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Polyglutamine (polyQ) tract polymorphism within the human androgen receptor (AR) shows population heterogeneity. African American men possess short polyQ tracts significantly more frequently than Caucasian American men. The length of polyQ tracts is inversely correlated with the risk of prostate cancer, age of onset, and aggressiveness at diagnosis. Aberrant activation of Wnt signaling also reveals frequently in advanced prostate cancer, and an enrichment of androgen and Wnt signaling activation has been observed in African American patients. Here, we assessed aberrant expression of AR bearing different polyQ tracts and stabilized β-catenin in prostate tumorigenesis using newly generated mouse models. We observed an early onset oncogenic transformation, accelerated tumor cell growth, and aggressive tumor phenotypes in the compound mice bearing short polyQ tract AR and stabilized β-catenin. RNA sequencing analysis showed a robust enrichment of Myc-regulated downstream genes in tumor samples bearing short polyQ AR versus those with longer polyQ tract AR. Upstream regulator analysis further identified Myc as the top candidate of transcriptional regulators in tumor cells from the above mouse samples with short polyQ tract AR and β-catenin. Chromatin immunoprecipitation analyses revealed increased recruitment of β-catenin and AR on the c-Myc gene regulatory locus in the tumor tissues expressing stabilized β-catenin and shorter polyQ tract AR. These data demonstrate a promotional role of aberrant activation of Wnt/β-catenin in combination with short polyQ AR expression in prostate tumorigenesis and suggest a potential mechanism underlying aggressive prostatic tumor development, which has been frequently observed in African American patients.

Microchromosome maintenance protein 7 (MCM7), a DNA replication permitting factor, plays an essential role in initiating DNA replication. MCM7 is reported to be involved in tumor formation and progression, whereas the expression profile and molecular function of MCM7 in colorectal cancer (CRC) remain unknown. In this study, we aimed to evaluate the clinical significance and biological function of MCM7 in CRC and investigated whether MCM7 can be used for a differential diagnosis in CRC and whether it may serve as a more sensitive proliferation marker for CRC evaluation. Moreover, immunohistochemical analysis of MCM7 was performed in a total of 89 specimens, and high MCM7 expression levels were associated with worse overall survival (OS) in CRC patients. Furthermore, the cell functional test suggested that lentivirus-mediated silencing of MCM7 with shRNA in CRC cells significantly inhibited cellular proliferation and promoted apoptosis in vitro and inhibited tumor growth in vivo. Additionally, mechanistic studies further demonstrated that P21-activated protein kinase 2 (PAK2) was regulated by MCM7 via microarray analysis and cell functional recovery tests, and miR-107 played a role in regulating expression MCM7 via miRNA microarray analysis and 3’UTR reporter assays. Taken together, our results suggest that the miR-107/MCM7/PAK2 pathway may participate in cancer progression and that MCM7 may serve as a prognostic biomarker in CRC

Androgens/androgen receptor (AR)-mediated signaling pathways are essential for prostate development, morphogenesis and regeneration. Specifically, stromal AR signaling has been shown to be essential for prostatic initiation. However, the molecular mechanisms underlying AR-initiated mesenchymal-epithelial interactions in prostate development remain unclear. Here, using a newly generated mouse model, we have directly addressed the fate and role of genetically marked AR-expressing cells during embryonic prostate development. Androgen signaling-initiated signaling pathways were identified in mesenchymal niche populations at single-cell transcriptomic resolution. The dynamic cell-signaling networks regulated by stromal AR were additionally characterized in relation to prostatic epithelial bud formation. Pseudotime analyses further revealed the differentiation trajectory and fate of AR-expressing cells in both prostatic mesenchymal and epithelial cell populations. Specifically, the cellular properties of Zeb1-expressing progenitors were assessed. Selective deletion of AR signaling in a subpopulation of mesenchymal rather than epithelial cells dysregulated the expression of the master regulators and significantly impaired prostatic bud formation. These data provide novel, high-resolution evidence demonstrating the important role of mesenchymal androgen signaling in the cellular niche controlling prostate early development by initiating dynamic mesenchyme-epithelia cell interactions.

Prostate embryonic development, pubertal and adult growth, maintenance, and regeneration are regulated through androgen signaling-mediated mesenchymal-epithelial interactions. Specifically, the essential role of mesenchymal androgen signaling in the development of prostate epithelium has been observed for over 30 years. However, the identity of the mesenchymal cells responsible for this paracrine regulation and related mechanisms are still unknown. Here, we provide the first demonstration of an indispensable role of the androgen receptor (AR) in sonic hedgehog (SHH) responsive Gli1-expressing cells, in regulating prostate development, growth, and regeneration. Selective deletion of AR expression in Gli1-expressing cells during embryogenesis disrupts prostatic budding and impairs prostate development and formation. Tissue recombination assays showed that urogenital mesenchyme (UGM) containing AR-deficient mesenchymal Gli1-expressing cells combined with wildtype urogenital epithelium (UGE) failed to develop normal prostate tissue in the presence of androgens, revealing the decisive role of AR in mesenchymal SHH responsive cells in prostate development. Prepubescent deletion of AR expression in Gli1-expressing cells resulted in severe impairment of androgen-induced prostate growth and regeneration. RNA-sequencing analysis showed significant alterations in signaling pathways related to prostate development, stem cells, and organ morphogenesis in AR-deficient Gli1-expressing cells. Among these altered pathways, the transforming growth factor β1 (TGFβ1) pathway was up-regulated in AR-deficient Gli1-expressing cells. We further demonstrated the activation of TGFβ1 signaling in AR-deleted prostatic Gli1-expressing cells, which inhibits prostate epithelium growth through paracrine regulation. These data demonstrate a novel role of the AR in the Gli1-expressing cellular niche for regulating prostatic cell fate, morphogenesis, and renewal, and elucidate the mechanism by which mesenchymal androgen-signaling through SHH-responsive cells elicits the growth and regeneration of prostate epithelium., Author summary Prostate formation, growth, and regeneration, as well as tumorigenesis, depend on androgens and androgen receptor (AR)-mediated signaling pathways. Tissue recombination assays done more than 30 years ago demonstrated a decisive role for stromal androgen signaling in prostatic epithelium development. However, in the intervening time, the identity of the mesenchymal cells in the urogenital sinus mesenchyme that convey androgen signaling and control prostate epithelium development, morphogenesis, and regeneration has not been determined. In this study, using mouse genetic tools, we demonstrate for the first time that selective deletion of AR in mesenchymal Gli1-expressing cells abolishes early development of prostate tissue and normal prostate formation, and diminishes prostate pubertal growth and regeneration. In addition, using tissue recombination assays, we directly determined an essential requirement for AR expression in mesenchymal Gli1-expressing cells during prostate epithelium development. Our results not only resolve a 30-year-old scientific puzzle by identifying the mesenchymal cell properties of androgen-responsive cells that elicit development of the embryonic prostate epithelium, but also explore a new regulatory mechanism for androgen and Shh signaling-mediated cellular niches in regulating prostatic cell fate, growth, and renewal through paracrine regulation. Given the importance of sex hormone and hedgehog signaling pathways in human development and tumorigenesis, this study extends beyond the field of prostate biology, raising new questions underlying sex hormone and SHH signaling in development and tumorigenesis.

Co-occurrence of aberrant hepatocyte growth factor (HGF)/MET proto-oncogene receptor tyrosine kinase (MET) and Wnt/β-catenin signaling pathways has been observed in advanced and metastatic prostate cancers. This co-occurrence positively correlates with prostate cancer progression and castration-resistant prostate cancer development. However, the biological consequences of these abnormalities in these disease processes remain largely unknown. Here, we investigated the aberrant activation of HGF/MET and Wnt/β-catenin cascades in prostate tumorigenesis by using a newly generated mouse model in which both murine Met transgene and stabilized β-catenin are conditionally co-expressed in prostatic epithelial cells. These compound mice displayed accelerated prostate tumor formation and invasion compared with their littermates that expressed only stabilized β-catenin. RNA-Seq and quantitative RT-PCR analyses revealed increased expression of genes associated with tumor cell proliferation, progression, and metastasis. Moreover, Wnt signaling pathways were robustly enriched in prostate tumor samples from the compound mice. ChIP-qPCR experiments revealed increased β-catenin recruitment within the regulatory regions of the Myc gene in tumor cells of the compound mice. Interestingly, the occupancy of MET on the Myc promoter also appeared in the compound mouse tumor samples, implicating a novel role of MET in β-catenin–mediated transcription. Results from implanting prostate graft tissues derived from the compound mice and controls into HGF-transgenic mice further uncovered that HGF induces prostatic oncogenic transformation and cell growth. These results indicate a role of HGF/MET in β-catenin–mediated prostate cancer cell growth and progression and implicate a molecular mechanism whereby nuclear MET promotes aberrant Wnt/β-catenin signaling–mediated prostate tumorigenesis.

YZG-331 is a synthetic adenosine analogue which exhibits the sedative and hypnotic effects by binding to the adenosine receptor. The present study was designed to investigate the effects of P-glycoprotein (P-gp) on the intestine and brain distribution of YZG-331 in vitro and in vivo as well as related binding mechanisms. The activity of P-gp ATPase was both induced by YZG-331 and verapamil, a typical P-gp inhibitor, but affinity of YZG-331 for P-gp was lower than that of verapamil. The docking analyses further elucidated the binding relationship of YZG-331 and P-gp. The directional transport of YZG-331 was disappeared in Caco-2 and MDCK-MDR1 cells when the P-gp activity was blocked. However, the penetration of digoxin, a P-gp known substrate, was not change in MDCK-MDR1 cells along with YZG-331. In the everted intestinal sac model, the influx of YZG-331 was significantly reduced in the presence of verapamil in all the segments except for the colon. In the in situ and in vivo study, the brain exposure of YZG-331 was promoted after co-administered of verapamil. Furthermore, the Kp value changed from 0.03 to 0.05 after drug combination. Taken together, these results indicated that YZG-331 is a substrate but may not an inhibitor of P-gp. The intestine and brain permeability of YZG-331 can be restricted, at least in part, by P-gp. The drug interactions should be awarded when YZG-331 and other P-gp-related drugs used together.

Busulfan is a widely used chemotherapeutic drug for chronic myelogenous leukemia and bone marrow transplantation. As a cell cycle nonspecific alkylation agent, busulfan has a severe side effect on germ cells, especially on spermatogonia before meiosis. Studies have revealed that busulfan causes DNA strand crosslinks in spermatogonia and induces apoptosis, and many corresponding strategies have been developed to ameliorate the side effects. However, fertility maintenance after busulfan treatment is still a challenging project in the clinic. Here, we demonstrated that continuous injection of melatonin effectively alleviated germline cytotoxicity both in recipient mice and cultured spermatogonia, and busulfan/melatonin recipient mice produced normal litters. We further revealed that melatonin rescues spermatogonia from apoptosis by neutralizing reactive oxidative species (ROS) induced by busulfan and recovered the phosphorylation of ATM and p53 to normal levels, and as a result apoptosis in spermatogonial progenitor cells was avoided. This study reports that pineal gland hormone melatonin effectively protects spermatogonia from the stress of chemotherapy and oxidation and reveals the underlying molecular mechanisms, which will provide an important hint for fertility protection in clinic.

Emerging evidence has shown that the hepatocyte growth factor (HGF) and its receptor, MET proto-oncogene, receptor tyrosine kinase (MET), promote cell proliferation, motility, morphogenesis, and angiogenesis. Whereas up-regulation of MET expression has been observed in aggressive and metastatic prostate cancer, a clear understanding of MET function in prostate tumorigenesis remains elusive. Here, we developed a conditional Met transgenic mouse strain, H11(Met/+):PB-Cre4, to mimic human prostate cancer cells with increased MET expression in the prostatic luminal epithelium. We found that these mice develop prostatic intraepithelial neoplasia after HGF administration. To further assess the biological role of MET in prostate cancer progression, we bred H11(Met/+)/Pten(LoxP/LoxP):PBCre4 compound mice, in which transgenic Met expression and deletion of the tumor suppressor gene Pten occurred simultaneously only in prostatic epithelial cells. These compound mice exhibited accelerated prostate tumor formation and invasion as well as increased metastasis compared with Pten(LoxP/LoxP):PB-Cre4 mice. Moreover, prostatic sarcomatoid carcinomas and lesions resembling the epithelial-to-mesenchymal transition developed in tumor lesions of the compound mice. RNA-Seq and qRT-PCR analyses revealed a robust enrichment of known tumor progression and metastasis-promoting genes in samples isolated from H11(Met/+)/Pten(LoxP/LoxP):PB-Cre4 compound mice compared with those from Pten(LoxP/LoxP):PB-Cre4 littermate controls. HGF-induced cell proliferation and migration also increased in mouse embryonic fibroblasts (MEFs) from animals with both Met transgene expression and Pten deletion compared with Pten-null MEFs. The results from these newly developed mouse models indicate a role for MET in hastening tumorigenesis and metastasis when combined with the loss of tumor suppressors.

In this study, we systematically investigated the plasma pharmacokinetics, tissue distribution, and elimination of three active alkaloids after oral administration of the effective fraction of alkaloids from Ramulus Mori (SZ–A)—an innovative hypoglycemic agent—in rats. Moreover, the influences of other components in SZ–A on dynamic process of alkaloids were explored for the first time. The results showed that 1-deoxynojirimycin (DNJ), fagomine (FGM) and 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) exhibited nonlinear pharmacokinetics following oral administration of SZ–A (40–1000 mg/kg). The prolonged t1/2 and greater area under concentration-time curve (AUC) versus time (AUC0–t) of DNJ for SZ–A than for purified DNJ has been observed after both oral and intravenous administration. It was found that other components in SZ–A could enhance the absorption of DNJ through the intestinal barrier. The major distribution tissues of DNJ, FGM, and DAB were the gastrointestinal tract, liver, and kidney. Three alkaloids were mainly excreted into urine and feces, but less into bile. Interestingly, the excess excretion of FGM was revealed to be partly due to the biotransformation of other components in SZ–A via gut microbiota. These information provide a rational basis for the use of SZ–A in clinical practice.

The present study was performed to investigate the effect of bicyclol, a synthetic anti-hepatitis drug with anti-oxidative and anti-inflammatory properties, on anti-tuberculosis (anti-TB) drug-induced liver injury and related mechanisms in rats. Bicyclol was given to rats by gavage 2 h before the oral administration of an anti-TB drug once a day for 30 days. Liver injury was evaluated by biochemical and histopathological examinations. Lipid peroxidation, mitochondrial function, and the activity of antioxidants were measured by spectrophotometric methods. Cytokines expression and CYP2E1 activity were determined by ELISA assay and liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis. The expressions of hepatic CYP2E1 and hepatocyte growth factor (HGF) were assessed by Western blotting. As a result, bicyclol significantly protected against anti-TB drug-induced liver injury by reducing the elevated serum aminotransferases levels and accumulation of hepatic lipids. Meanwhile, the histopathological changes were also attenuated in rats. The protective effect of bicyclol on anti-TB drug-induced hepatotoxicity was mainly due to its ability to attenuate oxidative stress, suppress the inflammatory cytokines and CYP2E1 expression, up-regulate the expression of HGF, and improve mitochondrial function. Furthermore, administration of bicyclol had no significant effect on the plasma pharmacokinetics of the anti-TB drug in rats.

The tumor suppressor p16Ink4a, encoded by the INK4a gene, is an inhibitor of cyclin D-dependent kinases 4 and 6, CDK4 and CDK6. This inhibition prevents the phosphorylation of the retinoblastoma protein (pRb), resulting in cellular senescence through inhibition of E2F-mediated transcription of S phase genes required for cell proliferation. The p16Ink4a plays an important role in tumor suppression, whereby its deletion, mutation, or epigenetic silencing is a frequently observed genetic alteration in prostate cancer. To assess its roles and related molecular mechanisms in prostate cancer initiation and progression, we generated a mouse model with conditional deletion of p16Ink4a in prostatic luminal epithelium. The mice underwent oncogenic transformation and developed prostatic intraepithelial neoplasia (PIN) from eight months of age, but failed to develop prostatic tumors. Given the prevalence of aberrant androgen signaling pathways in prostate cancer initiation and progression, we then generated R26hARL/wt:p16L/L: PB-Cre4 compound mice, in which conditional expression of the human AR transgene and deletion of p16Ink4a co-occur in prostatic luminal epithelial cells. While R26hARL/wt:PB-Cre4 mice showed no visible pathological changes, R26hARL/wt:p16L/L: PB-Cre4 compound mice displayed an early onset of high-grade PIN (HGPIN), prostatic carcinoma, and metastatic lesions. Strikingly, we observed tumors resembling human sarcomatoid carcinoma with intermixed focal regions of signet ring cell carcinoma (SRCC) in the prostates of the compound mice. Further characterization of these tumors showed they were of luminal epithelial cell origin, and featured characteristics of epithelial to mesenchymal transition (EMT) with enhanced proliferative and invasive capabilities. Our results not only implicate a biological role for AR expression and p16Ink4a deletion in the pathogenesis of prostatic SRCC, but also provide a new and unique genetically engineered mouse (GEM) model for investigating the molecular mechanisms for SRCC development.

Syl930 is a novel sphingosine-1-phosphate receptor subtype 1 (S1PR

A sensitive and specific UPLC-MS/MS method was developed and validated for the simultaneous determination of 2-amino-2-(2-(4'-(2-propyloxazol-4-yl)-[1,1'-biphenyl]-4-yl)ethyl)propane-1,3-diol (SYL930), phosphorylated metabolite (SYL930-P) and hydroxylated metabolite (SYL930-M) in dog blood using SYL927 and SYL927-P, analogues of SYL930, as the internal standards. Analytes were extracted with protein precipitation followed by chromatographic separation on a ZorbaxSB-C

A sensitive and specific LC-MS/MS method was developed and validated for simultaneous determination of 2-amino-2-(2-(4'-(2-propyloxazol-4-yl)-[1,1'-biphenyl]-4-yl)ethyl)propane-1,3-diol (SYL930) and its active phosphate metabolite (SYL930-P) in rat blood using SYL927, an analogue of SYL930 as the internal standard. Blood samples were prepared by a simple protein precipitation with acetonitrile. The chromatographic separation was performed on a ZorbaxSB-C18 column (3.5 μm, 2.1 × 100 mm) with a gradient mobile phase of methanol/water containing 0.1 % formic acid (v/v) at a flow rate of 0.2 mL/min. The detection was carried out on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization (ESI) in multiple reactions monitoring mode (MRM). The monitored transitions were 381.2 → 364.2 for SYL930, 461.2 → 334.2 for SYL930-P, and 367.1 → 350.4 for the internal standard, respectively. Good linearity was obtained for the analytes over the range of 0.2-100 ng/mL for SYL930 and 0.5-100 ng/mL for SYL930-P. The lower limits of quantitation (LLOQs) for SYL930 and SYL930-P were 0.2 and 0.5 ng/mL, respectively. The intra-day and inter-day precisions (RSD, %) of analytes were within 9.87 %, and the accuracy (RE, %) ranged from -7.04 to 13.15 %. The mean recoveries for two compounds in rat blood were 87.9-109 %. The analytes were proved to be stable during all sample storage, preparation, and analytic procedures. The validated method was successfully applied to pharmacokinetic and PK/PD studies of SYL930 and SYL930-P in rats after oral administration of SYL930. Graphical Abstract Quantitative determination of SYL930 and its active phosphorylated metabolite in rat blood by LCMS/MS and application to PK/PD analysis.

A sensitive LC–ESI-MS/MS method for simultaneous determination of MTC-220 and its metabolites (paclitaxel and MDA-linker) in dog plasma has been developed and validated. After addition of docetaxel (internal standard), plasma samples containing MTC-220, paclitaxel and MDA-linker were prepared based on a simple protein precipitation by adding two volumes of acetonitrile. The separation was performed on a ZorbaxSB-C18 column (3.5 μm, 2.1 mm × 100 mm) at a flow rate of 0.2 ml/min, using acetonitrile/water containing 0.1% formic acid (v/v) as mobile phase. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization (ESI) by selected reaction monitoring (SRM). The MS/MS ion transit ions monitored were 1444.4 → 623.8 for MTC-220, 876.4 → 307.9 for paclitaxel, 631.2 → 531.2 for MDA-linker and 830.2 → 549.1 for the internal standard. Linear detection responses were obtained for MTC-220, paclitaxel and MDA-linker ranging from 10 to 5000, 5 to 2500 and 5 to 500 ng/ml, respectively. The lower limits of quantitation (LLOQs) for MTC-220, paclitaxel and MDA-linker were 10, 5 and 5 ng/ml, respectively. The intra-day and inter-day precisions (RSD, %) of the three analytes do not exceed 10.9% except for LLOQs (≤17.50), and the accuracy (RE, %) were within ±17.5% for LLOQs and ±12.6% for the others. The average recoveries of three compounds were greater than 85.0%. The analytes were proved to be stable during all sample storage, preparation and analytic procedures. The validated method was successfully applied to pharmacokinetic studies of MTC-220 and its metabolites in beagle dogs after intravenous infusion of MTC-220 at 2.5 mg/kg.