20-Hydroxycholecalciferol, Product of Vitamin D3 Hydroxylation by P450scc, Decreases NF-κB Activity by Increasing IκBα Levels in Human Keratinocytes

The side chain of vitamin D3 is hydroxylated in a sequential manner by cytochrome P450scc (CYP11A1) to form 20-hydroxycholecalciferol, which can induce growth arrest and differentiation of both primary and immortalized epidermal keratinocytes. Since nuclear factor-κB (NF-κB) plays a pivotal role in the regulation of cell proliferation, differentiation and apoptosis, we examined the capability of 20-hydroxycholecalciferol to modulate the activity of NF-κB, using 1,25-dihydroxycholecalciferol (calcitriol) as a positive control. 20-hydroxycholecalciferol inhibits the activation of NFκB DNA binding activity as well as NF-κB-driven reporter gene activity in keratinocytes. Also, 20-hydroxycholecalciferol induced significant increases in the mRNA and protein levels of the NF-κB inhibitor protein, IκBα, in a time dependent manner, while no changes in total NF-κB-p65 mRNA or protein levels were observed. Another measure of NF-κB activity, p65 translocation from the cytoplasm into the nucleus was also inhibited in extracts of 20-hydroxycholecalciferol treated keratinocytes. Increased IκBα was concomitantly observed in cytosolic extracts of 20-hydroxycholecalciferol treated keratinocytes, as determined by immunoblotting and immunofluorescent staining. In keratinocytes lacking vitamin D receptor (VDR), 20-hydroxycholecalciferol did not affect IκBα mRNA levels, indicating that it requires VDR for its action on NF-κB activity. Comparison of the effects of calcitrol, hormonally active form of vitamin D3, with 20-hydrocholecalciferol show that both agents have a similar potency in inhibiting NF-κB. Since NF-κB is a major transcription factor for the induction of inflammatory mediators, our findings indicate that 20-hydroxycholecalciferol may be an effective therapeutic agent for inflammatory and hyperproliferative skin diseases

Characterization of a new pathway that activates lumisterol <i>in vivo</i> to biologically active hydroxylumisterols

Abstract Using LC/qTOF-MS we detected lumisterol, 20-hydroxylumisterol, 22-hydroxylumisterol, 24-hydroxylumisterol, 20,22-dihydroxylumisterol, pregnalumisterol, 17-hydroxypregnalumisterol and 17,20-dihydroxypregnalumisterol in human serum and epidermis, and the porcine adrenal gland. The hydroxylumisterols inhibited proliferation of human skin cells in a cell type-dependent fashion with predominant effects on epidermal keratinocytes. They also inhibited melanoma proliferation in both monolayer and soft agar. 20-Hydroxylumisterol stimulated the expression of several genes, including those associated with keratinocyte differentiation and antioxidative responses, while inhibiting the expression of others including RORA and RORC. Molecular modeling and studies on VDRE-transcriptional activity excludes action through the genomic site of the VDR. However, their favorable interactions with the A-pocket in conjunction with VDR translocation studies suggest they may act on this non-genomic VDR site. Inhibition of RORα and RORγ transactivation activities in a Tet-on CHO cell reporter system, RORα co-activator assays and inhibition of (RORE)-LUC reporter activity in skin cells, in conjunction with molecular modeling, identified RORα and RORγ as excellent receptor candidates for the hydroxylumisterols. Thus, we have discovered a new biologically relevant, lumisterogenic pathway, the metabolites of which display biological activity. This opens a new area of endocrine research on the effects of the hydroxylumisterols on different pathways in different cells and the mechanisms involved

Metformin inhibits melanoma development through autophagy and apoptosis mechanisms

Metformin is the most widely used antidiabetic drug because of its proven efficacy and limited secondary effects. Interestingly, recent studies have reported that metformin can block the growth of different tumor types. Here, we show that metformin exerts antiproliferative effects on melanoma cells, whereas normal human melanocytes are resistant to these metformin-induced effects. To better understand the basis of this antiproliferative effect of metformin in melanoma, we characterized the sequence of events underlying metformin action. We showed that 24 h metformin treatment induced a cell cycle arrest in G0/G1 phases, while after 72 h, melanoma cells underwent autophagy as demonstrated by electron microscopy, immunochemistry, and by quantification of the autolysosome-associated LC3 and Beclin1 proteins. In addition, 96 h post metformin treatment we observed robust apoptosis of melanoma cells. Interestingly, inhibition of autophagy by knocking down LC3 or ATG5 decreased the extent of apoptosis, and suppressed the antiproliferative effect of metformin on melanoma cells, suggesting that apoptosis is a consequence of autophagy. The relevance of these observations were confirmed in vivo, as we showed that metformin treatment impaired the melanoma tumor growth in mice, and induced autophagy and apoptosis markers. Taken together, our data suggest that metformin has an important impact on melanoma growth, and may therefore be beneficial in patients with melanoma

Catalytic Iodination of the Aliphatic C-F Bond by YbI3(THF)(3): Mechanistic Insight and Synthetic Utility

A facile iodination protocol, of unactivated alkyl fluorides: using catalytic amounts of YbI3(THF)(3) in the presence of iodotrimethylsilane as a stoichiometric fluoride trapping agent is presented. H-1 NMR spectroscopy demonstrates a two-step :catalytic cycle where TMSI regenerates active YbI3(THF)(3). Finally, the catalytic reaction is extended a one-pot procdure to demonstrate a potential application of the method Overall, the findings present a distinct strategy for C-F bond transformations in the presence of catalytic YbI3(THF)(3)

Effects of Intracerebroventricularly (ICV) Injected Ghrelin on Cardiac Inducible Nitric Oxide Synthase Activity/Expression in Obese Rats

The aim of this study was to examine the effects of ghrelin on regulation of cardiac inducible nitric oxide synthase (iNOS) activity/expression in high fat (HF), obese rats. For this study, male Wistar rats fed with HF diet (30 \% fat) for 4 weeks were injected every 24 h for 5 days intracerebroventriculary (ICV) with ghrelin (0.3 nmol/5 mu l) or with an equal volume of phosphate buffered saline (PBS). Control rats were ICV injected with an equal volume of PBS. Glucose, insulin and nitric oxide (NO) concentrations were measured in serum, while arginase activity and citrulline concentrations were measured in heart lysate. Protein iNOS and regulatory subunit of nuclear factor-kappa B (NF kappa B-p65), phosphorylation of enzymes protein kinase B (Akt) at Ser(473), and extracellular signal-regulated kinases 1/2 (ERK1/2) at Tyr(202)/Tyr(204) were determined in heart lysate by Western blot. For gene expression of iNOS qRT-PCR was used. Results show significantly (p < 0.01) higher serum NO production in ghrelin treated HF rats compared with HF rats. Ghrelin significantly reduced citrulline concentration (p < 0.05) and arginase activity (p < 0.01) in HF rats. In ghrelin treated HF rats, gene and protein expression of iNOS and NF kappa B-p65 levels were significantly (p < 0.05) increased compared with HF rats. Increased phosphorylation of Akt (p < 0.01) and decreased (p < 0.05) ERK1/2 phosphorylation were detected in HF ghrelin treated rats compared with HF rats hearts. Results from this study indicate that exogenous ghrelin induces expression and activity of cardiac iNOS via Akt phosphorylation followed by NF kappa B activation in HF rats.Ministry of Education, Science and Technology, Republic of Serbia {[}173033, 41025

Catalytic Iodination of the Aliphatic C–F Bond by YbI₃(THF)₃: Mechanistic Insight and Synthetic Utility

A facile iodination protocol of unactivated alkyl fluorides using catalytic amounts of YbI₃(THF)₃ in the presence of iodotrimethylsilane as a stoichiometric fluoride trapping agent is presented. ¹H NMR spectroscopy demonstrates a two-step catalytic cycle where TMSI regenerates active YbI₃(THF)₃. Finally, the catalytic reaction is extended into a one-pot procedure to demonstrate a potential application of the method. Overall, the findings present a distinct strategy for C–F bond transformations in the presence of catalytic YbI₃(THF)₃