Differential Effect of ITE on the Aryl Hydrocarbon Receptor Signaling in Breast Cancer Tissue: A Histopathological Study / Efecto Diferencial de ITE en la Señalización del Receptor de Hidrocarburo de Arilo en Tejido de Cáncer de Mama: Un Estudio Histopatológico

SUMMARY: Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is highly expressed in various types of cancers including breast cancer. However, the role of AhR with its endogenous ligand 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) on the progression of breast cancer remains poorly understood. We aimed to investigate cell proliferation and migration states in breast cancer after activating AhR with the endogenous ligand ITE. Breast cancer tissue was evaluated by cell lines, immunohistochemistry, reverse transcription-polymerase chain reaction, cell proliferation, flow cytometry, migration assays and western blot techniques. We found that AhR was widely expressed in breast cancer tissues and metastasis lymph node tissues, but not in normal tissues. The expression AhR was independent between the age, grades and TNM classifications for breast cancer tissues. ITE treatment significantly induced the activation of AhR in a time-dependent manner in both MCF-7 and T47D breast cancer cell lines. Meanwhile, ITE did not affect the cell migration but significantly suppressed the cell proliferation in estrogen receptor positive (ER+) MCF-7 andT47D cells, which probably attribute to the induction of cell cycle arrest in G1 phase and shortened S phase. Further mechanism study showed that ERK1/2 and AKT signaling were required for the activation of AhR in MCF-7 cells. These data suggest that AhR is a potential new target for treating patients with breast cancer. ITE may be more potentially used for therapeutic intervention for breast cancer with the kind of ER(+).

El receptor de hidrocarburo de arilo (AhR) es un factor de transcripción activado por ligando que se expresa en gran medida en varios tipos de cáncer, incluido el cáncer de mama. Sin embargo, el papel de AhR con su ligando endógeno 2- (1'H-indol-3'-carbonil)-tiazol-4-ácido carboxílico metil éster (ITE) en la progresión del cáncer de mama sigue siendo poco conocido. Nuestro objetivo fue investigar la proliferación celular y los estados de migración en el cáncer de mama después de activar AhR con el ligando endógeno ITE. El tejido de cáncer de mama se evaluó mediante líneas celulares, inmunohistoquímica, reacción en cadena de la polimerasa con transcriptasa inversa, proliferación celular, citometría de flujo, ensayos de migración y técnicas de transferencia Western. Descubrimos que AhR se expresó ampliamente en tejidos de cáncer de mama y en linfonodos con metástasis, pero no en tejidos normales. La expresión AhR fue independiente entre la edad, grados y clasificaciones TNM para tejidos de cáncer de mama. El tratamiento con ITE indujo significativamente la activación de AhR de manera dependiente del tiempo en las líneas celulares de cancer de mama MCF-7 y T47D. Mientras tanto, ITE no afectó la migración celular, pero suprimió significativamente la proliferación celular en células MCF-7 y T47D con receptor de estrógeno positivo (ER+), lo que probablemente se atribuye a la inducción de la detención del ciclo celular en la fase G1 y la fase S acortada. Un estudio adicional del mecanismo mostró que las señales de ERK1/2 y AKT eran necesarias para la activación de AhR en las células MCF-7. Estos datos sugieren que AhR es un nuevo objetivo potencial para el tratamiento de pacientes con cáncer de mama. ITE puede ser utilizado más potencialmente en la intervención terapéutica para el cáncer de mama con el tipo de ER (+).

Characterisation of the in vitro activity of a Nitazoxanide-N-methyl-1H-benzimidazole hybrid molecule against albendazole and nitazoxanide susceptible and resistant strains of Giardia intestinalis and its in vivo giardicidal activity

BACKGROUND It was previously demonstrated that CMC-20, a nitazoxanide and N-methyl-1H-benzimidazole hybrid molecule, had higher in vitro activity against Giardia intestinalis WB strain than metronidazole and albendazole and similar to nitazoxanide. OBJETIVES To evaluate the in vitro activity of CMC-20 against G. intestinalis strains with different susceptibility/resistance to albendazole and nitazoxanide and evaluate its effect on the distribution of parasite cytoskeletal proteins and its in vivo giardicidal activity. METHODS CMC-20 activity was tested against two isolates from patients with chronic and acute giardiasis, an experimentally induced albendazole resistant strain and a nitazoxanide resistant clinical isolate. CMC-20 effect on the distribution of parasite cytoskeletal proteins was analysed by indirect immunofluorescence and its activity was evaluated in a murine model of giardiasis. FINDINGS CMC-20 showed broad activity against susceptible and resistant strains to albendazole and nitaxozanide. It affected the parasite microtubule reservoir and triggered the parasite encystation. In this process, alpha-7.2 giardin co-localised with CWP-1 protein. CMC-20 reduced the infection time and cyst load in feces of G. muris infected mice similar to albendazole. MAIN CONCLUSIONS The in vitro and in vivo giardicidal activity of CMC-20 suggests its potential use in the treatment of giardiasis.

Characterisation of the in vitro activity of a Nitazoxanide-N-methyl-1H-benzimidazole hybrid molecule against albendazole and nitazoxanide susceptible and resistant strains of Giardia intestinalis and its in vivo giardicidal activity

BACKGROUND It was previously demonstrated that CMC-20, a nitazoxanide and N-methyl-1H-benzimidazole hybrid molecule, had higher in vitro activity against Giardia intestinalis WB strain than metronidazole and albendazole and similar to nitazoxanide. OBJETIVES To evaluate the in vitro activity of CMC-20 against G. intestinalis strains with different susceptibility/resistance to albendazole and nitazoxanide and evaluate its effect on the distribution of parasite cytoskeletal proteins and its in vivo giardicidal activity. METHODS CMC-20 activity was tested against two isolates from patients with chronic and acute giardiasis, an experimentally induced albendazole resistant strain and a nitazoxanide resistant clinical isolate. CMC-20 effect on the distribution of parasite cytoskeletal proteins was analysed by indirect immunofluorescence and its activity was evaluated in a murine model of giardiasis. FINDINGS CMC-20 showed broad activity against susceptible and resistant strains to albendazole and nitaxozanide. It affected the parasite microtubule reservoir and triggered the parasite encystation. In this process, alpha-7.2 giardin co-localised with CWP-1 protein. CMC-20 reduced the infection time and cyst load in feces of G. muris infected mice similar to albendazole. MAIN CONCLUSIONS The in vitro and in vivo giardicidal activity of CMC-20 suggests its potential use in the treatment of giardiasis.

Selective COX-2 Inhibitor (Meloxicam) and Tooth-Supporting Bone Quality. A Histomorphometric Study in Rats

Abstract The effects of the non-steroidal anti-inflammatory drugs (NSAIDs) on bone quantity and quality were investigated for years. However, there is lack of information on the impact of NSAIDs on the quality of tooth-supporting alveolar bone in absence of periodontal inflammation. Thus, the aim of this study was to evaluate histometrically the influence of a selective COX-2 NSAID (Meloxicam) on the inter-radicular bone mineral density in rats. Forty-nine adult male Wistar rats were randomly divided into four experimental groups: Subcutaneous injection of 0.9% sterile saline for 15 days (G1; n=12) and 45 days (G2; n=11); and subcutaneous injection of Meloxicam for 15 days (G3; n=13) and 45 days (G4; n=13). Mineral density was histometrically determined in the inter-radicular area of the 1st mandibular molars and data analysis performed by two-way ANOVA (a=5%). Results showed no interaction between time and treatment (p>0.05) and that meloxicam did not affect the alveolar bone density. In contrast, it was found that inter-radicular alveolar bone density increased with time (91.88±3.08% and 92.86±2.38% for groups 15 and 45 days, respectively) (p<0.05). Within the limits of this study, daily administration of a selective COX-2 inhibitor (Meloxicam) did not affect the quality of the inter-radicular alveolar bone in absence of periodontal infection.

Resumo Os efeitos dos fármacos anti-inflamatórios não esteroidais (AINEs) sobre a quantidade e qualidade óssea tem sido investigados ao longo dos anos.Entretanto, há falta de informação sobre o impacto dos AINEs na qualidade do osso alveolar de suporte na ausência de inflamação periodontal. Assim, o objetivo deste estudo foi avaliar, histometricamente, a influência de um AINE seletivo para COX-2 (Meloxicam) na densidade mineral óssea inter-radicular em ratos. Quarenta e nove ratos Wistar, machos e adultos foram divididos aleatoriamente em quatro grupos experimentais: injeções subcutâneas de 0,9% de solução salina estéril por 15 dias (G1, n=12) e 45 dias (G2, n=11); e injeções subcutâneas de Meloxicam por 15 (G3, n=13) e 45 dias (G4, n=13). A densidade mineral foi determinada histometricamente na área inter-radicular dos primeiros molares mandibulares e a análise dos dados realizada por meio de ANOVA (a=5%). Os resultados mostraram nenhuma interação entre tempo e tratamento (p>0,05) e que o meloxicam não afetou a densidade óssea alveolar. Em contraste, foi encontrado que a densidade óssea alveolar inter-radicular aumentou ao longo do tempo (91,88±3,08% e 92,86±2,38% para os grupos 15 e 45 dias, respectivamente) (p<0,05). Dentro dos limites deste estudo, a administração diária de um inibidor seletivo para COX-2 (Meloxicam) não afetou a qualidade do osso alveolar inter-radicular na ausência de infecção periodontal.

Meloxicam as a Radiation-Protective Agent on Mandibles of Irradiated Rats

Abstract This study evaluated the action of ionizing radiation and the possible radioprotective effect of the non-steroidal anti-inflammatory drug meloxicam on the bone physiology of rat mandibles by assessing the alveolar socket healing and bone strength. Forty male Wistar rats were divided in 4 groups (n=10): control (CG), irradiated (IG), meloxicam (MG), meloxicam irradiated (MIG). A dose of 0.2 mg/kg meloxicam was administered to MG and MIG. After this, IG and MIG were irradiated with 15 Gy radiation dose in the mandible. Forty days after the above procedures, the mandibular first molars were extracted and the animals were killed after 15 or 30 days (n=5). Micro-computed tomography and bending test were used to evaluate alveolar socket healing and bone strength, respectively. At 15 days, bone volume, bone volume fraction and trabecular thickness were higher in the CG and MG than in the IG and MIG; and trabecular separation was higher in the IG compared with the others. At 30 days, there was a difference only in trabecular separation, which was higher in IG than in CG and MG, and MIG did not differ from the others. Bone strength was lower in IG compared with CG and MG, and MIG did not differ from the others. In conclusion, the ionizing radiation affected the bone physiology of rat mandibles, delaying the alveolar socket healing and reducing the bone strength. Moreover, the meloxicam had a positive effect on the trabecular separation in alveolar socket healing and on the bone strength.

Resumo Este estudo avaliou a ação da radiação ionizante e o possível efeito radioprotetor do anti-inflamatório não esteroide meloxicam na fisiologia óssea de mandíbulas de rato por meio da análise da reparação alveolar e da resistência óssea. Quarenta ratos Wistar machos foram divididos em 4 grupos (n=10): controle (GC), irradiado (GI), meloxicam (GM), meloxicam irradiado (GMI). Administrou-se uma dose única de 0,2 mg/kg de meloxicam no GM e GMI. Posteriormente, o GI e GMI foram irradiados com dose de 15 Gy na região de mandíbula. Decorridos 40 dias dos procedimentos acima, extraiu-se os primeiros molares inferiores dos animais, que foram mortos após 15 e 30 dias (n=5). Utilizou-se a microtomografia computadorizada e o teste de flexão para avaliação da reparação alveolar e da resistência óssea, respectivamente. Aos 15 dias, o volume ósseo, a fração de volume ósseo e a espessura trabecular foram maiores no GC e GM comparados ao GI e GMI; já a separação trabecular foi maior no GI em relação aos demais. Aos 30 dias, houve diferença apenas na separação trabecular, que foi maior no GI em comparação ao GC e GM, não tendo o GMI diferido dos demais. A resistência óssea no GI foi menor em relação ao GC e GM, não tendo o GMI diferido dos demais. Concluiu-se que a radiação ionizante afetou a fisiologia óssea das mandíbulas de rato, promovendo atraso na reparação alveolar e redução da resistência óssea; além disso, o meloxicam, apresentou efeito positivo na separação trabecular da reparação alveolar e na resistência óssea.

Antimicrobial susceptibility of Brazilian Clostridium difficile strains determined by agar dilution and disk diffusion

Abstract Clostridium difficile is a leading cause of diarrhea in hospitalized patients worldwide. While metronidazole and vancomycin are the most prescribed antibiotics for the treatment of this infection, teicoplanin, tigecycline and nitazoxanide are alternatives drugs. Knowledge on the antibiotic susceptibility profiles is a basic step to differentiate recurrence from treatment failure due to antimicrobial resistance. Because C. difficile antimicrobial susceptibility is largely unknown in Brazil, we aimed to determine the profile of C. difficile strains cultivated from stool samples of inpatients with diarrhea and a positive toxin A/B test using both agar dilution and disk diffusion methods. All 50 strains tested were sensitive to metronidazole according to CLSI and EUCAST breakpoints with an MIC90 value of 2 μg/mL. Nitazoxanide and tigecycline were highly active in vitro against these strains with an MIC90 value of 0.125 μg/mL for both antimicrobials. The MIC90 were 4 μg/mL and 2 μg/mL for vancomycin and teicoplanin, respectively. A resistance rate of 8% was observed for moxifloxacin. Disk diffusion can be used as an alternative to screen for moxifloxacin resistance, nitazoxanide, tigecycline and metronidazole susceptibility, but it cannot be used for testing glycopeptides. Our results suggest that C. difficile strains from São Paulo city, Brazil, are susceptible to metronidazole and have low MIC90 values for most of the current therapeutic options available in Brazil.

Immediate postoperative evaluation of enteric anastomosis after the use of meloxicam in rats

ABSTRACT PURPOSE : To compare ileal anastomoses in the immediate postoperative healing period after meloxicam use. METHODS: Forty two male Wistar rats were randomly divided into two groups of 21, COX and control group. To COX meloxicam in combination with morphine was given in 3 days period. Control group received only morphine during the same period. Each group was divided into three sub-groups of 7, which were euthanized at 5, 10, and 21 days postoperatively. Comparison was based in histological evaluation of collagen type I and III using sirius red, immunohistochemical through vascular endothelial growth factor and matrix metalloproteinase-9. RESULTS: Healing process in scheduled periods did not show significant differences (p>0.05) between the COX and control groups during any of the periods. CONCLUSION: The use of meloxicam in the postoperative period following ileal anastomosis did not affect healing.

Copaiba oil effect on experimental jaw defect in Wistar rats

PURPOSE: To evaluate the effects of copaiba oil on jaw defects repair in Wistar rats treated with bioglass or adipose tissue. METHODS: A jaw defect was randomly created in forty-two rats and filled with bioglass or adipose tissue. The two groups (Gbio and Gcell) were subdivided in three subgroups with seven animals each according to gavage administration: control (distillated water), oil (copaiba oil) and melox (meloxicam). Euthanasia was performed after forty post-operative days. The bone formation was analyzed regarding the histological aspects. RESULTS: The osteoclasts activity was observed only in four subgroups (p=0.78). Regarding the osteoblasts presence, it was very similar between the subgroups, the difference was due to Gcell-melox (p=0.009) that presented less osteoblastic activity. The inflammatory cells were more evident in Gcell-melox subgroup, however, there was no difference in comparison with the other subgroups (p=0.52). Bone formation was observed in all subgroups, just two animals showed no bone formation even after 40 days. More than 50% of bone matrix mineralization was observed in 56% (23 animals) of the analyzed areas. The bone matrix mineralization was not different between subgroups (p=0.60). CONCLUSIONS: The subgroups that received copaiba oil showed bone repair, although not statistically significant in comparison to subgroups treated whit meloxicam or controls. Copaiba oil administered by gavage had no effect on bone repair in this experimental model. .

Reposicionamento de fármacos no câncer de boca: Identificação de possíveis agentes / Drug repositioning for oral cancer: Identifying candidate therapeutic agents

O objetivo deste estudo foi o de identificar compostos seletivamente tóxicos ao carcinoma espinocelular de boca in vitro por meio do reposicionamento de fármacos. Material e Métodos: Por meio de um escaneamento baseado na viabilidade celular de 1.280 fármacos, nós selecionamos três princípios ativos (luteolin, metixene hydrochloride e nitazoxanide) letais às células de câncer de boca SCC-25 e pouco tóxicos às células de queratinócitos cutâneos imortalizados HaCaT. Os fármacos candidatos foram investigados quanto à sua dose- e tempo-resposta bem como comparados e combinados à agentes quimioterápicos padrão por meio do ensaio por colorimetria com brometo de tiazolil azul de tetrazolio (MTT). O impacto dos fármacos na motilidade do SCC-25 e do HaCaT foi verificado pelo ensaio de migração celular e seus mecanismos de ação também foram explorados por meio da verificação dos níveis das proteínas fosforiladas pelo western blotting. Todos os experimentos foram realizados em triplicata e, pelo menos, três vezes independentes. O teste t de student foi utilizado para confrontar as variáveis e nível de significância de 5% foi estabelecido para todos os testes. Resultados: O flavonoide natural luteolin exerceu citotoxicidade potente contra as células de câncer de boca in vitro, apresentando baixa toxicidade ao HaCaT e alta eficiência quando comparado a quimioterápicos como a cisplatina e o AG1478. Do ponto de vista molecular, a luteolin ativou a via de sinalização do dano do DNA e, combinada com um inibidor do Chk, apresentou efeitos potencializados. Além disso, nós demonstramos que a nitazoxanide e o metixene hydrochloride são capazes de destruir células SCC-25 porém não as HaCaT de maneira proporcional à dose e ao tempo de tratamento. As combinações entre os três fármacos hit e com a cisplatina ou o AG1478 potencializaram seus efeitos contra as células malignas. Conclusões: O presente estudo traz a luteolin, o metixene hydrochloride e a nitazoxanide como...

Here we aimed at identifying and reposition approved drugs that could be selectively toxic towards oral squamous cell carcinoma cells. Materials and Methods: Through a cell-based drug screening of 1,280 chemical molecules, we selected 3 compounds (luteolin, metixene hydrochloride, and nitazoxanide) lethal to oral cancer SCC-25 cells, while sparing immortalized keratinocyte HaCaT cells. The drugs were then further challenged for their time- and dose-responses, as well as their comparison and combination to standard chemotherapeutic agents by colorimetric assay 1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan, Thiazolyl blue formazan (MTT). The impact on SCC-25 and HaCaT motility as well as the mode of action of the drugs was then further explored by scratching assay and western blotting, respectively. All the experiments were performed in triplicated and, at least, three independent times. Students t test was performed to verify the differences among the variables and the level of significance was set at 5%. Results: The natural flavonoid luteolin was a potent cytotoxic agent against oral cancer cells in vitro, presenting low toxicity against HaCaT cells and high efficiency as compared to standard-of-care, such as cisplatin and AG1478. From a molecular standpoint, luteolin coopted the DNA-damage pathway and could be efficiently combined with Chk pharmacological inhibitor. Moreover, we demonstrated that nitazoxanide and metixene hydrochloride kill the SCC-25 but not the HaCaT cells in a dose- and time-dependent. The combinations among the three drugs hit and with cisplatin and AG1478 improved their effect against the malignant cells. Conclusions: Luteolin, metixene hydrochloride, and nitazoxanide emerge as strong cytotoxic and/or adjuvant therapy in oral cancer, as these compounds present higher efficiency and lower toxicity against oral cancer cells in vitro than conventional chemotherapeutic agents...

Reposicionamento de fármacos no câncer de boca: Identificação de possíveis agentes / Drug repositioning for oral cancer: Identifying candidate therapeutic agents

Objetivos: O objetivo deste estudo foi o de identificar compostos seletivamente tóxicos ao carcinoma espinocelular de boca in vitro por meio do reposicionamento de fármacos. Material e Métodos: Por meio de um escaneamento baseado na viabilidade celular de 1.280 fármacos, nós selecionamos três princípios ativos (luteolin, metixene hydrochloride e nitazoxanide) letais às células de câncer de boca SCC-25 e pouco tóxicos às células de queratinócitos cutâneos imortalizados HaCaT. Os fármacos candidatos foram investigados quanto à sua dose- e tempo-resposta bem como comparados e combinados à agentes quimioterápicos padrão por meio do ensaio por colorimetria com brometo de tiazolil azul de tetrazolio (MTT). O impacto dos fármacos na motilidade do SCC-25 e do HaCaT foi verificado pelo ensaio de migração celular e seus mecanismos de ação também foram explorados por meio da verificação dos níveis das proteínas fosforiladas pelo western blotting. Todos os experimentos foram realizados em triplicata e, pelo menos, três vezes independentes. O teste t de student foi utilizado para confrontar as variáveis e nível de significância de 5% foi estabelecido para todos os testes. Resultados: O flavonoide natural luteolin exerceu citotoxicidade potente contra as células de câncer de boca in vitro, apresentando baixa toxicidade ao HaCaT e alta eficiência quando comparado a quimioterápicos como a cisplatina e o AG1478. Do ponto de vista molecular, a luteolin ativou a via de sinalização do dano do DNA e, combinada com um inibidor do Chk, apresentou efeitos potencializados. Além disso, nós demonstramos que a nitazoxanide e o metixene hydrochloride são capazes de destruir células SCC-25 porém não as HaCaT de maneira proporcional à dose e ao tempo de tratamento. As combinações entre os três fármacos hit e com a cisplatina ou o AG1478 potencializaram seus efeitos contra as células malignas. Conclusões: O presente estudo traz a luteolin, o metixene hydrochloride e a nitazoxanide como...

Objectives: Here we aimed at identifying and reposition approved drugs that could be selectively toxic towards oral squamous cell carcinoma cells. Materials and Methods: Through a cell-based drug screening of 1,280 chemical molecules, we selected 3 compounds (luteolin, metixene hydrochloride, and nitazoxanide) lethal to oral cancer SCC-25 cells, while sparing immortalized keratinocyte HaCaT cells. The drugs were then further challenged for their time- and dose-responses, as well as their comparison and combination to standard chemotherapeutic agents by colorimetric assay 1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan, Thiazolyl blue formazan (MTT). The impact on SCC-25 and HaCaT motility as well as the mode of action of the drugs was then further explored by scratching assay and western blotting, respectively. All the experiments were performed in triplicated and, at least, three independent times. Students t test was performed to verify the differences among the variables and the level of significance was set at 5%. Results: The natural flavonoid luteolin was a potent cytotoxic agent against oral cancer cells in vitro, presenting low toxicity against HaCaT cells and high efficiency as compared to standard-of-care, such as cisplatin and AG1478. From a molecular standpoint, luteolin coopted the DNA-damage pathway and could be efficiently combined with Chk pharmacological inhibitor. Moreover, we demonstrated that nitazoxanide and metixene hydrochloride kill the SCC-25 but not the HaCaT cells in a dose- and time-dependent. The combinations among the three drugs hit and with cisplatin and AG1478 improved their effect against the malignant cells. Conclusions: Luteolin, metixene hydrochloride, and nitazoxanide emerge as strong cytotoxic and/or adjuvant therapy in oral cancer, as these compounds present higher efficiency and lower toxicity against oral cancer cells in vitro than conventional chemotherapeutic agents.

Compounds used to produce cloned animals are genotoxic and mutagenic in mammalian assays in vitro and in vivo

The compounds 6-dimethylaminopurine and cycloheximide promote the successful production of cloned mammals and have been used in the development of embryos produced by somatic cell nuclear transfer. This study investigated the effects of 6-dimethylaminopurine and cycloheximide in vitro, using the thiazolyl blue tetrazolium bromide colorimetric assay to assess cytotoxicity, the trypan blue exclusion assay to assess cell viability, the comet assay to assess genotoxicity, and the micronucleus test with cytokinesis block to test mutagenicity. In addition, the comet assay and the micronucleus test were also performed on peripheral blood cells of 54 male Swiss mice, 35 g each, to assess the effects of the compounds in vivo. The results indicated that both 6-dimethylaminopurine and cycloheximide, at the concentrations and doses tested, were cytotoxic in vitro and genotoxic and mutagenic in vitro and in vivo, altered the nuclear division index in vitro, but did not diminish cell viability in vitro. Considering that alterations in DNA play important roles in mutagenesis, carcinogenesis, and morphofunctional teratogenesis and reduce embryonic viability, this study indicated that 6-dimethylaminopurine and cycloheximide utilized in the process of mammalian cloning may be responsible for the low embryo viability commonly seen in nuclear transfer after implantation in utero.

Expression and role of miR-34a in bladder cancer.

To investigate the biological function of microRNA-34a (miR-34a) in bladder cancer, the expression of miR-34a was determined using quantitative real-time polymerase chain (qRT-PCR) reaction in 42 cases of bladder cancer. The relationship between the expression of miR-34a and development of bladder cancer was also studied. The mature mimics of miR-34a were chemically synthesized and transiently transfected into human bladder cancer T24 cells. The effects of miR-34a on apoptosis, cell cycle and proliferation in T24 cells were evaluated by flow cytometry and MTT, respectively. The results showed that the low expression rate of miR-34a was correlated with the malignancy and tumor size of bladder cancer. The up-regulation of miR-34a in T24 cells contributed to cell growth and cell cycle arrest, but not caspase-3 pathway. These findings suggest that the relative low expression of miRNA-34a might be involved in the tumorigenesis of bladder cancer.

Mechanisms involved in the cytotoxic effects of berberine on human colon cancer HCT-8 cells

Berberine, a constituent of some traditional Chinese medicinal plants, has been reported to have cytotoxicity effects on different human cancer cell lines. There is no available information about the effects and mechanism of action of berberine on human colon cancer cell line HCT-8. In this paper, the cytotoxicity of berberine on HCT-8 cancer cells was investigated by MTT assay, fluorescence microscopy and flow cytometry analysis. Our data revealed that berberine could significantly inhibit the growth of HCT-8 cells in a dose- and time-dependent manner. Morphology of apoptotic cells was studied with acridine orange/ethidium bromide staining. The concentrations of lactate dehydrogenase and both acid and alkaline phosphatases were significantly increased in cell supernatants after berberine treatment, suggesting cell death. Furthermore, flow cytometry analysis showed that berberine could arrest HCT-8 cells at S phase in a time-dependent manner. To further investigate the apoptotic molecular mechanism, reverse transcription-polymerase chain reaction (RT-PCR) and western blotting methods were used. The up-regulated mRNA and/or protein expressions of Fas, FasL, TNF-a, caspase-3 and down-regulation of pro-caspase-3 suggest that the death receptor pathway may be involved in the apoptotic pathway induced by berberine. Decrease of Bcl-2 and increase of Bax in mRNA and/or protein expressions showed that the Bcl-2 family proteins were involved in berberine-induced apoptosis. We also found that berberine-induced apoptosis was associated with an up-regulated expressions of p53 and prohibitin (PHB), and decreased vimentin expression. These results suggest that berberine can suppress cell growth and reduce cell survival by arresting the cell-cycle and by inducing apoptosis of HCT-8 cells.

Peroxisome proliferator-activated receptor delta agonist attenuates hepatic steatosis by anti-inflammatory mechanism

Although peroxisome proliferator receptor (PPAR)-alpha and PPAR-gamma agonist have been developed as chemical tools to uncover biological roles for the PPARs such as lipid and carbohydrate metabolism, PPAR-delta has not been fully investigated. In this study, we examined the effects of the PPAR-delta agonist GW0742 on fatty liver changes and inflammatory markers. We investigated the effects of PPAR-delta agonist GW0742 on fatty liver changes in OLETF rats. Intrahepatic triglyceride contents and expression of inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and monocyte chemo-attractant protein-1 (MCP-1) and also, PPAR-gamma coactivator (PGC)-1alpha gene were evaluated in liver tissues of OLETF rats and HepG2 cells after GW0742 treatment. The level of TNF-alpha and MCP-1 was also examined in supernatant of Raw264. 7 cell culture. To address the effects of GW0742 on insulin signaling, we performed in vitro study with AML12 mouse hepatocytes. Rats treated with GW0742 (10 mg/kg/day) from 26 to 36 weeks showed improvement in fatty infiltration of the liver. In liver tissues, mRNA expressions of TNF-alpha, MCP-1, and PGC-1alpha were significantly decreased in diabetic rats treated with GW0742 compared to diabetic control rats. We also observed that GW0742 had inhibitory effects on palmitic acid-induced fatty accumulation and inflammatory markers in HepG2 and Raw264.7 cells. The expression level of Akt and IRS-1 was significantly increased by treatment with GW0742. The PPAR-delta agonist may attenuate hepatic fat accumulation through anti-inflammatory mechanism, reducing hepatic PGC-1alpha gene expression, and improvement of insulin signaling.

Aptitud biológica de poblaciones de Bemisia tabaci (Gennadius) Biotipo B (Hemiptera: Aleyrodidae) con diferente susceptibilidad al insecticida thiametoxam / Fitness of Bemisia tabaci (Gennadius) B Biotype (Hemiptera: Aleyrodidae) populations with different levels of susceptibility to the thiametoxam insecticide

In two field-collected populations of de Bemisia tabaci (Gennadius) B biotype previously selected with the neonicotinoid insecticide thiamethoxam (NEO-R, NEO-N), and a population susceptible to insecticides (SUSC), the level of response to the insecticide thiametoxam, as well as the life and fertility tables were determined. At LC95, the NEO-R population showed a resistance ratio (RR95) value of 8.8-fold, an intrinsic rate of increase (r m) of 0.72. The fitness of the NEO-R and NEO-N populations in relation to the SUSC was 1.5 and 2.0, respectively. The RR95 value for NEO-N was 1.9-fold; it's r m value was 0.082, while in the SUSC population was 0.041. The developmental time of egg and adult were longer in NEO-R population, while the nymph and pupa lasted longer in the NEO-N and SUSC populations. The developmental time was different in the three NEO-R, NEO-N, and SUSC populations with 19.7 d (315.84 degree days or dd), 15.7 d (250.4 dd) and 18.5 d (296.6 d), respectively. The populations previously selected with thiamethoxam did not reproduce faster than their susceptible counterpart.

Anti-parasitic action and elimination of intracellular Toxoplasma gondii in the presence of novel thiosemicarbazone and its 4-thiazolidinone derivatives

Toxoplasma, which infects all eukaryotic cells, is considered to be a good system for the study of drug action and of the behavior of infected host cells. In the present study, we asked if thiosemicarbazone derivatives can be effective against tachyzoites and which morphological and ultrastructural features of host cells and parasites are associated with the destruction of Toxoplasma. The compounds were tested in infected Vero cell culture using concentration screens (0.1 to 20 mM). The final concentration of 1 mM was chosen for biological assay. The following results were obtained: 1) These new derivatives decreased T. gondii infection with an in vitro parasite IC50 percent of 0.2-0.7 mM, without a significant effect on host cells and the more efficient compounds were 2, 3 (thiosemicarbazone derivatives) and 4 (thiazolidinone derivative); 2) The main feature observed during parasite elimination was continuous morphological disorganization of the tachyzoite secretory system, progressive organelle vesiculation, and then complete disruption; 3) Ultrastructural assays also revealed that progressive vesiculation in the cytoplasm of treated parasites did not occur in the host cell; 4) Vesiculation inside the parasite resulted in death, but this feature occurred asynchronously in different intracellular tachyzoites; 5) The death and elimination of T. gondii was associated with features such as apoptosis-like stage, acidification and digestion of parasites into parasitophorous vacuoles. Our results suggest that these new chemical compounds are promising for the elimination of intracellular parasites by mainly affecting tachyzoite development at 1 mM concentration for 24 h of treatment.

Pulsed Electromagnetic Field Stimulates Cellular Proliferation in Human Intervertebral Disc Cells

PURPOSE: The purpose of this study is to investigate the mechanism of cellular proliferation of electromagnetic field (EMF) on human intervertebral disc (IVD) cells. MATERIALS AND METHODS: Human IVD cells were cultured three-dimensionally in alginate beads. EMF was exposed to IVD cells with 650Omega, 1.8 millitesla magnetic flux density, 60 Hz sinusoidal wave. Cultures were divided into a control and EMF group. Cytotoxicity, DNA synthesis and proteoglycan synthesis were measured by MTT assay, [3H]-thymidine, and [35S]-sulfate incorporation. To detect phenotypical expression, reverse transcription-polymerase chain reactions (RT-PCR) were performed for aggrecan, collagen type I, and type II mRNA expression. To assess action mechanism of EMF, IVD cells were exposed to EMF with NG-Monomethyl-L-arginine (NMMA) and acetylsalicylic acid (ASA). RESULTS: There was no cytotoxicity in IVD cells with the EMF group in MTT assay. Cellular proliferation was observed in the EMF group (p < 0.05). There was no difference in newly synthesized proteoglycan normalized by DNA synthesis between the EMF group and the control. Cultures with EMF showed no significant change in the expression of aggrecan, type I, and type II collagen mRNA compared to the control group. Cultures with NMMA (blocker of nitric oxide) or ASA (blocker of prostaglandin E2) exposed to EMF demonstrated decreased DNA synthesis compared to control cultures without NMMA or ASA (p < 0.05). CONCLUSION: EMF stimulated DNA synthesis in human IVD cells while no significant effect on proteoglycan synthesis and chondrogenic phenotype expressions. DNA synthesis was partially mediated by nitric oxide and prostaglandin E2. EMF can be utilized to stimulate proliferation of IVD cells, which may provide efficient cell amplification in cell therapy to degenerative disc disease.

The impact of antipsychotic drugs on food intake and body weight and on leptin levels in blood and hypothalamic ob-r leptin receptor expression in wistar rats

OBJECTIVES: The aim of our study was to investigate the impact of typical and atypical antipsychotic drugs on leptin concentration in blood and changes in the receptor expression in the hypothalamus of male Wistar rats. METHODS: From the age of 13 to 18 weeks, three groups of 20 animals were fed an average dose of 3.5 + 0.03 mg/ kg body weight (BW) haloperidol; 30.6 + 0.22 mg/kg BW clozapine; or 14.9 + 0.13 mg/kg BW ziprasidone in ground food pellets containing 15 percent fat. Twenty control animals received no drugs. Blood samples were taken at week 14, 16, and 19. Locomotor activity and exploratory behavior were measured using the alcove test at weeks 15 and 17. The expression of the hypothalamic leptin receptor in rat brains was determined by using a Western blot. RESULTS: Rats medicated with haloperidol and ziprasidone showed a significantly decreased percentage weight gain and food consumption. We observed no differences in the alcove test, but locomotor activity was significantly reduced in the haloperidol group. Except for rats in the clozapine and ziprasidone groups, after 2 weeks of drug application, we found no changes in the leptin blood concentrations among the four groups or animals within each group. Moreover, we did not find specific differences in hypothalamic leptin receptor expression among the groups. CONCLUSION: We concluded that in male Wistar rats during this treatment period, the tested drugs did not act directly on the leptin regulatory system. We recommend further studies using long-term treatment of different rat strains.

Effect of anti-inflammatory agents on the integration of autogenous bone graft and bovine bone devitalized matrix in rats

PURPOSE: To study the repair of bone defect filled with autograft or bovine bone devitalized matrix in rats under anti-inflammatory action. METHODS: Two hundred and forty Wistar rats were distributed to two groups of 120 animals each. A 2mm-diameter defect was created in the femoral diaphysis. Animals of Group I had the bone defect filled with autograft and those of Group II, with bovine bone devitalized matrix. Animals of each group were redistributed to four subgroups according to the intramuscular administration of anti-inflammatory drug or saline solution: A - diclofenac sodium; B - dexamethasone; C - meloxicam; D - saline solution. Evaluation periods were 7, 14 and 30 days. Histological evaluation consisted of quantifying the inflammatory process, the bone neoformation, the collagen formation and the presence of macrophages. RESULTS: Animals of Group I did not show significant difference considering inflammatory reaction. Significant and progressive increase of bone neoformation was observed in both groups. The animals that received meloxicam and autograft showed less collagen formation at 14 and 30 days. The number of macrophages was higher in Group II than in Group I. The animals treated with dexamethasone and saline solution did not show statistically significant difference. CONCLUSIONS: Diclofenac sodium and meloxicam delayed bone graft repair and dexamethasone did not interfere in it.

OBJETIVO: Estudar o reparo do defeito ósseo preenchido com enxerto ósseo autógeno ou matriz óssea bovina desvitalizada sob ação de antiinflamatórios em ratos. MÉTODOS: 240 ratos Wistar, distribuídos em dois grupos de 120 animais. Confeccionou-se defeito de 2 mm de diâmetro na diáfise femoral. Os animais do Grupo I tiveram o defeito ósseo preenchido com enxerto ósseo autógeno e os do Grupo II com matriz óssea bovina desvitalizada. Cada grupo foi redistribuído em quatro subgrupos segundo a administração intramuscular de antiinflamatório ou solução salina: A - diclofenaco de sódio; B - dexametasona; C - meloxicam; D - solução salina. Os períodos de avaliação foram de 7, 14 e 30 dias. A avaliação histológica constou da quantificação do processo inflamatório, osso neoformado, formação de colágeno e macrófagos. RESULTADOS: Os animais do Grupo I não mostraram diferença significante em relação à reação inflamatória. Observou-se aumento significante e progressivo da neoformação óssea nos Grupos I e II. Os animais que receberam meloxicam e enxerto autógeno mostraram menor aporte de colágeno aos 14 e 30 dias de observação. Os macrófagos apresentaram-se em maior quantidade no Grupo II que no Grupo I. Os animais tratados com dexametasona e solução salina não demonstraram diferença estatisticamente significante entre os Grupos I e II. CONCLUSÕES: O diclofenaco de sódio e o meloxicam retardam a reparação do enxerto ósseo. A dexametasona não interfere na reparação do enxerto ósseo.