Mr. Lihui Yang, Department of Nursing, Guangxi Medical University, 8 Shuangyong Road, Nanning, Guangxi Zhuang Autonomous Region 530021, P.R. China
*Contributed equally
Aerobic glycolysis is commonly observed in tumor cells, including triple-negative breast cancer (TNBC) cells, and the rate of aerobic glycolysis is higher in TNBC cells than in non-TNBC cells. Hexokinase 2 (HK2) is a key enzyme in the glycolytic pathway and a target of the transcription factor c-Myc, which is highly expressed in TNBC and promotes aerobic glycolysis by enhancing HK2 expression. As an inhibitor of HK2, 3-bromopyruvic acid (3-BrPA) exhibits good therapeutic efficacy in intrahepatic and extrahepatic tumors and inhibits the proliferation of human tumor cells with high expression levels of c-Myc
Breast cancer is one of the most frequent malignant tumors that occurs in females worldwide, and its incidence is increasing annually (
Hexokinase 2 (HK2) serves an important role in the first step of glycolysis as a key enzyme (
Thioredoxin-interacting protein (TXNIP), a binding partner with negative regulatory effect of thioredoxin, could inhibit glucose uptake and aerobic glycolysis (
To confirm this scientific hypothesis, the present study analyzed the effects of 3-BrPA on aerobic glycolysis in TNBC using human TNBC (HCC1143) and non-TNBC (MCF-7) cell lines. After treatment with 3-BrPA at different concentrations and for different durations, the effects of 3-BrPA were evaluated in terms of cell viability using a Cell Counting Kit-8 (CCK-8) assay and apoptosis using flow cytometry. HK activity, lactate generation, ATP generation and the expression levels of TXNIP, GLUT1, HK2 and c-Myc, as well as mitochondria-mediated apoptosis pathway proteins were examined by western blotting to further clarify the internal mechanism by which 3-BrPA may inhibit TNBC cell viability and induce apoptosis.
TNBC (HCC1143) and non-TNBC (MCF-7) cell lines were purchased from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences. Cells were cultured in complete growth medium consisting of DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin (Beijing Solarbio Science & Technology Co., Ltd.). Cells were incubated in an incubator (Thermo Fisher Scientific, Inc.) at 37˚C with 5% CO2 and passaged every 3-4 days. Morphological changes were observed under an inverted fluorescence microscope (IX73; Olympus).
CCK-8 (Dojindo Laboratories, Inc.) was used to detect the viability of cells. Briefly, ~1x104 cells/well were added to 96-well plates and cultured in an incubator with 5% CO2 at 37˚C. Subsequently, cells were treated with 3-BrPA (Sigma-Aldrich; Merck KGaA) at 37˚C at different concentrations (0, 20, 30, 40, 50 and 60 µM) and for different durations (24 and 48 h). CCK-8 reagent (10 µl) was added to each well, mixed and then incubated for an additional 2 h. The absorbance values of the cells were immediately detected at 450 nm using a microplate reader (Thermo Fisher Scientific, Inc.). The inhibitory rate was calculated as (1-Asample/Acontrol)x100%, where Asample is the absorbance value of the sample treated with 3-BrPA at different concentrations and time points and Acontrol is the absorbance value of the control group (untreated cells) recorded at the same time point. Each experiment was performed in triplicate. The viability curves and IC50 of 3-BrPA for each cell line were calculated by GraphPad Prism 8.0.1 (GraphPad Software, Inc.).
Cells were treated with 0, 20, 40 and 60 µM 3-BrPA for 24 h. Approximately 1x105 cells were collected after centrifugation at 300 x g with pre-cooled phosphate buffer saline for 10 min at 4˚C. Then cells were stained with 5 µl Annexin V-allophycocyanin (Annexin V-APC) and 10 µl 7-amino-actinomycin D (7-AAD) according to the manufacturer's instructions for the Annexin V-APC/7-AAD cell apoptosis kit (Hangzhou Lianke Biotechnology Co., Ltd.). After gentle vortexing, cells were incubated at room temperature for 5 min in the dark. Flow cytometric analyses were performed on a FACSCalibur (BD Biosciences) with CellQuest Pro 5.2.1 (BD Biosciences). Each experiment was performed in triplicate.
Cells were seeded into 6-well plates at a density of 2x106 cells/well and were then incubated with 3-BrPA (0, 20 and 40 µM) for both 24 and 48 h at 37˚C in 5% CO2. HK activity detection was performed the Hexokinase Activity Detection kit (Beijing Solarbio Science & Technology Co., Ltd.) according to the manufacturer's instructions. The absorbance values at 340 nm at 20 sec (A1) and 320 sec (A2) after sample addition were measured using a spectrophotometer (Thermo Fisher Scientific, Inc.). Then, A1 and A2 were plugged into the formula HK(U/104 cell)=[(ΔA)(Vtotal/(εxd)x109]/(500xVsample/Vsample_total)/t=2.226xΔA (ΔA=A2-A1), where Vtotal refers to the total volume of the reaction system (1.038x10-3 l), ε is the NADPH molar extinction coefficient (6.22x103 l/mol/cm), d represents the diameter of the cuvette (1 cm), Vsample is the sample volume (0.03 ml), Vsample_total is the extract volume (1 ml), t is the reaction time (5 min), and 500 is the total number of cells (500x104). These factors were used to calculate HK activity. An enzyme activity unit (1 unit) is defined as 1 nmol of NADPH produced per min per 10,000 cells.
The two cell lines were seeded into 96-well plates at a density of 1x104 cells/well. After attachment, the cells were treated with different concentrations (0, 20 and 40 µM) of 3-BrPA for 24 or 48 h. The culture medium was collected to measure the level of lactate generation using the lactate assay kit (Sigma-Aldrich; Merck KGaA) according to the manufacturer's protocol. The optical density at 530 nm was measured using a microplate reader (Multiskan FC; Thermo Fisher Scientific, Inc.).
ATP generation was measured using the ATP Assay Kit (Beijing Solarbio Science & Technology Co., Ltd.). The two cell lines were seeded into 96-well plates at a density of 1x105 cells/well and cultured overnight at 37˚C. Subsequently, the cells were incubated with 3-BrPA (0, 20 and 40 µM) for 4 or 8 h (based on our preliminary experiments, the ATP generation after 24/48 h incubation with 3-BrPA is already very low and cannot be measured accurately, therefore 4/8 h was selected). Then, the cells were treated according to the manufacturer's protocol. The absorbance values at 340 nm at 10 sec (A1) after sample addition and 3 min after a 25˚C water bath (A2) were measured using a spectrophotometer (Thermo Fisher Scientific, Inc.). Both A1 and A2 were plugged into the formula ATP (µmol/106 cell)=(ΔAsample)/(ΔAstandard/Cstandard)V/n(ΔA=A2-A1), where Cstandard refers to the concentration of the standard solution (0.625 µmol/ml), V denotes the added volume of extraction solution (1 ml), and n indicates the number of cells (x106). These factors were used to calculate ATP production.
The two cell lines were treated with 0, 20 and 40 µM 3-BrPA for 24 or 48 h. Total protein was extracted using a Total Protein Extraction kit (Beijing Solarbio Science & Technology Co., Ltd.) for western blot analysis. The protein concentration was determined using a BCA Protein Assay kit (cat. no. 23227; Thermo Fisher Scientific, Inc.). The proteins (30 µg per lane) were separated on a denaturing 12% SDS-PAGE gel and then transferred to a polyvinylidene fluoride membrane. Then the membrane was cultured in QuickBlock Blocking Buffer for Western Blot (Beyotime Institute of Biotechnology) at room temperature for 1 h. The membrane was incubated with antibodies against GLUT1 (1:1,000; cat. no. TA312796; OriGene Technologies, Inc.), c-Myc (1:1,000; cat. no. TA150121; OriGene Technologies, Inc.), TXNIP (1:1,000; cat. no. TA349090; OriGene Technologies, Inc.), HK2 (1:1,000; cat. no. TA500856; OriGene Technologies, Inc.), Bcl-2 (1:2,000; cat. no. TA806591; OriGene Technologies, Inc.), Bax (1:2,000; cat. no. TA810334; OriGene Technologies, Inc.), cytochrome
All results were presented as the means ± standard deviations and images were analyzed using SPSS 20.0 statistical software (IBM Corp.) and GraphPad Prism 8.0.1 software (GraphPad Software, Inc.). Statistical analysis was performed using one-way ANOVA and Bonferroni's correction. P<0.05 was considered to indicate a statistically significant difference. The experiments were performed in triplicate.
The effects of 3-BrPA on cell viability in TNBC (HCC1143) and non-TNBC (MCF-7) cell lines were examined using a CCK-8 assay. Morphological changes were observed under an inverted fluorescence microscope, and numerous morphological changes occurred in cells after treatment with 3-BrPA. Untreated cells attached closely to one another, and were polygonal in shape (
Flow cytometry was performed to evaluate cell apoptosis. The results demonstrated that the early apoptosis rates of the TNBC cell line (HCC1143) were significantly increased in a dose-dependent manner compared with those of the corresponding untreated control cells (
Lactate is a vital final product in glycolysis that reflects glycolytic metabolism in cells (
The ATP generation level was considered to be a favorable indicator of the glycolysis rate in cells because it is the main ultimate product in glycolysis (
As a key enzyme in aerobic glycolysis, HK activity reflects the strength of glycolytic metabolism in tumor cells (
To confirm the effect of 3-BrPA on c-Myc, TXNIP, HK2 and GLUT1 expression, TNBC (HCC1143) and non-TNBC (MCF-7) cells were treated with 3-BrPA at different doses for different durations. Subsequently, protein expression levels were detected through western blotting. The protein expression levels of c-Myc and HK2 in the TNBC group were dose and time-dependently decreased following treatment with 3-BrPA (
To examine the effect of 3-BrPA on the mitochondria-mediated apoptosis pathway, the relative expression levels of anti-apoptotic Bcl-2 and pro-apoptotic Bax, Cyt-C and Caspase-3 were analyzed. Both TNBC and non-TNBC cells were exposed to 3-BrPA at different doses and for different durations. Subsequently, the expression levels of mitochondria-mediated apoptosis pathway proteins were detected through western blotting. Treatment with 3-BrPA significantly decreased the protein expression levels of anti-apoptotic Bcl-2 at 40 µM at 24 h, and 20/40 µM at 48 h; while it increased the protein expression levels of pro-apoptotic Bax and Caspase-3 at both 20/40 µM at 24/48 h; and Cyt-C at 40 µM at 24 h and 20/40 µM at 48 h in TNBC cells (
TNBC is a notable type of breast cancer because of its unique molecular type. The 5-year survival rate of patients with TNBC is poorer than that of patients with other types of breast cancer (
3-BrPA is receiving increasing attention given the increasing interest in research on antitumor drugs. 3-BrPA is an analog of pyruvate with high tumor selectivity (
HK is a key rate-limiting enzyme in the first step of glycolysis in tumor tissues, and its expression and activity increase in tumor tissues to promote effective anaerobic glycolysis even under anaerobic conditions (
An increasing number of researchers have recognized the prognostic and predictive power of TXNIP, which is a potent negative regulator of glucose uptake, in human breast cancer. Shen
Among various oncogenes, c-Myc, which is a strong transcription factor participating in several aspects of the oncogenic process, acts as a vital member of the Myc family (
The following mechanisms may be involved in the inhibition of TXNIP caused by c-Myc. Myc, which is a member of the basic region helix-loop-helix leucine zipper (bHLHZip) family of transcription factors, operates by binding to another member of the same family, MYC associated factor X (MAX) (
Apoptosis is a form of programmed cell death that occurs in multicellular organisms and is induced by a variety of physical and chemical factors. Although there are three predominant signaling pathways in apoptosis, apoptotic signaling usually occurs at the mitochondrial-mediated pathway level, which is mainly regulated by Bcl-2 family genes (
The results obtained by different teams were dissimilar despite the same drug and the same cell line being used. For example, Kwiatkowska
In conclusion, in the present study, 3-BrPA reduced the HK2 expression and promoted the TXNIP protein expression in TNBC cells by downregulating the expression of c-Myc, thereby inhibiting glycolysis including suppressing lactate generation, intracellular ATP generation and HK activity, inducing mitochondria-regulated apoptosis, and eventually limiting TNBC cell proliferation. These findings contribute to the field of breast cancer therapy, and 3-BrPA is expected to become an effective drug in breast cancer therapy in the future. The concept of using glycolysis inhibitors combined with chemotherapeutic drugs has already been proposed (
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
JL and JP participated in the preliminary experimental design, preliminary experiment, main experiment operation, data analysis, manuscript writing and revision. YL, XL, CY, WX and QL participated in data analysis, manuscript writing and revision. LY and XZ participated in early experimental design, including selecting drugs and designing possible signal pathways. JL and JP confirm the authenticity of all the raw data. All authors read and approved the final manuscript.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
3-BrPA inhibits TNBC cell viability. (A) Cell morphology changes were observed using an inverted microscope. Representative microscopy images showing the difference in size (magnification, x200) of untreated and 3-BrPA-treated HCC1143 and MCF-7 cells. Untreated cells attached closely to each other. However, the cells treated with 3-BrPA became round or floated up with larger zones between cells and the number of viable cells decreased. (B) Cell viability was measured using a Cell Counting Kit-8 assay. Cell viability was inhibited by 3-BrPA. Furthermore, the inhibitory rate in TNBC cells was higher than that in non-TNBC cells (*P<0.05 vs. non-TNBC group at the same time and dose). 3-BrPA, 3-bromopyruvic acid; TNBC, triple-negative breast cancer.
3-BrPA induces TNBC cell apoptosis. (A) Cell apoptosis was analyzed using flow cytometry. The upper right quadrant shows late apoptotic cells, the lower right quadrant shows early apoptotic cells, the upper left quadrant shows necrotic cells and the lower left quadrant shows normal cells. (B) Percentages of the early apoptosis in TNBC cells induced by 3-BrPA were significantly increased. However, this trend was not identified in non-TNBC cells. The percentages of the late apoptosis in both TNBC cells and non-TNBC cells induced by 3-BrPA were only significantly increased at 40 µM. *P<0.05 vs. corresponding control group. 3-BrPA, 3-bromopyruvic acid; 7-AAD, 7-amino-actinomycin D; APC, allophycocyanin; TNBC, triple-negative breast cancer.
Effect of 3-BrPA on glycolysis-related indicators. (A) 3-BrPA induced a reduction in intracellular lactate production in TNBC cells. (B) TNBC cells treated with 3-BrPA exhibited reduced intracellular ATP production. (C) HK activity was significantly reduced by 3-BrPA treatment in TNBC cells. However, the glycolysis-related indicators were not affected by 3-BrPA treatment in non-TNBC cells. *P<0.05 vs. corresponding control group. 3-BrPA, 3-bromopyruvic acid; HK, hexokinase; TNBC, triple-negative breast cancer.
3-BrPA regulates expression of c-Myc, TXNIP and HK2. (A) Expression levels of c-Myc, TXNIP, HK2 and GLUT1 were detected through western blot analysis. Grayscale values of the bands were determined using software. (B) 3-BrPA downregulated c-Myc and HK2 protein expression, whereas it upregulated TXNIP protein expression in TNBC cells (*P<0.05 vs. control group, the same cell line with 0 µM 3-BrPA). (C) However, in non-TNBC cells, the expression levels of c-Myc, TXNIP, HK2 and GLUT1 were not significantly affected. The expression levels of GLUT1 were not significantly affected in both TNBC and non-TNBC cells. TXNIP, thioredoxin-interacting protein; HK2, hexokinase 2; GLUT1, glucose transporter 1; 3-BrPA, 3-bromopyruvic acid; TNBC, triple-negative breast cancer.
3-BrPA regulates expression of mitochondria-regulated apoptosis pathway proteins. (A) Expression levels of mitochondria-regulated apoptosis pathway proteins, including Bcl-2, Bax, Cyt-C and Caspase-3 were detected through western blot analysis. (B) Bcl-2 protein expression was decreased, while Bax, Cyt-C and Caspase-3 protein expression was increased following 3-BrPA treatment in TNBC cells (*P<0.05 vs. control group, the same cell line with 0 µM 3-BrPA). (C) However, in non-TNBC cells, the expression levels of Bcl-2, Bax, Cyt-C and Caspase-3 were not significantly affected. 3-BrPA, 3-bromopyruvic acid; Cyt-C, cytochrome