Melanoma is an aggressive cancer with highly metastatic ability. We propose cofilin-1, a key prot... more Melanoma is an aggressive cancer with highly metastatic ability. We propose cofilin-1, a key protein in the regulation of actin dynamics and migration, as a prognostic marker. We determined cofilin-1 levels in a retrospective cohort of patients with melanomas and benign lesions of melanocytes (nevi) by immunohistochemistry. Higher cofilin-1 levels were found in malignant melanoma (MM) with Breslow Index (BI)>2 vs MM with BI<2, melanoma (MIS) and nevi and also in MM with metastasis vs MM without detected metastasis. Kaplan-Meier survival curves were performed, clustering patients according to either the type of melanocytic lesions or cofilin-1 level. Survival curves demonstrated worse prognosis of patients with high vs low cofilin-1 levels. TCGA database analysis of melanoma also showed low survival in patients with upregulated cofilin-1 mRNA vs patients without alteration in CFL1 mRNA expression. As cofilin-1 has a dual function depending on its intracellular localization, we ...
Advanced melanoma is the most aggressive form of skin cancer. It is highly metastatic and dysfunc... more Advanced melanoma is the most aggressive form of skin cancer. It is highly metastatic and dysfunctional in melanogenesis; two processes that are induced by H 2 O 2. This work presents a melanoma cell model with low levels of H 2 O 2 induced by catalase overexpression to study differentiation/dedifferentiation processes. Three clones (A7, C10 and G10) of human A375 amelanotic melanoma cells with quite distinct phenotypes were obtained. These clones faced H 2 O 2 scavenging by two main strategies. One developed by clone G10 where ROS increased. This resulted in G10 migration and metastasis associated with the increased of cofilin-1 and CAP1. The other strategy was observed in clone A7 and C10, where ROS levels were maintained reversing malignant features. Particularly, C10 was not tumorigenic, while A7 reversed the amelanotic phenotype by increasing melanin content and melanocytic differentiation markers. These clones allowed the study of potential differentiation and migration markers and its association with ROS levels in vitro and in vivo, providing a new melanoma model with different degree of malignancy.
Introdução: As espécies reativas de oxigênio estão relacionadas com a patologia de epilepsia. Exi... more Introdução: As espécies reativas de oxigênio estão relacionadas com a patologia de epilepsia. Existem vários modelos que induzem o estado epilétio em ratos, entre estes um dos mais utilizados é o do ácido caínico. Objetivos: Demonstrar uma possível associação entre espécies reativas de oxigênio e o estado epilético induzido por ácido caínico. Metodologia:O estado epilético foi induzido por uma injeção intraperitonial de ácido caínico (12 mg/Kg). Os hipocampos e a córtex foram isolados de ratos 28h, 9 dias, 80 dias e 150 dias após a indução. Os TBARs (indicadores de peroxidação lipídica), as relações lipidio/proteínas e o perfil lipídico por TLC foram determinados. Resultados: Foram observadas alterações no conteúdo de TBARs 24h após a indução,as relações lipídio/proteína estavam alteradas 80 e 150 dias após a indução e foram também observadas modificações no perfíl lipídico por TLC. Conclusões: O modelo do ácido caínico induziu modificações nas vias metabólicas de lipídios.
Trabalhos anteriores de nosso grupo demonstraram que o tratamento com retinol aumenta a produção ... more Trabalhos anteriores de nosso grupo demonstraram que o tratamento com retinol aumenta a produção de radicais livres, provavelmente via Reação de Fenton, causando danos à estrutura do DNA, levando as células a apoptose. Para manter a propagação da Reação de Fenton, uma fonte de elétrons deve estar disponível para regenerar o Fe +3 reduzido, sendo que o NADH é a molécula mais importante no desempenho desta função. O aumento nas concentrações de radicais livres desencadeiam na célula uma série de defesas no intuito de diminuir os danos produzidos. Dentre estas defesas destaca-se a catalase, uma enzima induzida por aumento nos radicais livres derivados do H 2 O 2. Neste trabalho, determinamos a quantidade de NADH intracelular e a atividade da catalase em células de Sertoli tratadas com retinol, além do efeito de baixas doses de etanol, um scavenger de radicais livres, na quantidade de NADH. Utilizamos em nossas culturas células de Sertoli de ratos Wistar de 15 dias tratadas ou não com retinol (7uM) por 24 horas. Para avaliar o efeito do etanol sobre o NADH intracelular foram utilizadas doses crescentes de etanol na diluição do retinol. A quantidade de NADH foi avaliada através da absorbância em espectrofotômetro (340 nm). A atividade da catalase foi medida através da decomposição do H 2 O 2 observada pelo decaimento da absorbância em espectrofotômetro (240 nm). O tratamento com retinol aumentou a concentração de NADH, efeito revertido pelo etanol. A atividade da catalase nas células tratadas foi significativamente maior em relação às células controle. O aumento no NADH intracelular induzido pelo tratamento com retinol provavelmente mantém ativa a Reação de Fenton, aumenta a produção de radicais livres, ativando assim a defesa antioxidante celular.
e21042 Background: Germline mutations in the TP53 gene are the underlying genetic defect of Li-Fr... more e21042 Background: Germline mutations in the TP53 gene are the underlying genetic defect of Li-Fraumeni Syndrome (LFS) and its variant, Li-Fraumeni-like (LFL) Syndrome, autosomal dominant disorders characterized by predisposition to multiple early-onset cancers. More recently, p53 is emerging as an important player in redox metabolism and important enzymes involved in defenses against oxidative stress have shown to be regulated by p53, including glutathione peroxidase 1 (GPX1) and mitochondrial superoxide dismutase 2 (SOD2). The aim of the present study was to investigate the redox profile parameters in blood of p.R337H mutation carriers and non-carriers individuals. Methods: A total of 34 individuals were included in the study and they were divided in two groups: mutation carriers (n=17) and non-carriers (n=17). Antioxidant enzyme activities (SOD, CAT, GPx) and oxidative stress parameters (Protein carbonyl content, Sulfhydryl content and TBARS) were measured in plasma, erythrocytes...
The present study analyzed the quality of bovine ovarian tissue after vitrification in a metal cl... more The present study analyzed the quality of bovine ovarian tissue after vitrification in a metal closed chamber, in terms of putative changes in tissue viability (lactate dehydrogenase -LDH- release), anti-oxidant defenses, and redox parameters caused by cryopreservation. Small and large fragmented bovine ovarian tissue specimens were vitrified in a metal chamber. After rewarming, tissue samples were fixed or cultured for 48 hours. Glutathione (GSH), protein sulfhydryl content, Total Radical Trapping Antioxidant Potential (TRAP), and lactate dehydrogenase were analyzed immediately after rewarming and after tissue culture. No changes in antioxidant parameters or viability of rewarmed tissue samples were found immediately or 48h after vitrification. The method of vitrification in a metal closed chamber used in this study preserved the quality of bovine ovarian tissue. Furthermore, our data showed that the size of the tissue specimens did not affect post-vitrification biochemical viabili...
To evaluate the consequences of plasma from individuals with obesity on parameters associated wit... more To evaluate the consequences of plasma from individuals with obesity on parameters associated with immunosenescence in unrelated healthy peripheral blood mononuclear cells (PBMC). Freshly isolated PBMC were incubated in media supplemented with 10% of plasma from individuals with obesity or control subjects for the first 4 hours of 24 to 120 hours of culture. Plasma from individuals with obesity modulated the phenotype of healthy PBMC, leading to a higher rate of apoptosis, lower amounts of phospho-γH2AX and -p53, and mitochondrial dysfunction. After 120 hours, there was a higher secretion of inflammatory cytokines IL-1β and IL-8. CD8(+) T lymphocytes presented decreased expression of CD28, which is associated with the immunosenescent phenotype. CD14(+) macrophages showed increased expression of CD80 and CD206, suggesting a modulation in the activation of macrophages. These results demonstrate that chronic systemic inflammation observed in obesity induces dysfunctional features in PB...
Background: To investigate the current status of radiotherapy (RT) trends in the definitive treat... more Background: To investigate the current status of radiotherapy (RT) trends in the definitive treatment of lung cancer in Turkey. Methods: A questionnaire consisting of 46 questions about the technical facilities, and indications regarding the definitive radiotherapy of lung cancer was sent to 62 centers in Turkey, and was answered by 47 centers.
Burns are associated with activation of the innate immunity that can contribute to complications.... more Burns are associated with activation of the innate immunity that can contribute to complications. Damage-associated molecular patterns (DAMPs) released after tissue injury play a critical role in the activation of the innate immunity, which appears to be mediated via toll-like receptors (TLRs). Previous findings have shown that TLRs and TLR-mediated responses are up-regulated after burn. Nonetheless, it is unclear what impact burn injury has on circulating levels of DAMPs. To study this, male C57BL/6 mice were subjected to a major burn injury or sham procedure. Three hours to 7 days thereafter, plasma was collected and assayed for the representative DAMPs (i.e., HMGB1, cytochrome C, DNA and S100A) and extracellular cleavage products (fibronectin and hyaluronan). HMGB1, cytochrome C, fibronectin and hyaluronan levels were elevated in a timedependent manner after burn as compared to sham levels. A significant elevation in TNF-α, IL-6 and IL-10 cytokine plasma levels was also found after burn. All cytokine levels were increased as early as 3 hr and remained elevated up to 24 hr. Circulating CD11b + monocytes were increased at 24 hr after burn and showed increased expression of TLR-2. In conclusion, these findings support the concept that burn-induced elevations in circulating DAMPs are in part responsible for monocyte activation and the development of inflammatory complications under such conditions and warrants further investigation.
Diabetes is one of the most prevalent chronic non-communicable diseases and is characterized by h... more Diabetes is one of the most prevalent chronic non-communicable diseases and is characterized by hyperglycemia and increased oxidative stress. These two alterations are also responsible for the main diabetic complications: cardiovascular disease, retinopathy, nephropathy and peripheral neuropathy. Diabetes progression is governed by pancreatic β-cell failure, and recent studies showed that sulforaphane (SFN) might be able to prevent this change, preserving insulin production. Consequently, our goal was to test the effects of SFN on metabolic parameters related to diabetic complications and antioxidant defenses (superoxide dismutase, catalase and sulfhydryl groups) in the pancreas, liver and kidney of non-diabetic and diabetic rats. Male Wistar rats were treated with water or 0.5 mg kg(-1) SFN i.p. for 21 days after diabetes induction. In diabetic animals treated with SFN, the serum levels of total cholesterol, non-HDL cholesterol and triacylglycerols were similar to those of non-diab...
Achyrobichalcone is a biflavonoid recently found in Achyrocline satureioides. This substance has ... more Achyrobichalcone is a biflavonoid recently found in Achyrocline satureioides. This substance has unprecedented chemical structure and occurrence, but resembles other bioactive bichalcones, which have important pharmacological properties, such as anticancer activity. The major challenge for evaluation of the physicochemical and biological properties of this new molecule is the isolation step, which affects the purity and yield of the isolated product. Thus, the objective of this work was to develop a semi-preparative method for achyrobichalcone isolation from Achyrocline satureioides by high-speed countercurrent chromatography. The high-speed countercurrent chromatography separation was achieved in two steps. In the first step, an enriched fraction of achyrobichalcone from the freeze-dried extract was obtained, using the solvent system hexane-ethyl acetate-methanol-water 0.8:1:0.8:1, v/v. The purification of achyrobichalcone from the enriched fractions was achieved by further high-speed countercurrent chromatography fractionation with hexane-ethyl acetate-methanol-water 0.9:0.9:0.8:1, v/v. The final isolated product was obtained using preparative thin layer chromatography and crystallization procedure. A yellow semi-crystalline solid with purity close to 90% was obtained as the final product. The mass recovery of achyrobichalcone isolation was near 67%. The structural identification from spectroscopic and chromatographic techniques confirmed the achyrobichalcone structure. This is the first report of achyrobichalcone isolation on a semi-preparative scale by high-speed countercurrent chromatography. This method afforded achyrobichalcone in good yield and purity for further biopharmaceutical studies.
Oxidative stress is involved in many cancer-related processes; however, current therapeutics are ... more Oxidative stress is involved in many cancer-related processes; however, current therapeutics are unable to benefit from this approach. The lungs have a very exquisite redox environment that may contribute to the frequent and deadly nature of lung cancer. Very few studies specifically address lung large-cell carcinoma (LCC), even though this is one of the major subtypes. Using bioinformatic (in silico) tools, we demonstrated that a more aggressive lung LCC cell line (HOP-92) has an overall increase activity of the human antioxidant gene (HAG) network (P=0.0046) when compared to the less aggressive cell line H-460. Gene set enrichment analysis (GSEA) showed that the expression of metallothioneins (MT), glutathione peroxidase 1 (GPx-1), and catalase (CAT) are responsible for this difference in gene signature. This was validated in vitro, where HOP-92 showed a pro-oxidative imbalance, presenting higher antioxidant enzymes (superoxide dismutase (SOD), CAT, and GPx) activities, lower reduced sulfhydryl groups and antioxidant potential, and higher lipoperoxidation and reactive species production. Also, HAG network is upregulated in lung LCC patients with worst outcome. Finally, the prognostic value of genes enriched in the most aggressive cell line was assessed in this cohort. Isoforms of metallothioneins are associated with bad prognosis, while the thioredoxin-interacting protein (TXNIP) is associated with good prognosis. Thus, redox metabolism can be an important aspect in lung LCC aggressiveness and a possible therapeutic target.
Vitamin A (retinol) exerts a major role in several biological functions. However, it was observed... more Vitamin A (retinol) exerts a major role in several biological functions. However, it was observed that retinol induces oxidative stress on different cellular types. Catalase (EC 1.11.1.6; CAT) is a hydrogen peroxide metabolizing enzyme, and its activity and expression is widely used as an index to measure oxidative stress and perturbations in the cellular redox state. The aim of this study was to investigate the effects of retinol and its major biologically active metabolite, all-trans retinoic acid (RA), on CAT regulation. For this purpose, cultured Sertoli cells (a physiological target of vitamin A) were treated with retinol or RA. Retinol (7 lM, 14 lM) and RA (100 nM, 1 lM) enhanced intracellular reactive species production and increased CAT activity after 24 h of treatment. Retinol increased CAT immunocontent but did not alter CAT mRNA expression, while the increase in CAT activity by RA was not related to alterations in immunocontent or mRNA expression. In vitro incubation of purified CAT with retinol or RA did not alter enzyme activity.
Germline mutations in TP53 are the underlying defect of Li-Fraumeni Syndrome (LFS) and Li-Fraumen... more Germline mutations in TP53 are the underlying defect of Li-Fraumeni Syndrome (LFS) and Li-Fraumeni-like (LFL) Syndrome, autosomal dominant disorders characterized by predisposition to multiple early onset cancers. In Brazil, a variant form of LFS/LFL is commonly detected because of the high prevalence of a founder mutation at codon 337 in TP53 (p.R337H). The p53 protein exerts multiple roles in the regulation of oxidative metabolism and cellular anti-oxidant defense systems. Herein, we analyzed the redox parameters in blood samples from p.R337H mutation carriers (C, n = 17) and non-carriers (NC, n = 17). We identified a significant increase in erythrocyte GPx activity and in plasma carbonyl content,an indicator of protein oxidative damage, in mutation carriers compared to non-carriers (P = 0.048 and P = 0.035, respectively). Mutation carriers also showed a four-fold increase in plasma malondialdehyde levels, indicating increased lipid peroxidation (NC = 40.2060.71, C = 160.560.88, P,0.0001). Finally, carriers showed increased total antioxidant status but a decrease in plasma ascorbic acid content. The observed imbalance could be associated with deregulated cell bioenergetics and/or with increased inflammatory stress, two effects that may result from loss of wild-type p53 function. These findings provide the first evidence that oxidative damage occurs in carriers of a germline TP53 mutation, and these may have important implications regarding our understanding of the mechanisms responsible for germline TP53 p.R337H mutation-associated carcinogenesis.
It is well established that oxidative stress plays a major role in several neurodegenerative cond... more It is well established that oxidative stress plays a major role in several neurodegenerative conditions, like Parkinson disease (PD). Hence, there is an enormous effort for the development of new antioxidants compounds with therapeutic potential for the management of PD, such as synthetic organoselenides molecules. In this study, we selected between nine different synthetic organoselenides the most eligible ones for further neuroprotection assays, using the differentiated human neuroblastoma SH-SY5Y cell line as in vitro model. Neuronal differentiation of exponentially growing human neuroblastoma SH-SY5Y cells was triggered by cultivating cells with DMEM/F12 medium with 1% of fetal bovine serum (FBS) with the combination of 10 lM retinoic acid for 7 days. Differentiated cells were further incubated with different concentrations of nine organoselenides (0.1, 0.3, 3, 10, and 30 lM) for 24 h and cell viability, neurites densities and the immunocontent of neuronal markers were evaluated. Peroxyl radical scavenging potential of each compound was determined with TRAP assay. Three organoselenides tested presented low cytotoxicity and high antioxidant properties. Pre-treatment of cells with those compounds for 24 h lead to a significantly neuroprotection against 6-hydroxydopamine (6-OHDA) toxicity, which were directly related to their antioxidant properties. Neuroprotective activity of all three organoselenides was compared to diphenyl diselenide (PhSe) 2 , the simplest of the diaryl diselenides tested. Our results demonstrate that differentiated human SH-SY5Y cells are suitable cellular model to evaluate neuroprotective/neurotoxic role of compounds, and support further evaluation of selected organoselenium molecules as potential pharmacological and therapeutic drugs in the treatment of PD.
Melanoma is an aggressive cancer with highly metastatic ability. We propose cofilin-1, a key prot... more Melanoma is an aggressive cancer with highly metastatic ability. We propose cofilin-1, a key protein in the regulation of actin dynamics and migration, as a prognostic marker. We determined cofilin-1 levels in a retrospective cohort of patients with melanomas and benign lesions of melanocytes (nevi) by immunohistochemistry. Higher cofilin-1 levels were found in malignant melanoma (MM) with Breslow Index (BI)>2 vs MM with BI<2, melanoma (MIS) and nevi and also in MM with metastasis vs MM without detected metastasis. Kaplan-Meier survival curves were performed, clustering patients according to either the type of melanocytic lesions or cofilin-1 level. Survival curves demonstrated worse prognosis of patients with high vs low cofilin-1 levels. TCGA database analysis of melanoma also showed low survival in patients with upregulated cofilin-1 mRNA vs patients without alteration in CFL1 mRNA expression. As cofilin-1 has a dual function depending on its intracellular localization, we ...
Advanced melanoma is the most aggressive form of skin cancer. It is highly metastatic and dysfunc... more Advanced melanoma is the most aggressive form of skin cancer. It is highly metastatic and dysfunctional in melanogenesis; two processes that are induced by H 2 O 2. This work presents a melanoma cell model with low levels of H 2 O 2 induced by catalase overexpression to study differentiation/dedifferentiation processes. Three clones (A7, C10 and G10) of human A375 amelanotic melanoma cells with quite distinct phenotypes were obtained. These clones faced H 2 O 2 scavenging by two main strategies. One developed by clone G10 where ROS increased. This resulted in G10 migration and metastasis associated with the increased of cofilin-1 and CAP1. The other strategy was observed in clone A7 and C10, where ROS levels were maintained reversing malignant features. Particularly, C10 was not tumorigenic, while A7 reversed the amelanotic phenotype by increasing melanin content and melanocytic differentiation markers. These clones allowed the study of potential differentiation and migration markers and its association with ROS levels in vitro and in vivo, providing a new melanoma model with different degree of malignancy.
Introdução: As espécies reativas de oxigênio estão relacionadas com a patologia de epilepsia. Exi... more Introdução: As espécies reativas de oxigênio estão relacionadas com a patologia de epilepsia. Existem vários modelos que induzem o estado epilétio em ratos, entre estes um dos mais utilizados é o do ácido caínico. Objetivos: Demonstrar uma possível associação entre espécies reativas de oxigênio e o estado epilético induzido por ácido caínico. Metodologia:O estado epilético foi induzido por uma injeção intraperitonial de ácido caínico (12 mg/Kg). Os hipocampos e a córtex foram isolados de ratos 28h, 9 dias, 80 dias e 150 dias após a indução. Os TBARs (indicadores de peroxidação lipídica), as relações lipidio/proteínas e o perfil lipídico por TLC foram determinados. Resultados: Foram observadas alterações no conteúdo de TBARs 24h após a indução,as relações lipídio/proteína estavam alteradas 80 e 150 dias após a indução e foram também observadas modificações no perfíl lipídico por TLC. Conclusões: O modelo do ácido caínico induziu modificações nas vias metabólicas de lipídios.
Trabalhos anteriores de nosso grupo demonstraram que o tratamento com retinol aumenta a produção ... more Trabalhos anteriores de nosso grupo demonstraram que o tratamento com retinol aumenta a produção de radicais livres, provavelmente via Reação de Fenton, causando danos à estrutura do DNA, levando as células a apoptose. Para manter a propagação da Reação de Fenton, uma fonte de elétrons deve estar disponível para regenerar o Fe +3 reduzido, sendo que o NADH é a molécula mais importante no desempenho desta função. O aumento nas concentrações de radicais livres desencadeiam na célula uma série de defesas no intuito de diminuir os danos produzidos. Dentre estas defesas destaca-se a catalase, uma enzima induzida por aumento nos radicais livres derivados do H 2 O 2. Neste trabalho, determinamos a quantidade de NADH intracelular e a atividade da catalase em células de Sertoli tratadas com retinol, além do efeito de baixas doses de etanol, um scavenger de radicais livres, na quantidade de NADH. Utilizamos em nossas culturas células de Sertoli de ratos Wistar de 15 dias tratadas ou não com retinol (7uM) por 24 horas. Para avaliar o efeito do etanol sobre o NADH intracelular foram utilizadas doses crescentes de etanol na diluição do retinol. A quantidade de NADH foi avaliada através da absorbância em espectrofotômetro (340 nm). A atividade da catalase foi medida através da decomposição do H 2 O 2 observada pelo decaimento da absorbância em espectrofotômetro (240 nm). O tratamento com retinol aumentou a concentração de NADH, efeito revertido pelo etanol. A atividade da catalase nas células tratadas foi significativamente maior em relação às células controle. O aumento no NADH intracelular induzido pelo tratamento com retinol provavelmente mantém ativa a Reação de Fenton, aumenta a produção de radicais livres, ativando assim a defesa antioxidante celular.
e21042 Background: Germline mutations in the TP53 gene are the underlying genetic defect of Li-Fr... more e21042 Background: Germline mutations in the TP53 gene are the underlying genetic defect of Li-Fraumeni Syndrome (LFS) and its variant, Li-Fraumeni-like (LFL) Syndrome, autosomal dominant disorders characterized by predisposition to multiple early-onset cancers. More recently, p53 is emerging as an important player in redox metabolism and important enzymes involved in defenses against oxidative stress have shown to be regulated by p53, including glutathione peroxidase 1 (GPX1) and mitochondrial superoxide dismutase 2 (SOD2). The aim of the present study was to investigate the redox profile parameters in blood of p.R337H mutation carriers and non-carriers individuals. Methods: A total of 34 individuals were included in the study and they were divided in two groups: mutation carriers (n=17) and non-carriers (n=17). Antioxidant enzyme activities (SOD, CAT, GPx) and oxidative stress parameters (Protein carbonyl content, Sulfhydryl content and TBARS) were measured in plasma, erythrocytes...
The present study analyzed the quality of bovine ovarian tissue after vitrification in a metal cl... more The present study analyzed the quality of bovine ovarian tissue after vitrification in a metal closed chamber, in terms of putative changes in tissue viability (lactate dehydrogenase -LDH- release), anti-oxidant defenses, and redox parameters caused by cryopreservation. Small and large fragmented bovine ovarian tissue specimens were vitrified in a metal chamber. After rewarming, tissue samples were fixed or cultured for 48 hours. Glutathione (GSH), protein sulfhydryl content, Total Radical Trapping Antioxidant Potential (TRAP), and lactate dehydrogenase were analyzed immediately after rewarming and after tissue culture. No changes in antioxidant parameters or viability of rewarmed tissue samples were found immediately or 48h after vitrification. The method of vitrification in a metal closed chamber used in this study preserved the quality of bovine ovarian tissue. Furthermore, our data showed that the size of the tissue specimens did not affect post-vitrification biochemical viabili...
To evaluate the consequences of plasma from individuals with obesity on parameters associated wit... more To evaluate the consequences of plasma from individuals with obesity on parameters associated with immunosenescence in unrelated healthy peripheral blood mononuclear cells (PBMC). Freshly isolated PBMC were incubated in media supplemented with 10% of plasma from individuals with obesity or control subjects for the first 4 hours of 24 to 120 hours of culture. Plasma from individuals with obesity modulated the phenotype of healthy PBMC, leading to a higher rate of apoptosis, lower amounts of phospho-γH2AX and -p53, and mitochondrial dysfunction. After 120 hours, there was a higher secretion of inflammatory cytokines IL-1β and IL-8. CD8(+) T lymphocytes presented decreased expression of CD28, which is associated with the immunosenescent phenotype. CD14(+) macrophages showed increased expression of CD80 and CD206, suggesting a modulation in the activation of macrophages. These results demonstrate that chronic systemic inflammation observed in obesity induces dysfunctional features in PB...
Background: To investigate the current status of radiotherapy (RT) trends in the definitive treat... more Background: To investigate the current status of radiotherapy (RT) trends in the definitive treatment of lung cancer in Turkey. Methods: A questionnaire consisting of 46 questions about the technical facilities, and indications regarding the definitive radiotherapy of lung cancer was sent to 62 centers in Turkey, and was answered by 47 centers.
Burns are associated with activation of the innate immunity that can contribute to complications.... more Burns are associated with activation of the innate immunity that can contribute to complications. Damage-associated molecular patterns (DAMPs) released after tissue injury play a critical role in the activation of the innate immunity, which appears to be mediated via toll-like receptors (TLRs). Previous findings have shown that TLRs and TLR-mediated responses are up-regulated after burn. Nonetheless, it is unclear what impact burn injury has on circulating levels of DAMPs. To study this, male C57BL/6 mice were subjected to a major burn injury or sham procedure. Three hours to 7 days thereafter, plasma was collected and assayed for the representative DAMPs (i.e., HMGB1, cytochrome C, DNA and S100A) and extracellular cleavage products (fibronectin and hyaluronan). HMGB1, cytochrome C, fibronectin and hyaluronan levels were elevated in a timedependent manner after burn as compared to sham levels. A significant elevation in TNF-α, IL-6 and IL-10 cytokine plasma levels was also found after burn. All cytokine levels were increased as early as 3 hr and remained elevated up to 24 hr. Circulating CD11b + monocytes were increased at 24 hr after burn and showed increased expression of TLR-2. In conclusion, these findings support the concept that burn-induced elevations in circulating DAMPs are in part responsible for monocyte activation and the development of inflammatory complications under such conditions and warrants further investigation.
Diabetes is one of the most prevalent chronic non-communicable diseases and is characterized by h... more Diabetes is one of the most prevalent chronic non-communicable diseases and is characterized by hyperglycemia and increased oxidative stress. These two alterations are also responsible for the main diabetic complications: cardiovascular disease, retinopathy, nephropathy and peripheral neuropathy. Diabetes progression is governed by pancreatic β-cell failure, and recent studies showed that sulforaphane (SFN) might be able to prevent this change, preserving insulin production. Consequently, our goal was to test the effects of SFN on metabolic parameters related to diabetic complications and antioxidant defenses (superoxide dismutase, catalase and sulfhydryl groups) in the pancreas, liver and kidney of non-diabetic and diabetic rats. Male Wistar rats were treated with water or 0.5 mg kg(-1) SFN i.p. for 21 days after diabetes induction. In diabetic animals treated with SFN, the serum levels of total cholesterol, non-HDL cholesterol and triacylglycerols were similar to those of non-diab...
Achyrobichalcone is a biflavonoid recently found in Achyrocline satureioides. This substance has ... more Achyrobichalcone is a biflavonoid recently found in Achyrocline satureioides. This substance has unprecedented chemical structure and occurrence, but resembles other bioactive bichalcones, which have important pharmacological properties, such as anticancer activity. The major challenge for evaluation of the physicochemical and biological properties of this new molecule is the isolation step, which affects the purity and yield of the isolated product. Thus, the objective of this work was to develop a semi-preparative method for achyrobichalcone isolation from Achyrocline satureioides by high-speed countercurrent chromatography. The high-speed countercurrent chromatography separation was achieved in two steps. In the first step, an enriched fraction of achyrobichalcone from the freeze-dried extract was obtained, using the solvent system hexane-ethyl acetate-methanol-water 0.8:1:0.8:1, v/v. The purification of achyrobichalcone from the enriched fractions was achieved by further high-speed countercurrent chromatography fractionation with hexane-ethyl acetate-methanol-water 0.9:0.9:0.8:1, v/v. The final isolated product was obtained using preparative thin layer chromatography and crystallization procedure. A yellow semi-crystalline solid with purity close to 90% was obtained as the final product. The mass recovery of achyrobichalcone isolation was near 67%. The structural identification from spectroscopic and chromatographic techniques confirmed the achyrobichalcone structure. This is the first report of achyrobichalcone isolation on a semi-preparative scale by high-speed countercurrent chromatography. This method afforded achyrobichalcone in good yield and purity for further biopharmaceutical studies.
Oxidative stress is involved in many cancer-related processes; however, current therapeutics are ... more Oxidative stress is involved in many cancer-related processes; however, current therapeutics are unable to benefit from this approach. The lungs have a very exquisite redox environment that may contribute to the frequent and deadly nature of lung cancer. Very few studies specifically address lung large-cell carcinoma (LCC), even though this is one of the major subtypes. Using bioinformatic (in silico) tools, we demonstrated that a more aggressive lung LCC cell line (HOP-92) has an overall increase activity of the human antioxidant gene (HAG) network (P=0.0046) when compared to the less aggressive cell line H-460. Gene set enrichment analysis (GSEA) showed that the expression of metallothioneins (MT), glutathione peroxidase 1 (GPx-1), and catalase (CAT) are responsible for this difference in gene signature. This was validated in vitro, where HOP-92 showed a pro-oxidative imbalance, presenting higher antioxidant enzymes (superoxide dismutase (SOD), CAT, and GPx) activities, lower reduced sulfhydryl groups and antioxidant potential, and higher lipoperoxidation and reactive species production. Also, HAG network is upregulated in lung LCC patients with worst outcome. Finally, the prognostic value of genes enriched in the most aggressive cell line was assessed in this cohort. Isoforms of metallothioneins are associated with bad prognosis, while the thioredoxin-interacting protein (TXNIP) is associated with good prognosis. Thus, redox metabolism can be an important aspect in lung LCC aggressiveness and a possible therapeutic target.
Vitamin A (retinol) exerts a major role in several biological functions. However, it was observed... more Vitamin A (retinol) exerts a major role in several biological functions. However, it was observed that retinol induces oxidative stress on different cellular types. Catalase (EC 1.11.1.6; CAT) is a hydrogen peroxide metabolizing enzyme, and its activity and expression is widely used as an index to measure oxidative stress and perturbations in the cellular redox state. The aim of this study was to investigate the effects of retinol and its major biologically active metabolite, all-trans retinoic acid (RA), on CAT regulation. For this purpose, cultured Sertoli cells (a physiological target of vitamin A) were treated with retinol or RA. Retinol (7 lM, 14 lM) and RA (100 nM, 1 lM) enhanced intracellular reactive species production and increased CAT activity after 24 h of treatment. Retinol increased CAT immunocontent but did not alter CAT mRNA expression, while the increase in CAT activity by RA was not related to alterations in immunocontent or mRNA expression. In vitro incubation of purified CAT with retinol or RA did not alter enzyme activity.
Germline mutations in TP53 are the underlying defect of Li-Fraumeni Syndrome (LFS) and Li-Fraumen... more Germline mutations in TP53 are the underlying defect of Li-Fraumeni Syndrome (LFS) and Li-Fraumeni-like (LFL) Syndrome, autosomal dominant disorders characterized by predisposition to multiple early onset cancers. In Brazil, a variant form of LFS/LFL is commonly detected because of the high prevalence of a founder mutation at codon 337 in TP53 (p.R337H). The p53 protein exerts multiple roles in the regulation of oxidative metabolism and cellular anti-oxidant defense systems. Herein, we analyzed the redox parameters in blood samples from p.R337H mutation carriers (C, n = 17) and non-carriers (NC, n = 17). We identified a significant increase in erythrocyte GPx activity and in plasma carbonyl content,an indicator of protein oxidative damage, in mutation carriers compared to non-carriers (P = 0.048 and P = 0.035, respectively). Mutation carriers also showed a four-fold increase in plasma malondialdehyde levels, indicating increased lipid peroxidation (NC = 40.2060.71, C = 160.560.88, P,0.0001). Finally, carriers showed increased total antioxidant status but a decrease in plasma ascorbic acid content. The observed imbalance could be associated with deregulated cell bioenergetics and/or with increased inflammatory stress, two effects that may result from loss of wild-type p53 function. These findings provide the first evidence that oxidative damage occurs in carriers of a germline TP53 mutation, and these may have important implications regarding our understanding of the mechanisms responsible for germline TP53 p.R337H mutation-associated carcinogenesis.
It is well established that oxidative stress plays a major role in several neurodegenerative cond... more It is well established that oxidative stress plays a major role in several neurodegenerative conditions, like Parkinson disease (PD). Hence, there is an enormous effort for the development of new antioxidants compounds with therapeutic potential for the management of PD, such as synthetic organoselenides molecules. In this study, we selected between nine different synthetic organoselenides the most eligible ones for further neuroprotection assays, using the differentiated human neuroblastoma SH-SY5Y cell line as in vitro model. Neuronal differentiation of exponentially growing human neuroblastoma SH-SY5Y cells was triggered by cultivating cells with DMEM/F12 medium with 1% of fetal bovine serum (FBS) with the combination of 10 lM retinoic acid for 7 days. Differentiated cells were further incubated with different concentrations of nine organoselenides (0.1, 0.3, 3, 10, and 30 lM) for 24 h and cell viability, neurites densities and the immunocontent of neuronal markers were evaluated. Peroxyl radical scavenging potential of each compound was determined with TRAP assay. Three organoselenides tested presented low cytotoxicity and high antioxidant properties. Pre-treatment of cells with those compounds for 24 h lead to a significantly neuroprotection against 6-hydroxydopamine (6-OHDA) toxicity, which were directly related to their antioxidant properties. Neuroprotective activity of all three organoselenides was compared to diphenyl diselenide (PhSe) 2 , the simplest of the diaryl diselenides tested. Our results demonstrate that differentiated human SH-SY5Y cells are suitable cellular model to evaluate neuroprotective/neurotoxic role of compounds, and support further evaluation of selected organoselenium molecules as potential pharmacological and therapeutic drugs in the treatment of PD.
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Papers by Fábio Klamt