Andin-3-glucoside and cyanidin-3rutinoside) and PCA; four fiber metabolites (acetic acid, butyric acid, propionic acid, and valeric acid); 4 lignans (lariciresinol, matairesinol, pinoresinol, and secoisolariciresinol); and three oligosaccharides (fructooligosaccharides, galactooligo-saccharides, and xylooligosaccharides). Of these 17 constituent molecules, only UA and UB suppressed endometrial cancer cell proliferation. UA, the more potent, arrested the cell cycle at the G2/M phase, though also exhibiting some estrogenic actions that may possibly contribute to the mechanism for preventing endometrial cancer.Author Manuscript Author Manuscript Author Manuscript Author Manuscript2 Materials and methods2.1 Reagents and chemical substances Cyanidin-3-glucoside, cyanidin-3-rutinoside, acetic acid, butyric acid, ellagic acid, propinoic acid, protocatechuic acid, valeric acid, matairesinol, lariciresinol, pinoresinol, secoisolariciresinol, and also other chemical substances were purchased from Sigma-Aldrich (St. Louis, MO). Fructooligosaccharides, xylooligosaccharides, and galactooligosaccharides had been obtained from Inventive Dynamics (Shirley, NY). Charcoal/dextran-treated fetal bovine serum (FBS) was procured from HyClone (Logan, Utah). Antibodies against cyclin-B1, cyclin-E2, p21, phospho-cdc2, Myt1, CDC25B, and -actin had been obtained from Cell Signaling Technology (Danvers, MA). Primers for RT-qPCR and siRNA for estrogen receptor- (ER) were purchased from Thermo Fisher Scientific (Grand Island, NY).Mol Nutr Meals Res. Author manuscript; obtainable in PMC 2017 November 01.Zhang et al.Page2.2 Urolithins A and B synthesisAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptUrolithin A and B were synthesized within the laboratory of Dr. Chung-Wai Shiau. Briefly, resorcinol and 2-bromo-5-methoxybenzoic acids have been condensed inside the presence of copper sulphate and NaOH to generate dibenzopyranone, from which the methyl group was removed (by means of catalysis, utilizing boron tribromide) to yield urolithin A. Resorcinol was reacted with 2-bromobenzoic acid within the presence of NaOH and copper sulphate, to create urolithin B.IL-2 Protein supplier Chemical structures and reactions are described in Supporting Details Fig.GPVI, Mouse (HEK293, His) 1.PMID:34645436 The purities of both urolithin A and B had been 94.34 (measured by NMR). 2.three Cell culture Endometrial cancer cells (HEC1A and Ishikawa) were kindly offered by Dr. Paul Goodfellow (Ohio State University). HEC1A cells were grown in DMEM and RRMPI-1640 (1:1) medium with 10 FBS. Ishikawa cells were cultured in DMEM medium with ten FBS, 1 NEAA. ECC-1 cells were obtained from ATCC (Manassas, VA) and grown in RPMI-1640 medium with five FBS, 1 HEPES, 1 glucose, and 1 sodium pyruvate. T HESCs cells from ATCC were maintained in DMEM/F12 medium with 1 ITS+ Premix (Becton Dickinson, Franklin Lakes, NJ), 500ng/mL puromycin, and 10 charcoal/dextrantreated FBS. Each of the cell lines have been maintained inside a 37 incubator with five CO2. Experiments had been performed within 6 months of the cell lines becoming thawed or obtained in the original sources. Cell line authentication was performed by IDEXX (Westbrook, ME), which utilizes brief tandem repeat (STR) profiling for characterization and authentication. To evaluate cell proliferation, endometrial cancer cells were cultured in full medium and treated with BRB components and metabolites as indicated. In experiments aimed at examining estrogenic effects, ECC-1 and Ishikawa cells had been grown in phenol redfree medium with five charcoal/dextran-treated FBS. two.4 C.
