Contributed equally
As an anti-diabetic drug, metformin has been demonstrated to exhibit antitumor effects. However, the mechanisms involved in decreasing tumor formation, including canine mammary gland tumors (CMGTs), are not well elucidated. The aim of the present study was to evaluate the ability of metformin to induce apoptosis and cell cycle arrest in CMGT cells, as well as identifying the pathways underlying these effects. Cell viability was assessed by Cell Counting Kit-8 analysis following treating with metformin. Subsequently, apoptosis and cell cycle progression were assessed by flow cytometry, and the expression of associated proteins was examined. Expression levels of classical AMP-activated protein kinase (AMPK), protein kinase B (AKT), mechanistic target of rapamycin (mTOR) and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) were then investigated using western blot analysis. Metformin inhibited the proliferation of CHMm cells in a concentration-dependent manner. Specifically, metformin induced cell cycle arrest in the G0/G1 phases, accompanied by increased expression of p21 and p27, and decreased expression of cyclin D1 and cyclin-dependent kinase 4. Marked levels of apoptosis were observed in CHMm cells alongside the activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase. Also, the level of Bcl-2 was decreased, and that of Bax was increased. The expression of associated signaling molecules revealed that metformin markedly increased the phosphorylation of AMPK in CHMm cells, and decreased the levels of phosphorylated (p-)AKT, p-mTOR and p-4E-BP1, while Compound C reversed these changes. These findings demonstrated that metformin may be a potential therapeutic agent for CMGTs, acting via the AMPK/AKT/mTOR signaling pathway.
Canine mammary gland tumors (CMGTs) are a serious threat to animal health, and are frequently seen in female dogs, 50% of which are malignant (
Metformin (1,1-dimethylbiguanide) is used to treat type 2 diabetes, and due to its safety and tolerability, has been the first-line hypoglycemic drug since the 1950s (as recommended by the American Diabetes Association and the European Association for the Study of Diabetes) (
The underlying molecular mechanisms involved in metformin-associated antitumor effects remain unknown. The AMP-activated protein kinase (AMPK)/mechanistic target of rapamycin (mTOR) signaling pathway is considered to be involved in the antitumor effect of metformin, similar to its hypoglycemic mechanism with regard to AMPK activation (
The CMGT cell line, CHMm, was derived from pleural effusion of canine spontaneous mammary adenocarcinomas, and obtained from the Laboratory of Veterinary Surgery, Graduate School of Agriculture and Life Sciences, University of Tokyo (Tokyo, Japan) (
The Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc.) was used to evaluate cell viability. CHMm cells were seeded into 96-well plates (1,000 cells per well) and cultured at 37°C (5% CO2) until 80% confluent. The cells were then treated with 0, 5, 10, 20 and 40 mM metformin (Sigma-Aldrich; Merck KGaA), and viability was assessed after 48 h incubation. After 1 h incubation with CCK-8 reagent, the absorbance was measured at 450 nm.
An Annexin V-fluorescein isothiocyanate (FITC) kit (Beyotime Institute of Biotechnology) was used to quantify apoptosis, according to the manufacturer's instructions. Cells were treated with or without 20 mM metformin for 48 h at 37°C after serum starvation for 24 h (to facilitate drug absorption). The cells were harvested, washed with PBS, resuspended in Annexin V-FITC binding buffer and stained with Annexin V-FITC/propidium iodide (PI). The processed samples were examined by FACSCalibur flow cytometry (BD Biosciences) using FACSDiva software (version 6.1.3; BD Biosciences).
To assess the effects of metformin on the cell cycle of CMGT cells, CHMm cells were treated with or without 20 mM metformin for 48 h after serum starvation for 24 h to synchronize the cell cycle at the G0/G1 phase of the life cycle. Sample cells were rinsed with PBS and fixed with ice-cold 70% ethanol overnight in 4°C, before being incubated with PI for 30 min at room temperature, as per the protocol of the Cell Cycle and Apoptosis kit (Beyotime Institute of Biotechnology). A flow cytometer and FACSDiva Version 6.1.3 software were used to assess cell cycle distribution.
CHMm cells were treated with 0 or 20 mM metformin for 48 h after 2 h pretreatment with 0 or 10 µM Compound C (CC; Selleck Chemicals). Cells were lysed at 4° using Cell lysis buffer for Western and IP without inhibitors with 1 mM PMSF (Beyotime Institute of Biotechnology) and the protein was collected. Protein determination was performed using the Enhanced BCA Protein Assay Kit (Beyotime Institute of Biotechnology). Equal amounts of protein (30 µg/lane) were separated using SDS-PAGE (10% separation gel and 5% concentrated gel), and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% fat-free milk including TBS-Tween (0.1%) for 2 h at room temperature, and then incubated overnight at 4°C with the following primary antibodies(Albumin Bovine V, Beijing Solarbio Science & Technology Co., Ltd.): Caspase-3 (cat. no. ab13847), poly(ADP-ribose) polymerase (PARP; cat. no. ab32071), cyclin D1 (cat. no. ab16663), cyclin-dependent kinase 4 (CDK4; cat. no. ab108357), p21 (cat. no. ab188224), p27 (cat. no. ab32034), AMPK (cat. no. ab3760), phosphorylated (p-)AMPK (Thr-183/172; cat. no. ab23875), AKT (cat. no. ab8805), p-AKT (Thr-308; cat. no. ab38449), mTOR (cat. no. ab2732), p-mTOR (Ser-2448; cat. no. ab84400) and β-actin (cat. no. ab6276) (all from Abcam), as well as 4E-BP1 (cat. no. bs-2559R) and p-4E-BP1(Thr-70; cat. no. bs-14550R) (both BIOSS). All primary antibodies were used at a 1:1,000 dilution in 5% fat-free milk with TBS-Tween (0.1%). Detection was performed using HRP secondary antibodies (goat anti-rabbit; cat. no. ZB-2306 or goat-anti mouse; cat. no. ZB-2305; both Wanleibio Co., Ltd.; 1:3,000) for 2 h at room temperature, and visualized using enhanced chemiluminescence reagents (Tanon High-sig ECL Western Blotting Substrate; Tanon Science and Technology Co., Ltd.). Signals were quantified using Tanon GIS software (version 210.060.1000; Tanon Science and Technology Co., Ltd.).
All statistical analysis was performed using GraphPad Prism (version 5.0; GraphPad Software, Inc.), and the data are presented as the mean ± SD of three experimental repeats. Comparisons among ≥3 groups were analyzed using one-way ANOVA, and Tukey's test was used for further pairwise analysis. Unpaired Student's t-test was used to compare two groups. P<0.05 was considered to indicate a statistically significant difference.
To determine the inhibitory effect of metformin on CMGT cell proliferation, cell viability was evaluated using the CCK-8 assay following treatment with 0, 5, 10, 20 or 40 mM metformin. The results demonstrated that metformin reduced the viability and density of CHMm cells in a dose-dependent manner, suggesting that metformin inhibits the proliferative ability of CMGT cells
To further investigate whether the cell cycle is associated with metformin-induced reduction in CMGT cell proliferation, cell cycle distribution was flow cytometrically assessed following treatment with 20 mM metformin. As presented in
To determine the effects of metformin on the apoptosis of CMGT cells, metformin-induced apoptosis was detected using Annexin V-FITC/PI staining and flow cytometry. Compared with the control group, 20 mM metformin markedly increased the percentage of apoptotic CHMm cells under serum-deprived conditions (
In order to determine the effects of metformin on AMPK/AKT/mTOR signaling, an AMPK inhibitor, CC, was selected, and expression of the associated signaling proteins was analyzed. CHMm cells were pretreated with or without 10 µM CC for 2 h, and then treated with 20 mM metformin for 48 h. As illustrated in
The results of the present study demonstrated that metformin decreased the proliferation of CHMm cells in a dose-dependent manner
G0/G1 cell cycle arrest has been recognized as one mechanism underlying the antitumor effects of metformin, which has been confirmed in several cancer cell lines, and its regulation is dependent on the sequential activation and inactivation of cyclin-CDK-CD inhibitors (
There may be different mechanisms that regulate cancer cell apoptosis. As a programmed cell death process, apoptosis can be induced by the mitochondrial, death receptor-mediated pathway and endoplasmic reticulum pathway (
It has been demonstrated that distinct subtypes of CMGT cells are inhibited by metformin to various degrees, as a result of distinct mechanisms, e.g. in an AMPK-independent or -dependent manner (
It has also been reported that metformin inhibited the proliferation of human breast cancer cells by decreasing AKT activation and repressing associated downstream molecules (
mTOR is an essential mediator of growth signals regulated by the AMPK/AKT signaling pathway; moreover, activating AMPK and inhibiting AKT may restrain the activity of mTOR and thereby exert an antitumor effect (
In conclusion, the results of the present study demonstrated that metformin was able to inhibit the proliferation of CMGT cells, potentially through cell cycle arrest, induced apoptosis, activation of AMPK and suppression of AKT/mTOR signaling pathways. These results suggest that metformin may be a potential chemotherapeutic agent in the treatment of canine mammary gland tumors, and may offer insights into potential avenues of human breast cancer research.
Not applicable.
The present study was supported by grants from the National Natural Science Foundation of China (grant no. 31672617), the National Key Research Projects (grant no. 2016YFD0501008), the Initial Scientific Doctoral Research Foundation in Henan University of Animal Husbandry and Economy (grant no. 2019HNUAHEDF025), and Key projects of Henan Province Colleges and Universities (grant no. 20B230004).
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
YL conceived the project and designed the experiments. YF and XR wrote and revised the manuscript. YF, XR, EX and SW analyzed the data. YW, XR, YF and RG conducted the experiments. YL and YW confirmed the authenticity of all the raw data. All authors have read and approved the final manuscript.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
Antiproliferative effects of metformin on canine mammary gland tumor cells
Effects of metformin on the canine mammary gland tumor cell cycle. (A) Percentages of G1, S and G2 phase CHMm cells 48 h after treatment with 20 mM metformin, assessed using flow cytometry. (B) Proportions of CHMm cells in the G1, S and G2 phases. (C) Cell cycle-associated proteins were analyzed using western blotting 48 h after treatment with 20 mM metformin. Expression levels of (D) cyclin D1, (E) CDK4, (F) p21 and (G) p27 following treatment with or without 20 mM metformin. *P<0.05 and **P<0.01 vs. control. Unpaired Student's t-test was performed, and the results are presented as the mean ± SD. CDK4, cyclin dependent kinase 4.
Effects of metformin on apoptosis in CHMm cells. (A) Flow cytometry was used to analyze apoptosis in CHMm cells 48 h after treatment with or without 20 mM metformin. (B) Histograms present the percentage of apoptotic CHMm cells. Annexin V-FITC-positive cells (Q2 + Q4) were regarded as apoptotic. (C) Apoptosis-associated proteins were analyzed using western blot analysis 48 h after treatment with 20 mM metformin. Expression levels of (D) cleaved-caspase-3, (E) cleaved-PARP, (F) Bax and (G) Bcl-2 following treatment with or without 20 mM metformin. Unpaired Student's t-test was performed, and the results are presented as the mean ± SD. **P<0.01 vs. control. PARP, poly(ADP-ribose) polymerase; FITC, fluorescein isothiocyanate.
Effects of metformin on AMPK and downstream proteins. (A) Viability of CHMm cells was determined following treatment with metformin with or without the AMPK inhibitor CC at the 48-h time point. (B) Western blot analysis of p-AMPK and AMPK levels, (C) the p-AMPK to AMPK ratio, (D) p-AKT and AKT levels, (E) the p-AKT to AKT ratio, (F) p-mTOR and mTOR levels, (G) the p-mTOR to mTOR ratio, (H) p-4E-BP1 and 4E-BP1 levels, (I) and the p-4E-BP1 to 4E-BP1 ratio are presented following treatment with 0 or 20 mM metformin, with or without CC. One-way ANOVA and Tukey's test were used for statistical analysis, and the results are presented as the mean ± SD; **P<0.01 vs. control; ##P<0.01 vs. MET. βAMPK, AMP-activated protein kinase; AKT, protein kinase B: p-, phosphorylated; mTOR, mechanistic target of rapamycin; Met, metformin; CC, Compound C.