Metformin impairs growth of endometrial cancer cells via cell cycle arrest and concomitant autophagy and apoptosis

Metformin (1,1-dimethylbiguanide hydrochloride), 3-methyladenine (3MA), chloroquine (CQ), and siRNA were purchased from Sigma Aldrich (St. Louis, MI, USA). Anti-actin antibody was purchased from Sigma; all other antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Modified Eagle’s medium (MEM), non-essential amino acids (NEAA), and trypsin/EDTA (0.25% trypsin, 1 mM EDTA) were purchased from Wako Pure Chemical Industries (Osaka, Japan). Antibiotics/antimycotics (ABAM) were purchased from Gibco (Carlsbad, CA, USA). Cell counting kit-8 (CCK-8) was purchased from Dojindo Laboratories (Tokyo, Japan). Caspase-Glo assay kits were purchased from Promega (Madison, WI, USA). FITC Annexin V apoptosis detection kit I, FITC BrdU Flow Kit, and BD MitoScreen (JC-1) were purchased from BD Pharmingen (San Diego, CA, USA). Acridine orange (AO) was purchased from Molecular Probes (Eugene, OR, USA). Lipofectamine 2000 was purchased from Invitrogen (Carlsbad, CA, USA).

The Ishikawa human endometrial adenocarcinoma cell line was purchased from the European Collection of Cell Culture (ECACC, Salisbury, UK). Ishikawa cells were cultured in MEM supplemented with l-glutamine (2 mM), 5% (v/v) FBS, 1% NEAA, and ABAM at 37°C in a humidified atmosphere with 5% CO₂.

We performed this work by using only cell line, but not clinical samples. Therefore, this work has been granted exemption from the Ethics Committee of Shiga University of Medical Science.

The WST-8 assay was used to measure cell viability. Cells were plated on 96-well plates at a density of 1 × 10⁴ cells/well in 100 μL medium. At 24 h after seeding, metformin (0, 0.01, 1, 5, 10, or 20 mM) was added to each well and cells were cultured for an additional 48 h. CCK-8 solution (10 μL) was then added to each well, and the plates were incubated at 37°C for 2 h. The absorbance of WST-8 formazan was measured at 450 nm using a microplate reader.

To measure colony formation, adherent Ishikawa cells were trypsinized and 1000 viable cells (depending on the experiment) were subcultured in 60-mm plates; each treatment was tested in triplicate. After 24 h, the medium was replaced with fresh culture medium containing metformin (0, 0.01, 1, 5, 10, or 20 mM) in a 37°C humidified atmosphere with 95% air and 5% CO₂ and grown for 2 weeks. The culture medium was replaced every 3 days. Cell clones were stained for 15 min with a solution containing 0.5% crystal violet and 25% methanol in water. Stained cells were rinsed three times with tap water to remove excess dye. Each dish was then washed and dried, and the number of colonies/plate was macroscopically counted. Colonies were defined as those containing >50 cells by microscopic examination.

To assess cell cycle progression, cells were seeded onto 60-mm plates and incubated for 24 h to allow for exponential growth. Ishikawa cells were incubated with or without metformin for an additional 48 h. All cells were incubated with 10 μM BrdU (BD Pharmingen) for 30 min; BrdU-labeled cells were then harvested, fixed, permeabilized, and stained with FITC-conjugated anti-BrdU antibody and 7-AAD, according to the manufacturer’s instructions. A flow cytometer (BD, FACSCalibur, San Jose, CA, USA) was used to assess DNA content and cell cycle phase.

Annexin V-FITC apoptosis detection kits were used according to the manufacturer’s instructions to measure apoptosis. Cells were incubated with or without metformin for 48 h, collected and washed with PBS, gently resuspended in annexin V binding buffer, and incubated with annexin V-FITC/7-AAD. Flow cytometry was performed using CellQuest Pro software (BD).

A mitochondrial membrane potential detection kit was used according to the manufacturer’s instructions to measure mitochondrial membrane potential (Δψm). In brief, cells were treated with or without metformin, resuspended in 0.5 mL of JC-1 solution, and incubated at 37°C for 15 min. Cells were then rinsed before flow cytometry. A dot plot of red (living cells with intact Δψm) versus green fluorescence (cells lacking Δψm) was generated. Data were expressed as the percentage of cells with intact Δψm.

The Caspase-Glo™ 3/7, Caspase-Glo™ 8 or Caspase-Glo™ 9 assay kit was used according to the manufacturer’s instructions to measure the activity of caspase 3/7, caspase-8 or caspase-9, respectively. In brief, 50 μL of cell lysate (cytosolic extracts, 20 μg) was incubated in 50 μL of Caspase-Glo reagent at room temperature for 1 h. After incubation, the luminescence of each sample was measured in a plate-reading luminometer (Tecan, Linz, Austria).

To identify autophagic cells, the volume of the cellular acidic compartment was visualized by AO staining [26]. Cells were seeded in 60-mm culture dishes and treated as described above. After 48 h of treatment with or without metformin, cells were incubated with medium containing 5 μg/mL AO for 15 min. The AO medium was then removed, cells were washed once with PBS, and fresh medium was added. Fluorescence micrographs were taken using an Olympus inverted fluorescence microscope (Olympus Corp., Tokyo, Japan). All images presented are at the same magnification. Flow cytometry was used to determine the number of cells with acidic vesicular organelles (AVOs). Cells were trypsinized and harvested; BD FACSCalibur and BD CellQuest Pro software was used to analyze the cells. A minimum of 10,000 cells within the gated region was analyzed for each treatment.

Lipofectamine 2000 reagent and the Invitrogen protocol were used to introduce Beclin-1 siRNA (Sigma-Aldrich; seq1 #SASI_Hs02_00336256) or a scramble control siRNA sequence (Sigma-Aldrich; #SIC001) into Ishikawa cells. Cells were then incubated for 48 h prior to metformin treatment (0, 5, or 10 mM).

Ishikawa cells (2 × 10⁶/dish) were seeded in 100-mm culture dishes and cultured for 24 h. After metformin treatment, cells were lysed in RIPA lysis buffer containing a protease-inhibitor cocktail (“Complete” protease inhibitor mixture; Roche Applied Science, Indianapolis, IN) on ice for 30 min. Suspensions of lysed cells were centrifuged at 14 000 × g at 4°C for 10 min; supernatants containing soluble cellular proteins were collected and stored at -80°C until use. BCA protein assay kits were used to measure protein concentration. Furthermore, 15 μg of protein was resuspended in sample buffer and separated on a 4%–20% tris-glycine gradient gel using the SDS-PAGE system. Resolved proteins were transferred to PVDF membrane, which was blocked with 5% milk in tris-buffered saline/0.1% Tween 20. Immunodetection was performed using each primary antibody. The membranes were incubated with donkey anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000 dilution). The ECL Western Blotting Detection System (GE Healthcare, Little Chalfont, UK) was used to detect signals, which were visualized using a LAS-4000 mini (GE Healthcare). Actin was used as the loading control.

All data points represent the mean of at least three independent measurements and are expressed as the mean ± standard deviation. SPSS ver. 20 was used to perform one-way ANOVA and Tukey’s post hoc test or Student’s t-test, as appropriate. A significance threshold of p < 0.05 was used.

WST-8 and colony formation assays were used to assess the effects of metformin on the viability of Ishikawa endometrial cancer cells. The number of viable cells decreased with increasing concentrations of metformin for 24- or 48-h treatments (Figure 1A). After 24 h, 20 mM of metformin significantly reduced the number of viable cells but 0.01–10 mM metformin did not. After 48 h, metformin at 5 mM or more significantly reduced the number of viable cells. At 48 h, IC50 of metformin was 6.78 mM.

The ability of metformin-treated and control Ishikawa cells to form colonies on 60-mm culture plates within two weeks was examined. Metformin at concentrations as low as 1 mM, significantly reduced colony formation (Figure 1B), and the inhibitory effect of metformin on colony formation was dose dependent. Metformin at 5 mM or more reduced colony formation to 10% of that of untreated control cells. Based on these results and those in several published reports [13, 17, 27, 28], 5 or 10 mM metformin was used in the following experiments.

To investigate the underlying mechanisms of metformin-induced growth inhibition in Ishikawa cells, we first evaluated the effect of metformin on cell proliferation and cell-cycle progression. Cell-cycle profiles were analyzed after 48 h of metformin treatment. There were significantly fewer S-phase cells and significantly more G2/M cells in metformin-treated cultures compared with those in control cultures, and these effects were dose dependent (Figure 2A). Furthermore, we used western blots to assess the effects of metformin on the expression of two cell cycle regulators, p53 and p21 (Figure 2B). Expression of p53 decreased in a dose-dependent manner with metformin treatment (Figure 2C). The induction of p21, a cell-cycle blocker, increased in a dose-dependent manner with metformin treatment (Figure 2D). These results indicate that metformin induced p21 expression, which led to cell cycle arrest in G1 and G2/M via a p53-independent pathway.

To assess whether the induction of apoptosis also contributed to metformin-mediated inhibition of Ishikawa cell growth, the proportion of apoptotic cells was measured. After cells were incubated with or without metformin (5 or 10 mM) for 48 h, the proportion of apoptotic cells was measured by flow cytometric of annexin V expression and JC-1 staining, which indicates the presence of a mitochondrial membrane potential (Figure 3A and 3B). Our results demonstrate that the proportion of apoptotic cells was higher in metformin-treated cultures compared with that in controls.To understand the mechanism by which metformin induced apoptosis in Ishikawa cells, we examined pro-apoptotic activity. Apoptosis can be activated through two main pathways: the intrinsic mitochondria-dependent pathway and the extrinsic death-receptor-dependent pathway. Caspase-8 is predominantly activated by signals from the extrinsic death-receptor pathway, while caspase-9 activation is dependent primarily on the intrinsic mitochondrial pathway. Together, pro-apoptotic Bax and anti-apoptotic Bcl-2 play an important role in mitochondrial outer membrane permeabilization. Metformin treatment induced a marked, dose-dependent increase in the Bax/Bcl-2 ratio (Figure 3D). Furthermore, metformin-mediated apoptotic death was accompanied by the activation of caspase, which is the principal apoptosis-executing enzyme. Fluorescence calorimetric analysis demonstrated that metformin treatment induced the activation of caspase-3/7, -8, and -9 (Figure 3E). Consistent with the induction of apoptosis, western blots revealed that metformin treatment led to cleavage of caspase-3 and PARP in Ishikawa cells in a dose-dependent manner (Figure 3C).

To determine whether metformin induced autophagy in Ishikawa cells, we used AO to stain AVOs, including autophagic vacuoles. Untreated Ishikawa cells exhibited bright green fluorescence in the cytoplasm and nuclei and lacked bright red fluorescence. In contrast, metformin-treated cells exhibited AVOs, identified as bright red compartments (Figure 4A). The number of AVOs was significantly higher in metformin-treated cells compared with that in untreated controls, and this effect was dose dependent (Figure 4A). Levels of LC3B (an autophagosome component) and p62 (an autophagosome target) positively and negatively correlate with autophagy, respectively. Therefore, we used western blots to assess LC3B-I to LC3B-II conversion and p62 protein levels. As expected, metformin treatment induced significant LC3 I to II conversion (Figure 4C and D) and a decrease in p62 levels (Figure 4C and E) in a dose-dependent manner. Taken together, these results demonstrate that metformin induced autophagy in Ishikawa cells.

To determine the relationship between apoptosis and autophagy in Ishikawa cells, we inhibited autophagy either pharmacologically (via 3MA or CQ) or genetically, and assessed the effects on metformin-mediated apoptosis. A WST-8 assay showed that 3MA and CQ treatment significantly enhanced the viability of metformin-treated (10 mM) cells (Figure 5A). On addition, flow cytometric analysis showed that 3MA treatment caused a marked decrease in the proportion of metformin-treated (10 mM) apoptotic cells (Figure 5B). Moreover, 3MA treatment caused a significant reduction in caspase activity in metformin-treated (10 mM) cells (Figure 5C). Thus, these findings revealed that inhibition of metformin-mediated autophagy reduced apoptosis in Ishikawa cells.To confirm these results, we used siRNA to repress expression of the autophagy regulator Beclin1 in Ishikawa cells. Beclin1 siRNA knocked down Beclin1 expression by approximately 75% (Figure 6A). Upon metformin treatment, significantly fewer Annexin-V-positive cells were observed in Beclin1siRNA cells compared with that in controls (Figure 6B). The inhibition of autophagy by Beclin1 siRNA resulted in decreases in caspase-3/7 activity (Figure 6C), PARP cleavage, and LC3-II and increases in p62 (Figure 6D), as did pharmacologic inhibition of autophagy by 3MA (Figure 5D). These results demonstrate that the inhibition of autophagy reduced apoptosis associated with metformin treatment.