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Effect of metformin on cell proliferation, apoptosis, migration and invasion in A172 glioma cells and its mechanisms

The purpose of the present study was to determine the effects of metformin on the inhibition of proliferation, apoptosis, invasion and migration of A172 human glioma cells in vitro and determine the underlying mechanism. The effects of metformin at different concentrations (0, 0.1, 1 and 10 mmol/l) on the inhibition of A172 cell proliferation were detected using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell apoptosis was detected by flow cytometry. Caspase-3 activity was analyzed by spectrophotometry. The invasion and migration of cells were detected by Transwell assays. The levels of Bcl-2-associated X protein (Bax), B-cell lymphoma 2 (Bcl-2), AMP-activated protein kinase (AMPK), phosphorylated-(p)AMPK and mechanistic target of rapamycin (mTOR) protein expression were detected by western blot analysis, and changes in the malondialdehyde (MDA) content and activity of superoxide dismutase (SOD) were determined. Compared with the control group, metformin significantly increased the inhibition of proliferation and apoptosis, and significantly reduced the invasion and migration of A172 cells in dose- and time-dependent manners (P<0.05). In addition, compared with the control group, metformin significantly enhanced the activity of caspase-3, increased the expression of AMPK/pAMPK/Bax proteins and reduced the expression of mTOR/Bcl-2 proteins (P<0.05). Metformin increased the MDA content and reduced the activity of SOD in a dose-dependent manner (P<0.05). Metformin may inhibit glioma cell proliferation, migration and invasion, and promote its apoptosis; the effects may be associated with the AMPK/mTOR signaling pathway and oxidative stress.


Introduction
Glioma is the most common type of malignant brain tumor, accounting for 40% of intracranial tumors. The main treatments for gliomas include surgery, radiotherapy and chemotherapy. With a high degree of malignancy, post-operative relapse and low 5-year survival rate, glioma poses a serious threat to the lives and health of patients (1). although there are currently new types of chemotherapeutic agents available, the outcome in clinical applications remains poor and thus identification of an effective anti-glioma drug is an important research goal for the treatment of gliomas.
Metformin (MeT) is a classic drug for the treatment of diabetes. in recent years, a number of studies have shown that MeT can reduce the incidence of cancer in patients with diabetes and even improve the survival rate of patients with type 2 diabetes mellitus (2)(3)(4), which has caught the attention of researchers. Studies have revealed that MeT has anti-tumor cell biological activity, inhibits the growth of tumor cells in vitro and in vivo (5)(6)(7)(8)(9), and enhances the sensitivity of tumor radiotherapy and chemotherapy (10). MeT has been studied in the clinical treatment of patients with a variety of cancers (2,4,11), and it has been identified that MeT combined with temozolomide can synergistically inhibit the growth of glioma stem cells and promote apoptosis in vivo and in vitro (12). MeT can inhibit the proliferation of brain tumor cells in vitro, but the mechanism is unknown (13). clinical studies have shown that MeT-treated diabetic patients treated with MeT for a long duration have a lower risk of developing glioma (14). Therefore, in order to observe the anti-tumor effect of MeT on gliomas, the present study used human glioma a172 cells and the effects of MeT on inhibition of proliferation, apoptosis, invasion and metastasis of glioma a172 cells were determined.
The aMP-activated protein kinase (aMPK)/mechanistic target of rapamycin (mTor) signaling pathway and oxidative stress serve an important role in tumor growth, metabolism

Effect of metformin on cell proliferation, apoptosis, migration and invasion in A172 glioma cells and its mechanisms
and apoptosis. it has been reported that MeT induces apoptosis in patients with lung and pancreatic cancers by regulating the aMPK/mTor signaling pathway and oxidative stress system (15,16). at present, the majority of studies involving the anti-tumor effect of MeT have been conducted in breast, pancreatic, gastric, lung and prostate cancer (3,10,16,17); however, few studies have focused on gliomas (6,18). The current study further observed the effect of MeT on glioma cells, and the association with the aMPK/mTor signaling pathway and oxidative stress, thus providing an experimental basis for further clinical application. Detecting cell proliferation with bromodeoxyuridine (BrdU). logarithmic growth phase cells (5x10 4 cells per well) were placed in 24-well plates with round coverslips and MeT (0, 0.1, 1 and 10 mmol/l) was inoculated for 48 hat 37˚C, then 10 µmol/l BrdU (Abcam) was added and incubated at 37˚C in a 5% CO 2 incubator for 1 h. Following washing with PBS, the cells were fixed in 4% paraformaldehyde for 30 min at room temperature. diluted anti-Brdu antibody (1:1,000; cat. no. ab8152; abcam) was added and incubated for 1 h at room temperature. Following washing with PBS, goat anti-mouse igG alexa Fluor ® 647-conjugatedsecondary antibody (1:500; cat. no. ab150115; abcam) was added and incubated at room temperature in the dark for 30 min. Following washing with PBS, daPi was added to the cells. The cell mixture was incubated in the dark at room temperature for 10 min. The cells were washed with PBS, mounted and observed with a fluorescence microscope with excitement wavelength of 649 nm (magnification, x100). A total of four randomly chosen microscopic fields were analyzed and the results are expressed as the average cell number. The cell proliferation rate was calculated as follows: Brdu positive labeled cells/total number of labeled cells x100%.

Culture
Flow cytometry using Annexin V-FITC/PI kits and a Gallios flow cytometer. cells in the logarithmic growth phase were inoculated into 6-well plates at a concentration of 5x10 4 /ml. The cells were treated with different concentrations of MeT (0, 0.1, 1 and 10 mmol/l) for 48 h at 37˚C, digested with trypsin, then collected. Subsequently, the annexin V-FiTc/Pi double-stain kit was used according to the manufacturer's protocols. The cells were resuspended in binding buffer at a density of 1x10 6 /ml. Then, 5 µl of Annexin V-FITC was added, the cell suspension was kept in the dark for 10 min, then centrifuged at 250 x g for 8 min at room temperature. The supernatant was discarded and buffer solution was added for resuspension, then 10 µl of PI staining solution was added. Following thorough mixing, the cells were incubated in the dark at 4˚C for 15 min and assessed within 30 min. The cells were acquired on a Gallios flow cytometer (Beckman Coulter, inc.) and the apoptosis rate was analyzed using FlowJo version 7. 6. 1 software (FlowJo llc, ashland, or, uSa). in total, 3 double-wells were established in each group and the experiments were repeated 3 times. apoptosis rate was the percentage of annexin V-FiTc + /Pi + cells.
Detection of caspase-3 by spectrophotometry. cells in the logarithmic growth phase were established at a concentration of 5x10 4 cells/ml and inoculated into 12-well plates. The cells were treated with different concentrations of MeT (0, 0.1, 1 and 10 mmol/l) for 48 h at 37˚C, digested with 0.25% trypsin at 37˚C for 5 min, then collected into EP tubes. Subsequently, the caspase-3 kit was used according to the manufacturer's protocols. The absorbance at 405 nm was read from a microplate reader. The data of the blank group were set to 0. a total of 6 double-wells were established in each group and the experiments were repeated 3 times.

Detection of cell invasion and migration by Transwell assay.
a172 cells were inoculated into 24-well plates and the cell concentration was adjusted to 2x10 5 cells/ml. Following treatment and culturing for 48 h as aforementioned, 50 µl Matrigel solution was coated on polycarbonate microporous membranes with a pore size of 8 µm between the upper and lower chambers of the Transwell chambers, which was then polymerized at 37˚C for 30 min. Cells were washed and digested with 0.25% trypsin at 37˚C for 5 min. Following collection, cells were added to the upper chamber and cultured at 37˚C in a 5% CO 2 incubator for 24 h, then 0.5% crystal violet solution was added and incubated at room temperature for 10 min. non-invaded and non-migrative cells in the upper chamber were wiped with a cotton swab, then the cells passing through the filter membrane were counted. Five wells were randomly selected, and cells were observed and counted under a light microscope (magnification, x200). For the determination of migration ability, the upper Transwell chamber did not require Matrigel solution coating; otherwise, the procedures were conducted in the same manner as aforementioned.
Western blot analysis. cells in the logarithmic growth phase at a concentration of 5x10 4 /ml were inoculated into cell culture flasks. Cells were treated with MET (0, 0. Statistical analysis. experiments were repeated at least three times. The experimental data are presented as the mean ± standard deviation and were analyzed using SPSS version 17. 0 software (SPSS, inc., chicago, il, uSa). one-way analysis of variance was performed followed by a post hoc Tukey's test for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference and P<0.01 was considered highly statistically significant.

Effect of MET on the viability of A172 cells by MTT assay.
MeT had an inhibitory effect on a172 human glioma cells; suppression of cell viability was enhanced with increasing concentrations of MeT and prolonged treatment time (Fig. 1). rate in the control group was 2.9±0.4%, while significant increases in response to MeT at 0.1, 1 and 10 mmol/l were 6.2±1.7, 23.5±3.8 and 35.4±2.8%, respectively (Fig. 4). This suggested that MeT induced apoptosis in a172 human glioma cells and the induced apoptotic effect increased as the concentration of MeT increased. Fig. 5, as the concentration of MeT increased, the activity of caspase-3 in a172 cells was induced in a concentration-dependent manner. There were significant differences in the caspase-3 activity between MeT concentrations at 0.1, 1 and 10 mmol/l compared with in the control. Fig. 6, as the concentration of MeT increased, the number of migrating and invading a172 glioma cells was significantly reduced than the control group. compared with the control group, there was a statistically significant difference at a MET concentration of 0.1 mmol/l (P<0.05); highly statistically significant differences when the MeT concentrations were 1 and 10 mmol/l (P<0.01; Fig. 5).

Effect of MET on the expression of AMPK/pAMPK/ mTOR/Bax/Bcl-2 proteins in A172 human glioma cells.
aMPK is an important serine/threonine protein kinase and an upstream regulator of key enzymes in cholesterol synthesis and fat metabolism. aMPK serves an important regulatory role in energy metabolism (15,16). aMPK is also referred to as an energy sensor (19). as presented in Fig. 7, the effect of MeT on protein expression of a172 cells was investigated. When a172 cells were treated with MeT at concentrations of 0, 0.1, 1 and 10 mmol/l, the expression levels of aMPK/paMPK/Bax were notably upregulated as the concentration of MeT increased, while the expression of mTor/Bcl-2 decreased as the concentration of MeT increased, showing a statistical difference compared with the control group.
Effect of MET on MDA content and SOD activity in A172 human glioma cells. The Mda content increased and the activity of Sod decreased as the concentration of MeT increased (Fig. 8). compared with the control group, there was a significant difference at a MeT concentration of 0.1 mmol/l and there was a highly significant difference at MeT concentrations of 1 and 10 mmol/l.

Discussion
Glioma is a common type of intracranial malignant tumor, the incidence of which is gradually increasing, thus posing a serious threat to human health (1). at present, a comprehensive therapeutic approach combining surgery, chemotherapy, radiotherapy and molecular targeted therapy has been adopted for the treatment of gliomas, but the therapeutic efficacy is poor, with a low 5-year survival rate and high mortality rate (20), giving rise to a heavy burden on society.
To find effective and low-toxicity anti-glioma medications with low toxicity remains as one of the critical areas of research, the goal of which is to improve the prognosis and treatment of gliomas.   MeT is a classic drug for the treatment of diabetes. in recent years, a number of studies have shown that MeT can reduce the incidence and mortality of cancers in patients with diabetes and even improve the survival rate of patients with malignant tumors (2,14,21). Studies have demonstrated   that MeT can inhibit the growth of tumor cells in vitro and in vivo (9,18,(21)(22)(23) and enhance the sensitivity of tumors to targeted drugs and radiotherapy (10). recently, MeT has been studied in the clinical application for the treatment of non-diabetic patients with cancer (3,4,21,24).
caspase-3, a key protease in apoptosis, in the core position of the apoptotic cascade, is the final implementer of the apoptosis program. The activated caspase-3 enzyme can directly lead to apoptosis by hydrolyzing the specific protein, including cyclic guanosine monophosphate (25). at the same time, it can destroy dna repair proteins to assist apoptosis (13). Bax and Bcl-2 are common proteins of the Bcl-2 gene family for promoting and inhibiting apoptosis, serving an important role in the process of tumor apoptosis (26). The current study demonstrated that when a172 glioma cells are treated with MeT (0.1, 1 and 10 mmol/l), the survival rate decreased, reductions in proliferation and apoptotic rate were promoted compared with the control group, presenting an apparent dose-response and time-effect association. in addition, MeT increased the activity of caspase-3, increased the expression of Bax protein and decreased that of Bcl-2 protein. as the concentration of MeT increased, the associated effects were promoted, suggesting that MeT exerts biological activity against glioma cells and inhibits proliferation, induces apoptosis, and inhibits the invasion and metastasis of glioma cells, consistent with the results of other studies (18,27).
aMPK is an important serine/threonine protein kinase and is an upstream regulator of key enzymes in cholesterol synthesis and fat metabolism. When the adenosine triphosphate (aTP) levels in the cells are decreased, the ratio of aMP/aTP is increased and aMP directly activates aMPK, which causes the cells to change from anabolic to catabolic metabolism, promoting glycolysis and fatty acid oxidation, preventing gluconeogenesis and protein and lipid synthesis (19). The aMPK/mTor signaling pathway also serves an important role in cell proliferation, survival, apoptosis, glucose metabolism, gene transcription and cell migration (12,21). aMPK, as a tumor suppressor gene, is one of the targets of tumor research. The activation of aMPK can inhibit mTor phosphorylation, providing an anti-tumor effect, which can affect a variety of biological behaviors, including tumor cell proliferation and apoptosis (27). Studies have shown that MeT can act on the aMPK/mTor pathway and serve a role in anti-gastric cancer, liver cancer, nasopharyngeal cancer and anti-aging (12,21,28,29). The present study demonstrated that MeT increases the expression of aMPK and paMPK proteins, and decreases the expression of mTor protein, which was statistically significant compared with the control group, suggesting that the effect of MeT on inhibiting proliferation and inducing apoptosis of glioma a172 cells may be associated with the aMPK/mTor signaling pathway.
Mda protects against the damage of oxygen free radicals to tissue cells and Sod can scavenge oxygen free radicals; both are markers of the oxidative stress system (30). The results of the present study indicated that MeT can increase Mda content and reduce Sod activity. it can be seen that the effect of MeT on gliomas may be associated with oxidative stress. in summary, the effect of MeT on glioma cell proliferation inhibition, apoptosis induction, and inhibition of invasion and migration may be associated with the aMPK/mTor signaling pathway and oxidative stress. The present study provided novel insight into the treatment of gliomas; however, further investigation is required.