*Contributed equally
Breast cancer is the most common malignant tumor in women and remains a major global challenge, with ~1.4 million cases per year, worldwide. Numerous studies have shown that changes in cell metabolism are associated with the regulation of tumor progression. In the present study, the anti-cancer properties of glyoxal (GO), which is the smallest dialdehyde formed in the oxidation-reduction reaction and involved in electron transfer and energy metabolism, in breast cancer was investigated. The biological functions and molecular mechanisms of GO were investigated in breast cancer cell lines using MTT and crystal violet assays, flow cytometry, western blot analysis, 3D laser scanning confocal microscopy and transmission electron microscopy. The results showed that GO strongly inhibited cell proliferation, promoted cell apoptosis and cell cycle G2/M arrest, induced the disappearance of cellular microvilli, and enlarged mitochondria. In addition, the protein expression level of AKT, mTOR and p70-S6K decreased in the AKT-mTOR pathway, accompanied by an increase in p-ERK and p-MEK in the MAPK pathway. The results from the present study indicate that GO suppressed breast cancer progression via the MAPK and AKT-mTOR pathways. Taken together, these results provide the basis for a potential therapeutic strategy for breast cancer.
Breast cancer is the most common malignancy in women and remains a major global challenge. Approximately 1.4 million cases occur per year, worldwide (
Glyoxal (GO) is the smallest dialdehyde formed in the oxidation-reduction reaction and is associated with electron transfer and metabolism (
The present study aimed to investigate the biofunctions of GO and investigate its molecular mechanisms in breast cancer progression.
The MDA-MB-231, SUM149 and SUM159 cell lines were used as representative triple-negative breast cancer cell models, while the EMT6 and MCF-7 cell lines were used as estrogen receptor-positive breast cancer cell models, and the MCF-10A cell line was used to represent a normal breast cell line. All the cells were purchased from the Shanghai Institutes for Biological Sciences and cultured at 37˚C in a humidified incubator with 5% CO2 in DMEM (HyClone; Cytiva) supplemented with 10% FBS (Natocor) and antibiotics (Sigma-Aldrich; Merck KGaA) (
GO (Sinopharm Chemical Reagent Co., Ltd.) is an organic compound with the chemical formula OCHCHO, and the Chemical Abstract Service number, 107-22-2. GO was filtered using a 0.22 µm filter (Millipore Sigma) and stored at 26˚C in the dark.
Cell viability was performed using the MTT assay (Sigma-Aldrich; Merck KGaA). The MDA-MB-231, SUM149, SUM159, EMT6, MCF-7 and MCF-10A cell lines (3x103 cells/well) were seeded in 96-well plates and treated with different concentrations of GO (0.129, 0.258, 0.516, 1.032, 2.065, 4.13 and 8.25 mmol/l for MDA-MB-231, SUM149 and SUM159; and 1.032, 2.065 and 4.13 mmol/l for EMT6, MCF-7 and MCF-10A). After 24 h, 20 µl 5 mg/ml MTT solution was added to each well and the plates were further incubated at 37˚C for 4 h. Following which, the medium was aspirated, and 200 µl dimethyl sulfoxide was added to each well. After the purple formazan crystals had dissolved, the absorbance was determined at 492 nm using an INFINITE F50 microplate reader (Tecan Group, Ltd.). According to the MTT data, IC50 values were computed by GraphPad Prism (v8.0; GraphPad Prism Software, Inc.). Meanwhile, the IC50 for GO was used for 24 h, and the morphology was captured by Nikon TS100 microscope (Nikon Corporation). The results were obtained from three independent experiments.
The MDA-MB-231, SUM149, SUM159, EMT6, MCF-7 and MCF-10A cell lines were seeded in 24-well plates, at a density of 1x103 cells/well and incubated for 24 h. The cells were then treated with different concentrations of GO (0.516, 1.032, 2.065, 4.13 and 8.25 mmol/l) continuously for 5 days. After fixation with 4% paraformaldehyde for 30 min at 26˚C, the cells were stained with crystal violet solution for 2 h at 26˚C. The results were obtained from three independent experiments.
Cell migration ability was performed using a wound-healing assay. Approximately 2x104 cells were seeded into each well of a 6-well plate without serum and a light microscope was used the following day to confirm that each well was coated with cells at ~90% confluence. A 1.0 ml pipette tip was used to remove the cells in the wound-healing region and the plates were washed with PBS three times to remove the displaced cells. The cells were treated with different concentrations of GO (3.550/4.130 mmol/l for MDA-MB-231 and SUM149; 1.437/2.065 mmol/l for SUM159; 1.85/4.13 mmol/l for EMT6; and 1.032/4.13 mmol/l for MCF-7 and MCF-10A). The control cells were incubated with serum-free medium at 37˚C with 5% CO2. At 0, 24 and 48 h, images were captured using a Nikon TS100 microscope (Nikon Corporation), at x40 magnification and the wound was measured using ImageJ software (v1.52a; National Institutes of Health). The experiment was repeated three times.
The MDA-MB-231, SUM149, SUM159 and EMT6 cell lines were treated with different concentrations of GO (3.550, 3.550, 1.437 and 2.22 mmol/l, respectively) for 24 h. For TEM, a total of 1x107 cells were pelleted by centrifugation at 2,683 x g for 5 min at 26˚C, then washed three times with PBS. The cells were then fixed in 2.5% glutaraldehyde at 4˚C for 24 h. Next, the cells were washed with PBS three times and post-fixed in 1% osmium tetroxide for 60 min at 4˚C, encapsulated in 1% agar and stained with uranyl acetate and phosphotungstic acid for 60 min at 4˚C. The cells were then dehydrated in a graded ethanol series and subsequently incubated in propylene oxide for 35 min at 26˚C. The TEM images were captured using a Hitachi TEM system (Hitachi High-Technologies Corporation).
For 3D micro-morphology, the volume and height of the SUM149, MDA-MB-231, SUM159 and EMT6 cell lines were measured using a VK-V150 laser microscopy system (Keyence Corporation). Phase-contrast observations of the cells were performed using an Olympus IX71 microscope (Olympus Corporation). The results were obtained from three independent experiments.
The protein expression level of ERK, phosphorylated (p)-ERK, MEK, p-MEK, AKT, p-AKT-Ser473, p-AKT-Thr308, mTOR and p70-S6k was measured using western blot analysis in the MDA-MB-231, SUM149 and SUM159 cell lines, which were each treated with the IC50 of GO. All cells were lysed in RIPA lysis buffer (cat. no. P0013B; Beyotime Institute of Biotechnology) and then centrifuged at 15,702 x g for 15 min at 4˚C. Protein concentrations were determined using a BCA kit (Beyotime Institute of Biotechnology). A total of 20 µg protein was separated on 6-10% gels using SDS-PAGE and transferred to PVDF membranes (MilliporeSigma). The membranes were blocked for 1 h at 26˚C with 5% bovine serum albumin containing 0.1% Tween-20. Immunoblotting was performed using the following primary antibodies: ERK (cat. no. 13-6200, 1:1,000), p-ERK (cat. no. 44-680G; 1:500), MEK (cat. no. PA5-116802; 1:500), p-MEK (cat. no. 44-452, 1:1,000), AKT (cat. no. MA191204; 1:1,000), mTOR (cat. no. A301-144A-T; 1:1,000), p70-S6k (cat. no. MA5-36267; 1:1,000) (all Invitrogen; Thermo Fisher Scientific, Inc.) and Tubulin (cat. no. AF1216; 1:1,000; Beyotime Institute of Biotechnology) overnight at 4˚C. The membranes were then washed with 1% TBS-Tween-20 three times and incubated with the corresponding secondary antibodies (cat. no. A0208; goat anti-rabbit; 1:5,000; Beyotime Institute of Biotechnology) at 37˚C for 2 h. The membranes were washed again with TBS, and the proteins were visualized using an enhanced chemiluminescence assay kit (Beyotime Institute of Biotechnology). Images were captured using a Bio-Rad Chemodoc XRS+ system and the Image-lab software (Version 6.0; Bio-Rad Laboratories, Inc.). The test was repeated three times.
The MDA-MB-231, SUM149 and SUM159 cell lines (5x104 cells/well) were seeded in 6-well plates and treated with GO (IC50: 3.78, 1.85 and 1.60 mmol/l, respectively) for 24 h. Next, the cells were collected and stored in pre-cooled alcohol overnight at 4˚C, then stained with PI (Shanghai Yeasen Biotechnology, Co., Ltd.) for 15 min in the dark at 4˚C. The samples were tested using a Guava EasyCyte Plus flow cytometer (Merck KGaA) and FlowJo VX (Becton-Dickinson and Company) was used to analyze the results. The test was repeated three times.
The MDA-MB-231, SUM149 and SUM159 cell lines (5x104 cells/well) were seeded in 6-well plates and treated with GO (IC50: 3.78, 1.85 and 1.60 mmol/l, respectively) for 24 h. The cells were then collected and stained using the Annexin V/PI kit (Shanghai Yeasen Biotechnology, Co., Ltd.) for 15 min in the dark at 4˚C. The samples were tested using a Guava EasyCyte Plus flow cytometer (Merck KGaA) and FlowJo VX (Becton, Dickinson and Company) was used to analyze the results. The test was repeated three times.
The data were expressed as the mean ± SD, and unpaired t-tests or repeated measures one-way ANOVA followed by Dunnett's multiple comparisons test were performed for statistical analyses using GraphPad Prism (v8.0; GraphPad Prism Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.
MTT and crystal violet assays were used to investigate the biofunctions of GO on cancer cell proliferation in different breast cancer cell lines. The results demonstrated that cell proliferation was inhibited in a concentration-dependent manner. As the GO concentration increased, a greater inhibitory effect was exerted in the breast cancer cell lines. Inhibition rates were up to 78.95±0.05, 88.83±1.35, 87.49±1.11, 88.98±9.90 and 71.77±1.29% for the MDA-MB-231, SUM149, SUM159, EMT6, and MCF-7, respectively (P<0.05 compared with cells without GO). However, GO only slightly reduced the proliferation rate in the MCF-10A normal breast cell lines, as the inhibition rate was <38.26%. The IC50 values of the MDA-MB-231, SUM149, SUM159, EMT6, MCF-7 and MCF-10A cell lines were 3.78, 1.85, 1.60, 1.29, 2.22, and 4.39 mmol/l, respectively (
To further elucidate the mechanisms underlying the action of GO, the protein expression level of the downstream kinases of the MAPK and AKT/mTOR pathways were investigated using western blot analysis. Consistent with the aforementioned results, GO was found to be involved in the regulation of the MEK-ERK and AKT/mTOR pathways. The results indicated that the protein expression level of AKT1 was suppressed in SUM149 and SUM159 group, and the expression level of mTOR and P70-S6K proteins was suppressed in MDA-MB-231, SUM149, and SUM159 cells (
To investigate the effect of GO on breast cancer and normal breast cell migration, differences in the wound healing rate in the MDA-MB-231, SUM149, SUM159, EMT6, MCF-7 and MCF-10A cell lines 48 h following GO treatment were observed. The results showed that the relative scratch width of the GO group was significantly wider compared with that in the control group at the 24 and 48 h time points. In the MDA-MB-231 group, the migration inhibition rates were 84.18 and 86.32% at 3.550 and 4.130 mmol/l, respectively. In the SUM149 group, the migration inhibition rates were 81.19 and 82.94% at 3.550 and 4.130 mmol/l, respectively. In the SUM159 group, the migration inhibition rates were 67.65 and 80.00% at 1.437 and 2.065 mmol/l, respectively. In the EMT6 group, the migration inhibition rate was 52.86 and 81.88% at 1.85 and 4.13 mmol/l, respectively. In the MCF-7 cells, the migration inhibition rate was 77.33 and 80.42% at 1.032 and 4.13 mmol/l, respectively. In the MCF-10A normal breast cells, the migration inhibition rate was 44.20 and 44.92% at 1.032 and 4.13 mmol/l, respectively. Under the above general tendency, the data indicated that GO suppressed cell migration in a concentration-dependent manner at both 24 and 48 h (P<0.05) (
The cellular ultrastructure was observed using TEM. A previous study has indicated that typical morphological features of apoptosis include chromatin condensation, nuclear fragmentation and the disappearance of surface microvilli (
To improve the understanding into the changes in cellular morphology following GO treatment, a 3D laser scanning confocal microscope was used, which is a valuable tool for obtaining high-resolution images and 3D reconstructions. Treatment with GO for 24 h notably altered cellular morphology, height and volume (
Next, the effects of GO on cell apoptosis and the cell cycle were investigated. As shown in
The morbidity rate of breast cancer has surpassed that of lung cancer so that breast cancer is now the most malignant tumor in the world. Thus, besides chemotherapy, targeted treatment and endocrine treatment, more treatments are required for breast cancer. Cytotoxic agents are no longer the only potential novel anticancer drugs. The ‘Warburg effect’ suggests that even under oxygen-sufficient conditions, tumor cells still take advantage of glycolysis metabolism, using oxidative phosphorylation, which is associated with the respiratory chain rather than producing ATP (
In addition, tumor cell proliferation is significantly inhibited by dichloroacetic acid, which inhibits mitochondrial pyruvate dehydrogenase kinase and activates potassium channels in all cancer cells, thereby inducing apoptosis (
Furthermore, metabolic reprogramming is an important hallmark of cancer cell proliferation (
Therefore, the present study aimed to further investigate the effects and mechanisms of GO stress on breast cancer cells. The results of functional analyses showed that GO treatment notably induced a decrease in cell proliferation, increased cell apoptosis, arrested the cell cycle in G2 phase and altered cellular ultrastructure. The western blot results indicated that GO treatment was involved in the MAPK and mTOR signaling pathways in breast cancer. Taken together, these findings suggest that GO is a potential anti-cancer therapeutic agent that inhibits breast cancer progression by regulating the MAPK and AKT/mTOR signaling pathways. Notably, measuring the increase in cellular levels of p-ERK has been shown to be an indirect indicator of the increase in bioavailable copper that causes cell apoptosis (
In conclusion, several compounds affect breast cancer cell lines via glycolysis, such as MGO and GO. As GO is the smallest dialdehyde and contains two adjacent reactive carbonyl groups, GO can cause increased cytotoxicity, and protein carbonylation than MGO. However, GO causes severe side-effects and toxicity than MGO; therefore, the concentration of GO must be strictly adjusted to be kept in a safe range. The results from the present study showed that GO could be a potential therapeutic agent for breast cancer; however, additional research is required to gain a more in-depth understanding of its mechanisms.
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
PR and LY designed the study. GN, LY and LQ contributed to the cell culture and experiments. PR and LX analyzed the data. LX and LY wrote the original paper. PR, LY, GN, LQ and LX confirm the authenticity of all the raw data. All authors have read and approved the manuscript.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
Inhibition ability at different doses of GO was analyzed using the MTT and crystal violet assays in the MDA-MB-231, SUM149, SUM159, EMT6, MCF-7 and MCF-10A cell lines. (A) Under the above general tendency, the results from the MTT assay showed that GO inhibited MDA-MB-231, SUM149, SUM159, EMT6 and MCF-7 cell proliferation in a concentration-dependent manner. GO at 4.13 mmol only notably inhibited the MCF-10A normal breast cell lines. (B) The MDA-MB-231, SUM149, SUM159, EMT6 and MCF-7 cell lines were treated with GO for 24 h and changes in cell morphology and cell death were observed. However, the cell morphology of the MCF-10A cell line was almost normal. GO, glyoxal.
(A) GO notably reduced the colony numbers in the MDA-MB-231, SUM159, MCF-7, MCF-10A and EMT6 cell lines following treatment with different concentrations. (B) The result of Western blot assay. a, TNBC cell lines were treated with GO. The protein expression level of AKT1 decreased in the SUM149 and SUM159 groups, and the expression level of mTOR and P70-S6K proteins decreased in all three cell lines. Meanwhile, p-ERK protein expression increased in all three cell lines, and p-MEK protein expression increased in MDA-MB-231 and SUM149 cell lines. b, Analysis of Western blot bands in each cell line by Image J software (National Institutes of Health, USA). GO, glyoxal; CTR, control; p, phosphorylated.
Wound healing assay using the MDA-MB-231, SUM149, SUM159, EMT6, MCF-7 and normal MCF-10A breast cell lines. The migration distance in the glyoxal groups was significantly shorter compared with that in the control group. Images were captured at 0, 24 and 48 h after scratching. *P<0.05, **P<0.01, ***P<0.001. Magnification, x40.
TEM analysis in the SUM149, SUM159 and MDA-MB-231 cell lines. Compared with that in the control group, glyoxal induced cellular surface microvillus disappearance (blue arrow), but the cell membrane remained intact (yellow arrow). Magnification, x5,000 and x20,000.
(A) The SUM149, SUM159, MDA-MB-231 and EMT6 cell lines were analyzed using 3D laser scanning confocal microscope. The cell morphology was changed to circular and flat structure following glyoxal treatment. Magnification, x400. Cellular (B) height and (C) volume decreased. *P<0.05, ***P<0.001.
Cell apoptosis and cell cycle was analyzed using flow cytometry. (Aa) Glyoxal increased the positive rate of Annexin V+/PI+ cells in the MDA-MB-231, SUM149 and SUM159 cell lines and the results were (Ab) statistically analyzed. (Ba) G2/M phase was arrested in the glyoxal group compared with that in the control group and the results were (Bb) statistically analyzed. *P<0.05, **P<0.01, ***P<0.001.