Osteoinductive effects of glyceollins on adult mesenchymal stromal/stem cells from adipose tissue and bone marrow.

BACKGROUND: While current therapies for osteoporosis focus on reducing bone resorption, the development of therapies to regenerate bone may also be beneficial. Promising anabolic therapy candidates include phytoestrogens, such as daidzein, which effectively induce osteogenesis of adipose-derived stromal cells (ASCs) and bone marrow stromal cells (BMSCs). PURPOSE: To investigate the effects of glyceollins, structural derivatives of daidzein, on osteogenesis of ASCs and BMSCs. STUDY DESIGN: Herein, the osteoinductive effects of glyceollin I and glyceollin II were assessed and compared to estradiol in ASCs and BMSCs. The mechanism by which glyceollin II induces osteogenesis was further examined. METHODS: The ability of glyceollins to promote osteogenesis of ASCs and BMSCs was evaluated in adherent and scaffold cultures. Relative deposition of calcium was analyzed using Alizarin Red staining, Bichinchoninic acid Protein Assay, and Alamar Blue Assay. To further explore the mechanism by which glyceollin II exerts its osteoinductive effects, docking studies of glyceollin II, RNA isolation, cDNA synthesis, and quantitative RT-PCR (qPCR) were performed. RESULTS: In adherent cultures, ASCs and BMSCs treated with estradiol, glyceollin I, or glyceollin II demonstrated increased calcium deposition relative to vehicle-treated cells. During evaluation on PLGA scaffolds seeded with ASCs and BMSCs, glyceollin II was the most efficacious in inducing ASC and BMSC osteogenesis compared to estradiol and glyceollin I. Dose-response analysis in ASCs and BMSCs revealed that glyceollin II has the highest potency at 10nM in adherent cultures and 1µM in tissue scaffold cultures. At all doses, osteoinductive effects were attenuated by fulvestrant, suggesting that glyceollin II acts at least in part through estrogen receptor-mediated pathways to induce osteogenesis. Analysis of gene expression demonstrated that, similar to estradiol, glyceollin II induces upregulation of genes involved in osteogenic differentiation. CONCLUSION: The ability of glyceollin II to induce osteogenic differentiation in ASCs and BMSCs indicates that glyceollins hold the potential for the development of pharmacological interventions to improve clinical outcomes of patients with osteoporosis.

Rational Design of a Boron-Modified Triphenylethylene (GLL398) as an Oral Selective Estrogen Receptor Downregulator.

Development of orally bioavailable nonsteroidal selective estrogen receptor downregulators (SERDs) provides clinical opportunities for the long-term treatment and adjuvant therapy of breast cancer at all stages. We describe the design, synthesis, and identification of a boron-modified GW7604 derivative (GLL398, 9), a SERD candidate, in which a boronic acid functional group replaces the phenolic hydroxyl group of GW7604. Compound 9 strongly binds to ERα in a fluorescence resonance energy transfer binding assay (IC50 = 1.14 nM) and potently degrades ERα in MCF-7 breast cancer cells (IC50 = 0.21 µM). Most importantly, the introduction of the boronic acid group confers superior oral bioavailability of 9 (AUC = 36.9 µg·h/mL) in rats as compared to GW7604 (AUC = 3.35 µg·h/mL). The strikingly favorable pharmacokinetic property of 9 makes it a promising oral SERD suitable for clinical evaluation.

Carcinogenic effects of oil dispersants: A KEGG pathway-based RNA-seq study of human airway epithelial cells.

The health impacts of the BP oil spill are yet to be further revealed as the toxicological effects of oil products and dispersants on human respiratory system may be latent and complex, and hence difficult to study and follow up. Here we performed RNA-seq analyses of a system of human airway epithelial cells treated with the BP crude oil and/or dispersants Corexit 9500 and Corexit 9527 that were used to help break up the oil spill. Based on the RNA-seq data, we then systemically analyzed the transcriptomic perturbations of the cells at the KEGG pathway level using two pathway-based analysis tools, GAGE (generally applicable gene set enrichment) and GSNCA (Gene Sets Net Correlations Analysis). Our results suggested a pattern of change towards carcinogenesis for the treated cells marked by upregulation of ribosomal biosynthesis (hsa03008) (p=1.97E-13), protein processing (hsa04141) (p=4.09E-7), Wnt signaling (hsa04310) (p=6.76E-3), neurotrophin signaling (hsa04722) (p=7.73E-3) and insulin signaling (hsa04910) (p=1.16E-2) pathways under the dispersant Corexit 9527 treatment, as identified by GAGE analysis. Furthermore, through GSNCA analysis, we identified gene co-expression changes for several KEGG cancer pathways, including small cell lung cancer pathway (hsa05222, p=9.99E-5), under various treatments of oil/dispersant, especially the mixture of oil and Corexit 9527. Overall, our results suggested carcinogenic effects of dispersants (in particular Corexit 9527) and their mixtures with the BP crude oil, and provided further support for more stringent safety precautions and regulations for operations involving long-term respiratory exposure to oil and dispersants.

Muscadine Grape Skin Extract Induces an Unfolded Protein Response-Mediated Autophagy in Prostate Cancer Cells: A TMT-Based Quantitative Proteomic Analysis.

Muscadine grape skin extract (MSKE) is derived from muscadine grape (Vitis rotundifolia), a common red grape used to produce red wine. Endoplasmic reticulum (ER) stress activates the unfolded protein response (UPR) that serves as a survival mechanism to relieve ER stress and restore ER homeostasis. However, when persistent, ER stress can alter the cytoprotective functions of the UPR to promote autophagy and cell death. Although MSKE has been documented to induce apoptosis, it has not been linked to ER stress/UPR/autophagy. We hypothesized that MSKE may induce a severe ER stress response-mediated autophagy leading to apoptosis. As a model, we treated C4-2 prostate cancer cells with MSKE and performed a quantitative Tandem Mass Tag Isobaric Labeling proteomic analysis. ER stress response, autophagy and apoptosis were analyzed by western blot, acridine orange and TUNEL/Annexin V staining, respectively. Quantitative proteomics analysis indicated that ER stress response proteins, such as GRP78 were greatly elevated following treatment with MSKE. The up-regulation of pro-apoptotic markers PARP, caspase-12, cleaved caspase-3, -7, BAX and down-regulation of anti-apoptotic marker BCL2 was confirmed by Western blot analysis and apoptosis was visualized by increased TUNEL/Annexin V staining upon MSKE treatment. Moreover, increased acridine orange, and LC3B staining was detected in MSKE-treated cells, suggesting an ER stress/autophagy response. Finally, MSKE-mediated autophagy and apoptosis was antagonized by co-treatment with chloroquine, an autophagy inhibitor. Our results indicate that MSKE can elicit an UPR that can eventually lead to apoptosis in prostate cancer cells.

Intestinal microbiota in pediatric patients with end stage renal disease: a Midwest Pediatric Nephrology Consortium study.

BACKGROUND: End-stage renal disease (ESRD) is associated with uremia and increased systemic inflammation. Alteration of the intestinal microbiota may facilitate translocation of endotoxins into the systemic circulation leading to inflammation. We hypothesized that children with ESRD have an altered intestinal microbiota and increased serum levels of bacterially derived uremic toxins. METHODS: Four groups of subjects were recruited: peritoneal dialysis (PD), hemodialysis (HD), post-kidney transplant and healthy controls. Stool bacterial composition was assessed by pyrosequencing analysis of 16S rRNA genes. Serum levels of C-reactive protein (CRP), D-lactate, p-cresyl sulfate and indoxyl sulfate were measured. RESULTS: Compared to controls, the relative abundance of Firmicutes (P = 0.0228) and Actinobacteria (P = 0.0040) was decreased in PD patients. The relative abundance of Bacteroidetes was increased in HD patients (P = 0.0462). Compared to HD patients the relative abundance of Proteobacteria (P = 0.0233) was increased in PD patients. At the family level, Enterobacteriaceae was significantly increased in PD patients (P = 0.0020) compared to controls; whereas, Bifidobacteria showed a significant decrease in PD and transplant patients (P = 0.0020) compared to control. Alpha diversity was decreased in PD patients and kidney transplant using both phylogenetic and non-phylogenetic diversity measures (P = 0.0031 and 0.0003, respectively), while beta diversity showed significant separation (R statistic = 0.2656, P = 0.010) between PD patients and controls. ESRD patients had increased serum levels of p-cresyl sulfate and indoxyl sulfate (P < 0.0001 and P < 0.0001, respectively). The data suggests that no significant correlation exists between the alpha diversity of the intestinal microbiota and CRP, D-lactate, or uremic toxins. Oral iron supplementation results in expansion of the phylum Proteobacteria. CONCLUSIONS: Children with ESRD have altered intestinal microbiota and increased bacterially derived serum uremic toxins.

Boronic prodrug of 4-hydroxytamoxifen is more efficacious than tamoxifen with enhanced bioavailability independent of CYP2D6 status.

BACKGROUND: Poor initial response to tamoxifen due to CYP2D6 polymorphism and adverse side effects are two clinical challenges in tamoxifen therapy. We report the development and preclinical testing of a boronic prodrug to orally deliver 4-OHT at therapeutically effective concentrations but at a fraction of the standard tamoxifen dose. METHODS: A mouse xenograft tumor model was used to investigate the efficacy of ZB497 in comparison with tamoxifen. Pharmacokinetic studies were conducted to evaluate the metabolism and bioavailability of the drug in mice. Drug and metabolites distribution in xenograft tumor tissues was determined by high performance liquid chromatography-tandem mass spectrometry. RESULTS: The boronic prodrug, ZB497, can not only be efficiently converted to 4-OHT in mice, but also afforded over 30 fold higher plasma concentrations of 4-OHT than in mice given either the same dose of 4-OHT or tamoxifen. Further, ZB497 was more effective than tamoxifen at lowered dosage in inhibiting the growth of xenograft tumors in mice. Consistent with these observations, ZB497 treated mice accumulated over 6 times higher total drug concentrations than tamoxifen treated mice. CONCLUSIONS: Our study demonstrates that ZB497 effectively delivers a markedly increased plasma concentration of 4-OHT in mice. The boronic prodrug was shown to have far superior bioavailability of 4-OHT compared to tamoxifen or 4-OHT administration as measured by the area under the plasma concentration time curve (AUC), plasma peak concentrations, and drug accumulation in tumor tissues. Further, ZB497 proves to be a more efficacious hormone therapy than tamoxifen administered at a reduced dose in mice.

Novel daidzein analogs enhance osteogenic activity of bone marrow-derived mesenchymal stem cells and adipose-derived stromal/stem cells through estrogen receptor dependent and independent mechanisms.

INTRODUCTION: Osteoporosis is a disease characterized by low bone mineral density (BMD) and increased risk of fractures. Studies have demonstrated the use of phytoestrogens, or plant-derived estrogens, such as genistein and daidzein, to effectively increase osteogenic activity of bone marrow-derived mesenchymal stem cells (BMSCs). Herein, the effects of daidzein analogs on the osteogenic differentiation efficiency of human BMSC and adipose-derived stromal/stem cells (ASC) were explored. METHODS: BMSCs and ASCs underwent osteogenic differentiation in the presence of vehicle, 17ß-estradiol (E2), phytoestrogens, or daidzein analogs. Cells were stained for alkaline phosphatase (ALP) enzymatic activity, calcium deposition by alizarin red s, and phosphate mineralization by silver nitrate. Gene expression analysis was conducted on cells treated with daidzein analogs. RESULTS: Cells treated with E2, daidzein, or genistein increased calcium deposition by 1.6-, 1.5-, and 1.4-fold, respectively, relative to vehicle-treated BMSCs and 1.6-, 1.7-, and 1.4-fold relative to vehicle-treated ASCs, respectively. BMSCs treated with daidzein analog 2c, 2g, and 2l demonstrated a 1.6-, 1.6-, and 1.9-fold increase in calcium deposition relative to vehicle-treated BMSCs, respectively, while ASCs treated with daidzein analog 2c, 2g, or 2l demonstrated a 1.7-, 2.0-, and 2.2-fold increase in calcium deposition relative to vehicle-treated ASCs, respectively. Additional analysis with BMSCs and ASCs was conducted in the more efficient compounds: 2g and 2l. ALP activity and phosphate mineralization was increased in 2g- and 2l-treated cells. The analysis of lineage specific gene expression demonstrated increased expression of key osteogenic genes (RUNX2, c-FOS, SPARC, DLX5, SPP1, COL1A1, IGF1, SOST, and DMP1) and earlier induction of these lineage specific genes, following treatment with 2g or 2l, relative to vehicle-treated cells. Estrogen receptor (ER) inhibitor studies demonstrated that ER antagonist fulvestrant inhibited the osteogenic differentiation of 2g in BMSCs and ASCs, while fulvestrant only attenuated the effects of 2l, suggesting that 2l acts by both ER dependent and independent pathways. CONCLUSIONS: These studies provide support for exploring the therapeutic efficacy of daidzein derivatives for the treatment of osteoporosis. Furthermore, the patterns of gene induction differed following treatment with each daidzein analog, suggesting that these daidzein analogs activate distinct ER and non-ER pathways to induce differentiation in BMSCs and ASCs.