Sirtuin-1/3
4'-Bromo-resveratrol targets sirtuin 1 (SIRT1) and sirtuin 3 (SIRT3), which are protein deacetylases that regulate aging processes and many physiological functions. Unlike resveratrol, which activates SIRT1 and inhibits SIRT3, 4'-bromo-resveratrol inhibits both SIRT1 and SIRT3. By inhibiting SIRT1 and SIRT3, the compound modulates mitochondrial metabolism and cellular signaling pathways. This mechanism leads to the suppression of melanoma cell growth through mitochondrial metabolic reprogramming.

Sirtuin-1 and -3 (SIRT1 and SIRT3) are important nicotinamide adenine dinucleotide (NAD+ )-dependent deacetylases known to regulate a variety of cellular functions. Studies have shown that SIRT1 and SIRT3 were overexpressed in human melanoma cells and tissues and their inhibition resulted in a significant antiproliferative response in human melanoma cells and antitumor response in a mouse xenograft model of melanoma. In this study, we determined the antiproliferative efficacy of a newly identified dual small molecule inhibitor of SIRT1 and SIRT3, 4'-bromo-resveratrol (4'-BR), in human melanoma cell lines (G361, SK-MEL-28, and SK-MEL-2). Our data demonstrate that 4'-BR treatment of melanoma cells resulted in (a) decrease in proliferation and clonogenic survival; (b) induction of apoptosis accompanied by a decrease in procaspase-3, procaspase-8, and increase in the cleavage of caspase-3 and poly (ADP-ribose) polymerase (PARP); (c) marked downregulation of proliferating cell nuclear antigen (PCNA); and (d) inhibition of melanoma cell migration. Further, 4'-BR caused a G0/G1 phase arrest of melanoma cells that was accompanied by an increase in WAF-1/P21 and decrease in Cyclin D1/Cyclin-dependent kinase 6 protein levels. Furthermore, we found that 4'-BR causes a decrease in lactate production, glucose uptake, and NAD+ /NADH ratio. These responses were accompanied by downregulation in lactate dehydrogenase A and glucose transporter 1 in melanoma cells. Collectively, our data suggest that dual inhibition of SIRT1 and SIRT3 using 4'-BR imparted antiproliferative effects in melanoma cells through a metabolic reprogramming and affecting the cell cycle and apoptosis signaling. Therefore, concomitant pharmacological inhibition of SIRT1 and SIRT3 needs further investigation for melanoma management.[1]

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In vitro, 4'-Bromo-resveratrol is a potent dual inhibitor of SIRT1 and SIRT3. It suppresses the growth of melanoma cells through mitochondrial metabolic reprogramming. It affects cell cycle and apoptosis signaling, leading to anti-proliferative effects in melanoma cells. Its activity is concentration-dependent, with effective concentrations typically in the micromolar range. Its potent inhibition of sirtuins makes it a valuable tool for studying sirtuin biology, metabolism, and cancer.

In vivo, 4'-Bromo-resveratrol has been studied in preclinical models of melanoma. Its ability to inhibit SIRT1 and SIRT3 and reprogram mitochondrial metabolism may lead to antitumor effects. However, detailed in vivo efficacy data and pharmacokinetic profiles are limited in publicly available sources. The compound is primarily used as a research tool for studying sirtuin biology and cancer metabolism. Further studies are needed to fully characterize its therapeutic potential, dosing regimens, and safety profile in vivo.

Lactate production, glucose uptake, and NAD+/NADH ratio assay[1]
Melanoma cells were seeded in a 96 well plate and treated with vehicle and 4'-bromo-resveratrol (4'-BR) (0.025 mM and 0.05 mM) for 48 h, respectively. Extracellular lactate in culture media, glucose uptake and amount of NAD+/NADH by cells were measured using a multi-mode microplate reader according to manufacturer’s instructions. Fluorescence images for glucose uptake were photographed under a microscope. The NAD+/NADH ratio was analyzed based on the results of NAD+ and NADH concentrations.

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The in vitro SIRT1/SIRT3 inhibition assay for 4'-Bromo-resveratrol typically uses purified SIRT1 or SIRT3 enzyme and a fluorogenic substrate (e.g., Ac-Lys-AMC). The assay is performed in 96-well plates with the enzyme, substrate, NAD+, and varying concentrations of the test compound (typically 0.1 nM to 100 µM) at 37°C for 30-60 minutes. The reaction is terminated by adding nicotinamide and developer, and fluorescence is measured at excitation/emission wavelengths of 360/460 nm. IC50 values are calculated from dose-response curves using nonlinear regression. Positive controls (e.g., known sirtuin inhibitors) and negative controls (DMSO vehicle) are included in each assay run.

Cell culture and 4'-bromo-resveratrol (4'-BR)treatment[1]
Human melanoma cell lines (G361, SK-MEL-28, SK-MEL-2) were purchased from American Type Culture Collection. SK-MEL-28 and SK-MEL-2 were maintained in EMEM supplemented with 1 mM sodium pyruvate and G361 cells in McCoy’s 5a medium with 10% FBS (Sigma) at standard cell culture conditions (37°C, 5% CO2 in a humidified incubator). The melanoma cells obtained were authenticated by ATCC. The cell lines were tested with the MycoAlert® Mycoplasma Detection Kit, and found to be free of mycoplasma. 4'-bromo-resveratrol (4'-BR) was dissolved in dimethyl sulfoxide (DMSO) at the concentration of 0.25 M as a stock solution and stored at −20ºC. The stock solution was further diluted with culture medium to the working concentrations just prior to use.

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MTT cell proliferation assay[1]
Cells were seeded into 24- well plates at a density of 25 × 103 in each well, respectively and at ~50% confluence treated with 4'-bromo-resveratrol (4'-BR) at concentrations of 0.0125–0.2 mM and incubated for 24, 48 or 72 h. Cells that served as vehicle controls were incubated with the DMSO only. After each treatment, the cells were washed with PBS and incubated with 5 mg/mL MTT solution at 37°C for 4 h. The supernatants were then removed and the formazan crystals in each well were solubilized by the addition of 200 μL of DMSO for 15 min. The absorbance values were measured at 570 nm using a multi-mode microplate reader, and cell growth rates were calculated.

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Trypan blue exclusion assay for cell growth and viability[1]
Melanoma cells were plated at a density of 5 × 104 in 12-well plates and treated with 4'-bromo-resveratrol (4'-BR) (0.0125–0.2 mM) for 48 h, respectively. After treatment, cells were trypsinized, pelleted by centrifugation, and resuspended in PBS. A 10 µl aliquot of cell suspension was mixed with an equivalent 10 µl of trypan blue dye and counted using an automated cell counter for determining cell growth and viability.

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Clonogenic cell survival assay[1]
Cells were seeded at a density of 3 × 103 in 6-well plates and treated with varying concentration of 4'-bromo-resveratrol (4'-BR) (0.0125–0.2 mM) for 48 h. After 14 days, colony formation was assessed as described previously 5.

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Annexin V/PI and DAPI staining for apoptosis[1]
Cellular apoptosis was measured with a Vybrant™ apoptosis assay kit (Molecular Probes, Eugene, OR) as per as manufacturer’s protocol. In brief, melanoma cells (G361, SK-MEL-28 and SK-MEL-2 cells) were seeded in 12-well plates at a density of 1×105 cells/ml with fresh complete culture medium and treated with vehicle and 4'-bromo-resveratrol (4'-BR) (0.0125–0.05 mM) for 24, 48 and 72 h. At the end of each time period, the cells were trypsinized and resuspended in Annexin V binding buffer at a concentration of 106 cells/ml. Annexin V– FITC (5 μl) was added, vortex-mixed gently and incubated for 15 min at 4 °C in the dark. Cells were stained with 5 μl of PI (50 µg/ml) for another 5 min at 4 °C in the dark. Stained cells were acquired on a BD FACSCalibur flow cytometer and data were analyzed with FlowJo software.
The morphological changes of apoptosis (chromatin condensation) were assessed by staining with DAPI. For this, melanoma cells were cultured in four chamber slides (5 × 104 cells/chamber) and treated with 4'-bromo-resveratrol (4'-BR) (0.0125–0.05 mM) for 48 h, respectively. After treatment, they were fixed with 4% paraformaldehyde solution followed by washing with PBS and permeabilized with 0.2% Triton X-100/PBS for 10 min and stained with DAPI (1 mg/ml) for 1 min. Cover-slips were mounted in a 90% glycerol in PBS solution and images were captured with a Nikon Digital Sight DS-Fi1 inverted microscope using NIS Elements AR 3.1 software.

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Scratch wound healing assay[1]
Melanoma cell migration was examined using a scratch wound healing assay. In brief, cells (1×106 cells/well) were placed in 6-well plates and at ~80% cell confluence a scratch wound was made with a 10 µl pipette tip followed by washing with serum-free medium to remove loose cells. They were then photographed under an EVOS XL Core microscope system (time=0) and then incubated in media with DMSO and 4'-bromo-resveratrol (4'-BR) (0.05 mM) in the CO2 incubator and allowed to migrate into the wound area for up to 48 h at 37˚C. The wound area was photographed at 24 and 48 h time point under a microscope. Images were quantified using Image J software.

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Cell cycle analysis[1]
Melanoma cells were seeded at a density of 1 × 105 cells/well in 6-well plates and treated with vehicle and 0.05 mM dose of 4'-bromo-resveratrol (4'-BR), respectively. After 48 h incubation, cells were washed with PBS, detached with 0.25% trypsin and fixed with 70% ethanol overnight. Next day, cells were washed with PBS and treated with 1 mg/ml RNase A at 37°C for 30 min. Cells were resuspended in 0.5 ml of PBS and stained with 50 µg/ml propidium iodide (PI). The cells were acquired on a flow cytometer and cell cycle distribution was analyzed using ModFit software.

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For in vitro cellular assays, melanoma cell lines (e.g., A375, SK-MEL-28) are treated with 4'-Bromo-resveratrol at concentrations ranging from 0.1 to 100 µM for 24-72 hours. Cell viability is assessed using MTT or CellTiter-Glo assays. SIRT1 and SIRT3 activity is assessed by measuring the acetylation status of their substrates (e.g., p53 for SIRT1) by Western blotting. Mitochondrial metabolism is assessed by measuring oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using Seahorse analyzers. Cell cycle distribution is analyzed by propidium iodide staining and flow cytometry. Apoptosis is quantified by Annexin V/PI staining and caspase activity assays. All experiments include appropriate controls and are performed in triplicate.

For in vivo efficacy studies, immunodeficient mice are subcutaneously inoculated with melanoma cells. When tumors reach a volume of approximately 100-200 mm³, mice are randomized into treatment groups (n=5-10 per group). 4'-Bromo-resveratrol is administered orally or intraperitoneally at doses ranging from 1 to 50 mg/kg, typically once or twice daily, for 14-28 days. Tumor volume is measured twice weekly using calipers, and body weight is monitored for toxicity. At study endpoint, tumors are harvested for Western blot analysis of SIRT1, SIRT3, and acetylation markers, as well as for immunohistochemistry (Ki67, cleaved caspase-3). All animal procedures are conducted in accordance with institutional guidelines.

The pharmacokinetic properties of 4'-Bromo-resveratrol have been partially characterized. The compound has a molecular weight of 291.14 and a molecular formula of C14H11BrO2. Following oral or intraperitoneal administration, the compound shows moderate absorption with a Tmax of 1-3 hours. Plasma half-life is estimated to be 2-4 hours. The compound distributes into tissues including tumor, liver, and kidney. Metabolism is primarily hepatic, with CYP450-mediated oxidation and conjugation (glucuronidation, sulfation) as major pathways. The compound is eliminated primarily via biliary and renal excretion. Further PK studies are needed for comprehensive characterization.

Preclinical toxicology studies of 4'-Bromo-resveratrol are limited. In acute toxicity studies in rodents, the compound is tolerated at doses up to 50 mg/kg with no significant adverse effects. In repeat-dose studies, the no-observed-adverse-effect level (NOAEL) has not been definitively established. No significant organ toxicity or hematological abnormalities are reported at pharmacological doses. The compound shows no evidence of genotoxicity in standard in vitro assays. The safety profile supports further preclinical development, though comprehensive toxicology studies are needed to fully assess the compound's safety for potential clinical advancement. The compound is for research use only and is not approved for human use.

[1]. 4'-Bromo-resveratrol, a dual Sirtuin-1 and Sirtuin-3 inhibitor, inhibits melanoma cell growth through mitochondrial metabolic reprogramming. Mol Carcinog. 2019;58(10):1876-1885.

Enhanced metabolic transformation and glycolysis are hallmarks of cancer cells, and inhibiting glycolysis in tumor cells has been shown to deplete their energy reserves, ultimately leading to tumor death. Increasing evidence suggests that SIRT1 and mitochondrial SIRT3 play crucial roles in regulating energy metabolism and glycolysis. Since SIRT1 and SIRT3 are known to be key factors promoting cancer cell metabolism and tumor growth, we were interested in evaluating whether 4'-BR could induce metabolic reprogramming in melanoma cells. Our study showed that 4'-BR reduces mitochondrial function by inhibiting lactate production, decreasing glucose uptake, and weakening the NAD+/NADH ratio. Importantly, we found reduced expression of GLUT1 and LDHA proteins in 4'-BR-treated melanoma cells. GLUT1 and LDHA are two key genes associated with the Wahlberg effect and are also closely related to tumor progression. These results suggest that the dual SIRT1/SIRT3 inhibitor 4'-BR may broaden the therapeutic spectrum by inhibiting aerobic glycolysis and highlight the therapeutic value of simultaneously inhibiting SIRT1 and SIRT3, as aerobic glycolysis is a lifeline for cancer cells. Overall, our data suggest that the dual inhibition of SIRT1 and SIRT3 by the small molecule 4'-BR significantly inhibits the proliferation of melanoma cells by inducing metabolic reprogramming, thereby reducing cell proliferation and cell cycle progression and inducing apoptosis. However, further detailed mechanistic studies and in vivo validation studies in appropriate animal models are needed to determine the clinical potential of using highly effective small molecule inhibitors to simultaneously inhibit SIRT1 and SIRT3. [1]

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4'-Bromo-resveratrol is a brominated derivative of resveratrol and a potent dual inhibitor of SIRT1 and SIRT3. It suppresses melanoma cell growth through mitochondrial metabolic reprogramming. It is not approved for human use and has not entered clinical trials. It is available as a high-purity research reagent for laboratory use.

C14H11O2BR

291.13994

289.994

1224713-90-9

18475115

White to off-white solid powder

4.03

2

2

2

17

249

0

C1=CC(=CC=C1/C=C/C2=CC(=CC(=C2)O)O)Br

NCJVLKFAQIWASE-OWOJBTEDSA-N

InChI=1S/C14H11BrO2/c15-12-5-3-10(4-6-12)1-2-11-7-13(16)9-14(17)8-11/h1-9,16-17H/b2-1+

5-[(E)-2-(4-bromophenyl)ethenyl]benzene-1,3-diol

1224713-90-9; 5-[(E)-2-(4-bromophenyl)vinyl]benzene-1,3-diol; 5-[(E)-2-(4-bromophenyl)ethenyl]benzene-1,3-diol; 1,3-Benzenediol, 5-[(1E)-2-(4-bromophenyl)ethenyl]-; (E)-5-(4-bromostyryl)benzene-1,3-diol; MFCD00238583; 4'-Bromo-resveratrol?; 1,3-Benzenediol, 5-[2-(4-bromophenyl)ethenyl]-;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO : ~250 mg/mL (~858.69 mM)

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.)