Microbiological profile, preclinical pharmacokinetics and efficacy of CRS0393, a novel antimycobacterial agent targeting MmpL3.

The benzothiazole amide CRS0393 demonstrated excellent in vitro activity against nontuberculous mycobacteria (NTM), including M. abscessus isolates from cystic fibrosis (CF) patients, with minimum inhibitory concentrations (MICs) of ≤0.03-0.5 µg/mL. The essential transport protein MmpL3 was confirmed as the target via analysis of spontaneous resistant mutants and further biological profiling. In mouse pharmacokinetic studies, intratracheal instillation of a single dose of CRS0393 resulted in high concentrations of drug in epithelial lining fluid (ELF) and lung tissue, which remained above the M. abscessus MIC for at least 9 hours post-dose. This exposure resulted in a penetration ratio of 261 for ELF and 54 for lung tissue relative to plasma. CRS0393 showed good oral bioavailability, particularly when formulated in kolliphor oil, with a lung-to-plasma penetration ratio ranging from 0.5 to 4. CRS0393 demonstrated concentration-dependent reduction of intracellular M. abscessus in a THP-1 macrophage infection model. CRS0393 was well tolerated following intranasal administration (8 mg/kg) or oral dosing (25 mg/kg) once daily for 28 days in dexamethasone-treated C3HeB/FeJ mice. Efficacy against M. abscessus strain 103 was achieved via the intranasal route, while oral dosing will need further optimization. CRS0393 holds promise for development as a novel agent with broad antimycobacterial activity.

ABO blood type, smoking status, other risk factors and prognosis of pancreatic ductal adenocarcinoma.

The aim of this observational study was to test whether ABO blood type was a prognostic factor for pancreatic ductal adenocarcinoma (PDAC) patients and whether other risk factors could influence pancreatic cancer patients' survival. This study included 610 patients who were diagnosed as pancreatic cancer and had undergone radical surgery. Patients' characteristics included age, gender, tumor stage, tumor grade, adenosquamous carcinoma (ASC) status, preoperative serum carbohydrate antigen 19-9 (CA19-9) levels, preoperative serum carcinoembryonic antigen (CEA) levels, ABO blood type, smoking status, and drinking status were analyzed in this study. Cox proportional hazards regression model and Kaplan-Meier method were used to evaluate the role of prognostic factors. For pancreatic cancer patients undergoing radical surgery, the overall survival was worse for ASC patients than PDAC patients (Log-rank = 11.315, P < .001). Compared with ASC patients (Log-rank < 0.001, P = .996), PDAC patients can benefit from chemotherapy (Log-rank = 17.665, P < .001). For PDAC patients, O blood type had better overall survival than non-O blood type (Log-rank = 4.153, P = .042). Moreover, the group with higher serum levels of CA19-9 had poor prognosis compared to another group with low serum CA19-9 (Log-rank = 4.122, P = .042). Higher CEA levels indicated poor prognosis (Log-rank = 13.618, P < .001). In conclusion, ASC status was associated with overall survival of pancreatic cancer patients and cannot benefit from postoperative chemotherapy. Non-O blood type was a prognostic factor for PDAC patients.

Regulation of human trophoblast syncytialization by histone demethylase LSD1.

A successful pregnancy is critically dependent upon proper placental development and function. During human placentation, villous cytotrophoblast (CTB) progenitors differentiate to form syncytiotrophoblasts (SynTBs), which provide the exchange surface between the mother and fetus and secrete hormones to ensure proper progression of pregnancy. However, epigenetic mechanisms that regulate SynTB differentiation from CTB progenitors are incompletely understood. Here, we show that lysine-specific demethylase 1 (LSD1; also known as KDM1A), a histone demethylase, is essential to this process. LSD1 is expressed both in CTB progenitors and differentiated SynTBs in first-trimester placental villi; accordingly, expression in SynTBs is maintained throughout gestation. Impairment of LSD1 function in trophoblast progenitors inhibits induction of endogenous retrovirally encoded genes SYNCYTIN1/endogenous retrovirus group W member 1, envelope (ERVW1) and SYNCYTIN2/endogenous retrovirus group FRD member 1, envelope (ERVFRD1), encoding fusogenic proteins critical to human trophoblast syncytialization. Loss of LSD1 also impairs induction of chorionic gonadotropin α (CGA) and chorionic gonadotropin ß (CGB) genes, which encode α and ß subunits of human chorionic gonadotrophin (hCG), a hormone essential to modulate maternal physiology during pregnancy. Mechanistic analyses at the endogenous ERVW1, CGA, and CGB loci revealed a regulatory axis in which LSD1 induces demethylation of repressive histone H3 lysine 9 dimethylation (H3K9Me2) and interacts with transcription factor GATA2 to promote RNA polymerase II (RNA-POL-II) recruitment and activate gene transcription. Our study reveals a novel LSD1-GATA2 axis, which regulates human trophoblast syncytialization.

Expression, Regulation, and Function of the Calmodulin Accessory Protein PCP4/PEP-19 in Myometrium.

OBJECTIVE: Calmodulin (CaM) plays a key role in the orchestration of Ca2+ signaling events, and its regulation is considered an important component of cellular homeostasis. The control of uterine smooth muscle function is largely dependent on the regulation of Ca2+ and CaM signaling. The objective of this study was to investigate the expression, function, and regulation of CaM regulatory proteins in myometrium during pregnancy. STUDY DESIGN: Myometrium was obtained from nonpregnant women and 4 groups of pregnant women at the time their primary cesarean delivery: (i) preterm not in labor, (ii) preterm in labor with clinical and/or histological diagnosis of chorioamnionitis, (3) term not in labor; and (4) term in labor. The effect of perinatal inflammation on pcp4/pep-19 expression was evaluated in a mouse model of Ureaplasma parvum-induced chorioamnionitis. Human myometrial cells stably expressing wild-type and mutant forms of PCP4/PEP-19 were used in the evaluation of agonist-induced intracellular Ca2+ mobilization. RESULTS: Compared to other CaM regulatory proteins, PCP4/PEP-19 transcripts were more abundant in human myometrium. The expression of PCP4/PEP-19 was lowest in myometrium of women with preterm pregnancy and chorioamnionitis. In the mouse uterus, pcp4/pep-19 expression was lower in late compared to mid-gestation and decreased in mice injected intra-amniotic with Ureaplasma parvum. In myometrial smooth muscle cells, tumor necrosis factor alpha and progesterone decreased and PCP4/PEP-19 promoter activity increased. Finally, the overexpression of PCP4/PEP-19 reduced agonist-induced intracellular Ca2+ levels in myometrial cells. CONCLUSION: The decreased expression of PCP4/PEP-19 in myometrium contributes to a loss of quiescence in response to infection-induced inflammation at preterm pregnancy.

High levels of serum glypican-1 indicate poor prognosis in pancreatic ductal adenocarcinoma.

Carbohydrate antigen 19-9 (CA19-9) fails to demonstrate the predictive value for early detection pancreatic ductal adenocarcinoma (PDAC). Glypican-1 (GPC1+) exosomes may serve as a noninvasive diagnostic tool to detect early stages of PDAC. Therefore, it is necessary to explore the serum GPC1 levels and determine whether serum GPC1 serves as a novel biomarker for PDAC patients. Blood samples were collected from 156 patients with PDAC, 199 non-cancer controls, and 240 patients with other cancers. Serological levels of GPC1 were examined by enzyme-linked immunosorbent assay (ELISA). Finally, a 5-year follow-up was monitored to evaluate the correlation between serum GPC1 levels and overall survival in 156 patients with PDAC. The results suggested that levels of serum GPC1 and CA19-9 were higher in PDAC patients than that of controls (P < 0.05). Serum GPC1 levels in PDAC were different from those in gallbladder carcinoma (P < 0.001), colorectal carcinoma (P < 0.001), gastric carcinoma (P < 0.001), and prostate cancer (P < 0.001), but not hepatocellular carcinoma (P = 0.395) and cholangiocarcinoma (P = 0.724). Receiver operating characteristic curve (ROC) analysis showed that serum CA19-9 was significantly better than serum GPC1 in distinguishing PDAC patients from the controls (AUC, 95% CI: 0.908, 0.868-0.947 vs 0.795, 0.749-0.841, respectively). The serum GPC1 cannot be used as a serum diagnostic biomarker for PDAC patients. The level of serum GPC1 decreased 2 days after surgery (P = 0.001), which were not different from serum GPC1 levels in healthy control (P = 0.381). The overall survival rate was shorter in patients with high levels of serum GPC1 compared to those with low levels of serum GPC1 (log-rank = 5.16, P = 0.023). Taken together, the results indicate that high levels of serum GPC1 predict poor prognosis in PDAC patients. Serum GPC1 may be a prognosis factor for PDAC patients.

p53-dependent CD51 expression contributes to characteristics of cancer stem cells in prostate cancer.

Castration-resistant prostate cancer (CRPC), which is considered to contain cancer stem cells (CSCs), leads to a high relapse rate in patients with prostate cancer (PCa). However, the markers of prostate CSCs are controversial. Here we demonstrate that CD51, in part, correlates with the poor prognosis of PCa patients. Further, we find that CD51 is a functional molecule that is able to promote the malignancy of PCa through enhancing tumor initiation, metastatic potential, and chemoresistance. Moreover, we find that elevated CD51 expression in PCa specimens correlates with p53 loss of function. Mechanistically, we demonstrate that p53 acts via Sp1/3 to repress CD51 transcription, and CD51 is required for PCa stemness and metastasis properties, and is downregulated by p53. Taken together, these results indicate that CD51 is a novel functional marker for PCa, which may provide a therapeutic target for the efficiently restricting PCa progression.

MicroRNA-205 promotes cell invasion by repressing TCF21 in human ovarian cancer.

BACKGROUND: Ovarian cancer is the leading lethal, gynecological malignancy in the United States. No doubt, the continued morbidity and mortality of ovarian cancer reflects a poor understanding of invasive mechanisms. Recent studies reveal that ovarian cancers express aberrant microRNAs (miRNAs or miRs), some of which have oncogenic or tumor suppressor properties. Several studies suggested that miR-205 is involved in tumorigenesis. Presently, we investigate the molecular mechanisms and target of miR-205 in ovarian cancer. METHODS: Quantitative real-time polymerase chain reaction and western blot were performed to assess miR-205 and transcription factor 21 (TCF21) expression in ovarian cancer and normal ovary samples. The effect of miR-205 on TCF21 was determined by luciferase reporter assay and western blot. The effect of miR-205 and TCF21 on cell invasion was quantitated using transwell invasion assay. RESULT: miR-205 expression was increased in ovarian cancer and it promoted the invasive behavior of ovarian cancer cell lines (OVCAR-5, OVCAR-8 and SKOV-3). miR-205 directly targeted TCF21, which was significantly decreased in ovarian cancer tissue. miR-205 inhibited TCF21 expression and as a consequence blunted the inhibitory effect of TCF21 on cell invasion. Matrix Metalloproteinases (MMPs) play an important role in cancer invasion and metastasis. TCF21 inhibited MMP-2 and MMP-10 and decreased ovarian cancer cell invasion. Co-transfection of TCF21 expression plasmid with miR-205 mimic diminished the inhibitory effect of TCF21 on MMP-2 and MMP-10 in ovarian cancer cells. CONCLUSION: miR-205 appears to have an important role in the spread of ovarian cancer by targeting TCF21. These findings offer a new mechanism of ovarian cancer tumorigenesis, which could be useful for the development of new therapeutic approaches to ovarian cancer treatment.

Autoantibodies against glucose-regulated protein 78 as serological biomarkers in metastatic and recurrent hepatocellular carcinoma.

PURPOSE: To identify Heptocellular carcinoma (HCC) associated antigens by proteomics, and validate whether autoantibodies against tumor-associated antigens (TAAs) could be used for diagnosis and conditional monitoring. RESULTS: The 78 kDa glucose regulated protein (GRP78) was selected as a candidate TAA. The titers of autoantibodies against 78 kDa glucose regulated protein (GRP78) from patients with HCC, liver cirrhosis (LC), and chronic hepatitis (CH) were significantly higher than that from normal controls (P<0.05, P<0.001, and P<0.01, respectively). The expression of autoantibodies against GRP78 was associated with clinical stage (P<0.01), portal vein invasion (P<0.05), and metastasis (P<0.05). The expression of anti-GRP78 antibodies was significantly higher 1 month after surgery in recurrent patients who had accepted hepatic resection 1 month after surgery compared to patients who had surgery before surgery or within 1 week after surgery (P<0.01 and P<0.001). Immunohistochemistry (IHC) showed higher expression of GRP78 in HCC compared to the non-HCC liver tissues (P <0.05). MATERIALS AND METHODS: HCC serum with high titer of autoantibodies against TAAs were screened and used for a proteome-based approach to identify HCC associated antigens. Indirect enzyme-linked immunoassay (ELISA) was used to detect the corresponding autoantibodies against TAAs. CONCLUSION: GRP78 is an autoantigen that could stimulate autoimmune responses and serve as a potential marker for recurrent and metastatic progression in HCC.

Effect of prostaglandin E2 on multidrug resistance transporters in human placental cells.

Prostaglandin (PG) E2, a major product of cyclooxygenase (COX)-2, acts as an immunomodulator at the maternal-fetal interface during pregnancy. It exerts biologic function through interaction with E-prostanoid (EP) receptors localized to the placenta. The activation of the COX-2/PGE2/EP signal pathway can alter the expression of the ATP-binding cassette (ABC) transporters, multidrug resistance protein 1 [P-glycoprotein (Pgp); gene: ABCB1], and breast cancer resistance protein (BCRP; gene: ABCG2), which function to extrude drugs and xenobiotics from cells. In the placenta, PGE2-mediated changes in ABC transporter expression could impact fetal drug exposure. Furthermore, understanding the signaling cascades involved could lead to strategies for the control of Pgp and BCRP expression levels. We sought to determine the impact of PGE2 signaling mechanisms on Pgp and BCRP in human placental cells. The treatment of placental cells with PGE2 up-regulated BCRP expression and resulted in decreased cellular accumulation of the fluorescent substrate Hoechst 33342. Inhibiting the EP1 and EP3 receptors with specific antagonists attenuated the increase in BCRP. EP receptor signaling results in activation of transcription factors, which can affect BCRP expression. Although PGE2 decreased nuclear factor κ-light chain-enhancer of activated B activation and increased activator protein 1, chemical inhibition of these inflammatory transcription factors did not blunt BCRP up-regulation by PGE2. Though PGE2 decreased Pgp mRNA, Pgp expression and function were not significantly altered. Overall, these findings suggest a possible role for PGE2 in the up-regulation of placental BCRP expression via EP1 and EP3 receptor signaling cascades.

Induction of xenobiotic receptors, transporters, and drug metabolizing enzymes by oxycodone.

Perturbations of the expression of transporters and drug-metabolizing enzymes (DMEs) by opioids can be the locus of deleterious drug-drug interactions (DDIs). Many transporters and DMEs are regulated by xenobiotic receptors [XRs; e.g., pregnane X receptor (PXR), constitutive androstane receptor (CAR), and Aryl hydrocarbon receptor (AhR)]; however, there is a paucity of information regarding the influence of opioids on XRs. The objective of this study was to determine the influence of oxycodone administration (15 mg/kg intraperitoneally twice daily for 8 days) on liver expression of XRs, transporters, and DMEs in rats. Microarray, quantitative real-time polymerase chain reaction and immunoblotting analyses were used to identify significantly regulated genes. Three XRs (e.g., PXR, CAR, and AhR), 27 transporters (e.g., ABCB1 and SLC22A8), and 19 DMEs (e.g., CYP2B2 and CYP3A1) were regulated (P < 0.05) with fold changes ranging from -46.3 to 17.1. Using MetaCore (computational platform), we identified a unique gene-network of transporters and DMEs assembled around PXR, CAR, and AhR. Therefore, a series of transactivation/translocation assays were conducted to determine whether the observed changes of transporters/DMEs are mediated by direct activation of PXR, CAR, or AhR by oxycodone or its major metabolites (noroxycodone and oxymorphone). Neither oxycodone nor its metabolites activated PXR, CAR, or AhR. Taken together, these findings identify a signature hepatic gene-network associated with repeated oxycodone administration in rats and demonstrate that oxycodone alters the expression of many transporters and DMEs (without direct activation of PXR, CAR, and AhR), which could lead to undesirable DDIs after coadministration of substrates of these transporters/DMEs with oxycodone.

Characterization of the transport, metabolism, and pharmacokinetics of the dopamine D3 receptor-selective fluorenyl- and 2-pyridylphenyl amides developed for treatment of psychostimulant abuse.

The recent discovery of novel high-affinity and selective dopamine D3 receptor (DA D3R) antagonists and partial agonists has provided tools with which to further elucidate the role DA D3R plays in substance abuse. The present study was conducted to evaluate the transport, metabolism, pharmacokinetics, and brain uptake of the DA D3R-selective fluorenyl amides, NGB 2904 [N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butyl)-9H-fluorene-2-carboxamide] fumarate) and JJC 4-077 [N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)-3-hydroxybutyl)-9H-fluorene-2-carboxamide hydrochloride], and the 2-pyridylphenyl amides, CJB 090 [N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butyl)-4-(pyridine-2-yl)benzamide hydrochloride] and PG 01037 [N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)-trans-but-2-enyl)-4-(pyridine-2-yl)benzamide hydrochloride], all of which have been studied in animal models of psychostimulant abuse. Additional screening with a panel of human and rat Supersomes was performed for NGB 2904 and PG 01037. Drug-stimulated ATPase activation assays and bidirectional transport and efflux assays were used to test for substrate specificity of NGB 2904 and PG 01037 for human and rat efflux transporters. All compounds exhibited moderate elimination half-lives, ranging from 1.49 to 3.27 h, and large volumes of distribution (5.95-14.19 l/kg). The brain-to-plasma ratios ranged from 2.93 to 11.81 and were higher than those previously reported for cocaine. Brain exposure levels of NGB 2904 and PG 01037 were significantly reduced after intraperitoneal administration compared with intravenous administration. The metabolism of these compounds was mediated primarily by CYP3A subfamilies. PG 01037 was a P-glycoprotein-transported substrate. Higher doses of these compounds are often required for in vivo action, suggesting decreased bioavailability via extravascular administration that may be attributed to high drug efflux and hepatic metabolism. These studies provide important preclinical information for optimization of next-generation D3R selective agents for the treatment of drug addiction.

Pregnancy and estradiol modulate myometrial G-protein pathways in the guinea pig.

OBJECTIVE: Coupled to hundreds of receptors, G-proteins modulate signal transduction pathways and are important hormonal targets. The first objective was to determine the effect of pregnancy and estradiol on myometrial guanosine triphosphatase activity. The second objective was to begin dissecting the molecular mechanism(s) underlying alterations in guanosine triphosphatase activity. STUDY DESIGN: Myometrial tissue was obtained from pregnant, nonpregnant, and ovariectomized untreated and estradiol-treated guinea pigs. Myometrial membranes were prepared by homogenization and differential centrifugation. Basal high-affinity specific guanosine triphosphatase activity was quantitated by enzymatic assay and expressed in rhomol 32Pi per milligram protein per minute. Guanosine triphosphatase activity was stimulated using oxytocin, isoproterenol, and prostaglandin F2alpha. Specific G-protein subunits were quantitated using Western blots. G-protein associated gene expression was semiquantitated using HGU133A gene array chips from Affymetrix. RESULTS: Basal myometrial guanosine triphosphatase activity was increased in pregnant compared with nonpregnant animals. Estradiol increased basal myometrial guanosine triphosphatase activity, compared with untreated controls. The effect of estradiol on stimulated activity was agonist dependent. Both Galphas and Galphai isoform 1 protein levels were increased in myometrium from late pregnant compared with nonpregnant animals. By late gestation, the messenger ribonucleic acid levels of those genes were unaltered, compared with the nonpregnant animal. In general, the impact of pregnancy on G-protein family member gene messenger ribonucleic acid expression was modest. Only the small guanosine triphosphatase Rap1b demonstrated altered expression more than 2-fold during either myometrial quiescence (midpregnancy) or activation (term pregnancy) (up 3-fold during quiescence). Genomic network analyses revealed that the expression of another small guanosine triphosphatase, Rab7, was exclusively up-regulated (80%) during quiescence. During late pregnancy, network analysis showed that only G-protein beta was exclusively altered (up-regulated). Estradiol mimicked the pregnancy effect on both transcription and translation of G-protein family members for some but not all potentially relevant genes. CONCLUSION: The increase in functional myometrial guanosine triphosphatase activity during pregnancy may reflect increased synthesis of 1 or more small guanosine triphosphatase.