s2fieldsofstudy: Biology Medicine

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Reversing chemoresistance of malignant glioma stem cells using gold nanoparticles

The low rate of survival for patients diagnosed with glioblastoma may be attributed to the existence of a subpopulation of cancer stem cells. These stem cells have certain properties that enable them to resist chemotherapeutic agents and ionizing radiation. Herein, we show that temozolomide-loaded gold nanostructures are efficient in reducing chemoresistance and destroy 82.7% of cancer stem cells compared with a 42% destruction rate using temozolomide alone. Measurements of in vitro cytotoxicity and apoptosis indicate that combination with gold facilitated the ability of temozolomide, an alkylating drug, to alter the resistance of these cancer stem cells, suggesting a new chemotherapy strategy for patients diagnosed with inoperable recurrent malignant glioma.

Introduction
Malignant gliomas are one of the most common primary tumors of the cerebrospinal axis and may occur in patients of any age, but tend to have two incidence peaks, ie, one at the age of 55-65 years in adults and another at 3-12 years in children. Malignant gliomas are responsible for approximately 11,000 patient deaths per year. 1 The standard of care for patients diagnosed with high-grade malignant glioma includes postoperative temozolomide adjuvant to radiation. However, this therapeutic strategy is associated with considerable toxicity and limited efficacy. The median length of survival for patients with high-grade primary glial tumors ranges from 11 to 33 months after initial diagnosis, with a mean survival time of 7 months following recurrence. 2,3 The failure of current approaches in the treatment of malignant glioma has been attributed to the existence of a subpopulation of cancer cells known as malignant glioma stem cells, which have the ability to withstand chemotherapeutics and ionizing radiation because of their unique properties of high antiapoptotic protein expression, high ABC pump expression, and a remarkable capacity for DNA repair. [4][5][6] Traditional therapies, such as DNA alkylating or methylating drugs, along with radiation, have poor efficacy against these types of cells. Therefore, more complex treatments capable of overcoming the ability of cancer stem cells to eliminate anticancer drugs and with other protective functions are urgently needed. For this reason, a combination of traditional treatments and nanotechnology-based approaches offers attractive possibilities. More efficient and less toxic therapeutics that can cross the protective barriers on cancer stem cells are also necessary. In this context, nanomaterials could play an important role because of their unique electronic, optical, magnetic, and structural properties that are found neither in bulk materials nor in single molecules and which are necessary to develop advanced cancer treatments.
The ability of various gold nanoparticles to cross the blood-brain barrier after intravenous administration has been reported. Particles with the greatest ability to cross the bloodbrain barrier are those in the 15-50 nm size range. 7,8 Herein, we demonstrate binding of temozolomide to the surface of 55 nm gold triangle wires and uptake of the drug in the first few hours, with progressive accumulation in the perinuclear region of glioma stem cells. This gold nanoparticle vector is a promising approach for increasing the efficacy of various chemotherapies, could increase the active life of temozolomide under physiological conditions, and may also facilitate crossing of the blood-brain barrier, thereby facilitating greater accumulation of the drug in brain cells and tissues.
Recently, gold nanoparticles have developed into an attractive candidate for the specific delivery of complex drugs, molecules, and/or biomolecules (proteins, DNA, and RNA) to their targets. Because of their ability to accumulate selectively in tumor tissue, gold nanoparticles can serve as detectors of cancer cells 9,10 or as targeted drug delivery agents. 11,12 Targeted delivery of anticancer drugs specifically to the tumor site will maximize the efficacy of the drug and minimize collateral damage by reducing systemic toxicity 13 and hence reduce the side effects typically associated with use of cytostatic agents in the treatment of cancer.
It is well known that a number of drugs when combined with gold nanoparticles significantly attenuate toxicity in various tumor cell lines. Several articles describing nanoparticle conjugations and their effects on different cancer types have been published. 14,15 Kahn et al have studied decoration of gold nanoparticles with anti-epidermal growth factor receptor in nasopharyngeal carcinoma (CNE2) and normal human lung fibroblast cells. 16 Localization of gold nanoparticles through confocal imaging increases the reflectance characteristics of the CNE2 cells, and the regions with higher reflectance values, represent areas with high epidermal growth factor receptor expression in cells. GNPs have also been conjugated with anticancer drugs, such as methotrexate and gemcitabine-epidermal growth factor receptor. 17,18 In vitro tests have demonstrated that these gold nanoparticle conjugates induce accumulation of methotrexate at higher rates and in higher concentrations in tumor cells, and inhibit proliferation of gemcitabine-treated pancreatic tumor cells. Methotrexate-loaded gold nanoparticles have been shown to achieve a higher cytotoxic response in various cancer cell lines when compared to similar doses of free methotrexate. Further, Liu has shown that gold nanoparticles modified with polyethylene glycol can induce toxicity in CT26 tumor cells exposed to irradiation. 19 The novel properties of nanomaterials need to be explored in order to improve traditional oncology management technologies and to develop high-profile treatments with low overall toxicity to the organism and high efficacy in targeting and destroying cancer stem cells. In the present study, we aimed to synthesize a drug delivery vector, by physically coupling an anticancer drug, temozolomide, to the surface of gold nanostructures treated with L-aspartate (GNP-Laspartate-TMZ) and to test their ability to kill malignant glioma-derived cancer stem cells. [20][21][22] The carboxyl group from L-aspartate reduced Au 3+ from tetrachloroauric acid (HAuCl 4 ) salt to Au, and the amine group capped the nanoparticles and organized them into high order GNP-L-aspartate nanostructures. Recently, Kora et al demonstrated that functional groups, such as COOH and OH from gum ghatti, have the ability to reduce silver nanoparticles. 23 Temozolomide was conjugated with gold nanoparticles in order to obtain the GNP-L-aspartate-TMZ delivery vector of interest. The morphological, optical, and structural characteristics of the vector were then investigated by transmission electron microscopy (TEM), along with Fourier transform infrared spectroscopy (FTIR), and x-ray photoelectron spectroscopy (XPS).
The in vitro effect of the GNP-L-aspartate-TMZ vector on malignant glioma stem cells 20 was tested using MTT proliferation and Annexin V/propidium iodide apoptosis assays and compared with the effect of temozolomide alone. Our results indicated that the GNP-L-aspartate-TMZ system produced highly statistically significant results. The conjugated drug appeared to be taken up by the cells in a pattern very different from that of unbound temozolomide, as visualized by optical microscopy. By binding the gold structure to the drug, we improved the treatment of World Health Organization grade III and IV tumors in the central nervous system owing to the increased efficacy of the newly formed compound. The dimer formed by the gold nanoparticles and temozolomide was shown to be more readily absorbed intracellularly by cancerous tissue than normal tissue because of the surrounding edema and increased neovascularization. It also had increased anticancer properties in comparison with the current standard of care.

Materials and methods
GNP-L-aspartate nanostructure preparation Aldrich Inc, St Louis, MO, USA). Pure temozolomide was obtained from Sigma-Aldrich (Munich, Germany). HAuCl 4 and L-aspartic acid solutions were prepared in concentrations of 0.5 × 10 −3 M and 1.5 × 10 −3 M, respectively. We slowly reduced a 50 mL solution of HAuCl 4 at room temperature by adding 15 mL of aspartic acid solution. During the reaction, three color changes were observed, ie, slightly pink, red, and, finally, after 9 hours, a persistent violet color. This indicates a complex reduction mechanism involving formation of small particles that act as nuclei for growth of triangular nanostructures on their {111} facets. The resulting product was then centrifuged at 15,000 rpm for 45 minutes and washed with Milli Q water (Millipore, Bedford, MA, USA). The purified nanostructures were redispersed in phosphate-buffered saline to give a final concentration of 2 × 10 −9 M. The purified aspartate nanostructures (GNP-L-aspartate) were analyzed by TEM, FTIR, UV-Vis and XPS spectroscopy.

Conjugation of temozolomide with gold nanostructures
The simple method described below provides highly monodispersed GNP-L-aspartate nanostructures that are required as intermediates in the preparation of the target delivery vector, GNP-L-aspartate-TMZ. First, 200 µL of temozolomide (50 µL/mL) was added to the GNP-L-aspartate nanostructure solution (10 mL, 2 nM) and stirred for one hour. The resulting vector (GNP-L-aspartate-TMZ) was analyzed by TEM, FTIR, UV-Vis and XPS spectroscopy.

Analytical characterization of multicomponent nanosystems
The optical properties of the GNP-L-aspartate nanostructures and GNP-L-aspartate-TMZ vector were monitored using an ultraviolet visible spectrophotometer (JASCO V-570, Jasco Inc, Easton, MD). The morphology of the GNP-L-aspartate and GNP-L-aspartate-TMZ was investigated by TEM under conventional beam conditions. Next, 30 µL droplets of suspension were pipetted on copper grids (3 mm diameter, 300 meshes) previously covered with parlodion and carbon films. After 2 minutes, the liquid was absorbed with filter paper. Examination of the grids was performed using TEM (JEM 1010, JEOL, Tokyo, Japan). The images were captured using a Mega VIEW III camera (Olympus, Soft Imaging System, Münster, Germany) and entered into a database using Soft Imaging System software (Münster, Germany). The diameter of the nanoparticles was analyzed using CellD software (Olympus Soft Imaging Solutions GMBH). Statistical analyses (mean value, standard deviation, and Student's t-test) were performed using Microsoft Office Excel software (Microsoft Corporation, Redmond, WA, USA). Fourier transform infrared spectroscopy measurements were performed with a JASCO 6100 spectrometer in the 4000-500 cm −1 spectral region with a resolution of 4 cm −1 using the KBr pellet technique. X-ray photoelectron spectroscopy associated with argon ion etching was used for qualitative and quantitative analysis of the nanocomposites using a SPECS custom-built system. Excitation was produced using the aluminum anode of the x-ray source (hυ = 1486.6 eV).

Cancer stem cells
The gold standard assay to determine whether a stem cell is or is not a cancer stem cell involves a series of transplantations in animal models. Potential surface markers of cancer stem cells include CD133, aldehyde dehydrogenase 1, CD44, and CD24. Efflux of Hoechst or rhodamine dyes (also referred to as side populations) have also been employed for the identification of putative cancer stem cells. Still, these markers have significant limitations given the fact that they fail to identify all cancer stem cells and mostly recognize a cell subpopulation with increased clonogenic and tumorigenic activity. Moreover, not all cells presenting cancer stem cell marker phenotypes present the same behavior as the cancer stem cells. Most markers for the selection of cancer stem cells are chosen because of being expressed in normal stem cells and, most interestingly, there are quite a few molecules which are commonly expressed in both normal and cancer stem cells and which guide to various phenomena in dependency to the particular environment. 24 The cancer stem cells used in this study were isolated from a glioblastoma multiforme biopsy as previously described. 20 Briefly, after mechanical dissociation of the tumor tissue, the fragments were placed in 1 mL of fetal calf serum. After 3 hours, 3 mL of Dulbecco's modified Eagle's medium/F-12 medium supplemented with 15% fetal calf serum was added to the dish. After reaching a subconfluent monolayer, the cells were detached using trypsinethylenediamine tetra-acetic acid (Merck) and resuspended in a serum-free medium, ie, Dulbecco's modified Eagle's medium/F-12 (1:1) medium supplemented with 15 ng/mL basic fibroblast growth factor, 20 ng/mL epidermal growth factor, 2 mM/L L-glutamine, 4 U/L insulin growth factor-1, and B-27 supplement 1:50 (Sigma Aldrich). Isolated and expanded cells revealed some stem cell-specific features, including expression of cellular markers (CD133, CD105, CD90, Nanog, Oct 3/4), immunocytochemistry expression of specific genes, ie, CXCR4, nestin, glial fibrillary acidic submit your manuscript | www.dovepress.com Dovepress Dovepress protein, and neurofilament protein (reverse transcriptase polymerase chain reaction). The cells also displayed a high proliferative potential despite chemotherapy and irradiation, and also had the ability to form spheroids in suspension.
All the studies were done by using exponentially growing cells with a doubling time of approximately 24 hours. For the passage, the medium was discarded and cells were washed with phosphate-buffered solution and afterwards detached with trypsin-ethylenediamine tetra-acetic acid 0.25%.
Cell survival was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, for which cells in a monolayer culture were incubated in complete medium before being washed twice with phosphatebuffered solution. Cells were then incubated with trypsinethylenediamine tetra-acetic acid, resuspended in culture medium with fetal calf serum, counted, and plated in 100 µL of medium at 15 × 10 3 cells/well in 96-well microtiter plates. After 24 hours, the cells were washed and treated with temozolomide alone or GNP-L-aspartate-TMZ. Temozolomide was added at a concentration of 5 µg/mL. The MTT activity of the GNP-L-aspartate-TMZ was compared with that of temozolomide in identical concentrations. Absorbance of the MTT was measured at 492 nm using a fluorescence microplate reader (Synergy 2, BioTek, Winooski, VT, USA).

Annexin V/propidium iodide staining apoptosis assay
Glioblastoma-derived stem cells were cultivated at subconfluence in complete medium in 6 cm Petri dishes and were exposed for 24 hours to temozolomide at a concentration of 5 µg/mL or to GNP-L-aspartate-TMZ. The controls were untreated cells and cells treated with GNP-L-aspartate alone.
Apoptosis was evaluated using flow cytometry and optical microscopy by staining with a FITC Annexin V and propidium iodide kit (Invitrogen-Molecular Probes, Carlsbad, CA, USA). After 24 hours of drug exposure, the cells were harvested by trypsinization and washed with cold phosphate-buffered solution and then resuspended in 100 µL of binding buffer. Next, 5 µL of FITC Annexin V and 1 µL of propidium iodide was added to each sample. The mixture was then incubated at room temperature in the dark for 15 minutes. Early and late apoptotic cells were identified using a BD FACSCanto™ II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) measuring fluorescence emission at 530 nm and 575 nm with 488 nm excitation. A similar protocol was used for staining adherent cells, and the samples were examined using an inverted phase microscope with a 488 nm fluorescence filter (Axiovert, Zeiss, Oberkochen, Germany). Image acquisition was performed using an AxioCam MRC camera, and cell counting of positive cells was performed using Axiovision Rel 4.6 image analysis software. Cells positive for Annexin V only are in an early stage of apoptosis, and cells positive for both Annexin V and propidium iodide are either dead or in a late stage of apoptosis.

Statistical analysis
Statistically significant values were obtained by using oneway variance analysis and a confidence level of 95% with a GraphPad Prism 5 statistics program (La Jolla, CA, USA). Data was analyzed with the Bonferroni multiple comparison test (Kruskal-Wallis as nonparametric). Statistical significance was set at P , 0.05, and all experiments were performed in triplicate.

Results and discussion
Synthesis and characterization of GNP-Laspartate-TMZ delivery vector Aspartate-stabilized gold nanoparticles were used for binding of temozolomide in order to form the desired final drug delivery system. Aspartate was used for reduction and stabilization in the synthesis of the nanostructures. Shape, size, and the surface ligand are important parameters with distinctive properties that must be chosen very carefully because they influence cellular binding, incorporation, and/or cytotoxicity. In order to establish the structure of the drug delivery system, we studied the conjugation of temozolomide with the GNP-Laspartate nanostructures. The binding of temozolomide to the surface of GNP-L-aspartate is an important parameter with a significant impact on the overall stability of the nanostructural multicomponent system.
First, the gold nanostructures were synthesized by reducing the gold salt, HAuCl 4  ) were present around the L-aspartate molecules. Nucleation of the gold atoms took place very slowly, with reduction occurring after 9 hours of reaction, and was evident visually by a change of color from pale yellow to pink to red violet. Triangular nanoparticles having a diameter of approximately 55 nm and aspartate as a ligand were synthesized by this method and used as a delivery vector for temozolomide. The resulting GNP-L-aspartate nanostructures were purified by centrifugation and used further in the reaction with temozolomide in order to obtain the desired compound, ie, GNP-L-aspartate. TEM ( Figure 1A) and vibrational spectroscopy ( Figure S1) were used to characterize the structures. Further, these nanostructures were used as building blocks for formation of the delivery vector, GNP-L-aspartate. Temozolomide was conjugated with GNP-L-aspartate by formation of electrostatic bonds between temozolomide and the aspartate molecules. Attachment was confirmed by TEM, vibrational spectroscopy, and x-ray photoelectron spectroscopy. TEM analysis along with diameter histograms are shown in Figure 1A-C. The images confirmed formation of the nanostructures and their triangular shape. The length of the chains ranged from 100 nm to 600 nm, with the majority in the range of 600 nm, and a mean diameter of 55 nm (see Figure 1A). A typical image showing the drug delivery system is seen in Figure 1B. A thin shell of temozolomide was seen to cover the nanostructure surface. It is interesting to note that, after coupling, no secondary effects such as nanoparticle aggregation appeared even one year after their preparation. The presence of temozolomide on the gold surfaces was also demonstrated by ultraviolet-visible spectroscopy ( Figure 1D) and vibrational spectroscopy (Fourier transform infrared, Figure 1E). Ultraviolet visible spectroscopy clearly showed absorption of the drug onto the surfaces of the nanostructure complexes. One peak at around 520 nm corresponds to the gold nanoparticles, and the other two at approximately 300 nm and 350 nm are attributed to aspartic acid and temozolomide, respectively. From the spectrum, we can also see that the drug absorbs to the nanostructure surface until equilibrium is reached. The quantity of drug that results in equilibrium is 200 µL ( Figure 1D).
The Fourier transform infrared spectrum for pure temozolomide has three broad bands at 3339, 3381, and 3526 cm −1 given by the stretching vibration modes of NH 2 and OH − , while the two bands at around 2935 cm −1 and 2899 cm −1 are related to the stretching vibrations of the aliphatic methylene groups. After interaction with gold, two single bands appear in this region, with one at 3389 cm −1 from the stretching vibration of NH 3 + groups and the other at 2934 cm −1 from the vibration of C−H and CH 3. This change is due to electrostatic interaction of these groups with the gold nanoparticles.
In the middle of the spectrum at 1746 cm −1 is a fourmember ring from the ketone band which is similar to that of pure temozolomide but more intense due to the presence of gold in the structure. The amide I is indirectly influenced by the NH 2 -nanostructure linkage and has a peak at 1682 cm −1 , whereas in the pure temozolomide the peak appears at 1672 cm −1 . There are no similarities between the Fourier transform infrared spectrum of pure temozolomide and that of the temozolomide-loaded gold nanoparticle sample (all peaks are rounded and shifted), suggesting that strong electrostatic interactions, such as hydrogen bonds, Van der Waals forces, or other mechanical or halogen bonds, can occur between temozolomide molecules and gold nanostructures.
X-ray photoelectron spectroscopic analysis was utilized to clarify the binding of temozolomide to the gold surface by studying the N 1s and Au 4f core-level lines.
The deconvoluted x-ray photoelectron spectroscopic lines of N 1s core-level 1 for the GNP-L-aspartate sample are presented in Figure 2A, while Figure 2B shows the deconvolution lines of Au 4f core-levels. For GNP-L-aspartate, the recorded x-ray photoelectron spectroscopic spectrum of N 1s core-level deconvoluted peaks and their assignment are shown in Figure 2C, while the corresponding Au 4f core-level deconvoluted doublet peaks are shown in Figure 2D.
The following lines are identifiable in Figure 2A: a deprotonated N of the amine group bound (attached) to the gold surface at 396.52 eV; an unattached N of the amine group at 401.67 eV; and a deprotonated positive N (not bound) at 402.88 eV. 25,26 Peak positions, line widths, and quantified area are summarized in Table S1.
Quantification of the data was done by taking into account real sensitivity, transmission, and mean free path factors. The molar ratio of aspartic acid to gold nanoparticles was found to be 0.22 by taking into account the attenuation lengths for L-aspartic acid (about 2.1 nm) and gold nanoparticles (1 nm). These values were calculated following the method of Cumpson and Seah. 27 The x-ray photoelectron spectra for GNP-L-aspartate-TMZ are presented in Figure 2C and D for N 1s core-level 1 and Au 4f core-levels, respectively. Comparing the x-ray photoelectron spectrum of the N 1s core-level from GNP-L-aspartate (Figure 2A) with the corresponding x-ray photoelectron spectrum from GNP-L-aspartate-TMZ ( Figure 2C), we can see that the intensities of the peaks and the positions of the spectra have changed. These data led us to conclude that the drug had bound to the surface of the nanostructured gold by forming electrostatic bonds.
The molar ratio value obtained for GNP-L-aspartate-TMZ was found to be 0.047. For its calculation, we took into consideration the molar ratio value of aspartic acid to gold nanoparticles (0.22) and the integrated intensity values. We also used the lengths corresponding to the total integrated intensity of Au 4f and N 1s.

Testing efficacy of GNP-L-aspartate-TMZ in glioblastoma stem cells
The resemblances of the self-renewal mechanisms of the cancer and stem cells, have resulted in the new concept of cancer stem cells. Over the course of the past 15 years, there has been increasing evidence to support the hypothesis of cancer stem cells, which indicates that these cells might be responsible for initiation, of tumors, metastasis, and their complex resistance to treatment. Therefore, it could be possible that a tumor might have its origins in the cancer stem cells, which could be originated from modified stem cells or progenitor cells with re-obtained self-renewal activity. 28 These rare cancer stem cells could play a major role in the control and treatment of this disease: due to asymmetric division, tumor development along with evade therapy given the characteristics shared by normal stem cells, that include quiescence, self-renewal capacity, and drug resistance through complex pump activity. 29 These cells were first identified in hematologic cancers, but have been recently isolated from submit your manuscript | www.dovepress.com Dovepress Dovepress solid tumors. Cancer stem cells are tumor-initiating cells in immunocompromised mice, and have the ability to generate heterogeneous cancer cell populations. 30,31 Based on these considerations, our goal was to use the properties of nanomaterials to evade activation of the complex self-renewal mechanism in cancer stem cells. Our study revealed that, by using conjugated nanomaterials, the rate of cell killing can be increased by up to 50% compared with the rate achieved by the anticancer drug alone. Our new synthesized GNP-L-aspartate compound was incubated for 24 hours with glioblastoma stem cells in vitro. In parallel, temozolomide was also incubated with a second cell line for use as a control. After 24 hours of internalization, we noticed that the GNP-L-aspartate-TMZ induced preapoptosis, leading to cell death. The number of viable cells decreased dramatically when exposed to GNP-L-aspartate-TMZ in comparison with exposure to the drug alone. The mechanism of uptake of the system into glioblastoma stem cells and their destruction is depicted in Figure 3.

MTT cytotoxicity assay
The interaction between gold nanoparticles and various cancer cell lines has been intensively studied, but very little is known about how these nanomaterials interact with derived stem cells. Generally, gold nanoparticles have been reported to have good biocompatibility with nonstem cells, but with effects dependent upon the particular cell line used in the experiment. [32][33][34] Moreover, the surface chemistry, 35 size, 35 and concentration have been shown to affect the internalization mechanism for such nanomaterials. 36 The surface modifiers used to stabilize gold nanoparticles include a range of anionic, cationic, and neutral groups, such as citrate, amine, and glucose. Thus, it has been concluded that the interaction depends upon how the biological cells respond to foreign materials in general, ie, the basic mechanism of incorporation, degradation, or bioaccumulation causing cell damage and changes in gene expression. 37,38 A very common method of evaluating the effects of different substances in cell cultures is the MTT assay, which is used to measure the mitochondrial function of cells in vitro ( Figure 4A). Herein, glioma cancer stem cells were found to interact strongly with our gold nanoparticle-based drug delivery vectors. Phase contrast microscopy showed that GNP-L-aspartate was internalized within the first hour (data not shown here). After 24 hours, the drug delivery vector affected the number of cells, their rate of proliferation, and their shape, suggesting that the cells entered a preapoptotic stage, as shown in Figure 4B. Hence, cells cultivated in the presence of the nonconjugated drug were not yet preapoptotic ( Figure 4C). Glioma-derived stem cells were initially resistant to temozolomide but, when GNP-L-aspartate-TMZ was added to the culture medium, the results showed a reduction in the rate of survival of the tumor cells.
Using Bonferroni's multiple comparison test, we found statistically significant (P < 0.05) decrease in proliferation for cells treated with the conjugated vector, GNP-L-aspartate-TMZ, compared with the cells treated with TMZ alone (95% confidence interval 0.09214-0.5345). The killing efficiency of the GNP-L-aspartate-TMZ system in glioblastoma-derived stem cells is approximately 50% higher compared with that of the alkaline drug, temozolomide, used alone. When one or several initiating genetic changes appeared at the progenitor level, all of the downstream cells continued this change. In one particular case, it is possible that a daughter cell acquired not only the characteristics of the stem cell, but also underwent additional alterations that allowed the glioma to progress further and invade adjacent tissues. Due to their small population of glioblastoma-derived stem cells, malignant gliomas have a negative response to various conventional treatments. 39

Flow cytometry apoptosis test
Annexin V binding to the cell membrane by conjugation of FITC was measured to identify and quantify apoptotic cells at the single-cell level by flow cytometry or fluorescence microscopy. Staining cells simultaneously with FITC-Annexin V (green fluorescence) and the nonvital dye, propidium iodide (red fluorescence) enables (bivariate analysis) to distinguish between intact cells (FITC negative, propidium iodide negative), early apoptotic (FITC positive, propidium iodide negative), and late apoptotic or necrotic cells (FITC positive, propidium iodide positive). We performed the apoptotic tests using the flow cytometry method and fluorescence microscopy.  Analyzing the results of the flow cytometry apoptosis assay, we observed a slight increase in apoptosis among cells treated with gold nanoparticles (apoptotic cells represent 10.7% of total cells by comparison with the level of 0.8% found in control cells), a fact that was not found in the microscopy apoptosis test (0.9% versus 1.24% for control cells). This observation might be explained by the mechanical stress affecting the cells loaded with nanoparticles during the centrifugation step for the flow cytometry method, a step which was not included in the microscopy test. Both apoptosis tests revealed an increase in apoptosis among cells exposed to gold nanoparticles conjugated with temozolomide. We noticed a dramatic enhancement of late apoptosis in the flow cytometry studies by 78.3%, represents a 1.9-fold increase within 24 hours compared with the rate of apoptosis in cells treated with temozolomide alone (31.1%). Similar results were obtained for the microscopic analysis, with the observation that the apoptotic index of cells treated with temozolomide-conjugated with gold nanoparticles was 3.41 times higher than that of cells treated with temozolomide alone (Table 1). Treatment with GNP-L-aspartate-TMZ induced translocation of plasma membrane phosphatidylserine as an early apoptosis event in cancer stem cells, and binding of Annexin V-FITC was visualized by fluorescence microscopy as shown in Figure 5. However, these cells retained a morphology and proliferation rate close to that observed in the control group. GNP-conjugated temozolomide-treated cells showed positivity for Annexin V-FITC, a rounded shape, and a decrease in cell numbers and adhesion (see Figure 5).
Our proposed nanoscale drug delivery system offers a promising chemotherapeutic strategy for patients diagnosed with unresectable recurrent malignant glioma. Current therapies are not yet curative, because cancer stem cells may survive as a result of the increased efflux of chemotherapy agents due to ABCG2 cell membrane proteins and enhanced DNA repair. 40 Multiple proteins of the ATP-binding cassette superfamily are being overexpressed by the cancer stem cells and produce a side population, with the capacity for enhanced efflux of the fluorescent dyes (Hoechst 33342 or rhodamine 123) and which are transported by the same ABC family proteins. 41 Because therapy-resistant, high-grade gliomas are one of the common causes of death due to cancer, and as a result, there is an urgent need for identification of alternative targeted and more efficient therapies. Such is the case with specific drug-delivery systems using nanoparticles. Further combination of various approaches to killing of individual cancer cells, such as photothermolysis 42,43 and radiofrequency-induced heating, 44,45 which combine more accurate drug release with additional biological damage produced by heated nanomaterials, could further increase the ability of nanostructured materials to serve as the foundation for more sophisticated approaches to cancer treatment.

Conclusion
Cancer cells are very complex biological structures that perform a variety of functions, ranging from invasion or metastasis to elimination of anticancer drugs from the cell membrane. Although the exact mechanisms need to be explored further, combining advances in fundamental oncology and nanotechnology offers the opportunity to impact future diagnostics and therapeutics in a significant way. We have shown that drug delivery vectors based on gold nanoparticles have the ability to deliver temozolomide, a cytostatic drug, to treat unresectable recurrent malignant glioma. Our studies have also shown that a novel drug delivery vector based on gold nanoparticles has low toxicity and the ability to internalize temozolomide. MTT and flow cytometry studies have clearly indicated the high bioactivity of the GNP-L-aspartate-TMZ nanostructure system, with the ability to induce apoptosis in almost 90% of high-grade glioma-derived cancer stem cells. Temozolomide alone induced death in only 42% of the cells. Our results indicate synergistic enhancement of the apoptosis-inducing activity of GNP-L-aspartate-TMZ vectors compared with GNP-L-aspartate or temozolomide alone. This approach could constitute a novel method for treating incurable recurrent malignant gliomas, and could be developed further for other types of cancer models, and potentially with great success.

Supplementary information
Fourier transform infrared spectra of pure temozolomide versus GNP-Laspartate and GNP-L-aspartate-TMZ The C−H stretching vibration groups at around 2750 cm −1 disappear when the drug is coupled with the nanostructures, and only one peak appears in the spectra. This explains the interaction of the drug with the gold surface. This is due to the chain length of the amino acids and its molecules which have a parallel orientation on the gold surface. These molecules pack into densities sufficient to form high-quality barriers for both electron and ion transfer processes. 46 Also, from the other regions of the Fourier transform infrared spectra, it is clear that the drug is linked directly to the gold surface. We are not denying the existence of aspartic acid in the molecules because there may be some interaction. Please see the other two regions of the Fourier transform infrared spectra ( Figure S1A-C).
Fourier transform infrared spectra of GNP-L-aspartate vector In the middle of the spectrum (2000 cm −1 ), we can see only a shoulder that could be related to a carbonyl group, the bending submit your manuscript | www.dovepress.com

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vibration of water at approximately 1650 cm −1 , and the amide and C−C double bond around 1500 cm −1 . These peaks are shifted to the right and their height is smaller compared with the pure amino acid. The bending deformation vibrations of C−H (methyl group) are given by a very weak peak at 1425 cm −1 (again because of their orientation parallel to the gold surface). As can be seen in Figure S2A and B, there is no similarity between the infrared spectrum of pure amino acid and the one that is bound to the gold nanoparticle surface on the gold nanoparticle-amino acid sample, suggesting that the L-aspartate is bound to the surface of the gold nanoparticles, most likely through the amino groups from the molecule. The International Journal of Nanomedicine is an international, peerreviewed journal focusing on the application of nanotechnology in diagnostics, therapeutics, and drug delivery systems throughout the biomedical field. This journal is indexed on PubMed Central, MedLine, CAS, SciSearch®, Current Contents®/Clinical Medicine, Journal Citation Reports/Science Edition, EMBase, Scopus and the Elsevier Bibliographic databases. The manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. Visit http://www.dovepress.com/ testimonials.php to read real quotes from published authors.


extfieldsofstudy: Medicine Biology

text:
Liver-derived endocrine IGF-I is not critical for activation of skeletal muscle protein synthesis following oral feeding

Background Insulin-like growth factor-1 (IGF-1) is produced in various tissues to stimulate protein synthesis under different conditions. It is however, difficult to distinguish effects by locally produced IGF-1 compared to liver-derived IGF-1 appearing in the circulation. In the present study the role of liver-derived endocrine IGF-I for activation of skeletal muscle protein synthesis following feeding was evaluated. Results Transgenic female mice with selective knockout of the IGF-I gene in hepatocytes were freely fed, starved overnight and subsequently refed for 3 hours and compared to wild types (wt). Liver IGF-I knockout mice had 70% reduced plasma IGF-I. Starvation decreased and refeeding increased muscle protein synthesis (p < 0.01), similarly in both IGF-I knockouts and wt mice. Phosphorylation of p70s6k and mTOR increased and 4EBP1 bound to eIF4E decreased in both IGF-I knockouts and wt mice after refeeding (p < 0.05). Muscle transcripts of IGF-I decreased and IGF-I receptor increased (p < 0.01) in wild types during starvation but similar alterations did not reach significance in knockouts (p>0.05). mTOR mRNA increased in knockouts only during starvation. Plasma glucose decreased during starvation in all groups in parallel to insulin, while plasma IGF-I and GH did not change significantly among the groups during starvation-refeeding. Plasma amino acids declined and increased during starvation-refeeding in wild type mice (p < 0.05), but less so in IGF-I (−/−) knockouts (p < 0.08). Conclusion This study demonstrates that re-synthesis of muscle proteins following starvation is not critically dependent on endocrine liver-derived IGF-I.

Background
Protein synthesis is rapidly increased in skeletal muscles after oral feeding due to intracellular signaling for activation of initiation or protein translation [1], which is quantitatively the most important alteration in the control of short term protein balance in skeletal muscles [2,3]. Meal feeding is a complex stimuli where nutrients are provided and appear simultaneously with changes in circulating levels of hormones and substrates as well as hormone binding proteins. Our earlier studies have suggested that amino acids are important substrate factor(s) behind postfeeding activation of muscle protein synthesis, whereas insulin seems rather permissive [4,5]. On the other hand, it can be expected that insulin-like growth factor I (IGF-I) should be involved since provision of anti IGF-I antibodies before meal feeding attenuated a subsequent rise in protein synthesis [5], besides its well-recognized effects on muscle cell proliferation and regeneration [6][7][8]. Furthermore, blood levels and muscle expression of IGF-I are significantly changed during starvation and feeding [5,9]. The role of endocrine liver-derived and locally tissue produced IGF-I to promote protein synthesis in response to feeding is however unclear, in part due to complex conditions with different IGF-binding proteins, which may also have independent control functions of net protein metabolism at tissue levels [10][11][12]. This complexity makes it uncertain to define roles of locally produced versus endocrine liverderived IGF-I for activation of protein translation and synthesis in skeletal muscle tissue following feeding [13,14]. The aim of the present study was therefore to evaluate the role of endocrine liver-derived IGF-I for activation of translation initiation of muscle proteins at refeeding in a transgenic mouse model with selectively and conditionally deleted liver IGF-I [15]. This deletion caused levels of circulating IGF-I to be reduced by 70% compared to wild type controls in combination with normal muscle expression of IGF-I [15].

Animals
Transgenic female mice (C57BL/6) with inducible inactivation of the Insulin-like growth factor-1 gene constructed with the Cre/LoxP system were used. Animals were bred as described earlier [15]. Recombination was induced by polyinosinic-polycytidylic acid (PiPc) treatment at 10-12 weeks of age in mice homozygous for loxP and heterozygous for Mx-Cre, [15,16]. These mice are referred to as liver IGF-I knockouts, Li-IGF-I (−/−) . PiPc treated siblings, homozygous for LoxP but lacking Mx-Cre, were used as controls and referred to as wild type animals, WT (+/+) . Adult weight stable Li-IGF-I (−/−) mice housed with 5 animals per cage, matured normally with normal body composition as compared to our previous experiments on partially IGF-I knockouts (+/−) [9]. Body weight was 22.2 ±0.1 g in knockouts and 23.8±0.10 g in wild types at the start of experiments, in 5 months old mice; which is before any alterations in body composition in knockouts [17]. Male animals were not used since they must be housed individually in this kind of experiment; a condition which is associated with stress reactions and adaptations. However, females are subjected to hormone alterations during the estrous cycle, which could theoretically induce alterations in muscle protein metabolism. However, comparisons of rates of muscle protein synthesis between phases of the menstrual cycle are limited. Miller et.al. found no effect of the menstrual cycle phases in human females [18], while Toth et.al. reported minor changes in the gastrocnemius fractional synthesis rate related to estradiol or progesterone replacement therapy in ovariectomized rats [19]. Therefore, all our experiments were carried out with mice randomly divided across cages housed simultaneously and close together, in order to promote estrous synchronization within and across cages.
Liver IGF-I knockouts and wild type mice were used across the groups; freely fed, starved and refed animals. Freely fed animals had continuous access to water ad lib. and standard rodent chow (2016 Global Tekland®, Netherlands). Starved mice had no access to food overnight for 12 hours before experiments, while refed animals were similarly starved overnight for 12 hours and had then free access to food during 3 hours before termination according to previous findings [4,9,20]. Animals were killed by cervical dislocation and blood samples were drawn by cardiac puncture. Mixed hind limb muscles from both legs were excised. Muscles from one leg were used for measurements of fractional protein synthesis and protein measurements. Muscle tissue from the other leg was used for RNA extraction and RNA expression analyses.
All animal procedures were performed in accordance to Swedish law (Animal welfare act, 1988:538) and national guidelines for care and use of research animals (DFS 2004:4) and approved by the regional ethics committee for animal research in Gothenburg (450-2008).

RNA expression
Hind limb mixed muscle tissue were snap-frozen in liquid nitrogen and kept in −70°C until analysis. RNA was extracted using RNeasy fibrous tissue kit (Qiagen) with DNAse step included. Total RNA concentrations were measured by spectrophotometer (Nanodrop ND-100) and RNA quality was checked by calculating the 18S/ 28S ratio using an Agilent 2100 bioanalyser. One μg of total RNA was reverse transcribed using oligo d(T)-primer according to kit instructions (Advantage RT for PCR kit,) for cDNA synthesis. Positive and negative controls were included in each run of cDNA synthesis. Realtime PCR was performed with a LightCycler 1.5 instrument (Roche) using the LightCycler FastStart DNA Master PLUS SYBR Green 1 kit. For analysis of Igf-1, 2 μl cDNA and 10 pmol of forward (GCT CTT CAG TTC GTG TGT GGA C) and reverse (CAT CTC CAG TCT CCT CAG ATC) primers were used to each reaction of 20 μl. PCR were performed with the following settings; denaturation 95°C for 10 sec., annealing 64°C, 4 sec. and extension 72°C, 6 sec. For analysis of Igf-1r (Quantitect Primer assay Nr QT00155351, Qiagen), 2 μl cDNA and 2 μl of premixed Quantitect primers were used for each reaction of 20 μl. PCR were performed with the following settings; denaturation 95°C for 10 sec., annealing 60°C, 6 sec. and extension 72°C, 5 sec. For analysis of Akt (QT00114401), mTOR (QT01532916) and PI3-kinase (QT00149709), 2 μl cDNA and 2 μl of premixed Quantitect primers were used for each reaction of 20 μl. Real time PCR were performed using QuantiFast™SYBR Green PCR kit (Qiagen). Quantitative results were produced by the relative standard curve method. All samples were analyzed in duplicates and negative controls were included in each run. All results are related to the expression of Gapdh as housekeeping gene according to separate evaluations among 12 gene alternatives, where Gapdh showed minimal alterations in response to starvation/refeeding with 8% variation among all the groups at extreme conditions as starvation/refeeding (0.15<p>0.80).

Protein synthesis
Fractional liver and skeletal muscle protein synthesis were measured by the flooding dose technique as described [21,22]. A single dose injection of L-[U-14 C] phenylalanine (0.4 μCi/g) in 150 mM phenylalanine was provided ip. 30 minutes before killing. Liver tissue and mixed hind limb muscles were rapidly excised and frozen in liquid nitrogen until analysis. Blood samples were heparinized and immediately centrifuged at +4°C and plasma was frozen until analysis. Fractional synthesis rate (% × hr -1 ) was calculated as described [9].

Western blotting
Mixed hind limb muscles were rapidly excised and frozen in liquid nitrogen until next day when biopsies were thawed and homogenized in seven volumes of ice-cold buffer A (20 mM Hepes, pH 7.4, 100 mM KCl, 0.2 mM EDTA, 2 mM EGTA, 1 mM DTT, 50 mM NaF, 50 mM β−glycerophosphate, 0.1 mM AEBSF, 1 mM bensamidine, 0.5 mM sodium vanadate). The homogenate was centrifuged at 10 000 × g for 10 min at 4°C. Aliquots of the supernatant were used for western blot analysis of phosphorylation state of 4EBP1 and p70s6 kinase or immunoprecipitated for analysis of 4EBP1 · eIF4E complexes as previously described [23]. Anti-eIF4E antibodies used were a kind gift from Dr Scot Kimball, Pennsylvania State University, USA. All blot membranes were exposed to Hyperfilm ECL (Amersham Biosciences, UK) and quantification was carried out with Quantity One software (Bio-Rad Laboratories AB, Sundbyberg, Sweden). For quantification of 4EBP1 · eIF4E complex the total optical density was measured and the results were corrected for eIF4E content. Optical density is expressed in arbitrary units. The phosphorylation state of 4EBP1 is expressed as percentage of the most phosphorylated γ-form compared to the total and calculated as optical density of the γ-band/optical density of all bands [(α+β+γ)*100]. Phosphorylation state of p70s6 kinase was measured by gel mobility shift and is expressed as percentage of the least phosphorylated α form compared to the total and calculated as optical density of the α-band/optical density of all bands [(α+β+γ)*100]. For measurement of mTOR and phosphorylated mTOR 2448 , aliquots of the homogenate supernatant were mixed with equal volumes of 2x SDS electrophoresis sample buffer and separated in a 3-8% NuPage Tris-acetate mini gel (Invitrogen). Proteins were transferred to PVDF membranes, which were incubated over night at +4°C with a rabbit anti mouse mTOR 2448 antibody (#2971, Cell Signaling Technology) after blocking in 10% nonfat dry milk in Tris-buffered Saline-Tween 20. The blots were then washed, incubated with secondary antibodies and developed using an ECL Western Blotting Kit according to the manufacturers description (Amersham Biosciences, UK) and exposed against Hyperfilm ECL (Amersham Biosciences, UK). After detection of signals, antibodies were removed by 45 minutes incubation at +50°C in stripping buffer (100 mM 2-mercaptoethanol, 2% sodium dodecyl sulfat (SDS), 62.5 mM Tris-HCl pH 7.6) and membranes were thereafter reprobed for measurement of total mTOR by incubating membranes 75 min at room temperature with a rabbit anti human mTOR antibody (sc-8319, Santa Cruz biotechnology, Santa Cruz, USA) Quantification of signals was carried out with GS-710 imaging densitometer and Quantity One software (Bio-Rad Laboratories AB, Sundbyberg, Sweden). Measured optical density was expressed as arbitrary units. On each gel, 2 lanes were loaded with MagicMark XP Western Protein Standards (Invitrogen). The average optical density for the standard band with most similar molecular size as the measured protein was used to normalize signal intensity between blots.

Western ligand blotting
IGF-I ligand blotting was performed under conditions described by Hosssenlopp et.al. [24]. Equal volumes from each supernatant with similar amount of proteins of the prepared muscle homogenates in one group were pooled and mixed with an equal volume of Laemmli sample buffer and heated to 95°C. 15 μl of the samples were separated by electrophoresis on 12% Tris-Glycine gels using non-reduced conditions and transferred to PVDF membranes. Membranes were blocked and incubated overnight with 125 I-labelled IGF-I (PerkinElmer). After washing, blots were exposed to Hyperfilm MP at −70°C. Films were scanned and band intensity was measured (Quantity One software, GS-710 densitometer, BioRad). The molecular weight of IGFBPs was estimated from prestained standards.

Plasma concentrations
Plasma IGF-I was measured by an IGF-I binding protein blocked RIA (Mediagnost). Glucose was measured with the glucose oxidase method according to the manufacturers' instructions (Roche). Plasma amino acids were analyzed by HPLC as described elsewhere [25,26], in order to relate alterations in Igf-I/Igf-Ir transcript expression to changes in plasma amino acids, which may be stimulators of muscle protein synthesis [20,27]. Growth hormone (GH) and insulin were measured with ELISA methods (Linco research). Plasma IGFBPs were not measured since IGFBPs were estimated in the muscle tissue compartment.

Statistics
Results are presented as mean ± SEM based on 8 animals in each group except for 7 mice in starved wild type and 9 in Li-IGF-I (−/−) . Factorial ANOVA was used to test within groups and among groups followed by Fisher PLSD test for multiple comparisons. P<0.05 was considered statistically significant and p < 0.10 a trend to significance in two tailed tests.

Plasma concentrations
Plasma levels of IGF-I were reduced by 70% in liver IGF-I knockout mice (Li-IGF-I (−/−) ) compared to wild type mice at all nutritional conditions (<0.001), while glucose and insulin levels were comparable among Li-IGF-I (−/−) and wild type mice. Overnight starvation caused a significant decrease in plasma glucose compared to freely fed mice in both knockout and wt mice (p<0.05) ( Table 1). Growth hormone did not change significantly within groups during freely feeding, starvation and refeeding and it was not significantly altered when comparing between groups. Branched chain and essential amino acids were significantly altered during starvation-refeeding in wild type mice, but not in liver IGF-I knockouts. These alterations were also reflected in concentrations of the sum of all amino acids (Table 2).

Protein synthesis
The magnitude of basal fractional synthesis rate in liver and muscle tissue was comparable in freely fed wild type and liver IGF-I knockout mice. Refeeding increased similarly liver (p < 0.10) and muscle protein synthesis (p < 0.01) compared to starvation in both wild type and liver IGF-I knockout animals ( Figure 1A,B).

RNA transcripts in skeletal muscles
mRNA transcript levels were comparable in freely fed wild type and knockout mice (Igf-I, Igf-Ireceptor, PI3kinase, Akt and mTOR). However, Igf-I transcripts decreased significantly in muscles from wild type mice during starvation but with a different time course in knockouts. Igf-Ir increased similarly during starvation in both wild type and knockout mice and remained increased during refeeding (Figure 2A . By contrast, mTOR expression was significantly increased in starved knockout animals compared to refed mice without any similar change in wild type mice ( Figure 2C). However, this increased transcript level did not correspond to altered mTOR protein levels among animal groups (not shown).
Translational and cell signaling proteins in skeletal muscle tissue The 4EBP1 · eIF4E complex, 4EBP1 phosphorylation state (% γ of total), p70s6k phosphorylation (% α of total) and mTOR posporylation (p-mTOR ser2448 /mTOR total ) were comparable among wild type and liver IGF-I knockout mice. Starvation significantly increased muscle content of 4EBP1 · eIF4E complexes with corresponding normalization in refed animals ( Figure 3A). The 4EBP1 phosphorylation state was significantly and similarly decreased in starved mice compared to freely fed mice with significant increase in refed wild type and liver IGF-I knockout mice ( Figure 3B). p70s6k and mTOR 2448 was less phosphorylated in starved wild type and liver IGF-I knockout mice with complete reversal in both groups during refeeding ( Figure 3C,D,E). IGFBP content was similar among all groups ( Figure 4) indicating comparable extracellular conditions of binding proteins among the groups.

Discussion
Anabolic effects by IGF-I are well recognized, particularly such as stimulation of protein synthesis related to cell proliferation, tissue growth and cell differentiation [6,28]. Parts of these effects are probably mediated by muscle tissue specific IGF-I isoforms such as the mechano growth factor [29]. We have earlier assumed that circulating IGF-I is involved in promotion of diurnal alterations of muscle protein synthesis in response to meal feeding [14]; where a similar role may be assumed for insulin [5]. Our earlier studies have, however, not confirmed an important and primary role of insulin in physiologic resynthesis of skeletal   [4], as well as in human skeletal muscles, where insulin only attenuated break down of non-myofibrillar proteins [2]. Therefore, we assumed that endocrine IGF-I may have a role in stimulation of diurnal variations of protein synthesis in skeletal muscles related to feeding with increased flux of substrates and amino acids across cell membranes [30]. A role of IGF-1 was also tempting, when we observed that locally expressed IGF-I decreased in muscle tissue following overnight starvation and increased in response to meal feeding [5]. However, the complex situation, with protein bound circulating IGF-I and locally produced IGF-I, makes   it difficult to demonstrate defined roles for the various IGF-I compartments, particularly when binding proteins themselves may have independent regulatory functions across muscle membranes [11,12]. Therefore, it seemed interesting to apply investigations in liver IGF-I knockout mice, where IGF-I in the circulation is low due to a lack in production of IGF-I in the liver and spleen, in order to reexamine the role of circulating level versus local tissue expression of IGF-I/IGF-IR for alterations in skeletal muscle protein synthesis during starvation-refeeding [5,9].
Earlier attempts to study nutritional effects by IGF-I have not provided unanimous and conclusive results [5,9,31]. We found that injection of anti IGF-I antibodies before feeding of mice attenuated a subsequent rise in protein synthesis by 25%, while ip. injections of IGF-I to overnight starved mice only increased protein synthesis    Figure 3 A-E Effects of starvation-refeeding on total 4E-BP1 · eIF4E complex (A), 4E-BP1 · phosphorylation (B) p70s6kinase phosphorylation (C) and mTOR phosphorylation in skeletal muscle tissue. Figure 3E is a representative blot, one of four, of each analysis. Proteins were immunoprecipitated with a monoclonal anti-eIF4E antibody and thereafter analyzed by Western blot with an antibody against 4E-BP1 in analysis of 4E-BP1 · eIF4E complex. Results are mean ± SEM with 7-9 animals per group. a-p<0.01 vs. freely fed and refed mice. All blot analyses were perfomed on the same protein extracts with the same amount of total protein applied among the groups (not shown).
marginally [5], and studies on genetically altered mice implied that fractional resynthesis of muscle protein was not clear-cut related to circulating GH, blood IGF-I and locally produced IGF-I mRNA [9]. Others have however reported that a one hour iv. infusion of IGF-I to overnight starved mice increased muscle protein synthesis to levels comparable to fed mice [32]. Furthermore, reduced free plasma IGF-I by 50% of normal levels, by infusion of IGF binding protein 1, decreased muscle protein synthesis by 25% with decreased phosphorylation of p70s6 kinase [33], while provision of IGF-I/IGFBP-3 complex improved sepsis induced muscle catabolism [34]. However, plasma IGF-I did not diverge from basal levels in humans who received a drink of essential amino acids at a time when muscle protein synthesis was stimulated by more than 100% [35]. Another study in humans evaluated effects by IGF-I when infused directly into arm muscle bed compared to systemically raised IGF-I [36], where protein synthesis was only increased subsequently to locally infused IGF-I. A study, based on microdialysis, revealed that concentrations of free IGF-I in muscle interstitial fluid were 20 fold higher than simultaneous plasma levels, supporting that circulating IGF-I is not directly determining rates of protein synthesis during feeding [37]; observations in line with our own findings that infused amino acids improved protein balance across human muscles independently of plasma insulin, IGF-I and IGF-I-binding proteins-1 and −3 [3]. A compensatory factor in the present study may be that increased levels of growth hormone (GH) in liver-IGF-I knockouts, observed in the present and a previous study, would counteract subnormal IGF-I levels in the circulation [15] since cell experiments indicate that GH phosphorylates similar or the same downstream targets for protein synthesis as amino acids and IGF-I through PI3-kinase dependent transduction [38]. Also, GH treatment increased fractional muscle protein synthesis in fed but not in starved pigs [39]. However, in a previous study we used mice where circulating levels of both GH and IGF-I were low and found that fractional muscle protein synthesis increased normally to expected levels after food intake [9]. Re-synthesis of muscle protein following starvation-refeeding did not relate to plasma GH concentrations in either knockouts or wild type mice. Therefore, we find it unlikely that GH explains rapid alterations in the feeding related muscle protein synthesis evaluated in the present model. We found no difference between liver IGF-I deficient and wild type mice regarding translational control of muscle protein synthesis. Both liver IGF-I knockouts and wild type mice showed increased phosphorylation of 4EBP1 protein and decreased association of the 4E-BP1 · eIF4E complex. Also, p70s6 kinase phosphorylation was increased similarly in both groups including upstream phosphorylation of mTOR ser2448 . Thus, effects by circulating IGF-I were less critical for the control of translation initiation compared to other possible factors such as plasma or extracellular amino acids, although consistent changes in plasma amino acids were not observed among IGF-I deficient and wild type mice at starvation-refeeding. However, we did not measure eIF4G · eIF4E complexes, which may be influenced by IGF-I as reported by others using a hindlimb perfusion model, where physiologic concentrations of IGF-I (10 nM) in the presence of amino acids were unable to dissociate 4E-BP1 from eIF4E, but increased the assembly of eIF4G-4E complex and stimulated protein synthesis [40].
Both liver IGF-I knockouts and wild type mice showed increased muscle expression of the IGF-IR during starvation, which usually reflects cellular processes related to regeneration and hypertrophy of muscle cells [6]. This increased receptor expression may reflect a positive feedback control of IGF-I signaling secondary to decreased IGF-I protein close to membranes; a conclusion supported by observations in cultured cells on increased IGF-IR number and promoted amino acid transport related to decreased extracellular concentration of amino acids, particularly glutamine [30,41]. Increased transcript and IGF-IR protein levels have also been reported in response to exercise [42][43][44], but information of similar changes in relation to feeding are sparse, although equivalent observations have been reported [45,46]. Thus, IGF-IR expression appears more tightly controlled than tissue IGF-I expression in skeletal muscle metabolism in response to starvationrefeeding. By contrast to feeding, a recent report in mice with non-functional IGF-IR showed that load- induced hypertrophy occurred [47], which suggested that increased protein synthesis following exercise was not entirely dependent on IGF-IR signaling. There were no changes in either PI3K or AKT expression during starvation-refeeding, while mTOR transcript expression was up-regulated in liver-IGF-I knockouts during starvation, but without apparent change in protein phosphorylation. This up-regulation in starved knockouts may therefore reflect compensatory effects unrelated to phosphorylation of mTOR, secondary to consistently decreased circulating IGF-I. If so, it remains to be explored which factor(s) that up-regulates mTOR expression in muscle tissue when endocrine IGF-I is declined, particularly during starvation of knockout mice ( Figure 2C).

Conclusion
In conclusion, our present results confirm previous reports that alteration in muscle protein synthesis during starvation-refeeding relates to alteration in muscle mTOR signaling, but not to Akt and PI3-kinase. Present findings also support our previous conclusions that muscle produced IGF-I/IGF-IR are related to translation initiation of muscle proteins at oral feeding. [5]. New information is that endocrine IGF-I is not, critical for this effect. It remains to determine to what extent local muscle IGF-I signaling is critical, since systemically IGF-I (+/−) knockout mice had normal fractional synthesis rate (%/hrs) in skeletal muscles at refeeding [9]. Whether a lack of effects by liver derived IGF-I may be overcome by alternative signals upstream to mTOR remains also to be determined, but insulin and GH are probably not such candidates.

Competing interests
The authors' declare that they have no competing interests.
Authors' contributions B.I carried out the analyses, calculated statistics, and drafted manuscript, JS CO generated the transgenic mice KL. Conceived of the study and drafted manuscript. All authors participated in study design and critical revision of manuscript. All authors have read and approved the manuscript.


Interleukin-13 +1923C/T Polymorphism Is Associated with Asthma Risk: A Meta-Analysis

There are controversies on the association between interleukin-13 (IL-13) +1923C/T polymorphism (rs1295686) and the risk of asthma. We performed this study to assess the association by the method of meta-analysis. A systematic search current to October 16, 2012, was conducted using PubMed, EMBASE, and China National Knowledge Infrastructure (CNKI) and identified ten studies comprising 13698 cases and 38209 controls. The pooled odds ratios (ORs) with 95% confidence intervals (CIs) were calculated. There was a significant association between IL-13 +1923C/T polymorphism and asthma risk in codominant model. When stratified by ethnicity, IL-13 +1923C/T polymorphism remained significantly associated with higher asthma risk in Asians and Caucasians. In the subgroup analysis by study quality, a significantly increased asthma risk was observed in high quality studies. Sensitivity analysis and cumulative analysis further strengthened the validity of the results. No publication bias was found in this meta-analysis. In conclusion, results from this meta-analysis suggested that IL-13 +1923C/T polymorphism was a risk factor of asthma.

Introduction
The prevalence of asthma has rapidly increased over the last few decades to epidemic proportions and there are approximately 300 million people worldwide [1]. The risk of developing asthma tends to run in families, and heritability of asthma has been estimated as 60% [2]. Thus, host genetic susceptibility may play a crucial role in the pathogenesis of asthma. Until now, many studies have focused on this field, and the interleukin-13 (IL-13) gene has been extensively studied.
IL-13 has been demonstrated to be the central mediator of allergic asthma [3,4]. In asthmatic patients, Huang et al. [5] found that the expression of IL-13 was increased in the allergen-challenged bronchoalveolar lavage (BAL). Similarly, a significant increase in the expression of IL-13 mRNA in BAL cells enriched for alveolar macrophages of the asthmatic patients was observed [6]. Recently, Saha and coworkers suggested that IL-13 overexpression in sputum and bronchial biopsy specimens was a feature of severe asthma [7]. Furthermore, IL-13 expression was related to asthma control and the intensity of eosinophilic inflammation [7]. Collectively, these results indicated that IL-13 might have an important role in the pathophysiology of asthma.
The IL-13 gene is located on chromosome 5q31. Two single nucleotide polymorphisms (SNPs) of IL-13 have been investigated in relation to asthma. One is located in the promoter region at position −1112, the other is a G>A transition at nucleotide +2044 in the coding region of exon 4 [8]. Two meta-analyses assessing associations between these polymorphisms and asthma risk have be published [9,10]. However, the role of IL-13 +1923C/T polymorphism (rs1295686) on risk of asthma was still unknown. Previous studies indicated that IL-13 +1923C/T polymorphism was not associated with susceptibility to asthma [11,12]. By contrast, recent studies suggested that this polymorphism 2 BioMed Research International played a critical role in the development of asthma [13,14]. Therefore, we conducted a meta-analysis of all available casecontrol studies to evaluate the association of IL-13 +1923C/T polymorphism with asthma risk.

Publication Search.
The electronic databases Pubmed, EMBASE, and China National Knowledge Infrastructure (CNKI) were searched using the following terms: (asthma or asthmatic) and (interleukin-13 or interleukin 13 or IL-13 or IL13) and (polymorphism or mutation or variant). Last search was updated in October 16, 2012. No publication date or language restrictions was imposed. We reviewed the bibliographies of all selection articles to identify additional relevant studies.

Inclusion and Exclusion Criteria.
All selected studies complied with the following three criteria: (1) evaluation of the +1923C/T polymorphism in IL-13 gene and asthma risk; (2) using a case-control design; (3) sufficient data for estimating for estimating an odds ratio (OR) and 95% confidence interval (CI).
Studies were excluded if one of the following existed: (1) not relevant to IL-13 +1923C/T polymorphism or asthma risk, (2) nonclinical study, (3) genotype frequencies or number not reported, and (4) reviews and abstracts. For the overlapping studies, the largest or most recent publication was selected.

Data Extraction. Two investigators (Yongan Liu and Tao
Liu) independently extracted the following data from each included study: the first author's name, year of publication, original country, ethnicity, age, atopic status, sample size, genotyping method, and genotype number in cases and controls. We verified accuracy of data by comparing collection forms from each investigator. Agreement was reached after discussion for conflicting data or a third author (Wei Nie) would assess these articles.

Quality
Assessment. The quality of included studies was assessed independently by two investigators (Wei Nie and Guoxiang Lai) using a quality scoring system. Table S1 in Supplementary Material available online at http://dx.doi.org/10.1155/2013/394316 shows the criteria for quality appraisal. The quality scoring system was based on traditional epidemiological considerations and asthma genetic issues [15]. The criteria covered the representativeness of cases and controls, the ascertainment of cases and controls, genotyping examination, Hardy-Weinberg equilibrium (HWE), association assessment, and response rate. Scores were ranged from 0 (worst) to 15 (best). We defined the study with scores >6 was high quality, and the study with scores ≤6 was low quality study.

Statistical
Analysis. When the data from at least three studies were available, a meta-analysis was performed. ORs with 95% CIs were computed to assess the strength of the correlation between the IL-13 +1923C/T polymorphism and asthma risk. The statistical significance of OR was analyzed by Z test. OR1, OR2, and OR3 regarding IL-13 +1923C/T polymorphism were calculated for genotypes TT versus CC, TC versus CC, and TT versus TC, respectively. These pairwise differences were used to indicate the most appropriate genetic model as follows: if OR1 = OR3 ̸ = 1 and OR2 = 1, then a recessive model was suggested; if OR1 = OR2 ̸ = 1 and OR3 = 1, then a dominant model was suggested; if OR2 = 1/OR3 ̸ = 1 and OR1 = 1, then a complete overdominant model was suggested; if OR1 > OR2 > 1 and OR1 > OR3 > 1 (or OR1 < OR2 < 1 and OR1 < OR3 < 1), then a codominant model was suggested [16][17][18]. Once the best genetic model was identified, this model was used to collapse the three genotypes into two groups (except in the case of a codominant model) and to pool the results again.
Departure from HWE in controls was tested by the Chisquare test. The Q statistic was used to test for heterogeneity between the studies, which is considered to be significant for < 0.10. 2 statistics were also used to investigate the degree of heterogeneity among studies. Subgroup analyses were performed by ethnicity and study quality. We defined the subjects from East Asia were Asians. We also defined people from Europe, Northern America, Oceania, North Africa, the Horn of Africa, Western Asia, and Central Asia were Caucasians. Sensitivity analysis was conducted by omitting each study in turn. We also performed cumulative metaanalysis to evaluate the trend of summary ORs (95% CIs) by the year of publication. Egger's test [19] was used to evaluate publication bias.

Sensitivity Analysis and Cumulative Meta-Analysis.
Sensitivity analyses were conducted repeatedly when each particular study was omitted. As shown in Figure 4, the results were not materially altered, with pooled ORs ranging from 1.37 to 1.56 for TT versus CC. Similarly, there was little modification of the estimates after exclusion of individual study, with pooled ORs ranging from 1.19 to 1.25 for TC versus CC ( Figure 5). We also performed cumulative meta-analyses by pooling data again, and each time a study was added. The results showed that the pooled ORs tended to be stable (Figures 6 and 7).

Discussion
Strong experimental evidence demonstrated that IL-13 could direct many of the important features of airway inflammation and remodeling in asthma. Introduction of exogenous IL-13 into murine airways resulted in lymphocytic and eosinophilic inflammation, airway remodeling, and airway hyperresponsiveness (AHR) [3,4]. By contrast, knocking out IL-13 in mice prevented the development of AHR after allergen exposure [26]. Furthermore, using a mouse model of chronic asthma, Yang et al. [27] found that IL-13 antibody significantly suppressed AHR, eosinophil infiltration, proinflammatory cytokine/chemokine production, serum IgE, and airway remodeling. In addition, Corren and colleagues demonstrated that lebrikizumab (a monoclonal antibody to IL-13) treatment was associated with improved lung function in patients with asthma. These results strongly suggested that IL-13 was a major effector of asthma. Recently, a genomewide association study (GWAS) conducted by Moffatt et al. [13] found that IL-13 +1923C/T polymorphism was associated with asthma risk and the total serum IgE concentration. More recently, another GWAS performed by Granada et al. [28] confirmed that IL-13 +1923C/T polymorphism was a risk factor for IgE dysregulation. Specifically, Maier and colleagues indicated that the IL-13 +1923T allele was associated with higher IgE level than the +1923C allele [29]. These results indicated the critical role of IL-13 +1923C/T polymorphism in the regulation of IgE level. IgE-dependent mechanisms play an important role in the development and maintenance of airway inflammation in asthma. It is biologically plausible that the IL-13 +1923C/T polymorphism which can affect IgE level could influence the susceptibility to asthma. However, it is currently unclear whether the IL-13 +1923C/T polymorphism causes overexpression or enhances function of IL-13. Therefore, the functional studies of this polymorphism are required. A number of studies have investigated the association between IL-13 +1923C/T polymorphism and asthma risk, but the results were controversial and underpowered. Metaanalysis offers a powerful means of overcoming problems such as small sample sizes and the inadequate statistical powers of genetic studies on complex disorders. Therefore, we performed this meta-analysis to assess the association between IL-13 +1923C/T polymorphism and asthma risk.
This meta-analysis, including a total of 13698 cases and 38209 controls, examined the association between the IL-13 +1923C/T polymorphism and asthma risk. We found that TT genotype and TC genotype were significantly associated with higher asthma risk, respectively. Compared with CC genotype, the carriers of the TT genotype had 47% increased asthma risk. As for the individuals carrying with TC genotype, they had 21% elevated asthma risk. These findings suggested that the carriers with the T allele of the IL-13 +1923C/T polymorphism might be predisposed to asthma. In the subgroup analysis by race, significant associations were observed in Asians and Caucasians. There were only two studies in African Americans for this polymorphism [12,21]. Therefore, subgroup analysis was not performed in African American subgroup. More studies with African American population are needed to evaluate the effect of IL-13 +1923C/T polymorphism on asthma risk. In addition, we carried out subgroup analysis by study quality. IL-13 +1923C/T polymorphism was still found to be associated with an elevated asthma risk in high quality studies. Because there were two studies with low quality, subgroup analysis was not performed.
We carried out sensitivity analysis to assess the stability of this meta-analysis. Removal of each study did not alter the conclusion of increased asthma risk, suggesting the reliability of these results. Additionally, the cumulative meta-analyses were conducted. Results from the cumulative meta-analyses showed a trend of more obvious association between IL-13 +1923C/T polymorphism and an increased risk of asthma as information accumulated by year. Therefore, we were convinced that the results of our meta-analysis were reliable.
We noted that there was moderate heterogeneity in the overall comparisons in codominant genetic model. Thus,    subgroup analysis was used to explore the sources of heterogeneity. We found that the I 2 values effectively reduced or disappeared when stratified by study quality. These results implicated that study quality may be the major source of the heterogeneity. Moreover, significant associations still existed in these high quality studies, which suggested that heterogeneity did not influence the results. In addition, it would be hard to interpret results, if significant publish bias was present. We assessed the publication biases by means of Egger's tests. However, we did not find obvious publication bias across the studies. Asthma is a complex inflammatory disease. It is unlikely that one SNP in one gene would be associated with asthma risk, without consideration of haplotype of the polymorphisms, environmental factors, or other polymorphic susceptible genes. For example, Sadeghnejad et al. [30] indicated that IL-13 +1923C/T, +2044A/G, and +2525A/G polymorphisms were in strong linkage disequilibrium and were associated with raised cord serum IgE. Wu et al. [23] found the haplotype of IL-13 +1923C/T polymorphism with the +2044A/G polymorphism affected susceptibility to asthma. In addition, there was a combined effect of IL-13 gene polymorphisms and tobacco smoke on persistent childhood wheezing and asthma [31]. Furthermore, many other genes were related to asthma, such as IL-4, IL-5, and PAI-1 [8,32]. However, haplotype analysis, gene-environment, and    gene-gene interactions could not be addressed in this metaanalysis due to the lack of sufficient information. Some limitations must be pointed out. First, although no publication bias was found, selection bias could have occurred because some articles published in other languages (not English or Chinese) were not obtained and included in this meta-analysis. Second, several lines of evidence supported a central role for IL-13 +1923C/T polymorphism in IgE dysregulation [27,28]. Thus, it was possible that this polymorphism might associate with the risk of atopic asthma. However, we could not evaluate the association between IL-13 +1923C/T polymorphism and atopic asthma because there was only one study performed in atopic asthmatic patients [11]. Third, there were only two studies of African American population in this meta-analysis and this population was not included in the subgroup analysis. Finally, different genotyping methods were used in the respective studies. This may be associated with different call rates.
To the best of our knowledge, this was the first metaanalysis to assess the relationship between the IL-13 +1923C/T polymorphism and asthma susceptibility. Results from our study suggested that the IL-13 +1923C/T polymorphism was significantly associated with the risk of asthma. Welldesigned multicentre studies with more ethnicities are needed to validate our findings and study the potential effect modification by atopic status. Moreover, further investigations should also consider gene-gene and gene-environment interactions.


extfieldsofstudy: Psychology Medicine

s2fieldsofstudy: Biology Education

text:
The impact of duration of one bout treadmill exercise on cell proliferation and central fatigue in rats

The purpose of this study was to investigate the impact of the duration-dependence of the one bout treadmill exercise on cell proliferation, stress, and central fatigue in rats. The animals were randomly divided into five groups: the non-exercise group, 1-h exercise group, 2-h exercise group, 4-h exercise group, and 6-h exercise group. The exercise load consisted of running at speed of 13 meters/min with the 0° inclination. Cell proliferation in the hippocampal dentate gyrus was increased in response to one bout moderate treadmill exercise in all exercise groups. But there was no statistical significance between the exercise duration and cell proliferation. The optical density of glucocorticoid (GR)-positive cells in the hippocampal dentate gyrus was not changed by treadmill exercise at any exercise duration. Expressions of serotonin (5-hydroxytryptamine, 5-HT) and tryptophan hyroxylase (TPH) were increased by treadmill exercise only at 6 h duration. It seemed like that there was no additional benefits on cell proliferation over 2 h exercise due to stressful factors with over exercise dose, and there was no change of GR expression due to early assessment point of time. It can be suggested that the one-bout of moderate treadmill exercise increased cell proliferation, but treadmill exercise prolonged to 6 h induced central fatigue in rats.

INTRODUCTION
The field of adult neurogenesis was rekindled in 1992, when Reynolds and Weiss showed that precursor cells isolated from the forebrain could differentiate into neurons in vitro (Reynolds and Weiss, 1992). These results and technical advances, including the development of immunocytochemical reagents identifying the phenotype of various neural cells more easily and accurately, led to an explosion of research in this field. Neurogenesis in the olfactory bulb (Tarozzo et al., 1995) and hippocampus (Eriksson et al., 1998) has been well-characterized in the adult mammalian brains nowadays.
It is well known that exercise enhances cell proliferation and/or neurogenesis (Kim et al., 2003;Kim et al., 2004b;Naylor et al., 2005). In the study of the effects of exercise intensity and duration on neurogenesis, Kim et al. (2003) reported that low-intensity treadmill exercise increased cell proliferation in the hippocampal dentate gyrus more potently compared to the moderate-intensity and high-intensity treadmill exercise, as low-intensity treadmill exercise exerted less stress. Naylor et al. (2005) reported that shortterm wheel running increased cell proliferation, however longterm wheel running resulted in negative effect on cell proliferation because of stress response, and this negative effect of wheel running on cell proliferation was prevented by restricting daily running distance. As a result, running distance and duration of exercise appeared an important factor for enhancing of cell proliferation.
Exercise activates hypothalamic-pituitary-adrenal (HPA) axis (Droste et al., 2003), and the most prominent product of HPA axis is cortisol (corticosterone in rodents). High level of glucocorticoids suppressed cell proliferation and adult neurogenesis (Gould et al., 1997;Tanapat et al., 2001), however longer-term elevation of glucocorticoids did not exert this effect (Heine et al., 2004b). In the brain, the actions of glucocorticoids are mediated by two types of adrenal steroid receptors: high-affinity mineralocorticoid receptor (MR) and low-affinity glucocorticoid receptor (GR). MR and GR coexist in the hippocampus. GR is mostly responsible for the neurogenesis. GR agonist dexamethasone inhibited neurogenesis both in vivo and in vitro (Kim et al., 2004a). Furthermore, pharmacological blockades of this receptor have a restorative effect against the glucocorticoids-mediated suppression on cell proliferation (Wong and Herbert, 2005). In relation to central fatigue, many evidences have suggested that prolonged moderate or exhaustive exercise increased serotonin (5-hydroxytryptamine, 5-HT) synthesis in the various brain regions (Bailey et al., 1993;Lim et al., 2001). Bailey et al. (1993) reported that 5-HT concentration in the brain was higher at exercise for 1 h than rest, and increment in 5-HT concentration caused central fatigue. The relation of exercise-induced central fatigue with expressions of 5-HT and tryptophan hyroxylase (TPH), the limiting enzyme for 5-HT synthesis, in the brain has been well documented (Bailey et al., 1993;Lim et al., 2001).
Exercise increases cell proliferation and/or neurogenesis, however, most of studies focused on the impact of exercise intensity using short-duration exercise, less than 2 h. The effects of the duration-dependency of the treadmill exercise on cell proliferation and central fatigue have not been clarified. Thus, we investigated the impact of the duration-dependence of the one bout treadmill exercise on cell proliferation, stress, and central fatigue using rats.

Animals and treatments
Male Sprague-Dawley rats weighing 245±10 g (n=20, 8 weeks old) were used. The experimental procedures were performed in accordance with the animal care guidelines of National Institutes of Health (NIH) and Korean Academy of Medical Sciences. All rats were housed under laboratory conditions for one week prior to experiment. The animals were housed under the controlled temperature (20±2°C) and maintained in light-dark cycles, each cycle consisting of 12 h of light and darkness (lights on from 07:00 h to 19:00 h). Food and water were made available ad libitum.

Experimental design
The animals were randomly divided into five groups (n=8): the control (non-exercise) group, 1-h exercise group, 2-h exercise group, 4-h exercise group, and 6-h exercise group. For adjusting the treadmill exercise, all rats in the exercise groups were forced to walk at speed of 13 meters/min with the 0° inclination during 30 min, for 3 days before the starting experiment. All rats were injected subcutaneously with 50 mg/kg 5-bromo-2´-deoxyuridine (BrdU, Sigma Chemical Co., St. Louis, MO, USA), 7 h prior to sacrifice. The rats in the exercise groups were started to walk in order to coincide the sacrifice time. For example, after 1 h injection, the rats in the 6-h exercise group were forced to walk on treadmill for 6 h. The exercise load consisted of walking at speed of 13 meters/min with the 0° inclination. This exercise regimen is a moderate-intensity exercise (Kim et al., 2003). The rats in the exercise groups took 10 min rest and hydration every 50 min exercise. All rats were sacrificed immediately after finishing exercise.

Tissue preparation
At the beginning of the sacrificial procedure, the animals were weighed and overdosed with Zoletil 50 ® (10 mg/kg, i.p.; Vibac Laboratories, Carros, France). After a complete lack of response was observed, the rats were transcardially perfused with 50 mM phosphate-buffered saline (PBS), and fixed with a freshly prepared solution consisting of 4% paraformaldehyde in 100 mM phosphate buffer (PB, pH 7.4). The brains were dissected and postfixed in the same fixative overnight, and transferred into 30% sucrose solution for cryoprotection. Serial coronal sections of 40 µm thick were made with a freezing microtome (Leica, Nussloch, Germany). Floating sections were stored at 4°C until processing for immunohistochemistry.

BrdU immunohistochemistry
For the detection of newly generated cells in the dentate gyrus, BrdU-specific immunohistochemistry was performed as previously described (Cho et al., 2013;Kim et al., 2013). Five sections on average were selected in each brain region spanning from Bregma -3.30 mm to -4.16 mm. Brain sections were permeabilized by incubation in 0.5% Triton X-100 in PBS for 20 min, then pretreated in 50% formamide-2 X standard saline citrate (SSC) at 65°C for 2 h, denaturated in 2 N HCl at 37°C for 30 min, and rinsed twice in 100 mM sodium borate (pH 8.5). Afterwards, the sections were incubated overnight at 4°C with BrdU-specific mouse monoclonal antibody (1:600; Roche, Mannheim, Germany). Then the sections were washed three times with PBS and incubated for 1 h with the biotinylated mouse secondary antibody (1:200; Vector Laboratories, Burlingame, CA, USA). Then the sections were incubated for another 1 h with avidin-peroxidase complex (1:100; Vector Laboratories). For visualization, the sections were incubated in 50 mM Tris-HCl (pH 7.6) containing 0.02% 3,3´-diaminobenzidine tetrahydrochloride (DAB), 40 mg/mL nickel chloride, and 0.03% hydrogen peroxide for 5 min. After BrdU-specific staining, counter-staining was performed on the same sections using a mouse anti-neuronal nucleic (NeuN) antibody (1:300; Chemicon International, Temecula, CA, USA). The sections were washed three times with PBS, incubated for l h with a biotinylated anti-mouse secondary antibody, and processed with VECTASTAIN ® Elite ABC Kit (Vector Laboratories). For visualization, the sections were incubated in a reaction mixture consisting of 0.02% DAB and 0.03% hydrogen peroxide for 5 min. The sections were finally mounted onto gelatin-coated slides. The slides were air dried overnight at room temperature, and coverslips were mounted using Permount ® (Fisher Scientific Co., Pittsburgh, PA, USA).

Glucocorticoid receptors immunohistochemistry
For the evaluation of optical density of GR-positive cells in the hippocampal dentate gyrus, GR-specific immunohistochemistry was performed as previously described (Park et al., 2013). Five sections on average were selected in each brain region spanning from Bregma -3.30 mm to -4.16 mm. Free-floating tissue sections were incubated overnight with rabbit anti-GR antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a dilution of 1:500 for visualization of GR distribution. Then the sections were incubated for 1 h with anti-rabbit secondary antibody (1:200, Vector Laboratories). The sections were subsequently incubated with an avidin-biotin-peroxidase complex (1:100, Vector Laboratories,) for 1 h at room temperature. Immunoreactivity was visualized by incubating the sections in a solution consisting of 0.02% DAB and 0.03% hydrogen peroxide in 50 mM Tris-HCl (pH 7.6) for approximately 5 min. Then the sections were mounted on gelatin-coated glass slides. The slides were air-dried overnight at room temperature, and coverslips were mounted using Permount ® (Fisher Scientific Co.).

5-HT and TPH immunohistochemistry
For detection of 5-HT-positive and TPH-positive cells in the dorsal raphe, immunohistochemistry was performed as previously described (Seo et al., 2011). Five sections on average were selected in each brain region spanning from Bregma -7.30 mm to -8.00 mm. Then the sections were incubated in PBS for 10 min and washed three times, again with PBS. The sections were then incubated in 1% hydrogen peroxide for 30 min. Next, the sections were incubated overnight with rabbit anti-5-HT antibody (Im-munoStar, Hudson, WI, USA) at dilution of 1:500 for visualization of 5-HT expression or mouse monoclonal anti-TPH antibody (Calbiochem, Darmstadt, Germany) at a dilution of 1:500 for visualization of TPH expression. Then the sections were incubated for 1 h with biotinylated anti-rabbit secondary antibody or anti-mouse secondary antibody (Vector Laboratories). The sections were subsequently incubated with avidin-biotin-peroxidase complex (Vector Laboratories) for 1 h at room temperature. Immunoreactivity was visualized by incubating the sections in a solution consisting of 0.05% DAB and 0.01% hydrogen peroxide in 50 mM Tris-buffer (pH7.6) for approximately 3 min. Then the sections were mounted on gelatin-coated glass slides. The slides were air-dried overnight at room temperature, and coverslips were mounted using Permount ® (Fisher Scientific Co.).

Data analysis
The area of the granular layer of the hippocampal dentate gyrus was measured using Image-Pro ® Plus image analyzer (Media Cybernetics Inc., Silver Spring, MD, USA). The number of BrdUpositive cells were counted hemilaterally and expressed as the number of cells per mm 2 of the cross-sectional area of the granular layer of the hippocampal dentate gyrus. The numbers of 5-HT-positive and TPH-positive cells were counted using Image-Pro ® Plus image analyzer (Media Cybernetics Inc.) and expressed as the number of cells per section of the dorsal raphe. For assessment of GR immunoreactivity, the staining intensities of the processed sections were assessed in a quantitative fashion according to a microdensitometrical method based on optical density using an image analyzer.
All data were analyzed using the statistical software SPSS (version 14.0). The data were expressed as the mean±standard error of the mean (SEM). For the comparison among the groups, oneway ANOVA and Tukey HSD post-hoc test were performed and differences were considered statistical significant at P<0.05.

Cell proliferation in the hippocampal dentate gyrus
The numbers of BrdU-positive cells in the exercise groups were increased significantly compared to the control group. However, there was no statistic difference among the exercise groups (Fig. 1). The present results showed that cell proliferation in the hippo-campal dentate gyrus was increased significantly in response to one bout treadmill exercise with all of exercise time (from 1 to 6 h) (P<0.05). But there was no additional benefit on cell proliferation over 2 h in one bout treadmill exercise at moderate-intensity.

Expression of GR in the hippocampal dentate gyrus
The optical density of GR-positive cells in the hippocampal dentate gyrus was not changed by treadmill exercise. There was no significant difference among the exercise groups (Fig. 2). The present results showed that one bout treadmill exercise at any duration exerted no effect on the GR expression in the hippocampal dentate gyrus.

Expressions of 5-HT and TPH in the dorsal raphe
One bout treadmill exercise in the duration from 1 to 4 h exerted no significant effect on the 5-HT synthesis in the dorsal raphe. In the 6-h exercise group, 5-HT synthesis in the dorsal raphe was increased significantly compared to the control group (P<0.05) (Fig. 3). The present results showed that one bout treadmill exercise until 4 h did not increased 5-HT synthesis, however one bout treadmill exercise during 6 h increased 5-HT synthesis.
One bout treadmill exercise in the duration from 1 to 4 h exerted no significant effect on the TPH expression in the dorsal raphe. In the 6-h exercise group, TPH expression in the dorsal raphe was increased significantly compared to the control group (P<0.05) (Fig. 4). The present results showed that one bout treadmill exercise until 4 h did not increased TPH expression, however one bout treadmill exercise during 6 h increased TPH expression. Holmes et al. (2004) reported that daily running activity was positively correlated with cell proliferation and cell survival. In the present results, the number of BrdU-positive cells was increased until 2 h, after then there was no more increment till 6 h (Fig.  1). So there was no dose-dependent tendency between exercise dose and cell proliferation. Ra et al. (2002) reported that forced swimming prolonged more than 5 min had no additional increment of cell proliferation due to the stress factors. Over-dose of exercise might induce higher levels of corticosterone synthesis and thus counteracted the beneficial effect of exercise on cell proliferation (Kim et al., 2003;Naylor et al., 2005).  The HPA axis is one of the primary stress systems and an integrated neural and endocrine system that plays a key role in releasing the glucocorticoids. Stress, induced by psychological and/or physical, activates the HPA axis and increases the secretion of glucocorticoids (Gould et al., 1997), and raised level of glucocorticoids suppresses proliferation of progenitor cells in the hippocampal dentate gyrus (Tanapat et al., 2001). Stress might prevent progenitors from cell cycle reentry by elevated levels of p27Kip1, an endogenous cell cycle inhibitor (Heine et al., 2004a). Considering these reports, the inhibiting factors (glucocorticoids and p27Kip1) on cell proliferation might be augmented gradually as increasing exercise time, and then cell proliferation of progenitors might be suppressed through accumulated effects of those factors. Thus it seemed like that the suppression on cell proliferation was initiated after 2 h in the present study.

DISCUSSION
GR expression underlies a feedback regulation, linking it tightly to the level of the glucocorticoids (Kalman and Spencer, 2002). Under the stressful situation, hippocampal GRs or their mRNA were down-regulated (Kitraki et al., 1999). In the present study, the optical density of GR-positive cells in the hippocampal dentate gyrus at any exercise time was not changed compared to the non-exercise group (Fig. 2). Considering the relation between exercise dose and glucocorticoids secretion, the plasma level of glucocorticoids might be increased. Regulation of GR mRNA and GR protein by glucocorticoids was dose-and time-dependent in vitro (Burnstein et al., 1990;Oalkey and Cidlowski, 1993) and in vivo (Hügin-Flores et al., 2004). Hügin-Flores et al. (2004) reported that a progressive decrement of GR mRNA in the hippocampus was related with increasing doses of corticosterone. Low plasma corticosterone level could require longer time to attain a critical number of activated receptors in the nucleus, so quantitatively significant changes in corticosteroid receptor mRNAs would appear later. Thus, the effect of corticosterone on GR mRNA in the hippocampus can be considered as dose-and time-dependent. In the present experiment, no change of GR expression with exercise prolonged to 6 h might be occurred due to relative low level of glucocorticoids which could not induce the significant alteration of GR expression at the time of ceasing exercise. However, the level of glucocorticoids secreted with one bout of 6 h exercise might not be a dose enough to down-regulate GR expression. In the present study, the change of GR expression was not occurred with 6 h exercise, however the level of glucocorticoid might be Most of the cell bodies of the serotonergic neurons in the brain arise from the dorsal raphe, and then they send projections to diverse target regions including the limbic system, hypothalamus, striatum, and cerebral cortex (Jacobs and Azmitia, 1992). So we investigated the expressions of 5-HT and TPH in the dorsal raphe to evaluate the effects of exercise on central fatigue. We found that both 5-HT and TPH expressions were increased gradually in response to exercise time. Increments of 5-HT and TPH expressions were detected only at 6 h exercise but not until 4 h exercise (Figs.  3, 4). These results mean that moderate exercise induced central fatigue by accumulated effect of prolonged exercise time.
The number of BrdU-positive cells in the hippocampal dentate gyrus was increased in response to one bout moderate treadmill exercise in all exercise groups. However, after 2 h, there was no more increase till 6 h. Expressions of 5-HT and TPH in the dorsal raphe were increased only in the 6 h exercise group. Based on the present results, one-bout of treadmill exercise at moderate intensity increased cell proliferation, meanwhile treadmill exercise prolonged to 6 h induced central fatigue in rats.