To analyze both differentially expressed genes and the Bcl-xL protein expression after acute and chronic treatment with fluoxetine in rat C6 glioma cells.
C6 glioma cells were cultured for 24 h or 72 h after treatment with 10 µM fluoxetine, and gene expression patterns were observed using microarray and qRT-PCR. Then, cells were cultured for 6 h, 24 h, 72 h or 96 h after treatment with 10 µM fluoxetine, and the expression of Bcl-xL protein was measured using western blot.
As determined by microarray, treatment with fluoxetine for 24 h up-regulated 33 genes (including Bcl-xL and NCAM140) and down-regulated 7 genes (including cyclin G-associated kinase). Treatment with fluoxetine for 72 h up-regulated 53 genes (including Gsα and Bcl-xL) and down-regulated 77 genes (including Gαi2 and annexin V). Based on the qRT-PCR results, there was an increase in Gsα mRNA and a decrease in Gαi2 mRNA at 72 h in fluoxetine-treated cells as compared to control, a result that was consistent with microarray. We also observed an increase in Bcl-xL mRNA (both at 24 h and at 72 h) in fluoxetine-treated cells as compared to control, demonstrating a tendency to increase gradually. Bcl-xL protein expression increased as the duration of fluoxetine treatment increased.
These results suggest that chronic treatment with fluoxetine not only initiates the cAMP pathway through inducing Gsα expression but also induces Bcl-xL expression, thus inhibiting apoptosis.
Fluoxetine, one of the selective serotonin reuptake inhibitors, shows a strong antidepressive effect and is widely used to augment the actions of serotonin (5-HT) in the nervous system.
Previous studies have shown that chronic treatment with antidepressants, including fluoxetine and tianeptine, regulates the expression of genes related to intracellular signal transduction pathways, including pathways involving cyclic adenosine monophosphate (cAMP) responsive element binding protein (CREB), brain-derived neurotropic factor (BDNF), mitogen-activated protein kinase and cell survival, indicating that antidepressants may keep nerve cells from being damaged and also help them regenerate.
In a previous study, we identified genes that may be involved in the therapeutic response of fluoxetine using microarray technology, which can simultaneously monitor the expression levels of thousands of genes.
The rat C6 glioma cells used in the present study are suitable for studying the effects of antidepressants
The rat C6 glioma cells (KCLB No;10107) were purchased from KTCC (Seoul, Korea), a rat brain 10K cDNA chip was obtained from Gaiagene (Seoul, Korea) and iQ SYBR green supermix was purchased from Bio-Rad (Hercules, CA, USA). BCL-xL antibody was purchased from Cell Signaling Technology (Beverly, MA, USA) and β-actin was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Fluoxetine was provided by Lilly Korea, Ltd. (Seoul, Korea).
C6 cells were grown overnight in Dulbecco's modified eagle medium containing L-glutamate, 4.5 g/L of glucose, 10% fetal bovine serum, 100 units of penicillin, and 100 µg/mL of streptomycin at 37℃ in humidified 5% CO2. As a control, the cells were fed with fresh media and cultured for 24 h and 72 h. For the fluoxetine treatment group, the cells were fed with fresh media containing 10 µM of fluoxetine and cultured for 24 h and 72 h, as was done in a previous study.
cDNA microarray analysis was performed using a chip containing 2,500 ESTs and 2,500 known genes. The total RNA from the control and fluoxetine-treated cells was isolated using TRIzol® Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Fluorescent (Cy3, Cy5)-labeled cDNA probes were made from 20 µg of total RNA from both the control cells and the fluoxetine-treated cells, using an amino-allyl cDNA labeling kit (Ambion, Austin, TX, USA). For each experiment, at least four replicates were performed, and two of these replicates were repeated with the fluorophores reversed to eliminate false-positive results. Cy3- and Cy5-labeled probes were mixed with water, 8 µg of poly (dA) (Pharmacia), 4 µg of Escherichia coli tRNA (Sigma-Aldrich, Saint Louis, MO, USA), 10 µg of mouse Cot1 DNA (Invitrogen, Carlsbad, CA, USA) and 60 µL of 2X EasyHyb buffer (U-vision Biotech Inc., Taipei, Taiwan) to a final volume of 120 µL. The resulting mixture was incubated at 95℃ for 5 min. Probes were then applied to the array for hybridization at 52℃ for 8 h, using a GeneTAG automatic hybridizer (Genomic Solutions, Boston, MA, USA). After hybridization, the slides were washed first in 2×SSC and 0.2% SDS at 52℃ for 5 min, then with 0.1×SSC and 0.2% SDS at 52℃ for 5 min and finally with 0.1×SSC for 5 min at room temperature. The slides were dried by spinning for 3 min. The dried slides were scanned with a ScanArray 5,000 fluorescence reader, and QuantArray image acquisition software (PerkinElmer Life Sciences) was used to quantify the signal and background intensity for each target element. The ratio of the two corrected signal intensities was calculated and used as the differential expression ratio (DE) for each specific gene in the two mRNA samples. The raw data of the repeated experiments were converted to log ratios. Negative control spots, small signal-to-noise ratio spots that were less than 2 and spots whose average sensitivity was less than 7.5 were removed, and the data were standardized by Lowess smoothing. Four repeated experiments were standardized by the significant analysis of microarray (SAM; t-statistics) method. Genes differentially expressed by fluoxetine were linkage-analyzed using Cluster 3.0 (open source clustering software) and visualized using JavaTreeview-1.1.3 software (Human Genome Center, Institute of Medical Science, Tokyo, Japan).
Total RNA from fluoxetine-treated and control cells was isolated using TRIzol® Reagent and reverse transcribed to cDNA in order to confirm the expression patterns of the genes Gsα, Gαi2, and Bcl-xL which were differentially expressed by fluoxetine treatment on cDNA microarray result. For qRT-PCR, 12.5 µL of SYBR® Premix Ex TaqTM II (Takara BIO, Shiga, Japan), 0.5 µL of ROX, 5 pmol of forward primer, 5 pmol of reverse primer and 0.5 µL of cDNA were mixed with water to a final volume of 25 µL. The mixture was amplified for 40 cycles with an ABI 7500 Real-Time PCR System (Applied Biosystems, Inc., Foster City, CA) with an initial melt at 95℃ for ten min followed by 40 cycles of 95℃ for 15 sec and 60℃ for one min. The cycle number at which a statistically significant increase in each gene was first detected (threshold cycle, Ct) was then normalized to the Ct for β-actin, which was used as an internal control. The relative expression differences between the control and fluoxetine-treated cells were calculated using the 2-ΔΔCT method.
The fluoxetine-treated cells were cultured in media containing 10 µM of fluoxetine for 6 h, 24 h, 72 h or 96 h. The cells were then washed in phosphate-buffered saline and harvested. Total protein was extracted from the cells using Pro-PrepTM (iNtRON Biotechnology, Korea) according to the manufacturer's instructions. The protein was quantified using the Bradford method (Bio-Rad Protein Assay Kit; Bio-Rad, Hercules, CA, USA). The protein was separated on 12% polyacrylamide gel through sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred onto a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). After blocking with 5% non-fat dry milk in TBST (20 mM Tris-HCl, pH 7.6; 500 mM NaCl, 0.1% Tween 20), the membrane was incubated overnight at 4℃ with the polyclonal rabbit anti-Bcl-xL antibody (1 : 1,000), followed by incubation with HRP-conjugated anti-mouse IgG (diluted 1 : 1,000) for 60 min. After washing in TBST, the reactions were detected using the enhanced chemiluminescence detection system (Amersham Biosciences, Sunnyvale, CA, USA). The concentration of Bcl-xL exposed on X-ray film was quantified with QuantityOne software (Bio-Rad, USA).
Data were expressed as the mean±standard error. A nonlinear-fitting program (OriginLab Corp., Northampton, MA, USA) was used. Paired and unpaired Student's t-tests were used as appropriate to evaluate the statistical significance of differences between the two groups (fluoxetine-treated and control groups). Values of p<0.05 were considered to indicate statistical significance.
The gene expression profile of control and 10 µM fluoxetine-treated cells after 24 h and 72 h was analyzed with a chip containing 2,500 ESTs and 2,500 known genes. After mining biological data using log_2 (a fold change cut-off of 1.5), a hierarchical dendrogram of 169 genes was completed by clustering differentially expressed genes at 24 h and 72 h after fluoxetine treatment (
Annexin V and Gαi2 were down-regulated in fluoxetine-treated cells at 72 h, whereas 14-3-3 eta and Gsα were up-regulated. Compared to the expression changes at 24 h, more genes showed a change in expression at 72 h.
To confirm the result of the microarray analysis, we performed qRT-PCR on RNA isolated from control and fluoxetine-treated cells. Gsα mRNA expression was 1.03±0.12-fold greater at 24 h and 2.63±0.24-fold greater at 72 h in fluoxetine-treated cells compared to the control cells as observed in our previous study (
After treating C6 cells with 10 µM fluoxetine and culturing them for 6 h, 24 h, 72 h or 96 h, we observed the expression pattern of Bcl-xL protein. Expression gradually increased as treatment time progressed (
Over the past few decades, theories on depression have suggested that atrophy of neurons from stress, apoptosis or depression may result from an impairment of the intracellular signaling pathway and a failure of neurons to appropriately adapt.
We analyzed the genes that were up- and down-regulated as a result of acute and chronic treatment with fluoxetine in C6 cells and detected expression changes in genes that engage in the intracellular signal transduction cascade, including the following: Gsα and Gαi2; neurite outgrowth, including NCAM140; and cell survival, such as annexin V and Bcl-xL. Depressed patients exhibit dysfunction of the cAMP signaling pathway, namely a decrease in adenylyl cyclases, the enzymes that generate intracellular cAMP.
NCAM, which has three major isoforms (NCAM180, 140 and 120), regulates structural and synaptic plasticity in the brain.
Annexin V is a Ca2+-dependent protein that combines with phospholipids phosphatidylserine (PS) after the translocation of PS from the inside to the outside of the plasma membrane. Annexin V expression increases in the early stages of cell death.
In the present study, Bcl-xL expression (both mRNA and protein) in the C6 cells was up-regulated by both acute and chronic treatment with fluoxetine, revealing a tendency for the expression level of Bcl-xL to rise as the duration of fluoxetine treatment grew longer. As already mentioned, neurotrophic factors (such as CREB and BDNF) are regulated by stress and antidepressants through cell survival pathways that also control the Bcl-2 family proteins.
In conclusion, our data not only confirmed that chronic treatment of fluoxetine induces Gsα and repressed Gαi2 expression but also demonstrated that expression of Bcl-xL (both mRNA and protein) is increased by chronic treatment with fluoxetine in C6 cells. These results suggest that these genes may play a critical role in delaying the clinical effects of antidepressants. However, additional studies are necessary to further analyze the other genes found in the microarray and their roles in the effects of antidepressants.
This work was supported by the research fund of Hanyang University (HY-2010-N).
Microarray analysis of fluoxetine-treated C6 glioma cells. Control and 10 µM fluoxetine-treated cells were cultured for 24 h or 72 h. Then, 169 differentially expressed genes in these cells were analyzed using cDNA microarray and clustered using a hierarchical dendrogram. In general, genes in the 72 h group were more up- or down-regulated than genes in the 24 h group. The gray color indicates that signal intensity was not identified. Three genes magnified in the hierarchical dendrogram were selected for confirmation of their mRNA expression using qRT-PCR.
mRNA expression of Gsα, Gαi2, and Bcl-xL. C6 glioma cells were incubated with 10 µM fluoxetine for 24 h or 72 h and the resulting mRNA from these cells was analyzed by qRT-PCR. A: mRNA levels of Gsα were significantly increased in fluoxetine-treated cells after 72 h compared to control cells. B: mRNA levels of Gαi2 were significantly decreased in fluoxetine-treated cells after 72 h compared to control cells. C: mRNA levels of Bcl-xL were significantly increased in fluoxetine-treated cells after both 24 h and 72 h compared to control cells. Values are expressed as mean±SEM (n=4). *significantly different from control (p<0.05). The data were analyzed by two sample t-test.
The effect of fluoxetine on Bcl-xL protein expression. C6 glioma cells were incubated with 10 µM fluoxetine for 6 h, 24 h, 72 h or 96 h. A: The result of western blotting using anti-Bcl-xL and anti-β-actin antibodies after separating proteins via 12% SDS-PAGE. B: The relative expression density of Bcl-xL in fluoxetine-treated cells compared to the control. The density of Bcl-xL in fluoxetine-treated cells and control cells was normalized with β-actin prior to comparison of Bcl-xL density between fluoxetine-treated cells and control cells. Results are shown as the mean±SEM (n=4). *significantly different from control (p<0.05). The data were analyzed by two sample t-test. SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Primers used in qRT-PCR
Summarized list of time-dependently expressed genes between control and fluoxetine-treated C6 cells
N/I (not identified) shows that signal intensity was not identified. A part of listed genes was already mentioned in our previous study