Previous studies have demonstrated that metformin (Met) may reduce the risk of cancer development. In the present study, the anti-cancer effects of Met in A498 cells were investigated. It was found that Met inhibited A498 cell proliferation in a time- and dose-dependent manner, as well as induced the activation of AMP-activated protein kinase. It was also demonstrated that Met promoted A498 cell apoptosis and mechanistic studies suggested that this was mediated by the downregulation of B-cell lymphoma 2 and concurrent upregulation of Bcl-2-associated X protein. In addition, it was observed that Met induced G1 cell cycle arrest by decreasing cyclin D1 expression. Furthermore, the results demonstrated that Met reduced A498 cell migration and invasion
Renal cell carcinoma (RCC) accounts for ~90–95% of all kidney neoplasms (
Metformin (Met) is a widely prescribed anti-diabetic agent and is used as first-line therapy for patients with type 2 diabetes mellitus (
In the present study, the anti-proliferative and anti-metastatic effects of Met on A498 cells were investigated
Human A498 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM; HyClone Laboratories, South Logan, UT, USA) with 10% fetal bovine serum (FBS) in 5% CO2 at 37°C. Met (1,1-dimethylbiguanide hydrochloride) was purchased from Sigma-Aldrich (St. Louis, MO, USA).
A498 cells (3×103 cells/well) in 100 μl medium were seeded in 96-well plates. Following 12 h, the medium in each well was replaced with medium containing different concentrations of Met (16 and 32 mM) and incubated for 24 h. Cell viability was then determined using the MTT assay as previously described (
When A498 cells reached 60% confluence, the cells were washed with phosphate-buffered saline and then exposed to 16 mM Met for 48 h. The morphology of treated A498 cells was observed under a microscope (Olympus, Tokyo, Japan) and photomicrographs were captured with an Olympus digital camera (Olympus).
A498 cells were seeded in 24-well plates. Following incubation with Met (16 mM) for 24 h, each well was scratched using a 200 μl pipette tip, and further incubated at 37°C with Met (16 mM). Images of the scratch area were then captured following 24 h and the distance between the two cell edges was analyzed using ImageJ software (National Institutes of Health, Bethesda, MA, USA).
The transwell system (24 wells, 8 μm pore size with polycarbonate membrane; Corning Costar, Lowell, MA, USA) coated with 2 mg/ml basement membrane Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) was used for the
For the migration assay, the cells were seeded in upper chambers without Matrigel. The rest of the assay was performed as the invasion assay. The number of cells on the lower surface was counted in five randomly selected fields and the cell number was then analyzed statistically.
Briefly, 1×105 cells/well were cultured in 6-well plates and incubated overnight. The cells were then treated with 0, 16 and 32 mM Met for 48 h. Following treatment, the cells were harvested and resuspended in 100 μl binding buffer. The cells were then incubated with RNase for 30 min at 37°C and 5 μl propidium iodide (PI) was added, followed by a 10 min incubation in the dark. The samples were subsequently analyzed using flow cytometry and the percentage of cells in each phase was determined using WinMDI V2.9 software (The Scripps Research Institute, San Diego, CA, USA).
Preparation of whole cell protein extracts and western blot analysis were conducted as previously described (
Student’s two-tailed t-test was used to determine statistical differences between the treatment and control cells. P<0.05 was considered to indicate a statistically significant difference. All data are presented as the mean ± standard deviation of three independent experiments.
The effect of Met on A498 cell proliferation was determined using the MTT assay. It was revealed that Met significantly inhibited the proliferation of A498 cells in a time- and dose-dependent manner (**P<0.01;
The alterations in the morphology of A498 cells following treatment with Met (16 mM) for 48 h are shown in
To investigate the effects of Met on cell cycle distribution, A498 cells were treated with 16 and 32 mM Met for 48 h and the cells were then analyzed using flow cytometry. As shown in
To detect and quantify the extent of apoptosis induced by Met, PI staining was used to analyze the percentage of apoptotic cells. As shown in
The scratch assay was performed to investigate the effect of Met on the migration of A498 cells. As shown in
To further investigate the effect of Met on cell migration and invasion, a transwell assay was performed on A498 cells treated with Met (16 and 32 mM). The results demonstrated that Met significantly inhibited the migration and invasion of A498 cells (**P<0.01;
To investigate the mechanisms underlying the anti-cancer effect of Met, the effect of Met on relevant cell signaling targets was investigated. Following treatment with varying concentrations of Met for 48 h, it was demonstrated using western blot analysis that Met increased the phosphorylation of AMPK in a dose-dependent manner (
Met is widely used for the treatment of diabetes mellitus (
AMPK is a therapeutic target for metabolic syndrome and type 2 diabetes since its activation stimulates fatty acid oxidation and enhances insulin sensitivity (
Cell cycle regulation is mainly controlled by cyclin-dependent kinases (
Apoptosis suppresses proliferation (
MMPs have been demonstrated to be crucial proteinases in the process of invasion and metastasis of RCC (
In conclusion, the present study demonstrated that Met inhibits A498 cell proliferation, induces apoptosis by regulating the expression of Bcl-2 family members and reduces the expression of cyclin D1, which contributes to G1 cell cycle arrest of A498 cells. Furthermore, it was demonstrated that Met inhibits A498 cell migration and invasion by decreasing MMP-2 expression. In combination, these results provide
This study was supported by the National Natural Science Foundation of China (grant no. 81172435).
Met suppresses the cell viability of A498 cells. Met significantly inhibited the cell viability of A498 cells in a dose- and time-dependent manner (**P<0.01). (A) Effect of Met on cell viability was measured using the MTT assay. A498 cells were treated with Met for 48, 72 and 96 h. (B) Morphological alterations of A498 cells induced by Met (0 and 16 mM; magnification, ×400). Met, metformin.
Met regulates the cell cycle distribution of A498 cells. (A) Flow cytometric analysis demonstrated that Met altered A498 cell cycle distribution following 48 h. (B) Met increased the number of cells in the G0/G1 phase and decreased the number of cells in the S phase compared with the control cells. Data are presented as the mean ± standard deviation of three independent experiments. **P<0.01. Met, metformin.
Met induces apoptosis of A498 cells. (A) A498 cells were treated with Met (0, 16 and 32 mM) for 48 h and stained with propidium iodide. (B) Percentage of apoptotic cells. Data are presented as the mean ± standard deviation of three independent experiments. **P<0.01. Met, metformin.
Cell migration and invasion ability are inhibited by Met. (A and B) Cell migration was investigated using the scratch assay. A498 cells were treated with Met (16 mM) for 24 h. The cells were then wounded by scratching with a pipette tip and then incubated with or without Met (16 mM) for 24 h. (B) Met-treated A498 cells demonstrated a lower rate of wound closure compared with the control cells. (C and D) Cell migration and invasion were further investigated using the transwell assay. Following treatment with Met, the number of A498 cells that successfully (C) migrated and (D) invaded were counted. The decreased number of A498 cells indicated the inhibitory effect of Met on cell mobility. Data are presented as the mean ± standard deviation of three independent experiments. **P<0.01. Met, metformin.
Effect of Met on the protein expression levels of p-AMPK, AMPK, Bcl-2, Bax, PARP, cyclin D1 and MMP-2 in A498 cells. Representative western blot showing changes in the protein levels of p-AMPK, AMPK, Bcl-2, Bax, PARP, cyclin D1 and MMP-2 in A498 cells following exposure to Met with GAPDH as a control. Met, metformin; p-AMPK, phosphorylated AMP-activated protein kinase; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; PARP, poly ADP ribose polymerase; MMP-2, matrix metalloproteinase-2.