Cytotoxicity evaluation of haloperidol, clozapine and a new molecule with antipsychotic potential, PT-31, in NIH-3T3 cells

Abstract Schizophrenia is an illness that affects 26 million people worldwide. However, conventional antipsychotics present side effects and toxicity, highlighting the need for new antipsychotics. We aimed to evaluate the cytotoxicity of haloperidol (HAL), clozapine (CLO), and a new molecule with antipsychotic potential, PT-31, in NIH-3T3 cells. The neutral red uptake assay and the MTT assay were performed to evaluate cell viability and mitochondrial activity, morphological changes were assessed, and intracellular reactive oxygen species (ROS) detection was performed. HAL and CLO (0.1 µM) showed a decrease in cell viability in the neutral red uptake assay and in the MTT assay. In addition, cell detachment, content decrease, rounding and cell death were also observed at 0.1 µM for both antipsychotics. An increase in ROS was observed for HAL (0.001, 0.01 and 1 µM) and CLO (0.01 and 1 µM). PT-31 did not alter cell viability in any of the assays, although it increased ROS at 0.01 and 1 µM. HAL and CLO present cytotoxicity at 0.1 µM, possibly through apoptosis and necrosis. In contrast, PT-31 does not present cytotoxicity to NIH-3T3 cells. Further studies must be performed for a better understanding of these mechanisms and the potential risk of conventional antipsychotics

PEG-PCL Nanocapsules Containing Curcumin: Validation of HPLC Method for Analyzing Drug-loading Efficiency, Stability Testing and Cytotoxicity on NIH-3T3 Cell Line

Abstract Curcumin (CUR) shows potential use for treating cancer. However, CUR has low solubility and reduced bioavailability, which limit its clinical effect. Therefore, the development of nanocarriers can overcome these problems and can ensure the desired pharmacological effect. In addition, it is mandatory to prove the quality, the efficacy, and the safety for a novel nanomedicine to be approved. In that sense, this paper aimed (a) to prepare CUR-loaded polyethylene glycol-poly(ε-caprolactone) nanocapsules; (b) to validate an analytical method by high performance liquid chromatography (HPLC) for quantifying CUR in these nanoformulations; (c) to evaluate the physicochemical stability of these formulations; and to investigate their cytotoxicity on NIH-3T3 mouse fibroblast cells. The HPLC method was specific to CUR in the loaded nanocapsules, linear (r = 0.9994) in a range of 10.0 to 90.0 µg.mL-1 with limits of detection and quantification of 0.160 and 0.480 µg.mL-1, respectively. Precision was demonstrated by a relative standard deviation lower than 5%. Suitable accuracy (102.37 ± 0.92%) was obtained. Values of pH, particle size, polydispersity index, and zeta potential presented no statistical difference (p > 0.05) for CUR-loaded nanoparticles. No cytotoxicity was observed against NIH-3T3 mouse embryo fibroblast cell line using both the tetrazolium salt and sulforhodamine B assays. In conclusion, a simple and inexpensive HPLC method was validated for the CUR quantification in the suspensions of nanocapsules. The obtained polymeric nanocapsules containing CUR showed suitable results for all the performed assays and can be further investigated as a feasible novel approach for cancer treatment.

Biological efficacy of perpendicular type-I collagen protruded from TiO / 国际口腔科学杂志·英文版

The aim of this study was to evaluate the biological efficacy of a unique perpendicular protrusion of type-I collagen (Col-I) from TiO

Efeitos citotóxicos da duloxetina nas linhagens celulares MKN45 e NIH3T3 e efeitos genotóxicos em linfócitos sanguíneos periféricos humanos / Cytotoxic effects of duloxetine on mkn45 and nih3t3 cell lines and genotoxic effects on human peripheral blood lymphocytes

ABSTRACT BACKGROUND: Gastric cancer is the second leading cause of cancer-related death globally. Unfortunately, the survival rate of the gastric cancer patients who underwent chemotherapy following surgery has been less than a half. Besides, chemotherapy has many side effects. Current evidence suggests that some antidepressants like duloxetine have growth-inhibiting effects against a number of cancer cell lines. OBJECTIVE: Thus, the aim of this study was to determine the cytotoxic and genotoxic effects of duloxetine on gastric cancer. METHODS: In this regard, the cytotoxicity and genotoxicity of duloxetine were investigated in MKN45 and NIH3T3 cell lines by MTT assay and on peripheral blood lymphocytes by MN assay. For this purpose, cells were cultured in 96 wells plate. Stock solutions of duloxetine and cisplatin were prepared. After cell incubation with different concentrations of duloxetine (1, 10, 25, 50, 100 and 200 μL), MTT solution was added. For micronucleus assay fresh blood was added to RPMI culture medium 1640 supplemented, and different concentrations of duloxetine (1, 10, 25, 50, 100 and 200 μL) were added. RESULTS: The cytotoxicity of duloxetine on MKN45 cancer cell line and NIH3T3 normal cell line were studied followed by MTT assay. duloxetine exhibited higher IC50 in the MKN45 cells in comparison with the NIH3T3 cells. In addition, genotoxic effect of duloxetine was evaluated by micronucleus assay. The results revealed that duloxetine induced more DNA damage at 100 and 200 μM and no significant difference at 200 μM with respect to cisplatin, but it had less genotoxic effects at 100 and 50 μM concentrations. CONCLUSION: Although, in this study, duloxetine had less genotoxicity than cisplatin in concentrations under 200 μM and showed cytotoxic effects as well, due to its IC50, it cannot be considered as a better choice for gastric cancer therapies with respect to cisplatin as a common anticancer drug.

RESUMO CONTEXTO: O câncer gástrico é a segunda principal causa de morte relacionada ao câncer globalmente. Infelizmente, a taxa de sobrevivência dos pacientes com câncer gástrico que se submeteram à quimioterapia após a cirurgia, tem sido inferior à metade. Além disso, a quimioterapia tem muitos efeitos colaterais. Evidências atuais sugerem que alguns antidepressivos como a duloxetina têm efeitos inibidores de crescimento contra um número de linhas de células cancerosas. OBJETIVO: Assim, o objetivo deste estudo foi determinar os efeitos citotóxicos e genotóxicos da duloxetina sobre o câncer gástrico. MÉTODOS: A este respeito, a citotoxicidade e a genotoxicidade da duloxetina foram investigadas em linhas celulares MKN45 e NIH3T3 por ensaio de MTT e por ensaio de MN em linfócitos periféricos de sangue. Para este efeito, as células foram cultivadas em 96 placas. Soluções de estoque de duloxetina e cisplatina foram preparadas. Após incubação celular com diferentes concentrações de duloxetina (1, 10, 25, 50, 100 e 200 μL), a solução de MTT foi adicionada. Para o teste do micronúcleo o sangue fresco foi adicionado ao meio de cultura RPMI 1640 suplementado, e as concentrações diferentes de duloxetina (1, 10, 25, 50, 100 e 200 μL) foram adicionadas. RESULTADOS: A citotoxicidade da duloxetina na linha celular cancerosa MKN45 e NIH3T3 linha celular normal foram estudadas e seguidas pelo ensaio de MTT. A duloxetina exibiu maior IC50 nas células MKN45 em comparação com as células NIH3T3. Além disso, o efeito genotóxico da duloxetina foi avaliado pelo ensaio de micronúcleos. Os resultados revelaram que a duloxetina induziu mais dano de DNA em 100 e 200 μM e não houve diferença significativa em 200 μM em relação à cisplatina, mas teve menos efeitos genotóxicos nas concentrações de 100 e 50 μM. CONCLUSÃO: Embora, neste estudo, a duloxetina tenha menos genotoxicidade do que a cisplatina em concentrações inferiores a 200 μm e também tenha mostrado efeitos citotóxicos, devido ao seu IC50, não pode ser considerada como uma escolha terapêutica melhor para o câncer gástrico no que diz respeito à cisplatina como uma droga anticâncer comum.

Efeitos citotóxicos do aripiprazol nas linhagens celulares MKN45 e NIH3T3 e efeitos genotóxicos nos linfócitos sanguíneos periféricos humanos / Cytotoxic effects of aripiprazole on mkn45 and nih3t3 cell lines and genotoxic effects on human peripheral blood lymphocytes

ABSTRACT BACKGROUND: Gastric cancer is known as the fourth most common cancer. Current treatments for cancer have damaged the sensitive tissues of the healthy body, and in many cases, cancer will be recurrent. Therefore, need for treatments that are more effective is well felt. Researchers have recently shifted their attention towards antipsychotic dopamine antagonists to treat cancer. The anticancer activities of aripiprazole remain unknown. OBJECTIVE: This study aimed to evaluate the efficacy and safety of aripiprazole on gastric cancer and normal cell lines. METHODS: In this regard, the cytotoxicity and genotoxicity of aripiprazole were investigated in MKN45 and NIH3T3 cell lines by methyl tetrazolium assay and on peripheral blood lymphocytes by micronucleus assay. For this purpose, cells were cultured in 96 wells plate. Stock solutions of aripiprazole and cisplatin were prepared. After cell incubation with different concentrations of aripiprazole (1, 10, 25, 50, 100 and 200 μL), methyl tetrazolium solution was added. For micronucleus assay fresh blood was added to RPMI culture medium 1640 supplemented, and different concentrations of aripiprazole (50, 100 and 200 μL) were added. RESULTS: The finding of present study showed that the IC50 of aripiprazole in the cancer cell line (21.36 μg/mL) was lower than that in the normal cell line (54.17 μg/mL). Moreover, the micronucleus assay showed that the frequency of micronuclei of aripiprazole at concentrations below 200 μM was much less than cisplatin. CONCLUSION: Aripiprazole can be a good cytotoxic compound and good candidate for further studies of cancer therapy.

RESUMO CONTEXTO: O câncer gástrico é conhecido como o quarto câncer mais comum. Os tratamentos atuais para o câncer danificaram os tecidos sensíveis do corpo saudável e, em muitos casos, o cancro será recorrente. Portanto, a necessidade de tratamentos que são mais eficazes é desejada. Recentemente, os pesquisadores mudaram sua atenção para os antagonistas antipsicóticos da dopamina para tratar o câncer. As atividades anticâncer de aripiprazol permanecem desconhecidas. OBJETIVO: Este estudo objetivou avaliar a eficácia e a segurança do aripiprazol no câncer gástrico e nas linhagens celulares normais. MÉTODOS: A este respeito, a citotoxicidade e a genotoxicidade do aripiprazol foram investigadas em linhas celulares MKN45 e NIH3T3 por ensaio de metil tetrazólio e em linfócitos periféricos de sangue por ensaio de micronúcleos. Para este efeito, as células foram cultivadas em 96 placas. As soluções de estoque de aripiprazol e cisplatina foram preparadas. Após incubação celular com diferentes concentrações de aripiprazol (1, 10, 25, 50, 100 e 200 μL), a solução de metil tetrazólio foi adicionada. Para o ensaio do micronúcleo o sangue fresco foi adicionado ao meio de cultura RPMI 1640 suplementado, e as concentrações diferentes de aripiprazole (50, 100 e 200 μL) foram adicionadas. RESULTADOS: O presente estudo mostrou que o IC50 de aripiprazol na linhagem celular cancerosa (21,36 μg/mL) foi menor do que na linha celular normal (54,17 μg/ mL). Além disso, o ensaio de micronúcleos demonstrou que a frequência de micronúcleos de aripiprazol em concentrações inferiores a 200 μM foi muito inferior à cisplatina. CONCLUSÃO: O aripiprazol pode ser um bom composto citotóxico e bom candidato para estudos adicionais da terapia do câncer.

A novel application of nano eggshell/titanium dioxide composite on occluding dentine tubules: an in vitro study

Abstract To synthesize Nano eggshell-titanium-dioxide (EB@TiO2) biocomposite and to evaluate its effectiveness in occluding opened dentine tubules. EB@TiO2 was synthesized and characterized using X-ray diffraction (XRD), and Transmission Electron Microscope (TEM). Sixteen simulated bovine dentine discs were prepared and randomly assigned into four groups according to the following treatment (n = 4): Group 1: No treatment; Group 2: eggshell powder; Group 3: EB@TiO2; Group 4: Sensodyne. These were then agitated in a solution of 1g powder and 40mL water for 3hours. Thereafter, each dentine discs from the respective groups were post-treated for 5 min with 2wt% citric acid to test their acid resistant characteristics. Scanning Electron Microscope (SEM) was used to observe the effectiveness of occluded dentine pre-and post-treatment. The cytotoxicity of the synthesized EB@TiO2 was tested using NIH 3T3 assay. ANOVA was used to evaluate the mean values of the occluded area ratio and the data of MTS assay. This was followed by a multi-comparison test with Bonferroni correction (α = .05). The XRD confirmed that EB@TiO2 was successfully modified through ball-milling. The TEM revealed the presence of both spherical and irregular particle shape powders. The SEM result showed that EB@TiO2 could effectively occlude open dentine tubules. Equally, the result demonstrated that EB@TiO2 exhibited the highest acid resistant stability post-treatment. NIH 3T3 assay identified that EB@TiO2 had little effect on the NIH 3T3 cell line even at the highest concentration of 100µg/ml. This study suggests that the application of EB@TiO2 effectively occluded dentine tubules and the occlusion showed a high acid resistant stability.

Histological Method to Study the Effect of Shear Stress on Cell Proliferation and Tissue Morphology in a Bioreactor

BACKGROUND: Tissue engineering represents a promising approach for the production of bone substitutes. The use of perfusion bioreactors for the culture of bone-forming cells on a three-dimensional porous scaffold resolves mass transport limitations and provides mechanical stimuli. Despite the recent and important development of bioreactors for tissue engineering, the underlying mechanisms leading to the production of bone substitutes remain poorly understood. METHODS: In order to study cell proliferation in a perfusion bioreactor, we propose a simplified experimental set-up using an impermeable scaffold model made of 2 mm diameter glass beads on which mechanosensitive cells, NIH-3T3 fibroblasts are cultured for up to 3 weeks under 10 mL/min culture medium flow. A methodology combining histological procedure, image analysis and analytical calculations allows the description and quantification of cell proliferation and tissue production in relation to the mean wall shear stress within the bioreactor. RESULTS: Results show a massive expansion of the cell phase after 3 weeks in bioreactor compared to static control. A scenario of cell proliferation within the three-dimensional bioreactor porosity over the 3 weeks of culture is proposed pointing out the essential role of the contact points between adjacent beads. Calculations indicate that the mean wall shear stress experienced by the cells changes with culture time, from about 50 mPa at the beginning of the experiment to about 100 mPa after 3 weeks. CONCLUSION: We anticipate that our results will help the development and calibration of predictive models, which rely on estimates and morphological description of cell proliferation under shear stress.

Effect of titanium tetrafluoride varnish on fibroblasts (NIH/3T3): viability assay, morphological analysis and cell signals for apoptotic pathways / Efeito do verniz de tetrafluoreto de titânio sobre fibroblastos (NIH/3T3): ensaios de viabilidade, morfologia e sinalização celular para apoptose

Current knowledge supports the application of TiF4 varnish to protect against tooth caries and erosion; however, it is indispensable to know its cytotoxic potential and the mechanism involved on it before applying in patients. Therefore, this study aimed to evaluate 1) The cytotoxic effect of titanium tetrafluoride (TiF4) varnish compared with sodium fluoride (NaF) varnish on murine fibroblast (NIH/3T3), varying the fluoride concentration and time of treatment and 2) The percentage of apoptosis and its mechanism (both mitochondrial mediated by the Bcl-2 family- and death receptorpathways) in human gingival fibroblasts (HGF) and murine fibroblasts (NIH/3T3) treated with TiF4 varnish compared to NaF varnish for 6 h. Step 1) NIH/3T3 were exposed to NaF or TiF4 varnishes containing 0.95, 1.95 or 2.45% F, for 6, 12 or 24 h. MTT viability (n=6) and Hoescht/PI stain assays (n=3) as well as the cells morphology (HE, only for 24 h, n=3) and stiffness (AFM, only for 2.45% F, 6 or 12 h) were analyzed. Both varnishes, at 1.90 and 2.45% F, reduced cells viability by similar extent (33-86% at 6 h, 35-93% at 12 h, and 87-98% at 24 h) compared to control, regardless of the type of fluoride. TiF4 and NaF (2.45% F) reduced cell stiffness to a similar extent, but only TiF4 differed from control. Step 2) HGF and NIH/3T3 were exposed to NaF or TiF4 (2.45% F) varnishes for 6 h. Cells were examined by the TUNEL method using fluorescence microscope. The caspases-3, -8 and -9 activities were assessed. The cDNA for cytocrome c, Bax, Bad, Bcl-2, VDAC-1 and Fas-L was amplified by quantitative PCR (qPCR). Bax, Bcl-2 and Fas-L were further detected by western blot. Both fluorides similarly increased the percentage of apoptosis, while they failed in activating caspases-3, -8 and -9 for both types of cells. Bax/Bcl-2 ratio, cytochrome C and VDAC-1 gene expressions were not altered by both fluoride treatments. However, NaF varnish increased the amplification of Fas-L gene for NIH/3T3 and HGF, while TiF4 varnish induced lower Bad/Bcl-2 ratio expression compared to control for NIH/3T3, but not for HGF. No effect of the fluorides was detected in the proteins analysis. TiF4 and NaF have similar cytotoxicity on NIH/3T3, which is dependent on the F concentration and the exposure time. Both fluorides, at the studied conditions, similarly induce a low percentage of apoptosis, with consequent modest activation of Bcl-2 and Fas-L-dependent signaling pathways.(AU)

Conhecimento atual suporta a aplicação de verniz de TiF4 para proteção contra cárie e erosão dentárias; entretanto, é indispensável conhecer o seu potencial citotóxico e o mecanismo envolvido antes de aplicá-lo em pacientes. Portanto, o objetivo deste estudo foi avaliar 1) o efeito citotóxico do verniz de tetrafluoreto de Titânio (TiF4) comparado ao fluoreto de sódio (NaF), em fibroblastos NIH/3T3, variando a concentração de fluoreto e o tempo de tratamento 2) a porcentagem de apoptose e seus mecanismos (ambos mitocondrial mediado pela família Bcl-2 e pelo receptor de morte celular) em fibroblastos gengivais humanos (FGH) e fibroblastos murinos (NIH/3T3) tratados com verniz de TiF4 comparado com verniz de NaF por 6 h. Etapa 1) NIH/3T3 foram expostos a vernizes de NaF e TiF4 contendo 0,95, 1,95 ou 2,45% F, por 6, 12 ou 24 h. Ensaios de viabilidade por MTT (n=6) e Hoechst 33342/iodeto de propídeo (n=3) bem como a morfologia (HE, apenas para 24 h, n = 3) e a rigidez celular (MFA, apenas para 2,45% F, 6 ou 12 h) foram realizados. Ambos os vernizes com 1,90 e 2,45% F reduziram a viabilidade das células de forma semelhante (33-86% em 6 h, 35-93% em 12 h e 87-98% em 24 h) em comparação com o controle, independentemente do tipo de fluoreto. TiF4 e NaF (2,45%) reduziram de forma similar a rigidez celular, mas somente TiF4 diferiu do controle no período de 6 h. Etapa 2) FGH e NIH/3T3 foram tratadas com verniz de NaF ou TiF4 por 6h. As células foram examinadas pelo método de TUNEL, usando microscopia de fluorescência. A atividade das caspases -3, -8 e -9 foram avaliadas. O cDNA para citocromo C, Bax, Bad, Bcl-2, VDAC-1 e Fas-L foi amplificado e quantificado por PCR em tempo real (qPCR). A expressão das proteínas Bax, Bcl-2 e Fas-L foi quantificada por western blot. Ambos os fluoretos aumentaram de forma semelhante a porcentagem de apoptose, enquanto falharam na ativação de caspases-3, -8 e -9 para ambos tipos celulares. A expressão gênica da relação Bax/Bcl-2, do citocromo C e do VDAC-1 não foram alteradas por ambos fluoretos. No entanto, o verniz NaF aumentou a amplificação do gene Fas-L para ambas as células, enquanto que o verniz TiF4 induziu menor expressão da razão Bad/Bcl-2 em comparação com o controle para NIH/3T3, mas não para FGH. Nenhum efeito foi detectado na análise de proteínas. TiF4 e NaF apresentam citotoxicidade similar em NIH/3T3, a qual é dependente da concentração de F e do tempo de exposição. Ambos os fluoretos, nas condições estudadas, induzem uma baixa porcentagem de apoptose, com consequente modesta ativação das vias de sinalização dependentes de Bcl-2 e Fas-L.(AU)

Extracellular matrix and fibroblast injection produces pterygium-like lesion in rabbits

BACKGROUND: Translational research to develop pharmaceutical and surgical treatments for pterygium requires a reliable and easy to produce animal model. Extracellular matrix and fibroblast are important components of pterygium. The aim of this study was to analyze the effect of the subconjunctival injection of fibroblast cells (NIH3T3 cell line) and exogenous extracellular matrix in rabbits in producing a pterygium-like lesion. METHODS: Six 3-month-old white New Zealand rabbits were injected with 20,000 NIH3T3 cells and 5 µL of Matrigel in the right conjunctiva, and with only 5 µL of Matrigel in the left conjunctiva. The eyes were photographed under a magnification of 16× using a 12-megapixel digital camera attached to the microscope on day 1,3 and 7. Conjunctival vascularization was measured by analyzing images to measure red pixel saturation. Area of corneal and conjunctival fibrovascular tissue formation on the site of injection was assessed by analyzing the images on day 3 and 7 using area measurement software. Histopathologic characteristics were determined in the rabbit tissues and compared with a human primary pterygium. RESULTS: The two treatments promoted growth of conjunctival fibrovascular tissue at day 7. The red pixel saturation and area of fibrovascular tissue developed was significantly higher in right eyes (p < 0.05). Tissues from both treatments showed neovascularization in lesser extent to that observed in human pterygium. Acanthosis, stromal inflammation, and edema were found in tissues of both treatments. No elastosis was found in either treatment. CONCLUSIONS: Matrigel alone or in combination with NIH3T3 cells injected into the rabbits' conjunctiva can promote tissue growth with characteristics of human pterygium, including neovascularization, acanthosis, stromal inflammation, and edema. The combination of Matrigel with NIH3T3 cells seems to have an additive effect on the size and redness of the pterygium-like tissue developed.

Efficacy of relacin combined with sodium hypochlorite against Enterococcus faecalis biofilms

Abstract Objective Relacin is a synthetic molecule that targets RelA, an essential protein in a conserved bacterial stress response system. It was shown to inhibit bacterial growth. The aims of this study were to evaluate the antimicrobial effect of relacin combined with sodium hypochlorite (NaOCl) on Enterococcus faecalis biofilms and to evaluate the cytotoxicity of relacin. Material and Methods 48-h E. faecalis OG1RF biofilms were treated by various concentrations of relacin in order to determine its inhibitory concentration. Then, the 48-h biofilms were treated either with 1-min NaOCl (0.01%, 0.05%) alone, or in combination of relacin. As a means of comparison, the biofilms of ΔrelA were also treated by 1-min NaOCl (0.01%, 0.05%, 0.25%). The treatment efficacy was determined by agar plate count assays. The cytotoxicity of relacin was examined on human gingival epithelial cells Ca9-22 and murine fibroblasts NIH-3T3 by a methyl thiazolyltetrazolium (MTT) assay and a lactate dehydrogenase assay. Statistical analysis was performed by one-way or two-way analysis of variance (ANOVA) with Bonferroni's post-hoc test and an independent Student's t-test. A significance level of p<0.05 was used. Results Relacin inhibited the growth of OG1RF biofilms partially at 8 mM and fully at 14 mM. The relacin (14 mM) and NaOCl combined treatment resulted in significantly higher treatment efficacy than NaOCl treatment alone. At 0.05% NaOCl, the combined treatment resulted in 5.65 (±0.19) log reduction in biofilm viability. The ΔrelA biofilms were more susceptible to NaOCl treatment than the wild type biofilms at 0.25% NaOCl. Relacin at 14 mM was not toxic to host epithelial cells and fibroblasts. Conclusions The combination of relacin with a low concentration of NaOCl was effective and not cytotoxic.

Preliminary Study on the Effect of Adipocytes on the Biological Behaviors of Lung Adenocarcinoma A549 Cells in Tumor Microenvironment / 中国肺癌杂志

BACKGROUND@#Adipocytes in the tumor microenvironment may provide the metabolic fuel or signal transduction through media and other means to promote a variety of malignant proliferation and invasion, of tumor cells, but their role in lung cancer progression is still unclear. The purpose of this study was to investigate the effect of adipocytes on lung cancer cell biology.@*METHODS@#3T3-L1 pre-adipocytes were induced into mature adipocytes. The cell morphology was observed by microscopy and Oil Red O staining. MTT assay, colony formation assay, wound-healing and Transwell methods were used to detect lung cancer cell proliferation, migration and invasion ability. The content of triglyceride in cells was determined by colorimetry.@*RESULTS@#The morphology of lung adenocarcinoma A549 cells became more slender after co-culture with mature adipocytes, and the proliferation and cloning ability were significantly enhanced (P<0.05). In addition, mature adipocytes can also promote the migration ability (P<0.05), invasion ability (P<0.01) and accumulation of intracellular lipid (P<0.05) of A549 cells.@*CONCLUSIONS@#These findings suggested that adipocytes in tumor microenvironment can promote the proliferation, migration and invasion of lung adenocarcinoma A549 cells, which may be related to lipid metabolism.

Cardamom extract induces cell proliferation by increasing potassium currents in NIH3T3 cell line

Amommum subulatum [Roxb.] or Cardamom extract is known to have anti-inflammatory and neuroprotective effects towards many gastrointestinal related problems. However, uptill now different fractions of cardamom extract on fibroblasts with respect to potassium channel activity have not been investigated. Therefore, present study investigated the effects of different tractions of cardamom extract on potassium channels in non-tumor NIH3T3 cell line. Phytochemical analysis of hydroalcoholic, n-hexane, butane and ethyl acetate fractions of cardamom extracts were purified and isolated by thin layer chromatography [TLC]. 3T3 cells were cultured and incubated with hydroalcohol [1-2 Hg/ml], n-hexane [1 microg/ml], butane [2 microg/ml] and ethyl acetate [1-2 microg/ml] for 5 hrs at 37°C. Modulation in potassium currents were recorded by whole-cell patch clamp method. The data showed two constituents Cineol [CioHigO] and Terpinyl acetate [CioHi[7]OOCCH[3]] by TLC method. The present study shows that the constituents in n-hexane, hydro alcohol [1 [microg/ml] and ethyl acetate [2 microg/ml] significantly increased [p<0.01] the potassium outward rectifying currents from NIH3T3 cells when compared to untreated controls cells. Where as, butanol fraction [2 microg/ml] significantly decreased [p<0.01] the inward rectifying currents when compared to controls. Moreover hydroalcoholic and n-hexane fractions have increased the proliferation in 3T3 cell line. On the other hand butanol and ethyl acetate did not induce proliferation in 3T3 cells. Taken together, our data suggested that cardamom extract contains constituents that increased K[+] currents, cell migration and proliferation and are involved in wound healing

Physical-chemical properties and cytotoxicity analysis of 5 different endodontic sealers / Propriedades físico-químicas e análise da citotoxicidade de 5 diferentes cimentos endodônticos

The aim of this study was to analyze the radiopacity, setting time, flowability, pH, calcium ion release, solubility and cytotoxicity of bioceramic cements Totalfill BC Sealer and Totalfill BC RRM, and compare them with AH Plus, MTA Fillapex and MTA Angelus. The groups were divided and compared among them according to the filling and retro-filling cement functions. Totalfill BC Sealer was compared with AH Plus and MTA Fillapex; and Totalfill BC RRM retrofilling cement with MTA Angelus. For radiopacity analysis, specimens were placed in metal rings measuring 10x1 mm placed on occlusal film together with the aluminum scale. Digora 1.51 software was used to evaluate the digitized images and determine radiographic density. Setting time was tested in accordance with the American Society for Testing and Materials C266-08 standard specifications, but specimens were fabricated in accordance with the International Organization for Standardization 6876: 2001. Flow was tested in accordance with ANSI/ADA No.57/200 specifications. In total 30 acrylic teeth were filled with filling-cements and 20, with (retrograde cavity) retro-filling cements. All teeth were immersed in ultrapure water for pH and calcium ion release measurement (atomic absorption spectrophotometer) for time intervals of 1, 3, 24, 72, 168 and 360 hours. Solubility was tested by scanning and digitizing 50 acrylic teeth twice by Micro- CT, before and after immersion in ultrapure water for time intervals of 168, 360 and 720 hours. The images were reconstructed and volume (mm3) values of samples obtained by means of CTan software (CTan v1.11.10.0, SkyScan). The in vitro effects on cells were analyzed at concentrations of 100, 50, 10, 5, 1 mg/mL, and 0 mg / mLnegative control group and recorded in time intervals of 24, 48 and 72 hours by MTT reduction assay. The results were statistically analyzed by the ANOVA, Tukey, Kruskal-Wallis and Dunn tests (P<0.05). All radiopacity values according to ISO 6876/2001, AH Plus (7.86 mm Al) being the most radiopaque followed by Totalfill BC Sealer (4.84 mm Al), MTA Fillapex (3.41 mm Al), Totalfill BC RRM (6.8 mm Al), and MTA Angelus (6.7 mm Al). The following values were the initial and final setting time (in hours), respectively: AH Plus (8 and 15); Totalfill BC Sealer (11 and 24); MTA Fillapex (13 and 26); MTA Angelus (10 and 120 minutes) and Totalfill BC RRM (3 hours and 22 hours). In flow analysis, the cements behaved as follows: MTA Fillapex (47 mm), Totalfill BC Sealer (41.5 mm), Totalfill BC RRM (33.5 mm), AH Plus (33 mm) e MTA Angelus (17.5 mm) (p < 0.05). pH analysis showed in general the lowest values for AH Plus cement, followed by Totalfill BC RRM, MTA Angelus, MTA Fillapex and Totalfill BC Sealer. AH Plus showed the highest Ca2+ release in time interval 1 hour (1.38 mg/L); MTA Fillapex, in 360 hours (3.81 mg/L); MTA Angelus, 1 hour (1.38 mg/L); Totalfill BC Sealer, 360 hours (6.77 mg/L) and Totalfill BC RRM, 360 hours (3.81 mg/L). Almost all the sealers presented solubility lower than 3% in all periods, as recommended by ISO 6876/2001. Whereas, the MTA Fillapex solubility value was higher than 5% in all periods. Relative to cytotoxicity, all the cements were shown to be toxic at the concentration of 100 mg/mL, however, Totalfill BC Sealer and Totalfill BC RRM showed the best cell viability result compared with the other cements tested. We concluded that all root canal filling and root retro-filling complied with the requisites of radiopacity, setting time, flow, pH, calcium ion release, solubility and cytotoxicity. With the exception of the MTA Fillapex that not only fulfilled the requirement of solubility. Of the sealers, Totalfill BC Sealer was outstanding: it showed the highest pH and Ca2+ release, and lowest cytotoxicity. Among the retrofilling cements, Totalfill BC RRM maintained its high pH, higher Ca2+ release, and lower cytotoxicity. (AU)

O objetivo do presente estudo foi analisar a radiopacidade, tempo de presa, escoamento, pH, liberação de íons cálcio, solubilidade e citotoxicidade dos cimentos biocerâmicos Totalfill BC Sealer e Totalfill BC RRM e compará-los ao AH Plus, MTA Fillapex e MTA Angelus. Os grupos foram divididos e comparados entre si de acordo com as funções dos cimentos de obturação e retro-obturação. Comparamos o cimento obturador Totalfill BC Sealer com os cimentos AH Plus e MTA Filapex, e o cimento retrobturador Totalfill BC RRM com o cimento retrobturador MTA Angelus. Para análise da radiopacidade, os espécimes foram colocados em anéis metálicos medindo 10x1 mm, dispostos sobre um filme oclusal com uma escala de alumínio. O software Digora 1.51 foi utilizado para avaliar as imagens digitalizadas e determinar a densidade radiográfica. O tempo de presa foi realizado de acordo com as especificações da American Society for Testing and Materials C266-08 standard specifications, mas os espécimes foram feitos de acordo com a International Organization for Standardization 6876: 2001. O escoamento foi realizado de acordo com as especificações ANSI/ADA N0 57/2000. Trinta dentes acrílicos foram preenchidos com cimentos obturadores e vinte dentes de acrílico (com cavidade retrógrada) foram preenchidos com cimentos retro-obturadores e imersos em água ultrapura para mensuração do pH e liberação de íons cálcio (espectrofotômetro de absorção atômica) no período de 1, 3, 24, 72, 168 e 360 horas. Para o teste de solubilidade, foram escaneados 50 dentes acrílicos e digitalizados duas vezes pelo Micro-CT, antes e após a imersão em água ultrapura nos períodos de 168, 360 e 720 horas. As imagens foram reconstruídas e o volume (mm3) das amostras foi obtido usando o software CTan (CTan v1.11.10.0, SkyScan). Os efeitos celulares in vitro foram analisados nas concentrações de 100, 50, 10, 5, 1 mg/mL e 0 mg / mLgrupo controle negativo e registados nos períodos de 24, 48 e 72 horas através do ensaio de redução de MTT. Os resultados foram analisados estatisticamente pelos testes ANOVA, Tukey, Kruskal-Wallis e Dunn (p < 0.05). Todos os valores de radiopacidade estavam de acordo com a norma ISO 6876/2001, sendo o AH Plus (7.86 mm Al) o mais radiopaco seguido dos demais cimentos; Totalfill BC Sealer (4.84 mm Al), MTA Filapex (3.41 mm Al), Totalfill BC RRM (6,8 mm Al), MTA Angelus (6,7 mm Al). Os valores obtidos para o tempo de presa inicial e final foram respectivamente, AH Plus (8 e 15 horas), Totalfill BC Sealer (11 e 24 horas), MTA Filapex (13 e 26 horas), Totalfill BC RRM (3 horas e 22 horas) e MTA Angelus (10 e 120 minutos). Na análise de escoamento os cimentos se comportaram da seguinte forma: AH Plus (33 mm), MTA Filapex (47 mm), Totalfill BC Sealer (41,5 mm), Totalfill BC RRM (33,5 mm), e MTA Angelus (17,5 mm) (p < 0.05). A análise do pH mostrou que o cimento AH Plus de um modo geral, foi o que apresentou os menores valores, seguido do Totalfill BC RRM, MTA Angelus, MTA Filapex e Totalfill BC Sealer. A maior liberação de Ca2+ do AH Plus foi no período de 1 hora (1.38 mg/L), MTA Filapex foi em 360 horas (3.81 mg/L), Totalfill BC Sealer 360 horas (6.77 mg/L), Totalfill BC RRM 360 horas (3.81 mg/L) e MTA Angelus em 1 hora (1.38 mg/L). Todos os cimentos apresentaram solubilidade menor que 3% em todos os períodos, como recomendado pela ISO 6876/2001. Entretanto, os valores de solubilidade do MTA Fillapex excedeu mais que 5% em todos os períodos. Com relação à citotoxicidade, todos os cimentos mostraram-se tóxicos na concentração de 100 mg/mL, porém o Totalfill BC Sealer e Totalfill BC RRM apresentaram melhor resultado de viabilidade celular comparado aos demais cimentos testados. Concluiu-se que os cimentos de obturação e retro-obturação cumpriram os requisitos de radiopacidade, tempo de presa, escomento, pH, liberação de íons cálcio, solubilidade e citotoxicidade. Com exceção do MTA Fillapex que não cumpriu somente o requisito de solubilidade. Dos cimentos obturadores, o que melhor se portou foi o Totalfill BC Sealer, apresentando maior pH e liberação de íons cálcio e menor citotoxicidade. Dentre os cimentos retro-obturadores, Totalfill BC RRM foi o que melhor se destacou, mantendo seu pH elevado, possuindo maior liberação de Ca2+ e menor citotoxicidade. (AU)

Induction of robust senescence-associated secretory phenotype in mouse NIH-3T3 cells by mitomycin C / 生理学报

Senescence-associated secretory phenotype (SASP) is often a concomitant result of cell senescence, embodied by the enhanced function of secretion. The SASP factors secreted by senescent cells include cytokines, proteases and chemokines, etc, which can exert great influence on local as well as systemic environment and participate in the process of cell senescence, immunoregulation, angiogenesis, cell proliferation and tumor invasion, etc. Relative to the abundance of SASP models in human cells, the in vitro SASP model derived from mouse cells is scarce at present. Therefore, the study aimed to establish a mouse SASP model to facilitate the research in the field. With this objective, we treated the INK4a-deficient mouse NIH-3T3 cells and the wildtype mouse embryonic fibroblasts (MEF) respectively with mitomycin C (MMC), an anticarcinoma drug which could induce DNA damage. The occurring of cell senescence was evaluated by cell morphology, β-gal staining, integration ratio of EdU and Western blot. Quantitative RT-PCR and ELISA were used to detect the expression and secretion of SASP factors, respectively. The results showed that, 8 days after the treatment of NIH-3T3 cells with MMC (1 μg/mL) for 12 h or 24 h, the cells became enlarged and the ratios of β-gal-positive (blue-stained) cells significantly increased, up to 77.4% and 90.4%, respectively. Meanwhile, the expression of P21 protein increased and the integration ratios of EdU significantly decreased (P < 0.01). Quantitative RT-PCR detection showed that the mRNA levels of several SASP genes, including IL-6, TNF-α, IL-1α and IL-1β increased evidently. ELISA detection further observed an enhanced secretion of IL-6 (P < 0.01). On the contrary, although wildtype MEF could also be induced into senescence by MMC treatment for 12 h or 24 h, embodied by the enlarged cell volume, increased ratios of β-gal-positive cells (up to 71.7% and 80.2%, respectively) and enhanced expression of P21 protein, the secretion of IL-6 displayed no significant change. Our study indicated that, although MMC could induce senescence in both mouse NIH-3T3 cells and wildtype MEF, only senescent NIH-3T3 cells displayed the canonical SASP phenomena. Current study suggested that senescent NIH-3T3 cells might be an appropriate in vitro SASP model of mouse cells.

Cloning and expression of CD19, a human B-cell marker in NIH-3T3 cell line

CD19 is a pan B cell marker that is recognized as an attractive target for antibody-based therapy of B-cell disorders including autoimmune disease and hematological malignancies. The object of this study was to stably express the human CD19 antigen in the murine NIH-3T3 cell line aimed to be used as an immunogen in our future study. Total RNA was extracted from Raji cells in which high expression of CD19 was confirmed by flow cytometry. Synthesized cDNA was used for CD19 gene amplification by conventional PCR method using Pfu DNA polymerase. PCR product was ligated to pGEM-T Easy vector and ligation mixture was transformed to DH5 alpha competent bacteria. After blue/white selection, one positive white colony was subjected to plasmid extraction and direct sequencing. Then, CD19 cDNA was sub-cloned into pCMV6-Neo expression vector by double digestion using Kpnl and HindIII enzymes. NIH-3T3 mouse fibroblast cell line was subsequently transfected by the construct using Jet-PEI transfection reagent. After 48 hours, surface expression of CD19 was confirmed by flow cytometry and stably transfected cells were selected by G418 antibiotic. Amplification of CD19 cDNA gave rise to 1701 bp amplicon confirmed by alignment to reference sequence in NCBI database. Flow cytometric analysis showed successful transient and stable expression of CD19 on NIH-3T3 cells [29 and 93%, respectively]. Stable cell surface expression of human CD19 antigen in a murine NIH-3T3 cell line may develop a proper immunogene which raises specific anti-CD19 antibody production in the mice immunized sera

Low concentrations of doxycycline attenuates FasL-induced apoptosis in HeLa cells

BACKGROUND: Doxycycline (DC) has been shown to possess non-antibiotic properties including Fas/Fas Ligand (FasL)-mediated apoptosis against several tumor types in the concentration range of 10-40 µg/mL. However, the effect of DC in apoptotic signaling at much low concentrations was not studied. METHODS: The present study investigated the attenuation effect of low dose of DC on FasL-induced apoptosis in HeLa cell by the methods of MTT assay, fluorescence microscopy, DNA fragmentation, flow cytometry analysis, and western blotting. RESULTS AND CONCLUSION: In the present findings we showed that low concentration of DC (<2.0 µg/mL) exhibited protective effects against FasL-induced apoptosis in HeLa cells. FasL treatment to HeLa cells resulted in a concentration-dependent induction of cell death, and treatment with low concentrations of DC (0.1-2 µg/mL) significantly (p < 0.001) attenuated the FasL-induced cell death as measured by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazo-lium bromide (MTT) assay. Further, the FasL-induced apoptotic features in HeLa cells, such as morphological changes, DNA fragmentation and cell cycle arrest was also inhibited by DC (0.5 µg/mL). Tetracycline and minocycline also showed similar anti-apoptotic effects but were not significant when compared to DC, tested at same concentrations. Further, DC (0.01-16 µg/mL) did not influence the hydrogen peroxide- or cisplatin-induced intrinsic apoptotic pathway in HeLa cells. Protein analysis using Western blotting confirmed that FasL-induced cleavage/activation of cas-pase-8 and caspase-3, were inhibited by DC treatment at low concentration (0.5 µg/mL). Considering the overall data, we report for the first time that DC exhibited anti-apoptotic effects at low concentrations in HeLa cells by inhibition of caspase activation via FasL-induced extrinsic pathway.

Construction and analysis of recombinant eukaryotic expression plasmids for SOX10, the causative gene of Warrdenburg syndrome / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the exogenous expression and subcellular localization of wild type (WT) and mutant SOX10 proteins in vitro through generation of expression plasmids in order to reveal the pathogenesis of Waardenburg syndrome (WS).</p><p><b>METHODS</b>The plasmids pECE-SOX10 and pCMV-Flag were ligated after they were subjected to double enzyme digestion using molecular cloning technique to generate recombinant eukaryotic expression plasmid pCMV-SOX10-Flag, which was as a template to generate expression plasmids for novel mutations G37fs, G38fs and E248fs of the SOX10 gene. The constructs were verified by direct sequencing. NIH3T3 cells were transiently transfected with the expression plasmids of wide type SOX10, G37fs, G38fs and E248fs, respectively. The exogenous expression of WT SOX10 protein and mutant G37fs, G38fs and E248fs proteins were analyzed using Western blot assay, while their subcellular distribution were observed with an immunofluorescence assay.</p><p><b>RESULTS</b>The DNA sequences of expression plasmids for SOX10 and its mutant G37fs, G38fs and E248f were all correct. Both WT and mutant SOX10 proteins were detected at the expected site. WT SOX10 and E248fs proteins have only localized in the nucleus, whereas G37fs and G38fs proteins showed aberrant localization in both cytoplasm and nucleus.</p><p><b>CONCLUSION</b>Recombinant eukaryotic expression plasmids for the SOX10 gene and its mutants were successfully constructed. Preliminary analysis showed that the mutations have affected the subcellular distribution of WT SOX10 proteins, which has laid a basis for further study of the molecular mechanism of WS caused by SOX10 gene mutations.</p>

Analysis of nuclear localization and signal function of MITF protein predisposing to Warrdenburg syndrome / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the role of dysfunction of nuclear localization signals (NLS) of MITF protein in the pathogenesis of Waardenburg syndrome.</p><p><b>METHODS</b>Eukaryotic expression plasmid pCMV-MITF-Flag was used as a template to generate mutant plasmid pCMV-MITF△NLS-Flag by molecular cloning technique in order to design the mutagenic primers. The UACC903 cells were transfected transiently with MITF and MITF△NLS plasmids, and the luciferase activity assays were performed to determine their impact on the transcriptional activities of target gene tyrosinase (TYR). The oligonucleotide 5'-GAACGAAGAAGAAGATTT-3' was subcloned into pEGFP-N1 to generate recombinant eukaryotic expression plasmid pEGFP-N1-MITF-NLS. The NIH3T3 cells were transfected separately with MITF, MITF△NLS, pEGFP-N1 and pEGFP-N1-NLS plasmids, and their subcellular distribution was observed by immunoflorescence assays.</p><p><b>RESULTS</b>Expression plasmids for the mutant MITF△NLS with loss of core NLS sequence and pEGFP-N1-NLS coupled with MITF△NLS were successfully generated. Compared with the wild-type MITF, MITF△NLS was not able to transactivate the transcriptional activities of promoter TYR and did not affect the normal function of MITF. MITF△NLS was only localized in the cytoplasm and pEGFP-N1 was found in both the cytoplasm and nucleus, whereas pEGFP-N1-NLS was mainly located in the nucleus.</p><p><b>CONCLUSION</b>This study has confirmed the localization function of NLS sequence 213ERRRRF218 within the MITF protein. Mutant MITF with loss of NLS has failed to transactivate the transcriptional activities of target gene TYR, which can result in melanocyte defects and cause WS.</p>

Effects of Zuogui pill, Yougui pill and relative compositions on differentiation towards germ cells of mouse embryonic stem cell 1B10 / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the effects of Zuogui pill, Yougui pill and the relative compositions on the differentiation towards germ cells of stem cells.</p><p><b>METHOD</b>The rat drug sera for Zuogui pill, Yougui pill and the common composition of Zuogui pill and Yougui pill were prepared respectively as the experimental drugs; the mouse embryonic stem cell 1B10 (MESC-1B10) was used as the representative of stem cells; the above rat drug sera were used to intervene MESC-1B10 and the process was traced by microscopy imaging; after 72 h of the intervention, the RNAs were extracted from the different intervened MESC-1B10, cDNAs were synthesized immediately and finally the Real-time quantitative PCR (qPCR) was used to measure the expression patterns of the 10 reproductive-differentiation-related genes for each intervention.</p><p><b>RESULT</b>The rat drug serum of Zuogui pill (ZGW-RS) significantly up-regulated Oct-4 and SCP3 and significantly down-regulated GDF-9 and Stra8; the rat drug serum of Yougui pill (ZGW-RS) significantly up-regulated Oct-4, GDF-9, Mvh and SCP3 and significantly down-regulated Stra8, Itga6 and Itgb1; the rat drug sera for the common composition of Zuogui pill and Yougui pill (ZGWYGW-RS) significantly up-regulated Oct-4, SCP3 and ZP3 and significantly down-regulated GDF-9, Stra8, Itga6 and Itgb1.</p><p><b>CONCLUSION</b>ZGW-RS can initiate the change towards meiosis, but can not start the reproductive differentiation of MESC-1B10; YGW-RS can initiate the change towards meiosis, and can also start the reproductive differentiation of MESC-1B10 towards female germ cells; ZGWYGW-RS can initiate the change towards meiosis, and can lightly start the reproductive differentiation of MESC-1B10 towards female germ cells but the inductive effect is smaller than YGW-RS. The experimental results, on one hand, strengthen the knowledge about the influence of the relative compositions of Zuogui pill and Yougui pill on the reproductive differentiation of stem cells, on the other hand, help to explain the mechanism of the treatment of the infertility by Zuogui pill and Yougui pill.</p>

The effect of nickel-smelting fumes on the expression of bcl-2 and bax in NIH/3T3 cells / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the effect of nickel-smelting fumes on the expression of bcl-2 and bax in mammalian cells.</p><p><b>METHODS</b>Logarithmic growth NIH/3T3 cells were exposed to venom for 24 h, which sample fumes concentration was respectively 0, 6.25, 12.50, 25.00, 50.00, 100.00 µg/ml. Cell viability was assessed by MTT assay and the level of extracellular LDH activity was detected with Lactate Dehydrogenase (LDH) kit. Morphological changes of apoptotic were observed with Hoechst33342, while Western blot was used to measure the expression of bcl-2 and bax.</p><p><b>RESULTS</b>In addition to 7 days of 6.25 µg/ml nickel-smelting fumes group, each time point and dose group's cell viability reduced with significant differences compared with the control group (P < 0.05). the extracellular LDH activity increased with increasing dose of nickel-smelting fumes, and the extracellular LDH activity of 6.25, 12.50, 25.00, 50.00, 100.00 µg/ml nickel-smelting fumes group increased as compared with the control group (P < 0.05). Simultaneously, the cells, treated with 100.00 µg/ml nickel-smelting fumes for 24 h, appeared obvious morphological changes of apoptosis, such as chromatin condensation and cell shrinkage. the expression of bcl-2 significantly increased in groups of 6.25, 12.50, 25.00 µg/ml nickel-smelting fumes (0.58 ± 0.01, 0.6 3± 0.01 and 0.57 ± 0.01) and decreased in groups of 50.00, 100.00 µg/m nickel-smelting fume (0.35 ± 0.01 and 0.27 ± 0.01) as compared with that of the control group (P < 0.05). And the expression of bax significantly decreased in group of 6.25 µg/ml nickel-smelting fumes (0.58 ± 0.00) and increased in groups of 50.00, 100.00 µg/m nickel-smelting fumes (0.71 ± 0.01 and 0.78 ± 0.02) as compared with that of the control group (P < 0.05).</p><p><b>CONCLUSION</b>Apoptosis was activated in NIH/3T3 cell after 24 h of exposure to Ni-smelting fumes, which may be induced by oxidative stress.</p>