Garcinol

Mouse kidney cells (TCMK-1) were a gift from Dr. T.J. Lee (Yeungnam University, Korea). Primary culture of human mesangial cells (Cryo NHMC) were purchased from Clonetics (San Diego, CA, USA), and other cell lines are from the ATCC (Manassas, VA, USA). All cells were maintained in Dulbecco’s modified Eagle’s medium, and supplemented with 10% FBS, 5% antibiotic solution, and 100 μg/mL gentamycin. Garcinol and lactacystin was purchased from Enzo Life Sciences (Ann Arbor, MI, USA). Recombinant human TRAIL and z-VAD-fmk were purchased from R&D (Minneapolis, MN, USA). Calbiochem supplied N-acetyl-l-cysteine (NAC), trolox, and MG132 (San Diego, CA, USA). The following antibodies were used: anti-XIAP, -Bcl-2, -Mcl-1, -survivin, -Cbl, and -Itch (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-c-FLIP (ALEXIS Corporation, San Diego, CA, USA); anti-PARP, -DR5, -PSMA5, -PSMD4/S5a, -cleaved caspase-3 (Cell Signaling Technology, Beverly, MA, USA); anti-caspase-3 (Enzo Life Sciences, Ann Arbor, MI, USA); anti-DR4 (Abcam, Cambridge, MA, USA); anti-actin (Sigma-Aldrich, St. Louis, MO, USA). Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Bioneer (Daejeon, Korea) supplied DR5 siRNA and GFP siRNA (control siRNA), respectively. Sigma Chemical Co. supplied other reagents (St. Louis, MO, USA).

Mouse studies were approved by the Italian Ministry of Health. Mice were group-housed (2–5 mice per cage) in standard mouse cages and maintained under a 12 h light/dark cycle with free access to water and standard mouse chow. Wild-type six-week-old male C57BL/6N mice (weight: 20–22 g) purchased from Charles River Laboratories were injected intraperitoneally (i.p.) with 0.5 μg/g of body weight of CD95-activating antibody (CD95-Ab; BD Biosciences), 0.6 μg/g of body weight of α-amanitin (Sigma-Aldrich), 0.4 mg/g of body weight of acetaminophen (APAP) (Sigma-Aldrich), or vehicle (saline). 20 mg/kg/day of garcinol (Enzo Life Sciences) or vehicle (saline) was injected i.p. for five days prior to the injection of CD95-Ab. Galloflavin at the doses of 10, 20, 40 or 80 mg/kg diluted in 40% dimethyl sulfoxide (DMSO) or vehicle (40% DMSO) was injected i.p. at the same time as CD95-Ab, α-amanitin, or APAP injections and was continued daily until mouse sacrifice by cervical dislocation at 72 h after CD95-Ab, α-amanitin, or APAP injections. Blood samples were collected by retro-orbital bleedings and were analyzed for alanine aminotransferase levels by colorimetric/fluorometric assay kit (Biovision) according to manufacturer’s instructions.

Garcinol (4) was purchased from Enzo Life Sciences (Villeurbanne, France), A485 was purchased from Bio-Techne (Rennes, France) and zoledronic acid (ZA), simvastatin (Sim), Vorinostat (SAHA), Trichostatin A (TSA) and C646 (4-[4-[[5-(4,5-Dimethyl-2-nitrophenyl)-2-furanyl]methylene]-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-1-yl]benzoic acid) were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France). Guttiferone J (1) has been kept in the in-house chemical library of our laboratory, which includes 139 polyphenols isolated from clusiaceous and calophyllaceous species. The extraction and purification of 1 have been previously described [16 (link)].

During non-constrictive cuff placement, ApoE*3-Leiden mice were treated with 10 μl pluronic gel F127 (40%, maintained at 0°C, Sigma Aldrich) ± 25 mg/ml garcinol (Enzo Life Sciences). In this way, garcinol was slowly released over a period of a couple of days at the site of injury. The pluronic gel with or without garcinol was lubricated around the isolated femoral artery and was allowed to harden out and settle around the cuff, which occurred within 20 seconds after application.

The procedures followed to perform this experiment were modified from previous reports.³⁶, ³⁷, ³⁸, ³⁹, ⁴⁰ The recombinant LymCBP HAT domain (200 ng) was incubated with histone H1 purified from calf thymus (1 μg, Sigma‐Aldrich) and 7.4 kBq of [¹⁴C]‐acetyl‐CoA (Moravek, Inc.) for 4 h in buffer containing 50 mM Tris–HCl (pH 8.0), 10% glycerol, 1 mM dithiothreitol, and 10 mM sodium butyrate. Curcumin (MedChemExpress), anacardic acid (MedChemExpress), and garcinol (Enzo) prepared in dimethyl sulfoxide (DMSO) were premixed at 50 μM each. Reactions were separated in 14% SDS‐PAGE gels. To detect β radiation released from radiocarbon, the gels were dried on pieces of filter paper and exposed to imaging plates for several days. Signals were observed using a fluoro image analyzer (FLA‐2000; Fuji Film).