β-alanine treatment at 100 mM for 24 hours significantly reduced basal glycolysis (extracellular acidification rate, ECAR) in both malignant MCF-7 and non-malignant MCF-10a breast epithelial cells compared to control and isonitrogenous valine control. Peak glycolytic capacity (induced by oligomycin) was significantly reduced in MCF-7 cells but unchanged in MCF-10a cells. Cellular acidity (proton production rate) was significantly reduced in both cell models. [1]
β-alanine treatment significantly reduced basal oxidative metabolism (oxygen consumption rate, OCR) in both MCF-7 and MCF-10a cells. MCF-10a cells exhibited increased oxidative reliance (ratio of OCR/ECAR). Total cellular ATP content was significantly elevated in MCF-10a cells but significantly reduced in MCF-7 cells following β-alanine treatment. [1]
β-alanine significantly reduced lactate dehydrogenase (LDH) mRNA levels in both cell models, but LDH protein expression was unaltered. Carnosine synthase (CS) protein was present in both cell models but unaltered by treatment. GLUT1 protein expression was significantly elevated in MCF-10a cells (measured by flow cytometry and confirmed by confocal microscopy). [1]
β-alanine significantly suppressed mRNA expression of PGC-1α in both cell models. PGC-1α protein expression was suppressed in MCF-7 cells but unaltered in MCF-10a cells. Mitochondrial content (measured by Mitotracker Green staining) was significantly reduced in MCF-7 cells but unchanged in MCF-10a cells. [1]
β-alanine at 100 mM significantly suppressed proliferation (confluency measured by live-cell imaging) of both MCF-7 and MCF-10a cells after 24 and 48 hours of treatment. Cell viability (measured by WST-1 assay) was not significantly altered in either cell model at any tested dose. [1]
β-alanine significantly reduced migration velocity (scratch-wound assay) of both MCF-7 and MCF-10a cells compared to controls. [1]
β-alanine at 100 mM combined with doxorubicin (Dox) for 24 hours significantly reduced MCF-7 cell viability at all tested Dox concentrations (10 pM to 10 μM) compared to DMSO control. Dox alone only reduced viability at high concentrations (1 μM–10 μM). Significant reductions in viability were observed at low Dox doses (e.g., 10 pM) when combined with β-alanine, indicating enhanced chemosensitivity. [1]

Cells (MCF-7 and MCF-10a) were seeded overnight at a density of 5 × 10^5 cells/well in a 24-well culture plate. After treatment with β-alanine at 100 mM for 24 hours, culture media was replaced with glucose-free assay media. Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were measured using an extracellular flux analyzer. Oligomycin (final concentration 1.0 μM) was injected to induce maximal glycolytic metabolism, followed by FCCP (final concentration 1.25 μM) to induce peak oxygen consumption, and finally rotenone (final concentration 1.0 μM) to reveal non-mitochondrial respiration. Measurements were taken over 24-minute intervals. [1]
Total cellular ATP content was measured by lysing cells in CHAPS lysis buffer, recovering the supernatant, and mixing 1:1 with ATP bioluminescence reagent in a 96-well plate; luminescence was then measured. [1]
For qRT-PCR, total RNA was extracted using an RNeasy kit, and cDNA was synthesized from 5000 ng total RNA. Primers for GLUT1, LDH, PGC-1α, and TBP (housekeeping) were used. SYBR Green-based PCR was performed in triplicate with 5000 ng cDNA per sample and 10 μM final primer concentration in a total volume of 30 μl. Cycling parameters: 95°C for 10 min, then 45 cycles of 95°C for 15 sec and 60°C for 1 min. Relative expression was determined by the ΔΔCp method. [1]
For flow cytometry, cells were resuspended in pre-warmed media with 200 nM Mitotracker Green and incubated for 45 min. Mean fluorescence was measured using a flow cytometer with 488 nm filter. For GLUT1 protein expression, cells were trypsinized, permeabilized, blocked with 0.1% Triton X-100 and 3.0% BSA, stained overnight with anti-GLUT1 antibody (1 μg/ml), then incubated with secondary antibody (Alexa Fluor 488, 1:200 dilution) for 1 hour, and fluorescence was quantified. [1]
For immunocytochemistry, cells were seeded on chamber slides, fixed with 3.7% formaldehyde, permeabilized with 0.1% Triton X-100, blocked with 3.0% BSA, stained overnight with anti-GLUT1 antibody (1 μg/ml), then with Alexa Fluor 633 secondary antibody (1:200). Slides were mounted with Prolong Gold with DAPI and imaged using a fluorescence microscope at 63× magnification with oil emulsion. [1]
For Western blotting, whole-cell lysates were collected in high salt lysis buffer (25 mM Tris base, 8 mM MgCl2, 1 mM DTT, 15% glycerol, 0.1% Triton) with protease inhibitor mix. After incubation on ice for 60 min and centrifugation at 17,500 × g for 3 min, protein concentrations were determined by Bradford assay. Total protein (120 μg per sample) was separated by 10% SDS-PAGE, transferred to nitrocellulose membranes, blocked in TBST-5% non-fat milk, and probed overnight at 4°C with antibodies against PGC-1α, GLUT1, carnosine synthase (CS), LDH, and β-actin. Bound antibodies were detected with HRP-conjugated secondary antibodies and chemiluminescence. Signal intensities were obtained by densitometry using ImageJ and normalized to β-actin. [1]
Cell proliferation was assessed by measuring confluency via phase-contrast live content imaging every hour for 48 hours after seeding cells in media with or without β-alanine at 100 mM. Cell migration was assessed by performing a scratch through confluent cells after 24-hour treatment, then measuring change in confluency for 24 hours. Cell viability was assessed by incubating cells for 1 hour in medium containing WST-1 reagent, then measuring fluorescence. For chemosensitivity, cells were treated with varying concentrations of doxorubicin (10 pM to 10 μM) with or without β-alanine at 100 mM for 24 hours (final DMSO concentration 0.1% for all groups). [1]

In vitro, β-alanine at concentrations up to 100 mM for 24 or 48 hours did not significantly alter cell viability in either MCF-7 or MCF-10a breast epithelial cells as measured by WST-1 assay. No cytotoxicity was observed at any tested dose. [1]

[1]. β-alanine suppresses malignant breast epithelial cell aggressiveness through alterations in metabolism and cellular acidity in vitro. Mol Cancer. 2014 Jan 24;13:14.

β-Alanine is a naturally occurring β-amino acid composed of propionic acid molecules with the amino group at the 3-position. It has multiple functions, including as an inhibitor, agonist, human metabolite, basic metabolite, and neurotransmitter. It is the conjugate acid of β-alanine and also the zwitterion tautomer of β-alanine. β-Alanine is an amino acid produced in vivo by the degradation of dihydrouracil and carnosine. Since neurons have been shown to take up β-alanine, and neuronal receptors are sensitive to β-alanine, this compound may be a pseudo-neurotransmitter alternative to γ-aminobutyric acid (GABA). A rare genetic disorder, hyperβ-alanineemia, has been reported. β-Alanine is present in or produced by Escherichia coli (K12 strain, MG1655 strain). It has also been reported in Neurospora alfalfa, potatoes, and other organisms with relevant data. β-Alanine is the only naturally occurring β-amino acid with the amino group at the - position of the carboxyl group. It is formed in vivo by the degradation of dihydrouracil and carnosine. It is a component of the natural peptides carnosine and anserine, and also a component of pantothenic acid (vitamin B5), which itself is a component of coenzyme A. Under normal circumstances, β-alanine is metabolized to acetic acid. Since neurons have been shown to take up β-alanine, and neuronal receptors are sensitive to β-alanine, this compound may be a pseudo-neuronalant that substitutes for γ-aminobutyric acid (GABA). There have been reports on a rare genetic disorder—hyperβ-alanineemia. β-alanine is an amino acid formed in vivo by the degradation of dihydrouracil and carnosine. Since neurons have been shown to take up β-alanine, and neuronal receptors are sensitive to β-alanine, this compound may be a pseudo-neuronalant that substitutes for GABA. There have been reports on a rare genetic disorder—hyperβ-alanineemia.

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Deregulated cellular energetics (Warburg effect) is a hallmark of cancer. β-alanine treatment increased the alkalinity of the cellular microenvironment, which may reduce activation of proteases used by malignant cells during migration and invasion and improve doxorubicin uptake and efficacy. Both cell models expressed carnosine synthase, but β-alanine treatment did not alter its expression. The anti-tumor effects of β-alanine may be mediated through its metabolite carnosine, although the precise mechanism remains ill-defined and tissue-specific. β-alanine reduced both glycolytic and oxidative metabolism, leading to reduced total metabolic rate without affecting cell viability. It also suppressed proliferation and migration, and enhanced the sensitivity of malignant breast cells to doxorubicin, suggesting a potential role as a co-therapeutic agent. [1]

C3H7NO2

89.0932

89.047

107-95-9

25513-34-2;16690-93-0 (mono-hydrochloride salt);36321-40-1 (calcium (2:1) salt);39748-53-3 (mono-potassium salt)

239

White to off-white solid powder

1.2±0.1 g/cm3

237.1±23.0 °C at 760 mmHg

202 °C (dec.)(lit.)

97.2±22.6 °C

0.0±1.0 mmHg at 25°C

1.463

-0.86

2

3

2

6

52.8

0

UCMIRNVEIXFBKS-UHFFFAOYSA-N

InChI=1S/C3H7NO2/c4-2-1-3(5)6/h1-2,4H2,(H,5,6)

3-aminopropanoic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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