Exacerbated vulnerability to oxidative stress in astrocytic C6 glioma cells with stable overexpression of the glutamine transporter slc38a1
Division of Pharmaceutical Sciences, Kanazawa University Graduate School of Natural Science and Technology, Kanazawa, Ishikawa, Japan. Neurochemistry International
(Impact Factor: 3.09).
03/2011; 58(4):504-11. DOI: 10.1016/j.neuint.2011.01.007
We have previously demonstrated the functional expression of glutamine (Gln) transporter (GlnT) believed to predominate in neurons for the neurotransmitter glutamate pool by rat neocortical astrocytes devoid of neuronal marker expression, with exacerbated vulnerability to oxidative stress after transient overexpression. To evaluate molecular mechanisms underlying the exacerbation, we established stable GlnT transfectants in rat astrocytic C6 glioma cells. In two different clones of stable transfectants with increased intracellular Gln levels, exposure to hydrogen peroxide (H(2)O(2)) and A23187, but not to tunicamycin or 2,4-dinitrophenol, led to significant exacerbation of the cytotoxicity compared to cells with empty vector (EV). Stable GlnT overexpression led to a significant increase in heme oxygenase-1 protein levels in a manner sensitive to H(2)O(2), whereas H(2)O(2) was significantly more effective in increasing NO(2) accumulation and reactive oxygen species (ROS) generation in stable GlnT transfectants than in EV cells. Moreover, exposure to A23187 led to a more effective increase in the generation of ROS in stable GlnT transfectants than in stable EV transfectants. These results suggest that GlnT may play a role in the mechanisms underlying the determination of cellular viability in astrocytes through modulation of intracellular ROS generation.
Available from: PubMed Central
- "In the present study, we found an increased expression of SLC38A1 in gastric carcinomas, relative to adjacent non-cancerous gastric mucosa, suggesting that SLC38A1 might play an important part in gastric cancer malignant transformation. Enhanced SLC38A1 expression has been observed in several other types of malignancies, including liver cancer
, Hilar cholangiocarcinoma
 and C6 glioma
. An elevated expression of SLC38A1 has been found to be closely linked to differentiation status, lymph node metastasis and TNM staging, and is thus implicated in the growth, invasion, metastasis and progression of gastric carcinomas. "
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ABSTRACT: Current literature has demonstrated that host glutamine depletion facilitates tumorigenesis. Likewise, the glutamine transporter SLC38A1 is putatively associated with malignant transformation and tumor progression. Taken together, this forms the premise for undertaking the current study. The twofold aim of this study was to provide insight into whether or not a variance in the expression of SLC38A1 exists between human gastric cancer and healthy human tissues, and to determine how silencing the SLC38A1 gene could affect the proliferation, viability, migration, and invasion of gastric cancer cells.
Immunohistochemical staining was used to analyze the expression of SLC38A1 in gastric cancer tissues and adjacent healthy mucosa in 896 patients with pathologically confirmed gastric cancer who had underwent R0 resection. SH-10-TC cells (a gastric cancer cell line) were used to examine whether silencing SLC38A1 with siRNA could affect cell viability, migration and invasion.
The SLC38A1 protein was very low or undetectable in healthy gastric mucosa. In contrast, strong staining of SLC38A1 protein was found in the cytoplasm in 495 out of the 896 gastric cancer samples. More pronounced SLC38A1 expression in gastric cancer tissues was significantly associated with age, differentiation status, lymph node metastasis, TNM stage and PCNA (proliferating cell nuclear antigen) expression. Upon univariate survival analysis, SLC38A1 expression was correlated with poor survival. Multivariate survival analysis revealed that SLC38A1 was an independent prognostic factor.
SLC38A1 is overexpressed in gastric cancer, which suggests that it is contributory to tumor progression. These results encourage the exploration of SLC38A1 as a target for intervention in gastric cancer.
BMC Gastroenterology 04/2014; 14(1):70. DOI:10.1186/1471-230X-14-70 · 2.37 Impact Factor
Available from: Yukio Yoneda
- "The full-length coding region of porcine Myo6, which was kindly donated by Dr. Tama Hasson (UCSD, San Diego, CA, USA), was inserted into pSI vector using the Ligation High reagent as described previously . Mouse embryonal carcinoma P19 cells were plated at a density of 1.5×105 cells/cm2, followed by culture in Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO BRL) with 10% FBS for 24 h and subsequent stable transfection with pSI-Myo6, pSI-GFP and pcDNA3.1 vectors, or pSI, pSI-GFP and pcDNA3.1 vectors, using 2 µg of DNA and Lipofectamine and Plus reagent (Invitrogen, San Diego, CA, USA) in 0.5 ml of Opti-MEM (Invitrogen) as described previously . "
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ABSTRACT: We have previously shown marked upregulation of the mRNA and corresponding protein for the cellular motor molecule myosin VI (Myo6) after an extremely traumatic stress experience, along with a delayed decrease in 5-bromo-2'-deoxyuridine incorporation in the murine hippocampus, a brain structure believed to undergo adult neurogenesis. In this study, we investigated the role of Myo6 in both proliferation and differentiation in pluripotent P19 cells by using stable transfection and RNA interference techniques.
Stable overexpression of Myo6 not only led to significant inhibition of the reducing activity of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and the size of clustered aggregates in P19 cells, but also resulted in selectively decreased mRNA expression of the repressor type proneural gene Hes5 without affecting the expression of neuronal and astroglial marker proteins. In P19 cells transfected with Myo6 siRNA, by contrast, a significant increase was found in the size of aggregate and MTT reduction along with increased Sox2 protein levels, in addition to marked depletion of the endogenous Myo6 protein. In C6 glioma cells, however, introduction of Myo6 siRNA induced a drastic decrease in endogenous Myo6 protein levels without significantly affecting MTT reduction. The Ca(2+) ionophore A23187 drastically increased the luciferase activity in P19 cells transfected with a Myo6 promoter reporter plasmid, but not in HEK293, Neuro2A and C6 glioma cells transfected with the same reporter.
These results suggest that Myo6 may play a predominant pivotal role in the mechanism underlying proliferation without affecting differentiation to progeny lineages in pluripotent P19 cells.
PLoS ONE 05/2013; 8(5):e63947. DOI:10.1371/journal.pone.0063947 · 3.23 Impact Factor
Available from: Yukio Yoneda
- "Cells were washed in cold PBS, followed by homogenization in 0.2 M perchrolic acid with a sonicator and subsequent centrifugation at 20,000 g for 10 min. An aliquot of supernatants was neutralized with 1 M KHCO3, followed by centrifugation and subsequent reaction for 2 min at room temperature with a derivatization reagent [5 mg/ml o-phthalaldehyde, 1% β-mercaptoethanol and 0.36 M potassium borate (pH 10.4)] for HPLC analysis as described previously . An aliquot was injected into an analysis column (Symmetry C18 3.5 µm, 4.6×75 mm). "
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ABSTRACT: We previously demonstrated the functional expression in newborn rat neocortical astrocytes of glutamine transporter (GlnT = slc38a1) believed to predominate in neurons over astroglia in the brain. In order to evaluate the possible role of this transporter in neurogenesis, we attempted to establish stable transfectants of GlnT in mouse embryonal carcinoma P19 cells endowed to proliferate for self-renewal and differentiate into progeny cells such as neurons and astroglia, in addition to in vitro pharmacological profiling of the green tea ingredient theanine, which is shown to be a potent inhibitor of glutamine transport mediated by GlnT in cultured neurons and astroglia.
The full-length coding region of rat GlnT was inserted into a vector for gene transfection along with selection by G418, followed by culture with all-trans retinoic acid under floating conditions and subsequent dispersion for spontaneous differentiation under adherent conditions. Stable overexpression of GlnT led to marked increases in the size of round spheres formed during the culture for 4 days and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide reduction, with concomitant promotion of subsequent differentiation into cells immunoreactive for a neuronal marker protein. In these stable GlnT transfectants before differentiation, drastic upregulation was seen for mRNA expression of several proneural genes with a basic helix-loop-helix domain such as NeuroD1. Although a drastic increase was seen in NeuroD1 promoter activity in stable GlnT transfectants, theanine doubled NeuroD1 promoter activity in stable transfectants of empty vector (EV), without affecting the promoter activity already elevated in GlnT transfectants. Similarly, theanine promoted cellular proliferation and neuronal differentiation in stable EV transfectants, but failed to further stimulate the acceleration of both proliferation and neuronal differentiation found in stable GlnT transfectants.
GlnT would promote both proliferation and neuronal differentiation through a mechanism relevant to the upregulation of particular proneural genes in undifferentiated P19 cells.
PLoS ONE 10/2012; 7(10):e48270. DOI:10.1371/journal.pone.0048270 · 3.23 Impact Factor
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