ABSTRACT
The in vitro growth of embryonic stem cells (ESCs) is usually obtained in the presence of murine embryonic fibroblasts (MEF), but new methods for in vitro expansion of ESCs should be developed due to their potential clinical use. This study aims to establish a culture system to expand and maintain ESCs in the absence of MEF by using murine embryonic stem cells (mECS) as a model of embryonic stem cell. Magnetic nanoparticles (MNPs) were used for growing mESCs in the presence of an external magnetic field, creating the magnetic field-magnetic nanoparticle (MF-MNP) culture system. The growth characteristics were evaluated showing a doubling time slightly higher for mESCs cultivated in the presence of the system than in the presence of the MEF. The undifferentiated state was characterized by RT-PCR, immunofluorescence, alkaline phosphatase activity and electron microscopy. Murine embryonic stem cells cultivated in presence of the MF-MNP culture system exhibited Oct-4 and Nanog expression and high alkaline phosphatase activity. Ultrastructural morphology showed that the MF-MNP culture system did not interfere with processes that cause structural changes in the cytoplasm or nucleus. The MF-MNP culture system provides a tool for in vitro expansion of mESCs and could contribute to studies that aim the therapeutic use of embryonic stem cells.
Subject(s)
Cell Culture Techniques/methods , Embryonic Stem Cells/cytology , Magnetics , Magnetite Nanoparticles/chemistry , Animals , Biomarkers/metabolism , Cell Differentiation/physiology , Cell Growth Processes/physiology , Cell Shape , Embryonic Stem Cells/metabolism , Embryonic Stem Cells/physiology , Histocytochemistry , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Mice , Nanog Homeobox Protein , Nanotechnology/methods , Octamer Transcription Factor-3/genetics , Octamer Transcription Factor-3/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The aim of this work was to study the effect of curcumin on cell cycle in the human SK-MEL-37 melanoma cell line. In addition, morphological and structural analyses were also performed. Flow cytometric analysis showed a G0/G1 arrest at 5 µM after 24 h exposure and a concentration-dependent increase in the proportion of sub-G0 hypodiploid cells. Typical apoptotic events were also observed by the fluorescence microscopy, transmission and scanning electronic microscopy. Loss of mitochondrial membrane potential was not detected. Results suggested that curcumin could arrest human melanoma cells at G0/G1 phase and induce a mitochondrial-independent apoptotic pathway.
O melanoma é um tipo agressivo de câncer cujo tratamento culmina com o estabelecimento de resistência aos quimioterápicos empregados. Portanto, é importante o desenvolvimento de novos agentes farmacológicos que sejam menos tóxicos e que não provoquem quimiorresistência. As inúmeras propriedades terapêuticas da curcumina vêm sendo confirmadas através de estudos sobre o seu mecanismo de ação em células cultivadas. No presente estudo, empregamos células de melanoma humano da linhagem SK-MEL-37, que desenvolveram resistência in vitro à doxorubicina e cisplatina, drogas normalmente utilizadas na clínica. Investigamos o efeito da curcumina sobre o ciclo celular através de citometria de fluxo. Além disso, análises morfológicas e estruturais também foram realizadas. Os resultados demonstraram que o tratamento com uma concentração de 5 ?M de curcumina provocou uma parada na subfase G0/G1. Além disso, observou-se um aumento dose-dependente na proporção de células hipodiplóides em sub-G0. Eventos apoptóticos típicos foram observados por microscopia de fluorescência, microscopia eletrônica de transmissão e microscopia eletrônica de varredura. Não foi detectada alteração no potencial de membrana mitocondrial. Os resultados indicam que futuros estudos poderão tornar possível a utilização da curcumina como um modulador para agentes quimioterápicos empregados na clínica no tratamento do melanoma.
ABSTRACT
Three magnetic fluid (MF) samples containing gamma-Fe2O3 (maghemite) nanoparticles surface-coated with either meso-2,3-dimercaptosuccinic acid (DMSA), citric acid or lauric acid were prepared, characterized, and assessed for their cytotoxic potential on the human SK-MEL-37 melanoma cell line. Ultra-structural analysis was also performed using transmission electron microscopy (TEM). In vitro cytotoxicity was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The inhibitory concentration (IC50) derived from the sigmoidal dose response curve was 254 microg-iron/mL (95% confidence interval 239-270 microg-iron/mL) for lauric acid-coated nanoparticles. DMSA-coated nanoparticles did not exhibit a clear trend toward toxicity (IC50 value is more than 2260 +/- 50 microg-iron/mL) and the IC50 value was about 433 +/- 14 microg-iron/mL for citric-acid coated nanoparticles. The cytotoxic response correlated with both the hydrodynamic diameter and the zeta potential suggests that the chain length of the carboxylic acid of the coating species may influence metabolic cellular process. Also the assayed nanoparticles can be considered non-cytotoxic to human melanoma cells since IC50 values are higher than plasma concentration usually observed in clinical use of contrast agents. Using TEM we verified that all assayed nanoparticles were internalized by cells through endocytic vesicles. Additionally, cells treated with lauric acid-coated nanoparticles at high concentration (588 or 840 microg-iron/mL) displayed morphological features of apoptosis (surface blebbing, intense vacuolization and chromatin condensation) or a typical DNA ladder pattern when analyzed by TEM or agarose gel electrophoresis, respectively. Apoptotic events may be operative, suggesting a promising therapeutic application for the lauric acid-coated nanoparticle in the treatment of cancer cells.
