Differential DNA-binding and cofactor recruitment are possible determinants of the synthetic steroid YK11-dependent gene expression by androgen receptor in breast cancer MDA-MB 453 cells.

Recently, selective androgen receptor modulators (SARMs), which bind to AR and act in a tissue/effect-specific manner, have been developed, but the selective mechanism is not well understood. In this study, we investigated the selective mechanism using the synthetic steroid YK11, which showed AR-mediated gene-selective transactivation. In the AR-positive human breast cancer MDA-MB-453 cells, different patterns of AR-mediated target gene expression and AR recruitment to their enhancer regions were observed between DHT and YK11. A docking study suggested the helices 11 and 12 was moved by the sterically hindered C17-group of YK11. Furthermore, the mutational studies of AR Gln902 and mammalian two-hybrid assays suggested different cofactor recruitment between DHT and YK11. The results of this study suggest that gene selective regulation by SARMs results from differential DNA-binding and/or cofactor recruitment by ligands. These results provide novel insights into the mechanism of action of SARMs.

High-Dose Vitamin C Preadministration Reduces Vancomycin-Associated Nephrotoxicity in Mice.

Vancomycin is recommended for treating severe infections caused by Gram-positive cocci, including methicillin-resistant Staphylococcus aureus. However, renal damage often occurs as a side effect because vancomycin is mainly excreted via the kidneys. The mechanism of vancomycin-associated nephrotoxicity is thought to involve the elevation of oxidative stress in the kidneys. Vitamin C (VC) has strong antioxidant properties; therefore, we evaluated the effect of high-dose VC preadministration on vancomycin-associated nephrotoxicity. Vancomycin was intraperitoneally injected into mice once daily for 7 d. Additionally, high-dose VC was intraperitoneally injected into mice at 30 min before vancomycin administration for 7 d. The plasma creatinine and urea nitrogen levels were increased by vancomycin treatment; however, high-dose VC preadministration suppressed the increase in these levels. Histological examination also revealed that high-dose VC preadministration reduced the characteristics of vancomycin-associated nephrotoxicity, such as dilated renal tubules with casts, the dilation of renal proximal tubules, and tubular epithelial desquamation. Furthermore, high-dose VC preadministration reduced the appearance of apoptotic cells presumably derived from the epithelial cells in the dilated proximal tubules. Thus, intraperitoneally injected high-dose VC preadministration reduced vancomycin-associated nephrotoxicity in mice. These novel findings may indicate that vancomycin-associated nephrotoxicity in humans may be reduced by high-dose VC preadministration.

Vitamin C deficiency causes muscle atrophy and a deterioration in physical performance.

L-Ascorbic acid (AsA) is a water-soluble antioxidant. We examined the effect of AsA deficiency on skeletal muscle using senescence marker protein-30 (SMP30)-knockout (KO) mice that are defective in AsA biosynthesis, which makes this mouse model similar to humans, to clarify the function of AsA in skeletal muscle. Eight-week-old female SMP30-KO mice were divided into the following two groups: an AsA-sufficient group [AsA(+)] that was administered 1.5 g/L AsA and an AsA-deficient group [AsA(-)] that was administered tap (AsA-free) water. At 4 weeks, the AsA content in the gastrocnemius muscle of AsA(-) mice was 0.7% compared to that in the gastrocnemius muscle of AsA(+) mice. Significantly lower weights of all muscles were observed in AsA(-) mice than those in AsA(+) mice at 12 and 16 weeks. The cross-sectional area of the soleus was significantly smaller in AsA(-) mice at 16 weeks than that in AsA(+) mice. The physical performance of AsA(-) mice was significantly less than that of AsA(+) mice at 12 weeks. Following AsA deficiency for 12 weeks, the expression of ubiquitin ligases, such as atrogin1/muscle atrophy F-box (MAFbx) and muscle RING-finger protein 1 (MuRF1), was upregulated. Furthermore, all detected effects of AsA deficiency on muscles of the AsA(-) group at 12 weeks were restored following AsA supplementation for 12 weeks. Thus, longer-term AsA deficiency is associated with muscle wasting, that this can be reversed by restoring AsA levels.

Selective Androgen Receptor Modulator, YK11, Up-Regulates Osteoblastic Proliferation and Differentiation in MC3T3-E1 Cells.

Androgens are key regulators that play a critical role in the male reproductive system and have anabolic effects on bone mineral density and skeletal muscle mass. We have previously reported that YK11 is a novel selective androgen receptor modulator (SARM) and induces myogenic differentiation and selective gene regulation. In this study, we show that treatment of YK11 and dihydrotestosterone (DHT) accelerated cell proliferation and mineralization in MC3T3-E1 mouse osteoblast cells. Further, YK11-treated cells increased osteoblast specific differentiation markers, such as osteoprotegerin and osteocalcin, compared to untreated cells. These observations were attenuated by androgen receptor (AR) antagonist treatment. To clarify the effect of YK11, we investigated rapid non-genomic signaling by AR. The phosphorylated Akt protein level was increased by YK11 and DHT treatment, suggesting that YK11 activates Akt-signaling via non-genomic signaling of AR. Because it is known Akt-signaling is a key regulator of androgen-mediated osteoblast differentiation, YK11 has osteogenic activity as well as androgen.

Alisol B 23-acetate from the rhizomes of Alisma orientale is a natural agonist of the human pregnane X receptor.

BACKGROUND: Pregnane X receptor (PXR) is a key regulator of the induction of drug metabolizing enzymes. PXR has been studied for its importance in drug-drug or herb-drug interactions, and it is also a molecular target for the treatment of inflammatory and metabolic diseases. PURPOSE: This study aims to determine new natural PXR-ligands from traditional plant medicines. METHODS: The PXR activation activity was measured by a mammalian one hybrid assay of PXR. Identification of the active compound from Alisma rhizome (the rhizomes of Alisma orientale) was carried out by bioassay-guided fractionation method. The transcriptional activity of the liver-enriched nuclear receptors was measured by the luciferase reporter assay. The interaction between the SRC-1 and PXR was measured by a mammalian 2-hybrid assay. The expression of endogenous CYP3A4 mRNA in both cultured hPXR-overexpressing hepatoma cells and human primary hepatocytes were measured by quantitative RT-PCR method. RESULTS: The extract of Alisma rhizome showed the most potent activation activity by screening of a library of medicinal plant extracts. Alisol B 23-acetate (ABA) was identified to be the active compound of Alisma rhizome. ABA caused a concentration-dependent increase on the PXR-dependent transactivation of a luciferase reporter gene, but did not affect the ligand binding activity of the liver-enriched nuclear receptors, such as CAR, LXR, FXR, PPARα, PPARδ and PPARγ, emphasizing that ABA is a potent and specific agonist of PXR. With ABA treatment, the direct interaction between the ligand-binding domain of PXR and the receptor interaction domain of SRC1 was observed. ABA also induced the expression of endogenous CYP3A4 mRNA in both cultured hPXR-overexpressing hepatoma cells and human primary hepatocytes. CONCLUSION: Since the rhizomes of Alisma orientale are used for a wide range of ailments in traditional Chinese medicine and Japanese Kampo medicine, this study could possibly extend into the clinical usage of these medicines via the mechanism of PXR activation.

Palladium(II) Catalyzed Cyclization-Carbonylation-Cyclization Coupling Reaction of (ortho-Alkynyl Phenyl) (Methoxymethyl) Sulfides Using Molecular Oxygen as the Terminal Oxidant.

An efficient Pd(II)/Pd°-p-benzoquinone/hydroquinone-CuCl2/CuCl catalyst system was developed that uses environmentally friendly molecular oxygen as the terminal oxidant to catalyze the cyclization-carbonylation-cyclization coupling reaction (CCC-coupling reaction) of (o-alkynyl phenyl) (methoxymethyl) sulfides.

Nigramide J is a novel potent inverse agonist of the human constitutive androstane receptor.

The constitutive androstane receptor (CAR, NR1I3) is very important for drug development and for understanding pharmacokinetic drug-drug interactions. We screened by mammalian one hybrid assay among natural compounds to discover novel ligands of human constitutive androstane receptor (hCAR). hCAR transcriptional activity was measured by luciferase assay and mRNA levels of CYP2B6 and CYP3A4 in HepTR-hCAR cells and human primary hepatocytes were measured by real-time RT-PCR. Nigramide J (NJ) whose efficacy is comparable to those of hitherto known inverse agonists such as clotrimazole, PK11195, and ethinylestradiol. NJ is a naturally occurring cyclohexane-type amide alkaloid that was isolated from the roots of Piper nigrum. The suppressive effect of NJ on the CAR-dependent transcriptional activity was found to be species specific, in the descending order of hCAR, rat CAR, and mouse CAR. The unliganded hCAR-dependent transactivation of reporter and endogenous genes was suppressed by NJ at concentrations higher than 5 µmol/L. The ligand-binding cavity of hCAR was shared by NJ and CITCO, because they were competitive in the binding to hCAR. NJ interfered with the interaction of hCAR with coactivator SRC-1, but not with its interaction with the corepressor NCoR1. Furthermore, NJ is agonist of human pregnane X receptor (hPXR). NJ is a dual ligand of hCAR and hPXR, being an agonist of hPXR and an inverse agonist of hCAR.

Nigramide C is a natural agonist of human pregnane x receptor.

Pregnane X receptor (PXR) is known as a xenosensor, playing a key role in response to xenochemical stimuli. Activation of PXR enhanced expression of various drug-metabolizing enzymes and transporters such as cytochrome P450 3A4 (CYP3A4). During a screening of a natural compounds library for novel ligands of human xenosensing receptors by the mammalian one-hybrid assay, two cyclohexene-type amide alkaloids were isolated, with nigramide C (NigC) showing the most potent activation of human PXR (hPXR). NigC-mediated hPXR activation was enhanced by overexpression of steroid receptor coactivator 1 (SRC1), peroxisome proliferator-activated receptor γ, coactivator 1α, and protein arginine methyltransferase 1. A direct interaction between the ligand-binding domain of hPXR and the receptor interaction domain of SRC1 was observed. NigC induced the expression of endogenous CYP3A4 mRNA and protein in both cultured hepatoma cells and primary hepatocytes. However, in primary hepatocytes, the relative agonist activity of NigC was not as potent as that of rifampicin, probably because of lower metabolic stability of NigC in these cells. In conclusion, NigC was found to be an effective agonist of hPXR. NigC is a useful tool for investigation of hPXR function.