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BACKGROUND: Despite considerable medical proceedings, cancer is still a leading cause of death. Major problems for tumor therapy are chemoresistance as well as toxic side effects. In recent years, the additional treatment with the antidiabetic drug metformin during chemotherapy showed promising results in some cases. The aim of this study was to develop an in vitro tumor therapy model in order to further investigate the potential of a combined chemotherapy with metformin. METHODS: Cytotoxic effects of a combined treatment on BALB/c fibroblasts were proven by the resazurin assay. Based on the BALB/c cell transformation assay, the BALB/c tumor therapy model was established successfully with four different and widely used chemotherapeutics from different categories. Namely, Doxorubicin as a type-II isomerase inhibitor, Docetaxel as a spindle toxin, Mitomycin C as an alkylating agent and 5-Fluorouracil as an antimetabolite. Moreover, glucose consumption in the medium supernatant was measured and protein expressions were determined by Western Blotting. RESULTS: Initial tests for the combined treatment with metformin indicated unexpected results as metformin could partly mitigate the cytotoxic effects of the chemotherapeutic agents. These results were further confirmed as metformin induced resistance to some of the drugs when applied simultaneously in the tumor therapy model. Mechanistically, an increased glucose consumption was observed in non-transformed cells as well as in the mixed population of malignant transformed cell foci and non-transformed monolayer cells, suggesting that metformin could also increase glucose consumption in transformed cells. CONCLUSION: In conclusion, this study suggests a cautious use of metformin during chemotherapy. Moreover, the BALB/c tumor therapy model offers a potent tool for further mechanistic studies of drug-drug interactions during cancer therapy.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Metformina/farmacologia , Neoplasias/tratamento farmacológico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Células 3T3 BALB , Carcinógenos/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Transformação Celular Neoplásica/induzido quimicamente , Meios de Cultura/metabolismo , Docetaxel/farmacologia , Docetaxel/uso terapêutico , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Interações Medicamentosas , Fluoruracila/farmacologia , Fluoruracila/uso terapêutico , Glucose/metabolismo , Humanos , Metformina/uso terapêutico , Metilcolantreno/toxicidade , Camundongos , Mitomicina/farmacologia , Mitomicina/uso terapêuticoRESUMO
The sirtuin 1/2 inhibitor tenovin-1 activates p53 and may have potential in the management of cancer. Here, we investigated the responsiveness of Ewing's sarcoma cells to tenovin-1. We examined its effects in two Ewing's sarcoma cell lines with different p53 status, i.e. in p53 wild-type and p53 null cells. Effects were assessed by flow cytometric analyses of cell death, mitochondrial membrane depolarization and reactive oxygen species (ROS) generation, by caspase 3/7 activity measurement, by mRNA expression profiling and by immunoblotting. Tenovin-1 elicited caspase-mediated cell death in p53 wild-type cells, but caspase-independent cell death in p53 null cells. Remarkably, it induced a nonlinear concentration response in the latter: low concentrations of tenovin-1 were much more effective than were higher concentrations. Tenovin-1's effects in p53 null cells involved gene expression changes of Bcl-2 family members, mitochondrial membrane depolarization, nuclear translocation of apoptosis-inducing factor, ROS formation and DNA damage; all these effects followed a bell-shaped pattern. In conclusion, our results provide new insights into tenovin-1's mode of action by demonstrating that it can induce different pathways of cell death.
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Acetanilidas/farmacologia , Fator de Indução de Apoptose/metabolismo , Apoptose/efeitos dos fármacos , Sarcoma de Ewing/patologia , Sirtuína 1/antagonistas & inibidores , Sirtuína 2/antagonistas & inibidores , Tioureia/análogos & derivados , Antineoplásicos/farmacologia , Caspases/metabolismo , Linhagem Celular Tumoral , Dano ao DNA , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Sirtuína 1/metabolismo , Sirtuína 2/metabolismo , Tioureia/farmacologia , Proteína Supressora de Tumor p53/metabolismoRESUMO
Friedreich's ataxia is an inherited neurodegenerative disease caused by the reduced expression of the mitochondrially active protein frataxin. We have previously shown that mice with a hepatocyte-specific frataxin knockout (AlbFxn(-/-)) develop multiple hepatic tumors in later life. In the present study, hepatic carbohydrate metabolism in AlbFxn(-/-) mice at an early and late life stage was analyzed. In young (5-week-old) AlbFxn(-/-) mice hepatic ATP, glucose-6-phosphate and glycogen levels were found to be reduced by â¼74, 80 and 88%, respectively, when compared with control animals. This pronounced ATP, G6P and glycogen depletion in the livers of young mice reverted in older animals: while half of the mice die before 30 weeks of age, the other half reaches 17 months of age and exhibits glycogen, G6P and ATP levels similar to those in age-matched controls. A key event in this respect seems to be the up-regulation of GLUT1, the predominant glucose transporter in fetal liver parenchyma, which became evident in AlbFxn(-/-) mice being 5-12 weeks of age. The most significant histological findings in animals being 17 or 22 months of age were the appearance of multiple clear cell, mixed cell and basophilic foci throughout the liver parenchyma as well as the development of hepatocellular adenomas and carcinomas. The hepatocarcinogenic process in AlbFxn(-/-) mice shows remarkable differences regarding carbohydrate metabolism alterations when compared with all other chemically and virally driven liver cancer models described up to now.
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Metabolismo dos Carboidratos , Proteínas de Ligação ao Ferro/genética , Neoplasias Hepáticas Experimentais/metabolismo , Proteínas Mitocondriais/genética , Trifosfato de Adenosina/metabolismo , Animais , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Glucose-6-Fosfato/metabolismo , Glicogênio/metabolismo , Glicogênio Sintase , Quinase 3 da Glicogênio Sintase/metabolismo , Hepatócitos/metabolismo , Proteínas de Ligação ao Ferro/metabolismo , Fígado/enzimologia , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas Experimentais/etiologia , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , FrataxinaRESUMO
AIM: The purpose of this study was to investigate whether a clinical effect of Lactobacillus reuteri in supportive periodontal therapy can already be detected with a minimum dose of one tablet a day and a minimum observation and intake period of 3 months. MATERIALS AND METHODS: 28 patients with stage III and IV periodontitis undergoing periodontal follow-up were randomly divided into two groups receiving a lozenge containing L. reuteri or a placebo preparation daily for 90 days. After 0, 4, 8, and 12 weeks, the parameters bleeding on probing (BoP), plaque control record (PCR), periodontal probing depth (PPD), and clinical attachment level (CAL) were recorded in the test and control groups. RESULTS: The results sed a different effect of L. reuteri on the respective patients. In certain patients, clinical parameters worsened or remained largely unchanged. However, in other patients, there were positive effects on the clinical parameters. In the overall analysis, BoP was the only clinical parameter that was statistically significantly reduced. CONCLUSIONS: The oral administration of one lozenge per day for 3 months with L. reuteri in supportive periodontal therapy might have a positive influence on clinical parameters in supportive periodontal therapy, depending on the individual.
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Cancer cells commonly exhibit increased nonoxidative D-glucose metabolism whereas induction of mitochondrial metabolism may impair malignant growth. We have first used an in silico method called elementary mode analysis to identify inhibition of ALAT (L-alanine aminotransferase) as a putative target to promote mitochondrial metabolism. We then experimentally show that two competitive inhibitors of ALAT, L-cycloserine and ß-chloro-L-alanine, inhibit L-alanine production and impair D-glucose uptake of LLC1 Lewis lung carcinoma cells. The latter inhibition is linked to an initial energy deficit, as quantified by decreased ATP content, which is then followed by an activation of AMP-activated protein kinase and subsequently increased respiration rates and mitochondrial production of reactive oxygen species, culminating in ATP replenishment in ALAT-inhibited LLC1 cells. Moreover, we observe altered phosphorylation of p38 MAPK (mitogen-activated protein kinase 14), ERK (extracellular signal-regulated kinase 1/2), and Rb1 (retinoblastoma 1) proteins, as well as decreased expression of Cdc25a (cell decision cycle 25 homolog A) and Cdk4 (cyclin-dependent kinase 4). Importantly, these sequelae of ALAT inhibition culminate in similarly reduced anchorage-dependent and anchorage-independent growth rates of LLC1 cells, together suggesting that inhibition of ALAT efficiently impairs cancer growth by counteracting the Warburg effect due to compensatory activation of mitochondrial metabolism.
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Alanina Transaminase/antagonistas & inibidores , Biologia Computacional , Inibidores Enzimáticos/farmacologia , Neoplasias Pulmonares/patologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Trifosfato de Adenosina/metabolismo , Adenilato Quinase/metabolismo , Alanina/biossíntese , Alanina Transaminase/metabolismo , Animais , Ligação Competitiva , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Respiração Celular/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/metabolismo , Glucose/metabolismo , Glutamina/metabolismo , Neoplasias Pulmonares/metabolismo , Camundongos , Oxirredução/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Metabolic adaptations can allow cancer cells to survive DNA-damaging chemotherapy. This unmet clinical challenge is a potential vulnerability of cancer. Accordingly, there is an intense search for mechanisms that modulate cell metabolism during anti-tumor therapy. We set out to define how colorectal cancer CRC cells alter their metabolism upon DNA replication stress and whether this provides opportunities to eliminate such cells more efficiently. METHODS: We incubated p53-positive and p53-negative permanent CRC cells and short-term cultured primary CRC cells with the topoisomerase-1 inhibitor irinotecan and other drugs that cause DNA replication stress and consequently DNA damage. We analyzed pro-apoptotic mitochondrial membrane depolarization and cell death with flow cytometry. We evaluated cellular metabolism with immunoblotting of electron transport chain (ETC) complex subunits, analysis of mitochondrial mRNA expression by qPCR, MTT assay, measurements of oxygen consumption and reactive oxygen species (ROS), and metabolic flux analysis with the Seahorse platform. Global metabolic alterations were assessed using targeted mass spectrometric analysis of extra- and intracellular metabolites. RESULTS: Chemotherapeutics that cause DNA replication stress induce metabolic changes in p53-positive and p53-negative CRC cells. Irinotecan enhances glycolysis, oxygen consumption, mitochondrial ETC activation, and ROS production in CRC cells. This is connected to increased levels of electron transport chain complexes involving mitochondrial translation. Mass spectrometric analysis reveals global metabolic adaptations of CRC cells to irinotecan, including the glycolysis, tricarboxylic acid cycle, and pentose phosphate pathways. P53-proficient CRC cells, however, have a more active metabolism upon DNA replication stress than their p53-deficient counterparts. This metabolic switch is a vulnerability of p53-positive cells to irinotecan-induced apoptosis under glucose-restricted conditions. CONCLUSION: Drugs that cause DNA replication stress increase the metabolism of CRC cells. Glucose restriction might improve the effectiveness of classical chemotherapy against p53-positive CRC cells. The topoisomerase-1 inhibitor irinotecan and other chemotherapeutics that cause DNA damage induce metabolic adaptations in colorectal cancer (CRC) cells irrespective of their p53 status. Irinotecan enhances the glycolysis and oxygen consumption in CRC cells to deliver energy and biomolecules necessary for DNA repair and their survival. Compared to p53-deficient cells, p53-proficient CRC cells have a more active metabolism and use their intracellular metabolites more extensively. This metabolic switch creates a vulnerability to chemotherapy under glucose-restricted conditions for p53-positive cells.
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DNA-repair mechanisms enable cells to maintain their genetic information by protecting it from mutations that may cause malignant growth. Recent evidence suggests that specific DNA-repair enzymes contain ISCs (iron-sulfur clusters). The nuclearencoded protein frataxin is essential for the mitochondrial biosynthesis of ISCs. Frataxin deficiency causes a neurodegenerative disorder named Friedreich's ataxia in humans. Various types of cancer occurring at young age are associated with this disease, and hence with frataxin deficiency. Mice carrying a hepatocyte-specific disruption of the frataxin gene develop multiple liver tumours for unresolved reasons. In the present study, we show that frataxin deficiency in murine liver is associated with increased basal levels of oxidative DNA base damage. Accordingly, eukaryotic V79 fibroblasts overexpressing human frataxin show decreased basal levels of these modifications, while prokaryotic Salmonella enterica serotype Typhimurium TA104 strains transformed with human frataxin show decreased mutation rates. The repair rates of oxidative DNA base modifications in V79 cells overexpressing frataxin were significantly higher than in control cells. Lastly, cleavage activity related to the ISC-independent repair enzyme 8-oxoguanine glycosylase was found to be unaltered by frataxin overexpression. These findings indicate that frataxin modulates DNA-repair mechanisms probably due to its impact on ISC-dependent repair proteins, linking mitochondrial dysfunction to DNA repair and tumour initiation.
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Dano ao DNA , Reparo do DNA/genética , Ataxia de Friedreich/genética , Proteínas de Ligação ao Ferro/genética , Animais , Linhagem Celular , Células Cultivadas , DNA Glicosilases/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Ataxia de Friedreich/metabolismo , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Proteínas de Ligação ao Ferro/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Mamíferos/genética , Mamíferos/metabolismo , Camundongos , Camundongos Knockout , Mutação , Estresse Oxidativo , Células Procarióticas/metabolismo , Salmonella enterica/genética , Transfecção , FrataxinaRESUMO
The BALB/c cell transformation assay (BALB-CTA) considers inter- and intra-tumor heterogeneities and affords the possibility of a direct comparison between untransformed and malignant cells. In the present study, we established monoclonal cell lines that originate from the BALB-CTA and mimic heterogeneous tumor cell populations, in order to investigate phenotype-specific effects of the anti-diabetic drug metformin and the short-chain fatty acid butyrate. Growth inhibitory effects were measured with a ViCell XR cell counter. The BALB/c tumor therapy model (BALB-TTM) was performed, and the extracellular glucose level was measured in the medium supernatant. Using a Seahorse Analyzer, the metabolic phenotypes of four selected clones were characterized, and effects on energy metabolism were investigated. Anti-carcinogenic effects and reduced glucose uptake after butyrate application were observed in the BALB-TTM. Metabolic characterization of the cell clones revealed three different phenotypes. Surprisingly, treatment with metformin or butyrate induced opposite metabolic shifts with similar patterns in all cell clones tested. In conclusion, the BALB-TTM is a relevant model for mechanistic cancer research, and the generation of monoclonal cell lines offers a novel possibility to investigate specific drug effects in a heterogeneous tumor cell population. The results indicate that induced alterations in energy metabolism seem to be independent of the original metabolic phenotype.
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Butiratos/farmacologia , Transformação Celular Neoplásica/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Glucose/metabolismo , Metformina/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Células Clonais/citologia , Células Clonais/efeitos dos fármacos , Meios de Cultura/química , Humanos , Camundongos , Modelos Biológicos , FenótipoRESUMO
INTRODUCTION: Ewing's sarcoma is an aggressive childhood malignancy whose outcome has not substantially improved over the last two decades. In this study, combination treatments of the HSP90 inhibitor AUY922 with either the ATR inhibitor VE821 or the ATM inhibitor KU55933 were investigated for their effectiveness in Ewing's sarcoma cells. METHODS: Effects were determined in p53 wild-type and p53 null Ewing's sarcoma cell lines by flow cytometric analyses of cell death, mitochondrial depolarization and cell-cycle distribution as well as fluorescence and transmission electron microscopy. They were molecularly characterized by gene and protein expression profiling, and by quantitative whole proteome analysis. RESULTS: AUY922 alone induced DNA damage, apoptosis and ER stress, while reducing the abundance of DNA repair proteins. The combination of AUY922 with VE821 led to strong apoptosis induction independent of the cellular p53 status, yet based on different molecular mechanisms. p53 wild-type cells activated pro-apoptotic gene transcription and underwent mitochondria-mediated apoptosis, while p53 null cells accumulated higher levels of DNA damage, ER stress and autophagy, eventually leading to apoptosis. Impaired PI3K/AKT/mTOR signaling further contributed to the antineoplastic combination effects of AUY922 and VE821. In contrast, the combination of AUY922 with KU55933 did not produce a cooperative effect. CONCLUSION: Our study reveals that HSP90 and ATR inhibitor combination treatment may be an effective therapeutic approach for Ewing's sarcoma irrespective of the p53 status.
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Late-stage colorectal cancer (CRC) is still a clinically challenging problem. The activity of the tumor suppressor p53 is regulated via post-translational modifications (PTMs). While the relevance of p53 C-terminal acetylation for transcriptional regulation is well defined, it is unknown whether this PTM controls mitochondrially mediated apoptosis directly. We used wild-type p53 or p53-negative human CRC cells, cells with acetylation-defective p53, transformation assays, CRC organoids, and xenograft mouse models to assess how p53 acetylation determines cellular stress responses. The topoisomerase-1 inhibitor irinotecan induces acetylation of several lysine residues within p53. Inhibition of histone deacetylases (HDACs) with the class I HDAC inhibitor entinostat synergistically triggers mitochondrial damage and apoptosis in irinotecan-treated p53-positive CRC cells. This specifically relies on the C-terminal acetylation of p53 by CREB-binding protein/p300 and the presence of C-terminally acetylated p53 in complex with the proapoptotic BCL2 antagonist/killer protein. This control of C-terminal acetylation by HDACs can mechanistically explain why combinations of irinotecan and entinostat represent clinically tractable agents for the therapy of p53-proficient CRC.
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Neoplasias Colorretais , Proteína Supressora de Tumor p53 , Acetilação , Animais , Apoptose , Benzamidas , Neoplasias Colorretais/tratamento farmacológico , Humanos , Irinotecano/farmacologia , Camundongos , Piridinas , Proteína Supressora de Tumor p53/metabolismoRESUMO
We have established an automated soft agar colony formation assay that can be used as a potent tool in experimental tumor therapy studies as well as anticancer compound screening. It allows the direct and simultaneous comparison of the effects of a high number of anticancer compounds on the anchorage-independent growth of a variety of tumor cell lines. By making use of a commercially available automated pipetting system, the user gets results of excellent quality within 1 week and does not need special cell culture practice.
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Ensaios de Seleção de Medicamentos Antitumorais/métodos , Ágar , Linhagem Celular Tumoral , HumanosRESUMO
The synthesis of a new nanogel drug carrier system loaded with the anti-cancer drug doxorubicin (DOX) is presented. Poly(2-oxazoline) (POx) based nanogels from block copolymer micelles were cross-linked and covalently loaded with DOX using pH-sensitive Schiff' base chemistry. DOX loaded POx based nanogels showed a toxicity profile comparable to the free drug, while unloaded drug carriers showed no toxicity. Hemolytic activity and erythrocyte aggregation of the drug delivery system was found to be low and cellular uptake was investigated by flow cytometry and fluorescence microscopy. While the amount of internalized drug was enhanced when incorporated into a nanogel, the release of the drug into the nucleus was delayed. For in vivo investigations the nanogel drug delivery system was combined with a metronomic treatment of DOX. Low doses of free DOX were compared to equivalent DOX loaded nanogels in a xenograft mouse model. Treatment with POx based nanogels revealed a significant tumor growth inhibition and increase in survival time, while pure DOX alone had no effect on tumor progression. The biodistribution was investigated by microscopy of organs of mice and revealed a predominant localization of DOX within tumorous tissue. Thus, the POx based nanogel system revealed a therapeutic efficiency despite the low DOX concentrations and could be a promising strategy to control tumor growth with fewer side effects.
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Selenoprotein H (SELENOH) is supposed to be involved in redox regulation as well as in tumorigenesis. However, its role in healthy and transformed cells of the gastrointestinal tract remains elusive. We analyzed SELENOH expression in cells depending on their selenium supply and differentiation status and found that SELENOH expression was increased in tumor tissue, in undifferentiated epithelial cells from mice and in colorectal cancer lines as compared to more differentiated ones. Knockdown studies in human colorectal cancer cells revealed that repression of SELENOH decreased cellular differentiation and increased proliferation and migration. In addition, SELENOH knockdown cells have a higher competence to form colonies or tumor xenografts. In parallel, they show a faster cell cycle transition. The high levels of SELENOH in tumors as well as in undifferentiated, proliferative cells together with its inhibitory effects on proliferation and G1/S phase transition suggest SELENOH as a key regulator for cell cycle progression and for prevention of uncontrolled proliferation. As SELENOH expression is highly dependent on the selenium status, effects of selenium supplementation on cancer initiation and progression appear to involve SELENOH.
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Adenocarcinoma/metabolismo , Neoplasias Colorretais/metabolismo , Proteínas de Ligação a DNA/metabolismo , Selenoproteínas/metabolismo , Adenocarcinoma/patologia , Animais , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Neoplasias Colorretais/patologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The identification of cancer preventive or therapeutic substances as well as carcinogenic risk assessment of chemicals is nowadays mostly dependent on animal studies. In vitro cell transformation assays mimic different stages of the in vivo neoplastic process and represent an excellent alternative to study carcinogenesis and therapeutic options. In the BALB/c-3T3 two-stage transformation assay cells are chemically transformed by treatment with MCA and TPA, along with the final Giemsa staining of morphological aberrant foci. In addition to the standard method we can show, that it is possible to apply other chemicals in parallel to identify potential preventive or therapeutic substances during the transformation process. Furthermore, we successfully combined the BALB/c cell transformation assay with several endpoint applications for protein analysis (immunoblot, subcellular fractionation and immunofluorescence) or energy parameter measurements (glucose and oxygen consumption) to elucidate cancer mechanisms in more detail. In our opinion the BALB/c cell transformation assay proves to be an excellent model to investigate alterations in key proteins or energy parameters during the different stages of transformation as well as therapeutic substances and their mode of action.
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Células 3T3 BALB/efeitos dos fármacos , Transformação Celular Neoplásica , Técnicas Citológicas/métodos , Animais , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/fisiopatologiaRESUMO
INTRODUCTION: Matrix Metalloproteinase (MMP) -8 plays crucial role in pathogenesis of periodontitis and is also a possible biomarker candidate in peri-implantitis. AIM: The aim of the study was to analyse MMP-8 levels in peri-Implant Sulcus Fluid (PISF) from peri-implantitis affected implants in smoking and non-smoking patients with different periodontal health status of natural teeth before and after peri-implantitis treatment. SETTINGS AND DESIGN: Altogether 29 patients with peri-implantitis were recruited and divided into two study groups (11 with healthy periodontium or gingivitis, i.e. no marginal bone loss, and 18 with chronic periodontitis). MATERIALS AND METHODS: PISF sample from one implant with peri-implantitis from each patient was collected at the baseline and six months after conservative and surgical peri-implantitis treatment, and clinical parameters were registered. Samples were analysed for MMP-8 with dento ELISA method applying a monoclonal antibody. Mucosal cell samples were also analysed for IL-1 gene polymorphism. PISF MMP-8 levels' differences between periodontal diagnosis groups and between smokers' and non-smokers' were analysed. Also, IL-1 polymorphism profiles were compared between study groups. RESULTS: PISF MMP-8 levels were higher at the baseline compared to and after the treatment when all sampled implant sites were analysed together (p = 0.001). MMP-8 levels' distribution was broader in periodontitis patients' PISF samples, and only in periodontitis patients' group levels decreased statistically significantly after the treatment (p = 0.005). Smokers'and non-smokers' PISF MMP-8 was at similar level both at the baseline and after the treatment. No difference between distributions of IL-1 genotypes was found between study groups. CONCLUSION: MMP-8 levels increase in peri-implantitis affected implants both in non-periodontitis and periodontitis patients, but levels still after treatment of the condition reflect intensified host response around implants and indicate challenges of controlling peri-Implantitis with any treatment modality.
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Friedreich ataxia is a neurodegenerative disease caused by a GAA triplet repeat expansion in the first intron of the frataxin gene, which results in reduced expression levels of the corresponding protein. Despite numerous animal and cellular models, therapeutic options that mechanistically address impaired frataxin expression are lacking. Here, we have developed a new mammalian cell model employing the Cre/loxP recombination system to induce a homozygous or heterozygous frataxin knockout in mouse embryonic fibroblasts. Induction of Cre-mediated disruption by tamoxifen was successfully tested on RNA and protein levels. After loss of frataxin protein, cell division, aconitase activity and oxygen consumption rates were found to be decreased, while ROS production was increased in the homozygous state. By contrast, in the heterozygous state no such changes were observed. A time-resolved analysis revealed the loss of aconitase activity as an initial event after induction of complete frataxin deficiency, followed by secondarily elevated ROS production and a late increase in iron content. Initial impairments of oxygen consumption and ATP production were found to be compensated in the late state and seemed to play a minor role in Friedreich ataxia pathophysiology. In conclusion and as predicted from its proposed role in iron sulfur cluster (ISC) biosynthesis, disruption of frataxin primarily causes impaired function of ISC-containing enzymes, whereas other consequences, including elevated ROS production and iron accumulation, appear secondary. These parameters and the robustness of the newly established system may additionally be used for a time-resolved study of pharmacological candidates in a HTS manner.
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The increased cancer mortality of diabetes type 2 patients is most likely an evidence of the tight connection between tumor development and energy metabolism. A major focus of today's research is still the identification of key proteins of both diseases and the development of corresponding inhibitors. In this study we combined the two-stage BALB/c-3T3 cell transformation assay (BALB-CTA) with the IR/IGF-1R inhibitor OSI-906 (linsitinib) and analyzed alterations in protein activity and energy parameters in non-transformed as well as transformed cells. OSI-906 successfully inhibited the phosphorylation of IR/IGF-1R and decreased cell growth in non-transformed cells. In the BALB-CTA, a permanent treatment with OSI-906 reduced cellular transformation dose-dependently, whereas a temporary treatment gave evidence for a preventive effect in the promotion phase. Furthermore, even though several key proteins were affected, it was possible to show that the phosphorylation of GSK3, Erk 1/2 and the S6 protein are not crucial for the cell foci reducing effect of OSI-906. Taken together, the BALB-CTA confirmed results of OSI-906 from animal studies and enhanced the knowledge of its mode of action. Therefore, the BALB-CTA offers the opportunity to analyze alterations in the transformation process more precisely and will be helpful to identify effective cancer treatments.
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Transformação Celular Neoplásica/metabolismo , Insulina/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas de Neoplasias/metabolismo , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/metabolismo , Somatomedinas/metabolismo , Animais , Células 3T3 BALB , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Insulina/genética , Camundongos , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas de Neoplasias/genética , Receptor IGF Tipo 1/genética , Somatomedinas/genéticaRESUMO
OBJECTIVE: The replacement of missing teeth with dental implants has been standard practice in dentistry for many years. The success of dental implants depends on many factors, among which the diagnosis, clinical severity, and treatment of peri-implant diseases play a key role. In this prospective case series, the influence of cumulative treatment modalities on peri-implantitis with and without pus formation on clinical outcome was assessed. METHOD AND MATERIALS: During 2010, 28 patients were referred for peri-implantitis treatment. They presented two different types of peri-implant diseases: peri-implantitis with (17 implants) or without pus formation (33 implants). After microbiologic diagnosis, all patients were treated at baseline with full-mouth scaling and root planing. Two months later, further full-mouth scaling and root planing and additional antimicrobial photodynamic therapy (aPDT) was applied. Four months after baseline, patients with pus formation additionally underwent access flap surgery. Active human matrix metalloproteinase-8 (aMMP-8) levels were measured in eluates before and after all treatment modalities and 7 months after baseline. RESULTS: Clinical parameters (probing depth, bleeding on probing) and aMMP-8-levels improved in both groups after treatment and the final examination. In periimplantitis patients without pus formation, all parameters decreased after full-mouth scaling and root planing and the additional aPDT and no surgery was necessary to improve the parameters. In patients with pus formation, the parameters decreased only after access flap surgery. CONCLUSION: The presence of pus influences the clinical outcome of the treatment of peri-implant diseases. Whereas peri-implantitis cases without pus formation can be successfully managed nonsurgically, peri-implantitis with pus formation can be effectively treated after an additional observation time of 3 months postoperatively only with additional flap surgery.
Assuntos
Peri-Implantite/terapia , Anti-Infecciosos Locais/uso terapêutico , Bacteroides/isolamento & purificação , Clorexidina/análogos & derivados , Clorexidina/uso terapêutico , Protocolos Clínicos , Raspagem Dentária/métodos , Feminino , Seguimentos , Fusobacterium nucleatum/isolamento & purificação , Líquido do Sulco Gengival/enzimologia , Retração Gengival/microbiologia , Retração Gengival/cirurgia , Retração Gengival/terapia , Humanos , Terapia com Luz de Baixa Intensidade/métodos , Masculino , Metaloproteinase 8 da Matriz/análise , Pessoa de Meia-Idade , Peri-Implantite/microbiologia , Peri-Implantite/cirurgia , Perda da Inserção Periodontal/microbiologia , Perda da Inserção Periodontal/cirurgia , Perda da Inserção Periodontal/terapia , Índice Periodontal , Bolsa Periodontal/microbiologia , Bolsa Periodontal/cirurgia , Bolsa Periodontal/terapia , Fotoquimioterapia/métodos , Porphyromonas gingivalis/isolamento & purificação , Estudos Prospectivos , Aplainamento Radicular/métodos , Supuração , Retalhos Cirúrgicos/cirurgia , Resultado do Tratamento , Treponema denticola/isolamento & purificaçãoRESUMO
Cardiac failure is the most prevalent cause of death at higher age, and is commonly associated with impaired energy homeostasis in the heart. Mitochondrial metabolism appears critical to sustain cardiac function to counteract aging. In this study, we generated mice transgenically over-expressing the mitochondrial protein frataxin, which promotes mitochondrial energy conversion by controlling iron-sulfur-cluster biogenesis and hereby mitochondrial electron flux. Hearts of transgenic mice displayed increased mitochondrial energy metabolism and induced stress defense mechanisms, while overall oxidative stress was decreased. Following standardized exposure to doxorubicin to induce experimental cardiomyopathy, cardiac function and survival was significantly improved in the transgenic mice. The insulin/IGF-1 signaling cascade is an important pathway that regulates survival following cytotoxic stress through the downstream targets protein kinase B, Akt, and glycogen synthase kinase 3. Activation of this cascade is markedly inhibited in the hearts of wild-type mice following induction of cardiomyopathy. By contrast, transgenic overexpression of frataxin rescues impaired insulin/IGF-1 signaling and provides a mechanism to explain enhanced cardiac stress resistance in transgenic mice. Taken together, these findings suggest that increased mitochondrial metabolism elicits an adaptive response due to mildly increased oxidative stress as a consequence of increased oxidative energy conversion, previously named mitohormesis. This in turn activates protective mechanisms which counteract cardiotoxic stress and promote survival in states of experimental cardiomyopathy. Thus, induction of mitochondrial metabolism may be considered part of a generally protective mechanism to prevent cardiomyopathy and cardiac failure.
Assuntos
Metabolismo Energético , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/prevenção & controle , Mitocôndrias/metabolismo , Animais , Antibióticos Antineoplásicos/efeitos adversos , Modelos Animais de Doenças , Doxorrubicina/efeitos adversos , Insuficiência Cardíaca/induzido quimicamente , Hemodinâmica , Humanos , Insulina/metabolismo , Proteínas de Ligação ao Ferro/metabolismo , Camundongos , Camundongos Transgênicos , Transdução de Sinais/fisiologia , FrataxinaRESUMO
The aim of the study was to establish a 96-well microtiter plate-based reporter gene assay to test the influence of natural compounds on the promoter activities of rat catalase, human glutathione peroxidase and human superoxide dismutase expressed in V79 cells. Luciferase expression vectors with the promoter regions of the genes coding for the three above-mentioned enzymes were constructed and transfected into V79 cells. Thereafter the ability of sodium ascorbate, L-carnitine, catechin, epigallocatechin gallate, genistein, paraquat, quercetin, 12-O-tetradecanoylphorbol-13-acetate and Trolox to enhance the promoter activities was evaluated. Genistein, paraquat and quercetin led to a statistically significant increase in the glutathione peroxidase and superoxide dismutase gene promoter activities. None of the compounds tested enhanced the catalase gene promoter activity. The reporter gene assay described in this report is easy to perform, fast and allows one to test a high number of compounds and different concentrations of a single compound at the same time.