DNA synthesis; Microbial Metabolite

Micromolar quantities of urolithin A cause cell closure and autophagy. In the human sw620 colorectum, urolithin A suppresses DNA synthesis and the development of the cell cycle [2]. T24 and Caco-2 cell growth is inhibited by urolithin A, which also has anti-proliferative effects. In G2/M and S phase, urolithin A was dose- and time-dependent when compared to control cells, with 50 and 100 μM at 24 and 48 hours, respectively. IC 50 values are 43.9 and 49 μM, in that order [3]. At 50 and 100 μM, it causes apoptosis [4]. HepG2 cells exhibit strong antiproliferative activity in response to urolithin A. TCF/LEF fast activation was markedly downregulated and the expression of β-catenin, c-Myc, and cyclin D1 was reduced when urolithin A promoted cell death. In addition, urolithin A upregulates the expression of caspase-3, p38-MAPK, and p53, while suppressing NF-κB, p65, and other neuromediators [5].

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The intestinal metabolites of ellagic acid (EA), urolithins are known to effectively inhibit cancer cell proliferation. This study investigates antiproliferative and antioxidant effects of Urolithin A (UA) on cell survival of the HepG2 hepatic carcinomas cell line. The antiproliferative effects of UA (0-500 μM) on HepG2 cells were determined using a CCK assay following 12-36 h exposure. Effects on β-catenin and other factors of expression were assessed by using real-time PCR and Western blot. We found that UA showed potent antiproliferative activity on HepG2 cells. When cell death was induced by UA, it was found that the expression of β-catenin, c-Myc and Cyclin D1 were decreased and TCF/LEF transcriptional activation was notably down-regulated. UA also increased protein expression of p53, p38-MAPK and caspase-3, but suppressed expression of NF-κB p65 and other inflammatory mediators. Furthermore, the antioxidant assay afforded by UA and EA treatments was associated with decreases in intracellular ROS levels, and increases in intracellular SOD and GSH-Px activity. These results suggested that UA could inhibit cell proliferation and reduce oxidative stress status in liver cancer, thus acting as a viably effective constituent for HCC prevention and treatment. [2]

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Autophagy is an evolutionarily conserved pathway in which cytoplasmic contents are degraded and recycled. This study found that submicromolar concentrations of urolithin A, a major polyphenol metabolite, induced autophagy in SW620 colorectal cancer (CRC) cells. Exposure to Urolithin A also dose-dependently decreased cell proliferation, delayed cell migration, and decreased matrix metalloproteinas-9 (MMP-9) activity. In addition, inhibition of autophagy by Atg5-siRNA, caspases by Z-VAD-FMK suppressed urolithin A-stimulated cell death and anti-metastatic effects. Micromolar urolithin A concentrations induced both autophagy and apoptosis. Urolithin A suppressed cell cycle progression and inhibited DNA synthesis. These results suggest that dietary consumption of urolithin A could induce autophagy and inhibit human CRC cell metastasis. Urolithins may thus contribute to CRC treatment and offer an alternative or adjunct chemotherapeutic agent to combat this disease. [3]

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IsoUro-A inhibited the proliferation of Caco-2 cells in a time- and dose-dependent manner, though it was significantly lower than Urolithin A/Uro-A (IC50 = 69.7 ± 4.5 and 49.2 ± 3.8 μM at 48 h, respectively). Both urolithins arrested Caco-2 cell cycle at S and G2/M phases and induced apoptosis at concentrations previously found in human colon tissues. Notably, Caco-2 cells glucuronidated more efficiently IsoUro-A than Uro-A (~50 vs. ~20 % of conversion after 48 h, respectively). Both Uro-A and IsoUro-A glucuronides did not exert antiproliferative effects. In addition, cell growth inhibition was higher in Caco-2 than in normal cells. Conclusions: IsoUro-A exerts strong antiproliferative activity, which is reduced by the extensive glucuronidation at 9-position in cancer cells. Further studies are needed to elucidate whether the in vitro structure-activity relationship found for Uro-A and IsoUro-A plays any role in humans [6].

Furthermore, an hour following the delivery of urolithin A in the vessel wall, the amount of paw edema decreased. Furthermore, the treated cell stems exhibited high oxygen radical antioxidant capacity (ORAC) scores an hour after urolithin A was added to the blood vessel wall, with the remaining unformed levels comparable to A [6].

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It was demonstrated that Urolithin A (UA) ameliorated cognitive impairment, prevented neuronal apoptosis, and enhanced neurogenesis in APP/PS1 mice. Furthermore, UA attenuated Aβ deposition and peri-plaque microgliosis and astrocytosis in the cortex and hippocampus. We also found that UA affected critical cell signaling pathways, specifically by enhancing cerebral AMPK activation, decreasing the activation of P65NF-κB and P38MAPK, and suppressing Bace1 and APP degradation. Conclusions: The results indicated that UA imparted cognitive protection by protecting neurons from death and triggering neurogenesis via anti-inflammatory signaling in APP/PS1 mice, suggesting that UA might be a promising therapeutic drug to treat AD. [1]

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Urolithin A (UA) is a major metabolite produced by rats and humans after consumption of pomegranate juice or pure ellagitannin geraniin. In this study, we investigated the anti-inflammatory effect of Urolithin A on carrageenan-induced paw edema in mice. The volume of paw edema was reduced at 1h after oral administration of Urolithin A. In addition, plasma in treated mice exhibited significant oxygen radical antioxidant capacity (ORAC) scores with high plasma levels of the unconjugated form at 1h after oral administration of urolithin A. These results indicate strong associations among plasma Urolithin A levels, the plasma ORAC scores, and anti-inflammatory effects and may help explain a mechanism by which ellagitannins confer protection against inflammatory diseases [4].

Cell culture and treatment [2]
HepG2 cell line was purchased from American Type Culture Collection. Cells were maintained in DMEM medium supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin sulfate in a 5% CO2, humidified atmosphere of 95%, at 37 °C. To perform the CCK assay, serial dilutions of Urolithin A/UA were prepared in ratios relative to the IC50 of cells and incubated for 12, 24 and 36 h, respectively. To perform real-time quantitative PCR and Western blot analysis, cells were treated with UA at different concentrations for 24 h added in culture medium with DMSO. The final concentration of DMSO in each well was 0.6% and it is no observable toxicity in our study and as previously described (Aiken et al., 2004). A DMSO-added group (the same volume as drug treated groups) was used as negative control.

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Measurement of intracellular ROS production, SOD, MDA and GSH-Px level [2]
The effects of Urolithin A/UA on ROS production, SOD, MDA and GSH-Px level were evaluated by using a hydrogen peroxide mediated oxidative stress model as described, with the appropriate modifications (Sohn et al., 2013). Briefly, after a 12 h treatment with UA and EA, HepG2 cells were gently washed with PBS 3 times, and incubated with 50 mM H2O2 for 3 h at 37 °C to form intracellular acute oxidative state. The protein concentration was measured using BCA Protein Assay Kit.

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RNA extraction and reverse transcription [2]
After treatment with IC50 of Urolithin A/UA, total RNA was extracted from cells by using a Trizol reagent according to the manufacturer’s recommendation. After DNA contamination was removed by RNase-free DNase I, first-strand cDNA synthesis was performed using MMLV reverse transcriptase and oligo dT according to the manufacturer’s protocol. The obtained cDNA was stored at −80 °C for further use.

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Chamber migration assay [3]
Transwell polycarbonate membrane inserts (8 µm poresize, 10 mm diameter) were utilized for chamber migration assays. SW620 cells with or without Urolithin A were suspended in serum‐free medium (5 × 105 cells/mL) and added to the upper compartment, while L‐15 medium containing 10% FBS was added to the lower compartment. After 4 h, non‐invaded cells on the upper side of the Transwell polycarbonate membrane were removed with a cotton tip applicator. Invaded cells on the bottom surface of the membrane were fixed with methanol and stained with 0.5% crystal violet.

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Western blot [3]
SW620 cells were incubated with or without 1.5 µM Urolithin A, harvested, washed with ice‐cold PBS and lysed for 30 min. Protein concentrations were determined using a BCA assay kit and 50 µg of protein per sample was used for Western blot analysis with the following antibodies: LC3‐I/II and anti‐mouse HRP‐conjugated IgG secondary antibody (1:2000). All samples were normalized to β‐actin.

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Flow cytometry [3]
SW620 cells were labeled with propidium iodide (PI) for cell cycle analysis. Cells were harvested by trypsinization after 24, 48, or 72 h treatment with Urolithin A and were suspended in PBS (0.1 M, pH 7.4). Cells were fixed in 70% ethanol for at least 30 min at −20°C. Before analysis, cells were washed twice in cold PBS and re‐suspended in 200 µL PBS (0.25 mg/mL RNase A, 0.1 mg/mL PI). After incubation in darkness for 30 min at 37°C, samples were analyzed via flow cytometry and histograms were calculated by Cell Quest software.

Mice (28 weeks old) were orally administered 300 mg/kg Urolithin A/UA dissolved in 0.5% carboxymethylcellulose at the same time each day for 14 days. Control mice (APP/PS1 transgenic mice and wild-type mice) were orally administered the same quantity of 0.5% carboxymethylcellulose (vehicle).[1]
Morris water maze [1]
After Urolithin A/UA treatment, the spatial learning and memory of mice were assessed by the Morris water maze. Briefly, the maze consisted of a stainless steel pool (120 cm in diameter and 50 cm in height) with a submerged escape-platform (10 cm in diameter) placed 1 cm below the water surface. The water temperature was maintained at 24 ± 1 °C. The spatial learning task consisted of four consecutive days of testing with four trials per day. In each trial, the time required to find the hidden platform was recorded as the escape latency. The mice were given a maximum of 60 s to find the hidden platform. If a mouse failed to locate the platform within 60 s, the session was terminated, a maximum escape-latency score of 60 s was assigned, and the mouse was manually guided to the hidden platform (10 s). To test spatial memory, a single probe trial was conducted 24 h after the last trial of the fourth day. The submerged platform was removed and the mice were placed into the pool from the quadrant opposite to the quadrant where the platform used to be (target quadrant). The mice were allowed to freely swim for 60 s. The time spent in the target quadrant and numbers of crossings through this quadrant were recorded. Swimming speed was also recorded.

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Morris water maze was used to detect the cognitive function. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was performed to detect neuronal apoptosis. Immunohistochemistry analyzed the response of glia, Aβ deposition, and neurogenesis. The expression of inflammatory mediators were measured by enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR). The modulating effects of Urolithin A on cell signaling pathways were assayed by Western blotting.[1]

[1]. Urolithin A attenuates memory impairment and neuroinflammation in APP/PS1 mice.

[2]. In vitro antiproliferative and antioxidant effects of urolithin A, the colonic metabolite of ellagic acid, on hepatocellular carcinomas HepG2 cells. Toxicol In Vitro. 2015 Aug;29(5):1107-15.

[3]. Metabolite of ellagitannins, urolithin A induces autophagy and inhibits metastasis in human sw620colorectal cancer cells. Mol Carcinog. 2018 Feb;57(2):193-200.

[4]. In vivo anti-inflammatory and antioxidant properties of ellagitannin metabolite urolithin A. Bioorg Med Chem Lett. 2011 Oct 1;21(19):5901-4.

[5]. In vitro antioxidant and antiproliferative effects of ellagic acid and its colonic metabolite, urolithins, on human bladder cancer T24 cells. Food Chem Toxicol. 2013 Sep;59:428-37.

[6]. Antiproliferative activity of the ellagic acid-derived gut microbiota isourolithin A and comparison with its urolithin A isomer: the role of cell metabolism.Eur J Nutr. 2017 Mar;56(2):831-841.

Urolithin A is a member of coumarins. It has a role as a geroprotector.
Urolithin A is a metabolite of ellagic acid. It has been demonstrated to stimulate mitophagy and improve muscle health in old animals and in preclinical models of aging.
urolithin A has been reported in Punica granatum and Trogopterus xanthipes with data available.

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In summary, our results in an AD mouse model demonstrated the protective effects of UA on AD pathology by its targeting of multiple pathological processes such as reactive gliosis, inflammatory signaling, AB plaque formation, and apoptosis. Our findings indicate that UA may serve as a promising therapeutic agent for AD.[1]

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In summary, the data in this study suggests that UA inhibits cell proliferation on HepG2 cells through suppressing β-catenin signaling and its congenerous mediators. Removing accumulation of β-catenin can increase the TP53 gene and its downstream gene expression by amending the p38-MAPK signaling, and suppressing the expression of NF-κB and other related inflammatory mediators. UA could also reduce the oxidative stress status in HepG2 cells. In Pfundstein et al.’s study, the data showed that the plasma concentrations of metabolism of ellagitannins in the volunteers elevated to 100–200 μM after walnut consumption (Pfundstein et al., 2014). This suggests that the concentration used in our study could be applied in an in vivo model. Moreover, urolithins are metabolites of polyphenols in diet and it seems that reasonable guidance of consuming polyphenols-rich diet could benefit individuals undergoing liver cancer chemotherapy, though an in vivo animal or human model is still further needed to confirm the beliefs. [2]

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In summary, we showed for the first time that treatment of colon cancer SW620 cells with dietary submicromolar urolithin A can induce autophagy and inhibit CRC cell growth and metastasis. Our in vitro findings provide novel insights into understanding the anti‐tumor functions of dietary urolithin A in CRC. [3]

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Purpose: Urolithins, metabolites produced by the gut microbiota from ellagic acid, have been acknowledged with cancer chemopreventive activity. Although urolithin A (Uro-A) has been reported to be the most active one, 10-50 % of humans can also produce the isomer isourolithin A (IsoUro-A). However, no biological activity for IsoUro-A has been reported so far. Herein, we describe for the first time the antiproliferative effect of IsoUro-A, compared to Uro-A, against both human colon cancer (Caco-2) and normal (CCD18-Co) cell lines. Methods: Cell proliferation was evaluated by MTT and Trypan blue exclusion assays. Cell cycle was analyzed by flow cytometry and apoptosis measured by the Annexin V/PI method. Finally, urolithins metabolism was analyzed by HPLC-DAD-MS/MS. [6]

C13H8O4

228.2002

228.042

C, 68.42; H, 3.53; O, 28.04

1143-70-0

5488186

Light yellow to brown solid powder

1.516g/cm3

527.9ºC at 760 mmHg

214.2ºC

9.24E-12mmHg at 25°C

1.717

2.357

2

4

0

17

317

0

RIUPLDUFZCXCHM-UHFFFAOYSA-N

InChI=1S/C13H8O4/c14-7-1-3-9-10-4-2-8(15)6-12(10)17-13(16)11(9)5-7/h1-6,14-15H

3,8-dihydroxybenzo[c]chromen-6-one

urolithin A; 1143-70-0; 3,8-dihydroxy-6H-benzo[c]chromen-6-one; 3,8-Dihydroxyurolithin; 3,8-dihydroxybenzo[c]chromen-6-one; 6H-Dibenzo[b,d]pyran-6-one, 3,8-dihydroxy-; 3,8-Hydroxydibenzo-alpha-pyrone; 3,8-Dihydroxy-6H-dibenzo(b,d)pyran-6-one;

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)

DMSO : ~30 mg/mL (~131.46 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (10.96 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (10.96 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (10.96 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 5 mg/mL (21.91 mM) in 0.5% CMC/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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