Affinity-tagged SMAD1 and SMAD5 mouse lines reveal transcriptional reprogramming mechanisms during early pregnancy.

Endometrial decidualization, a prerequisite for successful pregnancies, relies on transcriptional reprogramming driven by progesterone receptor (PR) and bone morphogenetic protein (BMP)-SMAD1/SMAD5 signaling pathways. Despite their critical roles in early pregnancy, how these pathways intersect in reprogramming the endometrium into a receptive state remains unclear. To define how SMAD1 and/or SMAD5 integrate BMP signaling in the uterus during early pregnancy, we generated two novel transgenic mouse lines with affinity tags inserted into the endogenous SMAD1 and SMAD5 loci (Smad1HA/HA and Smad5PA/PA). By profiling the genome-wide distribution of SMAD1, SMAD5, and PR in the mouse uterus, we demonstrated the unique and shared roles of SMAD1 and SMAD5 during the window of implantation. We also showed the presence of a conserved SMAD1, SMAD5, and PR genomic binding signature in the uterus during early pregnancy. To functionally characterize the translational aspects of our findings, we demonstrated that SMAD1/5 knockdown in human endometrial stromal cells suppressed expressions of canonical decidual markers (IGFBP1, PRL, FOXO1) and PR-responsive genes (RORB, KLF15). Here, our studies provide novel tools to study BMP signaling pathways and highlight the fundamental roles of SMAD1/5 in mediating both BMP signaling pathways and the transcriptional response to progesterone (P4) during early pregnancy.

Impaired bone morphogenetic protein (BMP) signaling pathways disrupt decidualization in endometriosis.

Endometriosis is linked to increased infertility and pregnancy complications due to defective endometrial decidualization. We hypothesized that identification of altered signaling pathways during decidualization could identify the underlying cause of infertility and pregnancy complications. Our study reveals that transforming growth factor ß (TGFß) pathways are impaired in the endometrium of individuals with endometriosis, leading to defective decidualization. Through detailed transcriptomic analyses, we discovered abnormalities in TGFß signaling pathways and key regulators, such as SMAD4, in the endometrium of affected individuals. We also observed compromised activity of bone morphogenetic proteins (BMP), a subset of the TGFß family, that control endometrial receptivity. Using 3-dimensional models of endometrial stromal and epithelial assembloids, we showed that exogenous BMP2 improved decidual marker expression in individuals with endometriosis. Our findings reveal dysfunction of BMP/SMAD signaling in the endometrium of individuals with endometriosis, explaining decidualization defects and subsequent pregnancy complications in these individuals.

Inhibition of CSF1R and KIT With Pexidartinib Reduces Inflammatory Signaling and Cell Viability in Endometriosis.

Endometriosis is a common and debilitating disease, affecting ∼170 million women worldwide. Affected patients have limited therapeutic options such as hormonal suppression or surgical excision of the lesions, though therapies are often not completely curative. Targeting receptor tyrosine kinases (RTKs) could provide a nonhormonal treatment option for endometriosis. We determined that 2 RTKs, macrophage-colony stimulating factor 1 receptor (CSF1R) and mast/stem cell growth factor receptor KIT (KIT), are overexpressed in endometriotic lesions and could be novel nonhormonal therapeutic targets for endometriosis. The kinase activity of CSF1R and KIT is suppressed by pexidartinib, a small molecule inhibitor that was recently approved by the US Food and Drug Administration. Using immunohistochemistry, we detected CSF1R and KIT in endometriotic tissues obtained from peritoneal lesions, colorectal lesions, and endometriomas. Specifically, we show that KIT is localized to the epithelium of the lesions, while CSF1R is expressed in the stroma and macrophages of the endometriotic lesions. Given the high epithelial expression of CSF1R and KIT, 12Z endometriotic epithelial cells were used to evaluate the efficacy of dual CSF1R and KIT inhibition with pexidartinib. We found that pexidartinib suppressed activation in 12Z cells of JNK, STAT3, and AKT signaling pathways, which control key proinflammatory and survival networks within the cell. Using quantitative real-time polymerase chain reaction, we determined that pexidartinib suppressed interleukin 8 (IL8) and cyclin D1 (CCND1) expression. Lastly, we demonstrated that pexidartinib decreased cell growth and viability. Overall, these results indicate that pexidartinib-mediated CSF1R and KIT inhibition reduces proinflammatory signaling and cell viability in endometriosis.

Affinity-tagged SMAD1 and SMAD5 mouse lines reveal transcriptional reprogramming mechanisms during early pregnancy.

Endometrial decidualization, a prerequisite for successful pregnancies, relies on transcriptional reprogramming driven by progesterone receptor (PR) and bone morphogenetic protein (BMP)-SMAD1/SMAD5 signaling pathways. Despite their critical roles in early pregnancy, how these pathways intersect in reprogramming the endometrium into a receptive state remains unclear. To define how SMAD1 and/or SMAD5 integrate BMP signaling in the uterus during early pregnancy, we generated two novel transgenic mouse lines with affinity tags inserted into the endogenous SMAD1 and SMAD5 loci (Smad1HA/HA and Smad5PA/PA). By profiling the genome-wide distribution of SMAD1, SMAD5, and PR in the mouse uterus, we demonstrated the unique and shared roles of SMAD1 and SMAD5 during the window of implantation. We also showed the presence of a conserved SMAD1, SMAD5, and PR genomic binding signature in the uterus during early pregnancy. To functionally characterize the translational aspects of our findings, we demonstrated that SMAD1/5 knockdown in human endometrial stromal cells suppressed expressions of canonical decidual markers (IGFBP1, PRL, FOXO1) and PR-responsive genes (RORB, KLF15). Here, our studies provide novel tools to study BMP signaling pathways and highlight the fundamental roles of SMAD1/5 in mediating both BMP signaling pathways and the transcriptional response to progesterone (P4) during early pregnancy.

Impaired bone morphogenetic protein (BMP) signaling pathways disrupt decidualization in endometriosis.

Endometriosis is linked to increased infertility and pregnancy complications due to defective endometrial decidualization. We hypothesized that identification of altered signaling pathways during decidualization could identify the underlying cause of infertility and pregnancy complications. Our study reveals that transforming growth factor ß (TGFß) pathways are impaired in the endometrium of individuals with endometriosis, leading to defective decidualization. Through detailed transcriptomic analyses, we discovered abnormalities in TGFß signaling pathways and key regulators, such as SMAD4, in the endometrium of affected individuals. We also observed compromised activity of bone morphogenetic proteins (BMP), a subset of the TGFß family, that control endometrial receptivity. Using 3-dimensional models of endometrial stromal and epithelial assembloids, we showed that exogenous BMP2 improved decidual marker expression in individuals with endometriosis. Our findings unveil a previously unidentified dysfunction in BMP/SMAD signaling in the endometrium of individuals with endometriosis, explaining decidualization defects and subsequent pregnancy complications in these individuals.

Impaired bone morphogenetic protein signaling pathways disrupt decidualization in endometriosis.

It is hypothesized that impaired endometrial decidualization contributes to decreased fertility in individuals with endometriosis. To identify the molecular defects that underpin defective decidualization in endometriosis, we subjected endometrial stromal cells from individuals with or without endometriosis to time course in vitro decidualization with estradiol, progesterone, and 8-bromo-cyclic-AMP (EPC) for 2, 4, 6, or 8 days. Transcriptomic profiling identified differences in key pathways between the two groups, including defective bone morphogenetic protein (BMP)/SMAD4 signaling (ID2, ID3, FST), oxidate stress response (NFE2L2, ALOX15, SLC40A1), and retinoic acid signaling pathways (RARRES, RARB, ALDH1B1). Genome-wide binding analyses identified an altered genomic distribution of SMAD4 and H3K27Ac in the decidualized stromal cells from individuals without endometriosis relative to those with endometriosis, with target genes enriched in pathways related to signaling by transforming growth factor ß (TGFß), neurotrophic tyrosine kinase receptors (NTRK), and nerve growth factor (NGF)-stimulated transcription. We found that direct SMAD1/5/4 target genes control FOXO, PI3K/AKT, and progesterone-mediated signaling in decidualizing cells and that BMP2 supplementation in endometriosis patient-derived assembloids elevated the expression of decidualization markers. In summary, transcriptomic and genome-wide binding analyses of patient-derived endometrial cells and assembloids identified that a functional BMP/SMAD1/5/4 signaling program is crucial for engaging decidualization.

Beclin-1-dependent autophagy, but not apoptosis, is critical for stem-cell-mediated endometrial programming and the establishment of pregnancy.

The human endometrium shows a remarkable regenerative capacity that enables cyclical regeneration and remodeling throughout a woman's reproductive life. Although early postnatal uterine developmental cues direct this regeneration, the vital factors that govern early endometrial programming are largely unknown. We report that Beclin-1, an essential autophagy-associated protein, plays an integral role in uterine morphogenesis during the early postnatal period. We show that conditional depletion of Beclin-1 in the uterus triggers apoptosis and causes progressive loss of Lgr5+/Aldh1a1+ endometrial progenitor stem cells, with concomitant loss of Wnt signaling, which is crucial for stem cell renewal and epithelial gland development. Beclin-1 knockin (Becn1 KI) mice with disabled apoptosis exhibit normal uterine development. Importantly, the restoration of Beclin-1-driven autophagy, but not apoptosis, promotes normal uterine adenogenesis and morphogenesis. Together, the data suggest that Beclin-1-mediated autophagy acts as a molecular switch that governs the early uterine morphogenetic program by maintaining the endometrial progenitor stem cells.

SMAD2/3 signaling in the uterine epithelium controls endometrial cell homeostasis and regeneration.

The regenerative potential of the endometrium is attributed to endometrial stem cells; however, the signaling pathways controlling its regenerative potential remain obscure. In this study, genetic mouse models and endometrial organoids are used to demonstrate that SMAD2/3 signaling controls endometrial regeneration and differentiation. Mice with conditional deletion of SMAD2/3 in the uterine epithelium using Lactoferrin-iCre develop endometrial hyperplasia at 12-weeks and metastatic uterine tumors by 9-months of age. Mechanistic studies in endometrial organoids determine that genetic or pharmacological inhibition of SMAD2/3 signaling disrupts organoid morphology, increases the glandular and secretory cell markers, FOXA2 and MUC1, and alters the genome-wide distribution of SMAD4. Transcriptomic profiling of the organoids reveals elevated pathways involved in stem cell regeneration and differentiation such as the bone morphogenetic protein (BMP) and retinoic acid signaling (RA) pathways. Therefore, TGFß family signaling via SMAD2/3 controls signaling networks which are integral for endometrial cell regeneration and differentiation.

Establishing 3D Endometrial Organoids from the Mouse Uterus.

Endometrial tissue lines the inner cavity of the uterus and is under the cyclical control of estrogen and progesterone. It is a tissue that is composed of luminal and glandular epithelium, a stromal compartment, a vascular network, and a complex immune cell population. Mouse models have been a powerful tool to study the endometrium, revealing critical mechanisms that control implantation, placentation, and cancer. The recent development of 3D endometrial organoid cultures presents a state-of-the-art model to dissect the signaling pathways that underlie endometrial biology. Establishing endometrial organoids from genetically engineered mouse models, analyzing their transcriptomes, and visualizing their morphology at a single-cell resolution are crucial tools for the study of endometrial diseases. This paper outlines methods to establish 3D cultures of endometrial epithelium from mice and describes techniques to quantify gene expression and analyze the histology of the organoids. The goal is to provide a resource that can be used to establish, culture, and study the gene expression and morphological characteristics of endometrial epithelial organoids.

Discovery of Highly Potent and BMPR2-Selective Kinase Inhibitors Using DNA-Encoded Chemical Library Screening.

The discovery of monokinase-selective inhibitors for patients is challenging because the 500+ kinases encoded by the human genome share highly conserved catalytic domains. Until now, no selective inhibitors unique for a single transforming growth factor ß (TGFß) family transmembrane receptor kinase, including bone morphogenetic protein receptor type 2 (BMPR2), have been reported. This dearth of receptor-specific kinase inhibitors hinders therapeutic options for skeletal defects and cancer as a result of an overactivated BMP signaling pathway. By screening 4.17 billion "unbiased" and "kinase-biased" DNA-encoded chemical library molecules, we identified hits CDD-1115 and CDD-1431, respectively, that were low-nanomolar selective kinase inhibitors of BMPR2. Structure-activity relationship studies addressed metabolic lability and high-molecular-weight issues, resulting in potent and BMPR2-selective inhibitor analogs CDD-1281 (IC50 = 1.2 nM) and CDD-1653 (IC50 = 2.8 nM), respectively. Our work demonstrates that DNA-encoded chemistry technology (DEC-Tec) is reliable for identifying novel first-in-class, highly potent, and selective kinase inhibitors.

The Abl/Abi signaling links WAVE regulatory complex to Cbl E3 ubiquitin ligase and is essential for breast cancer cell metastasis.

The family of Abelson interactor (Abi) proteins is a component of WAVE regulatory complex (WRC) and a downstream target of Abelson (Abl) tyrosine kinase. The fact that Abi proteins also interact with diverse membrane proteins and intracellular signaling molecules places these proteins at a central position in the network that controls cytoskeletal functions and cancer cell metastasis. Here, we identified a motif in Abi proteins that conforms to consensus sequences found in a cohort of receptor and non-receptor tyrosine kinases that bind to Cbl-tyrosine kinase binding domain. The phosphorylation of tyrosine 213 in this motif is essential for Abi degradation. Double knockout of c-Cbl and Cbl B in Bcr-Abl-transformed leukemic cells abolishes Abi1, Abi2, and WAVE2 degradation. Moreover, knockout of Abi1 reduces Src family kinase Lyn activation in Bcr-Abl-positive leukemic cells and promotes EGF-induced EGF receptor downregulation in breast cancer cells. Importantly, Abi1 depletion impeded breast cancer cell invasion in vitro and metastasis in mouse xenografts. Together, these studies uncover a novel mechanism by which the WRC and receptor/non-receptor tyrosine kinases are regulated and identify Abi1 as a potential therapeutic target for metastatic breast cancer.

BMP/SMAD1/5 Signaling in the Endometrial Epithelium Is Essential for Receptivity and Early Pregnancy.

The biological processes that control endometrial receptivity and embryo implantation are critical for the successful outcome of pregnancy. The endometrium is the complex inner lining of the uterine wall that is under the cyclical control of estrogen and progesterone and is a site of intimate contact between mother and blastocyst. The bone morphogenetic signaling (BMP) pathway is a highly conserved signaling pathway that controls key cellular processes throughout pregnancy and exerts intracellular effects via the SMAD1/5 transcription factors. To delineate the endometrial compartment-specific roles of BMP signaling, we generated mice with epithelial-specific conditional deletion of SMAD1/5 using Lactoferrin-icre (Smad1flox/flox;Smad5flox/flox;Lactoferrin-cre, "Smad1/5 cKO"). Histological analysis of the reproductive tracts showed that Smad1/5 cKO mice were developmentally normal and displayed no defects in glandular morphology. In fertility analyses, single SMAD1 or SMAD5 deletion had no effect on fertility; however, double-conditional deletion of SMAD1 and SMAD5 resulted in severe subfertility. Timed mating analyses revealed endometrial receptivity defects in the Smad1/5 cKO mice beginning at 3.5 days post coitum (dpc) that perturbed embryo implantation at 4.5 dpc, as demonstrated by the detection of unattached blastocysts in the uterus, decreased COX2 expression, and FOXO1 cytoplasmic mislocalization. We also found that defects that arose during peri-implantation adversely affected embryonic and decidual development at 5.5 and 6.5 dpc. Thus, uterine epithelial BMP/SMAD1/5 signaling is essential during early pregnancy and SMAD1/5 epithelial-specific deletion has detrimental effects on stromal cell decidualization and pregnancy development.

FcεRIγ-negative NK cells persist in vivo and enhance efficacy of therapeutic monoclonal antibodies in multiple myeloma.

Monoclonal antibodies (mAbs) are a central component of therapy for hematologic malignancies. Widely used mAb agents in multiple myeloma (MM) include daratumumab and elotuzumab. However, not all patients respond to these agents, and resistance is a significant clinical issue. A recently discovered subset of human natural killer (NK) cells lacking expression of FcεRIγ (g-NK cells) was found to have a multifold increase in antibody-dependent effector functions after CD16 crosslinking. In this study, we tested the capacity of g-NK cells to enhance the efficacy of therapeutic mAbs against MM. In vitro, we found that g-NK cells have strikingly superior anti-myeloma cytotoxicity compared with conventional NK (cNK) cells when combined with daratumumab or elotuzumab (∼sixfold; P < .001). In addition, g-NK cells naturally expressed minimal surface CD38 and SLAMF7, which reduced the incidence of therapeutic fratricide. In tumor-naïve murine models, the persistence of g-NK cells in blood and spleen was >10 times higher than that of cNK cells over 31 days (P < .001). In vivo efficacy studies showed that the combination of daratumumab and g-NK cells led to a >99.9% tumor reduction (by flow cytometry analysis) compared with the combination of daratumumab and cNK cells (P < .001). Moreover, treatment with daratumumab and g-NK cells led to complete elimination of myeloma burden in 5 of 7 mice. Collectively, these results underscore the unique ability of g-NK cells to potentiate the activity of therapeutic mAbs and overcome limitations of current off-the-shelf NK cell therapies without the need for cellular irradiation or genetic engineering.

Endometrial receptivity and implantation require uterine BMP signaling through an ACVR2A-SMAD1/SMAD5 axis.

During early pregnancy in the mouse, nidatory estrogen (E2) stimulates endometrial receptivity by activating a network of signaling pathways that is not yet fully characterized. Here, we report that bone morphogenetic proteins (BMPs) control endometrial receptivity via a conserved activin receptor type 2 A (ACVR2A) and SMAD1/5 signaling pathway. Mice were generated to contain single or double conditional deletion of SMAD1/5 and ACVR2A/ACVR2B receptors using progesterone receptor (PR)-cre. Female mice with SMAD1/5 deletion display endometrial defects that result in the development of cystic endometrial glands, a hyperproliferative endometrial epithelium during the window of implantation, and impaired apicobasal transformation that prevents embryo implantation and leads to infertility. Analysis of Acvr2a-PRcre and Acvr2b-PRcre pregnant mice determined that BMP signaling occurs via ACVR2A and that ACVR2B is dispensable during embryo implantation. Therefore, BMPs signal through a conserved endometrial ACVR2A/SMAD1/5 pathway that promotes endometrial receptivity during embryo implantation.

Interception Targets of Angelica Gigas Nakai Root Extract versus Pyranocoumarins in Prostate Early Lesions and Neuroendocrine Carcinomas in TRAMP Mice.

We reported efficacy of Angelica gigas Nakai (AGN) root ethanol extract and equimolar decursin (D)/decursinol angelate (DA) through daily gavage starting at 8 weeks of age (WOA) to male transgenic adenocarcinoma of mouse prostate (TRAMP) mice such that these modalities suppressed precancerous epithelial lesions in their dorsolateral prostate (DLP) to similar extent, but AGN extract was better than the D/DA mixture at promoting the survival of mice bearing prostate neuroendocrine carcinomas to 28 WOA. Here, we compared by microarray hybridization the mRNA levels in pooled DLP tissues and individual neuroendocrine carcinomas to characterize potential molecular targets of AGN extract and D/DA. Clustering and principal component analyses supported distinct gene expression profiles of TRAMP DLP versus neuroendocrine carcinomas. Pathway Enrichment, Gene Ontology, and Ingenuity Pathway Analyses of differential genes indicated that AGN and D/DA affected chiefly processes of lipid and mitochondrial energy metabolism and oxidation-reduction in TRAMP DLP, while AGN affected neuronal signaling, immune systems and cell cycling in neuroendocrine carcinomas. Protein-Protein Interaction Network analysis predicted and reverse transcription-PCR verified multiple hub genes common in the DLP of AGN- and D/DA-treated TRAMP mice at 28 WOA and select hub genes attributable to the non-D/DA AGN components. The vast majority of hub genes in the AGN-treated neuroendocrine carcinomas differed from those in TRAMP DLP. In summary, the transcriptomic approach illuminated vastly different signaling pathways and networks, cellular processes, and hub genes of two TRAMP prostate malignancy lineages and their associations with the interception efficacy of AGN and D/DA. PREVENTION RELEVANCE: This study explores potential molecular targets associated with in vivo activity of AGN root alcoholic extract and its major pyranocoumarins to intercept precancerous epithelial lesions and early malignancies of the prostate. Without an ethically-acceptable, clearly defined cancer initiation risk reduction strategy available for the prostate, using natural products like AGN to delay formation of malignant tumors could be a plausible approach for prostate cancer prevention.

Optimizing live-animal bioluminescence imaging prediction of tumor burden in human prostate cancer xenograft models in SCID-NSG mice.

BACKGROUND: Noninvasive live-animal longitudinal monitoring of xenograft tumor growth and metastasis by bioluminescent imaging (BLI) has been widely reported in cancer biology and preclinical therapy literature, mainly in athymic nude mice. Our own experience at calibrating BLI readout with tumor weight/volume in human prostate cancer xenograft models in haired, SCID-NSG mice through intraprostatic (orthotopic) and subcutaneous (SC) inoculations revealed either nonexistent or poor correlation (coefficient of determination, R 2 = ~0.01-0.3). The present work examined several technical and biological factors to improve BLI utility. METHODS: After ruling out promoter-luciferase (luc) specificity and luc gene loss in the cell inoculum with LNCaP-AR-luc cells expressing an androgen receptor (AR) and tagged with AR-responsive probasin promoter-luc gene, we evaluated different routes of d-luciferin administration, imaging time during the day, charge-coupled device camera image acquisition settings, and hair removal methods to improve the imaging protocol. For most imaging sessions, BLI was carried out within the same day of tumor volume measurement. After necropsy, histological and immunohistochemical (IHC) analyses were performed on the tumors to evaluate necrosis and expression of luciferase and AR, respectively. RESULTS: Injection of d-luciferin by SC route, robust image-capture setting (30 000 counts and autoexposure), imaging in the morning and thorough hair removal resulted in a substantial improvement of R2 to ~0.6. Histological analyses confirmed the lack of BLI signal in necrotic tumor masses consistent with luciferase-mediated light emission only in oxygenated adenosine triphosphate-producing viable cells. IHC staining detected heterogeneous expression of luciferase tracking generally with AR expression in nonnecrotic tumor tissues. CONCLUSIONS: Our body of work highlighted a framework to validate imaging protocols to ensure the acquisition of interpretable BLI data as an indicator of xenograft tumor burden. The vast tissue heterogeneity in prostate tumor xenografts and variable luciferase expression constrained this technology from achieving a high correlation.

Role of P53-Senescence Induction in Suppression of LNCaP Prostate Cancer Growth by Cardiotonic Compound Bufalin.

Bufalin is a major cardiotonic compound in the traditional Chinese medicine, Chansu, prepared from toad skin secretions. Cell culture studies have suggested an anticancer potential involving multiple cellular processes, including differentiation, apoptosis, senescence, and angiogenesis. In prostate cancer cell models, P53-dependent and independent caspase-mediated apoptosis and androgen receptor (AR) antagonism have been described for bufalin at micromolar concentrations. Because a human pharmacokinetic study indicated that single nanomolar bufalin was safely achievable in the peripheral circulation, we evaluated its cellular activity within range with the AR-positive and P53 wild-type human LNCaP prostate cancer cells in vitro Our data show that bufalin induced caspase-mediated apoptosis at 20 nmol/L or higher concentration with concomitant suppression of AR protein and its best-known target, PSA and steroid receptor coactivator 1 and 3 (SRC-1, SRC-3). Bufalin exposure induced protein abundance of P53 (not mRNA) and P21CIP1 (CDKN1A), G2 arrest, and increased senescence-like phenotype (SA-galactosidase). Small RNAi knocking down of P53 attenuated bufalin-induced senescence, whereas knocking down of P21CIP1 exacerbated bufalin-induced caspase-mediated apoptosis. In vivo, daily intraperitoneal injection of bufalin (1.5 mg/kg body weight) for 9 weeks delayed LNCaP subcutaneous xenograft tumor growth in NSG SCID mice with a 67% decrease of final weight without affecting body weight. Tumors from bufalin-treated mice exhibited increased phospho-P53 and SA-galactosidase without detectable caspase-mediated apoptosis or suppression of AR and PSA. Our data suggest potential applications of bufalin in therapy of prostate cancer in patients or chemo-interception of prostate precancerous lesions, engaging a selective activation of P53 senescence. Mol Cancer Ther; 17(11); 2341-52. ©2018 AACR.

Prostate Cancer Xenograft Inhibitory Activity and Pharmacokinetics of Decursinol, a Metabolite of Angelica gigas Pyranocoumarins, in Mouse Models.

We have previously shown that the ethanol extract of dried Angelica gigas Nakai (AGN) root exerts anticancer activity against androgen receptor (AR)-negative human DU145 and PC-3 prostate cancer xenografts and primary carcinogenesis in the transgenic adenocarcinoma of mouse prostate (TRAMP) model. The major pyranocoumarin isomers decursin (D) and decursinol angelate (DA), when provided at equi-molar intake to that provided by AGN extract, accounted for the inhibitory efficacy against precancerous epithelial lesions in TRAMP mice. Since we and others have shown in rodents and humans that D and DA rapidly and extensively convert to decursinol, here we tested whether decursinol might be an in vivo active compound for suppressing xenograft growth of human prostate cancer cells expressing AR. In SCID-NSG mice carrying subcutaneously inoculated human LNCaP/AR-Luc cells overexpressing the wild type AR, we compared the efficacy of 4.5[Formula: see text]mg decursinol per mouse with equi-molar dose of 6[Formula: see text]mg D/DA per mouse. The result showed that decursinol decreased xenograft tumor growth by 75% and the lung metastasis, whereas D/DA exerted a much less effect. Measurement of plasma decursinol concentration, at 3[Formula: see text]h after the last dose of respective dosing regimen, showed higher circulating level in the decursinol-treated NSG mice than in the D/DA-treated mice. In a subsequent single-dose pharmacokinetic experiment, decursinol dosing led to 3.7-fold area under curve (AUC) of plasma decursinol over that achieved by equi-molar D/DA dosing. PK advantage notwithstanding, decursinol represents an active compound to exert in vivo prostate cancer growth and metastasis inhibitory activity in the preclinical model.

Blood-brain barrier disruption in diabetic mice is linked to Nrf2 signaling deficits: Role of ABCB10?

Blood-brain barrier (BBB) damage is a critical neurovascular complication of diabetes mellitus that adversely affects the CNS health and function. Previously, we showed the protective role of NF-E2 related factor-2 (Nrf2), a redox sensitive transcription factor, in regulation of BBB integrity. Given the pathogenic role of mitochondrial oxidative stress in diabetes-related microvascular complications, we focused on assessing: 1) the impact of diabetes on brain Nrf2 in correlation with BBB permeability and 2) Nrf2-dependent regulation of the mitochondrial transporter ABCB10, an essential player in mitochondrial function and redox balance at BBB endothelium. Using live animal fluorescence imaging, we demonstrated a strong increase in BBB permeability to 70kDa dextran in db/db diabetic mice that correlated with significant down-regulation of brain Nrf2 protein. Further, Nrf2 gene silencing in human BBB endothelial cells markedly suppressed ABCB10 protein, while Nrf2 activation by sulforaphane up-regulated ABCB10 expression. Interestingly, ABCB10 knockdown resulted in a strong-induction of Nrf2 driven anti-oxidant responses as evidenced by increased expression of Nrf2 and its downstream targets. Nrf2 or ABCB10 silencing elevated endothelial-monocyte adhesion suggesting an activated inflammatory cascade. Thus, our results demonstrate a novel mechanism of ABCB10 regulation driven by Nrf2. In summary, Nrf2 dysregulation and ABCB10 suppression could likely mediate endothelial oxidative/inflammatory stress and BBB disruption in diabetic subjects.

Pyranocoumarin Tissue Distribution, Plasma Metabolome and Prostate Transcriptome Impacts of Sub-Chronic Exposure to Korean Angelica Supplement in Mice.

Herbal products containing Korean Angelica gigas Nakai (AGN) root extract are marketed as dietary supplements for memory enhancement, pain killing, and female menopausal symptom relief. We have shown the anticancer activities of AGN supplements in mouse models. To facilitate human anticancer translational research, we characterized the tissue distribution of AGN marker pyranocoumarin compounds decursin (D) and decursinol angelate (DA) ([Formula: see text]% in AGN) and their metabolite decursinol (DOH), assessed the safety of sub-chronic AGN dietary exposure in mice, and explored its impact on plasma aqueous metabolites and the prostate transcriptome. The data show that after a gavage dose, plasma contained readily detectable DOH, but little D and DA, mirroring patterns in the liver. Extra-hepatic tissues retained greater levels of DA and D than the liver did. For sub-chronic exposures, male mice were provided ad libitum AIN93M-pellet diets with 0.5 and 1% AGN for six weeks. No adverse effects were observed on the plasma biochemistry markers of liver and kidney integrity in spite of their enlargement. Histopathological examinations of the liver, kidney and other visceral organs did not reveal tissue abnormalities. Metabolomic assessment of plasma from mice fed the 1%-AGN diet suggested metabolic shifts of key amino acids especially in the methionine-cysteine cycle, purine cycle, and glycolysis-citrate cycle. Prostate transcriptomic profiling identified gene signature changes in the metabolisms of drugs, lipids and cellular energetics, neuro-muscular features, immunity and inflammation, and tumor suppressor/oncogene balance. The safety profile was corroborated with a daily [Formula: see text] injection of AGN extract (100-300[Formula: see text]mg/kg) for four weeks, which resulted in much greater systemic pyranocoumarin exposure than the dietary route did.