Hexokinase II/HXK II

3-Bromopyruvate/3-Bromopyruvic Acid improves the light exposure caused by TRAIL in breast cancer cells [2]. 3-Bromopyruvate increases the expression of DR5 while suppressing the synthesis of ATP. 3-Bromopyruvate raises AMPK, GRP78, and CHOP phosphorylation as well as the levels of Bax and caspase-3 triggered by TRAIL [2].3-BP enhances TRAIL-induced apoptosis in breast cancer cells. 3-BP/3-Bromopyruvic Acid inhibits ATP generation and upregulates the expression of DR5. 3-BP upregulates CHOP, GRP78 and the phosphorylation of AMPK and augments TRAIL-induced Bax and caspase-3 levels. The AMPK inhibitor Compound C reduces the TRAIL-synergizing effect of 3-BP on cell growth inhibition and apoptosis. Compound C inhibits 3-BP-induced upregulation of DR5, GRP78, CHOP and p-AMPK apoptosis-associated proteins.[2]

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Our results showed that 3-Bromopyruvic Acid/3-BrPA can induce growth inhibition in a dose-dependent pattern by cell apoptosis. 3-BrPA combined with rapamycin played a synergistic suppression role in NB cells, affected the cell apoptosis, cell cycle and the metabolic pathway. Up-regulated LC3-II accumulation was conscious in NB cells incubated with 3-BrPA and rapamycin. Rapamycin individually discourages the mTOR signaling pathway, while combined with 3-BrPA can enhance this phenomenon and influence cell metabolism of the NB cells. Conclusion: The results suggested that 3-BrPA combined with rapamycin could induce cell apoptosis in NB cells by inhibiting mTOR activity. In conclusion, our research proposed that the dual inhibitory effect of the mTOR signaling pathway and the glycolytic activity may indicate a valid therapeutic tactic for NB chemoprevention. [1]

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Hexokinase II (HKII), a key enzyme of glycolysis, is widely over-expressed in cancer cells. 3-Bromopyruvic Acid (3-BrPA), an inhibitor of HK II, has been proposed as a specific antitumor agent. Autophagy is a process that regulates the balance between protein synthesis and protein degradation. Autophagy in mammalian systems occurs under basal conditions and can be stimulated by stresses, including starvation, oxidative stress. Therefore, we hypothesized that 3-BrPA could induce autophagy. In the present study, we explored the mechanism of 3-BrPA and its combined action with chloroquine. Our results demonstrate that in MDA-MB-435 and in MDA-MB-231 cells, 3-BrPA induces autophagy, which can be inhibited by chloroquine. Furthermore, the combined treatment synergistically decreased the number of viable cells. Interestingly, the combined treatment triggered apoptosis in MDA-MB-435 cells, while it induced necroptosis in MDA-MB-231 cells. ROS mediated cell death when 3-BrPA and CQ were co-administered. Finally, CQ enhanced the anticancer efficacy of 3-BrPA in vivo. Collectively, our results show that 3-BrPA triggers autophagy, increasing breast cancer cell resistance to 3-BrPA treatment and that CQ enhanced 3-BrPA-induced cell death in breast cancer cells by stimulating ROS formation. Thus, inhibition of autophagy may be an innovative strategy for adjuvant chemotherapy of breast cancer.human skeletal muscle. Efficient Mirk depletion in SU86.86 pancreatic cancer cells by an inducible shRNA decreased expression of eight antioxidant genes. Thus both cancer cells and differentiated myotubes utilize Mirk kinase to relieve oxidative stress. [3]

3-Bromopyruvate (8 mg/kg; intraperitoneal injection; every 4 days for 28 days) in nude mice demonstrated excellent antitumor activity in MCF-7 cell xenografts [2].

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Antitumor efficacy of 3-Bromopyruvic Acid/3-BP and TRAIL in tumor xenografts [2]
MCF-7 cells were inoculated hypodermically into the forelimb of nude mice to induce the formation of xenograft tumors. Once the tumors reached a mean volume of ~100 mm3, mice matched for tumor volumes were divided into 4 groups and treated with either PBS, 3-BP, TRAIL or 3-BP and TRAIL. Following 28 days of treatment, the tumor volumes of mice treated with either PBS, 3-BP, TRAIL, or 3-BP and TRAIL were ~1,200±100, ~850±71, ~700±77 and ~232±40 mm3, respectively (Fig. 6A). To investigate the hepatotoxicity and nephrotoxicity of the aforementioned treatments, the levels of serological markers including aspartate aminotransferase (AST), alanine-aminotransferase (ALT), blood urea nitrogen (BUN) and creatinine (Cr) were determined. The results revealed that mice did not exhibit marked levels of toxicity following treatment with either PBS, 3-BP, TRAIL or 3-BP and TRAIL, as the expression of these markers was unchanged (Fig. 6B). The results of H&E staining demonstrated that necrosis occurred in the tumors of groups treated with 3-BP, TRAIL or both 3-BP and TRAIL, and the necrotic area in the group treated with both 3-BP and TRAIL was larger compared with the other treatment groups (Fig. 6C). H&E staining of the liver and kidney revealed that no evident damage was induced following the treatments (Fig. 6C). Finally, TUNEL staining demonstrated that the number of apoptotic cells in the group treated with 3-BP and TRAIL was increased compared with the other groups (Fig. 6D). Therefore, the results suggested that the antitumorigenic effect of 3-BP and TRAIL was associated with low hepatotoxicity and nephrotoxicity in vivo.

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CQ enhanced anti-tumor efficacy of 3-Bromopyruvic Acid/3-BrPA in nude mice [3]
To ascertain whether a combination of 3-BrPA and CQ could suppress tumor growth, MDA-MB-231 xenografts were examined. Tumors continued to grow in xenografted mice treated with vehicle, CQ, or 3-BrPA, or a combination of CQ and 3-BrPA. The combination of CQ plus 3-BrPA prevented tumor growth (Figure 6A). After treatment ended, mice were sacrificed, and tumors were excised and evaluated. Tumor weights of mice treated with vehicle, CQ, and 3-BrPA alone were greater than those from mice treated with CQ plus 3-BrPA (Figure 6B). Hematoxylin and eosin (HE) staining indicated greater necrotic areas in CQ plus 3-BrPA-treated mice. Thus, CQ enhanced the anti-tumor efficacy of 3-BrPA in vivo.

Cell Viability Assay[2]
Cell Types: MCF-7 and MDA-MB-231 Cell
Tested Concentrations: 40, 80, 160 or 320 µM
Incubation Duration: 24 hrs (hours)
Experimental Results: 3-bromopyruvate (80 and 160 µmol/l) and TRAIL (400 ng/ml) Dramatically inhibited cell viability.

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Western Blotting Analysis [1]
The human neuroblastoma cells were treated with 3-Bromopyruvic Acid/3-BrPA and/or rapamycin, total proteins were isolated on ice with RIPA lysing buffer, and the protein concentration was measured using a BCA protein assay kit. A total of 40μg proteins were submitted to SDS-PAGE and transferred to the PVDF membrane. The PVDF membranes were blocked with 5% skimmed milk supplemented with 0.05% Tween 20 in PBS for 1 hour, and then incubated with the primary antibody at 4°C overnight. The next day, the PVDF membranes were incubated with secondary antibody for 1 hour at the room temperature. Washing with TBST 3 times, the PVDF membranes were further developed with the ECL Plus Western Blotting Detection Reagents and Analysis System. To control the equal amount of protein loading, the PVDF membranes were calibrated with β-actin antibody.

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Annexin V-FITC/PI Staining [1]
The Annexin V-FITC Apoptosis Kit was used to detect the cell apoptosis in neuroblastoma cells (SH-SY5Y and SK-N-SH) treated with 3-BrPA and/or rapamycin. We treated SH-SY5Y and SK-N-SH with 3-BrPA and/or rapamycin, collected the supernatant and the cells, centrifuged and resuspended, added 1×Binding Buffer and 5uL Annexin V-FITC, mixed gently and incubated on ice for 10 minutes in dark place, added 10uL PI staining solution, then gently mixed and incubated on ice for 5 minutes. The data were performed on the Accuri C6, and the data were analyzed using Treestar FlowJo software.

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Flow Cytometric Evaluation of Cell Cycle [1]
The neuroblastoma cells (SH-SY5Y and SK-N-SH) were seeded into a 6-well plate at a density of 3×105 cells per well. The cells were treated with 3-BrPA and/or rapamycin, harvested with 0.25% trypsin (without EDTA), washed with pre-cooled PBS, fixed with 75% ethanol at 4°C for 2 hours or overnight, then washed twice with pre-cooled PBS, suspended in 100μL of 5 mg/mL RNase solution, incubated in darkness at the room temperature for 30 minutes, and then stained with 50 µg/mL Propidium Iodide (PI) solution for 30 minutes at 4°C in darkness. The analysis was performed with the FACS Calibur Flow Cytometer.

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Metabolites Analysis [1]
The neuroblastoma cells were cultured in RPMI-1640 medium with DMSO, 25μM rapamycin, 50μM 3-BrPA or 25μM rapamycin + 50μM 3-BrPA for 6 hours. The cellular glucose uptake, lactate production and ATP production were determined using respective assay kits obtained from BioVision (Milpitas). Data were an average of triplicate and presented as a percentage of the control group.

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Cell Viability Assay [1]
The cell proliferation of the NB cells treated with 3-Bromopyruvic Acid/3-BrPA and/or rapamycin was detected by Cell Counting Kit-8 assay. The NB cell lines (IMR-32, SH-SY5Y and SK-N-SH) were sown in 96-well plates with 5×103 cells per well overnight. And the cells were treated with different concentrations (0µM, 25µM, 50µM, 75µM and 100µM) of 3-BrPA for 24h, whereas DMSO was used as the blank group. The supernatant were thoroughly removed, subsequently the CCK-8 solutions were joined in the wells and incubated for 4 hours at 37°C in dark place. Finally, we use a microplate reader to measure the absorbance of all the wells at 450 nm. Assays were performed on three independent experiments.

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Cell viability assay [2]
Cells were seeded in 96-well plates (6×103 cells/well) and treated with different concentrations of 3-Bromopyruvic Acid/3-BP (0, 40, 80, 160 and 320 µmol/l) or TRAIL (25, 50, 100, 200 and 400 ng/ml) for 24 h. Phosphate-buffered saline (PBS) containing 5 mg/ml MTT (15 µl) was added to each well, and the cells were incubated for a further 4 h. Subsequently, the medium was then replaced with dimethyl sulfoxide (DMSO; 150 µl/well) in order to solubilize the formazan crystals. Finally, absorbance was determined at 490 nm using a plate reader.

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PI staining [2]
Cells were seeded in 12-well plates (1.5×105 cells/well) and incubated for 24 h, until the cells reached exponential phase. Subsequently, cells were treated for 24 h with various concentrations of 3-BP, TRAIL or both. Cells were subsequently stained using propidium iodide (PI; 600 µl/well) for 2 h and then analyzed using flow cytometry.

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ATP quantification [2]
CellTiter-Glo Luminescent Cell Viability Assay kit was used to investigate ATP levels, according to the manufacturer's instructions. Cells (1.5×105/well) were seeded in 12-well plates for 24 h and then treated with different concentrations of 3-BP for 4 h at 37°C. Following this, cells were collected and then lysed using radioimmunoprecipitation assay (RIPA) lysis buffer for 10 min on ice. Cell lysates were subsequently centrifuged at 13,225 × g for 5 min at 4°C. A nucleotide-releasing buffer (100 µl/well), ATP-monitoring enzyme (10 µl/well), and cell lysate (30 µl/well) were added to 96-well plates. Developed signals were then detected using a Luminoskan luminometer and a Varioskan™ Flash spectral scanning multimode reader.

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Cell surface staining [2]
Cells (2.5×105/ml) were seeded in 6-well plates, treated with PBS or 3-BP (80 µmol/l), and then incubated for 24 h. Subsequently, non-specific antibody binding sites were blocked using PBS containing 10% FBS for 20 min. Cells were then washed with PBS, re-suspended in 200 µl PBS, aliquoted into two tubes and incubated with 20 µl of an antibody-containing solution (rabbit anti-DR5 in PBS with 1% FBS; dilution 1:1,000) for 30 min on ice. Subsequently, the cells were washed twice with PBS, pelleted and incubated with 100 µl FITC-conjugated goat anti-rabbit IgG for 30 min on ice. Cell surface staining was investigated via flow cytometry using the Accuri™ C6 flow cytometer.

Animal/Disease Models: Female nude mice (BALB/c; 4-5 weeks old and 18-20 g) [2]
Doses: 8 mg/kg
Route of Administration: intraperitoneal (ip) injection; every 4 days for 28 days
Experimental Results: In tumors Demonstrated antitumor efficacy in xenografts.

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In vivo experiments [2]
In order to investigate the antitumor effect of 3-Bromopyruvic Acid/3-BP and TRAIL, female nude mice (BALB/c; 4–5-weeks old and 18–20 g) were used. Mice were maintained under specific pathogen-free conditions (26–28°C, air pressure difference was 10–20 kPa, 10-h light/14-h dark cycle, food and water were taken ad libitum). Mice were injected subcutaneously with MCF-7 cells (4×106 cells/mouse) to induce tumor formation. A total of 20 mice with tumors of >100 mm3 were randomly divided into four groups (five per group), and injected intraperitoneally with either PBS (0.2 ml), 3-BP (8 mg/kg), TRAIL (0.1 mg/kg) or both 3-BP (8 mg/kg) and TRAIL (8 mg/kg) every 4 days. Body weight was monitored prior to each injection. Tumor volume was calculated using the following formula: Length × width2/2. Mice were sacrificed by cervical dislocation after 28 days of treatment. Following treatment for 28 days, the solid tumors were resected from mice, stored in 4% formalin solution, cut into sections and subsequently subjected to with hematoxylin and eosin (H&E) or TUNEL staining.

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In vivo tumor experiment [3]
The nude mice (5-6 weeks) used in these studies were obtained from Beijing vitalriver and weighed 20–25 g at the time of tumor implantation. The mice were kept under a 12:12 h light–dark cycle, at 24 ± 2 °C and fed with clean food and water. Human MDA-MB-231 cells (107cells/ml) were inoculated subcutaneously to form tumors. Mice with tumors (100-200 mm3) assorted to four groups (5 mice/group). Vehicle (0.9% NS) or CQ (40mg/kg/d, 24 days) or 3-Bromopyruvic Acid/3-BrPA (5mg/kg/d, 24 days) alone or in combination was administrated intraperitoneally. Tumor growth was monitored every three days by two-dimensional measurements of individual tumors for each mouse. Tumor volume was calculated using the formula: length×width2/2. After treatment ended, mice from each group were sacrificed. Tumors were excised, calculated and fixed in 4% formalin solution, embedded in paraffin, and then stained with hematoxillin-eosin (H&E).

The intraperitoneal LD50 in mice was 72 mg/kg. Sensory organs and special senses: lacrimal production: eyes; lungs, pleural cavity, or respiration: respiratory stimulation; gastrointestinal tract: changes in salivary gland structure or function. Journal of Pharmacology and Experimental Therapeutics, 123(48), 1958 [PMID:13539790]

[1]. Synergistic Effect of 3-Bromopyruvate in Combination with Rapamycin Impacted Neuroblastoma Metabolism by Inhibiting Autophagy. Onco Targets Ther. 2020;13:11125-11137.

[2]. 3 Bromopyruvate sensitizes human breast cancer cells to TRAIL induced apoptosis via the phosphorylated AMPK mediated upregulation of DR5. Oncol Rep. 2018;40(5):2435-2444.

[3]. Hexokinase II inhibitor, 3-BrPA induced autophagy by stimulating ROS formation in human breast cancer cells. Genes Cancer. 2014;5(3-4):100-112.

3-Bromopyruvate is a 2-oxomonocarboxylic acid, a derivative of pyruvate where one methyl hydrogen atom is replaced by a bromine atom. It is a synthetic bromine derivative and structural analog of pyruvate, a highly reactive alkylating agent, and also an anticancer drug. It is both an alkylating agent and an antitumor drug. It is a 2-oxomonocarboxylic acid and an organic bromine compound, functionally related to pyruvate, and is the conjugate acid of 3-bromopyruvate. Our study also found that in MYCN-amplified neuroblastomas, the expression of autophagy protein SQSTM1/p62 was downregulated, indicating that autophagy activity is higher in high-risk neuroblastomas and may play a carcinogenic role. However, the autophagy pathway in neuroblastomas was impaired after the addition of the glucose metabolism inhibitor 3-BrPA. The combination of rapamycin and 3-bromopyruvate (3-BrPA) can produce a synergistic inhibitory effect on tumors, affecting not only related genes in the autophagy pathway but also intracellular glucose metabolism levels. This study provides a new therapeutic strategy for the combined treatment of high-risk neuroblastoma with 3-BrPA and rapamycin. Therefore, there may be a coupling relationship between metabolic reprogramming and autophagy. The idea that cancer cells reprogram their metabolism to cope with the biosynthetic challenges of cell growth and proliferation may affect autophagy, thus providing an opportunity to regulate cell metabolism for cancer treatment. [1] Previous studies have shown that the sensitivity of breast cancer cells to apoptosis induced by tumor necrosis factor-associated apoptosis-inducing ligand (TRAIL) is related to the expression of death receptors on the cell membrane. However, drug resistance limits the application of TRAIL in cancer treatment. Numerous studies have shown that endoplasmic reticulum (ER) stress response can upregulate death receptors that induce apoptosis. 3-bromopyruvate/3-bromopyruvate (3-BP) is an anticancer drug that inhibits cell growth and induces apoptosis by interfering with glycolysis. This study shows that 3-BP can synergistically enhance the sensitivity of breast cancer cells to TRAIL-induced apoptosis by upregulating death receptor 5 (DR5). Furthermore, we found that 3-BP treatment increased the protein levels of glucose-associated protein 78 (GRP78) and CCAAT enhancer-binding protein homolog (CHOP). Bax expression increased in MCF-7 cells, and caspase-3 expression increased in MDA-MB-231 cells. The expression of the anti-apoptotic protein Bcl-2 decreased after 3-BP combined with TRAIL treatment. To investigate the molecular mechanism regulating this effect, we examined the expression of 3-BP-activated adenosine monophosphate-activated protein kinase (AMPK). The results showed that phosphorylated AMPK expression was upregulated after 3-BP treatment. Notably, the AMPK inhibitor Compound C reversed the effect of 3-BP. Finally, a synergistic anti-tumor effect of 3-BP and TRAIL was observed in a nude mouse MCF-7 cell xenograft model. In summary, these results indicate that 3-BP enhances the sensitivity of breast cancer cells to TRAIL through AMPK-mediated DR5 upregulation. [2]

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In summary, 3-bromopyruvate/3-BrPA-induced autophagy in breast cancer cells may serve as a mechanism to resist cell death. Therefore, inhibiting autophagy may become a novel strategy for adjuvant therapy of breast cancer. Although the detailed mechanisms of ROS generation and autophagy in our model are not yet clear, our data will provide a reference for future research on 3-BrPA therapy for cancer cells. [3]

C3H3BRO3

166.96

165.926

1113-59-3

70684

White to light yellow solid powder

2.0±0.1 g/cm3

223.4±23.0 °C at 760 mmHg

77-82 °C

88.9±22.6 °C

0.0±0.9 mmHg at 25°C

1.521

-0.54

1

3

2

7

98.4

0

C(C(=O)C(=O)O)Br

PRRZDZJYSJLDBS-UHFFFAOYSA-N

InChI=1S/C3H3BrO3/c4-1-2(5)3(6)7/h1H2,(H,6,7)

3-bromo-2-oxopropanoic acid

3-bromopyruvic acid; bromopyruvic acid; 3-Bromo-2-oxopropionic acid; Propanoic acid, 3-bromo-2-oxo-; DTXSID7040940; CHEBI:131461; 3-BrPA cpd; ...; 1113-59-3;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~250 mg/mL (~1497.36 mM)

Solubility in Formulation 1: 100 mg/mL (598.95 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)