and proliferation, migration, and invasion, enhanced apoptosis, and modulated macrophage polarization associated with the tumoral immune-inflammatory response of tongue cancer cell lines (TSCC). PL treatment significantly decreased the expression of MMP1 (p < 0.01) and MMP2 (p < 0.001), and increased the expression of TIMP1 (p < 0.001) and TIMP2 (p < 0.0001) in these cells. The mesenchymal-epithelial transition phenotype was promoted in cells treated with PL, through upregulation of E-CAD (p < 0.001) and reduction of N-CAD (p < 0.05). PL restrained OSCC progression in vivo by inhibiting tumor volume growth and decreasing the number of severe dysplasia lesions and squamous cell carcinomas. Ki-67 was significantly higher expressed in tongue tissues of animals not treated with PL(p < 0.05), and a notable reduction in Bcl2 (p < 0.05) and Pcna (p < 0.05) cell proliferation-associated genes was found in dysplastic lesions and TSCCs of PL-treated mice. Finally, N-cad(Cdh2), Vim, and Twist were significantly reduced in tongue tissues treated with PL. Conclusions: PL significantly decreased OSCC carcinogenic processes in vitro and inhibited tumor progression in vivo. PL also appears to contribute to the modulation of immune-inflammatory oral tumor-associated responses. Taken together, these results suggest that PL plays an important antitumor role in processes associated with oral carcinogenesis and may be a potential phytotherapeutic target for the prevention and/or adjuvant treatment of TSCCs