Enzalutamide and CXCR7 inhibitor combination treatment suppresses cell growth and angiogenic signaling in castration-resistant prostate cancer models.

Previous studies have shown that increased levels of chemokine receptor CXCR7 are associated with the increased invasiveness of prostate cancer cells. We now show that CXCR7 expression is upregulated in VCaP and C4-2B cells after enzalutamide (ENZ) treatment. ENZ treatment induced apoptosis (sub-G1) in VCaP and C4-2B cells, and this effect was further increased after combination treatment with ENZ and CCX771, a specific CXCR7 inhibitor. The levels of p-EGFR (Y1068), p-AKT (T308) and VEGFR2 were reduced after ENZ and CCX771 combination treatment compared to single agent treatment. In addition, significantly greater reductions in migration were shown after combination treatment compared to those of single agents or vehicle controls, and importantly, similar reductions in the levels of secreted VEGF were also demonstrated. Orthotopic VCaP xenograft growth and subcutaneous MDA133-4 patient-derived xenograft (PDX) tumor growth was reduced by single agent treatment, but significantly greater suppression was observed in the combination treatment group. Although overall microvessel densities in the tumor tissues were not different among the different treatment groups, a significant reduction in large blood vessels (>100 µm2 ) was observed in tumors following combination treatment. Apoptotic indices in tumor tissues were significantly increased following combination treatment compared with vehicle control-treated tumor tissues. Our results demonstrate that significant tumor suppression mediated by ENZ and CXCR7 combination treatment may be due, in part, to reductions in proangiogenic signaling and in the formation of large blood vessels in prostate cancer tumors.

Pathogenicity of Bordetella avium under immunosuppression induced by Reticuloendotheliosis virus in specific-pathogen-free chickens.

Multiple infections of Bordetella avium (B. avium) with virus, especially immunosuppressive virus, have become more and more severe in chickens in China. The increasing morbidity and mortality of its complications have amplified concerns about the impact of B. avium on animal health. To evaluate the pathogenicity of B. avium under immunosuppression status, we developed four types of Reticuloendotheliosis virus (REV) infection models. After a comparison of body weight, relative immune organ index, Newcastle disease virus antibody titers and lymphocyte ratio, we chose the early age with low dose infection as our immunosuppressive model. To investigate the pathogenicity of B. avium under this model, a study was completed in which chickens were inoculated with REV-only, B. avium-only, both agents (REV -B. avium) or first inoculated with REV and 5 d later with B. avium (REV/B. avium). Results revealed that antibody titers to B. avium, concentrations of IFN-γ and SIgA were decreased in coinfected chickens when compared to the B. avium-only chickens, but the changing trend was similar between REV/B. avium and B. avium-only groups. Overall, REV did enhance the pathogenicity of B. avium. However, B. avium-only did not cause severe immune dysfunction unless chicks were coinfected with REV. REV preceding infection with B. avium showed mild impairment, which needs further exploration.

Comparative study of microRNA profiling in keloid fibroblast and annotation of differential expressed microRNAs.

Keloids are tumor-like skin scars that grow as a result of the aberrant healing of skin injuries, with no effective treatment. The molecular mechanism underlying keloid pathogenesis is still largely unknown. In this study, we compared microRNA (miRNA) expression profiles between keloid-derived fibroblasts and normal fibroblasts (including fetal and adult dermal fibroblasts) by miRNA microarray analysis. We found that the miRNA profiles in keloid-derived fibroblasts are different with those in normal fibroblasts. Nine miRNAs were differentially expressed, six of which were significantly up-regulated in keloid fibroblasts (KFs), including miR-152, miR-23b-3p, miR-31-5p, miR-320c, miR-30a-5p, and hsv1-miR-H7, and three of which were significantly down-regulated, including miR-4328, miR-145-5p, and miR-143-3p. Functional annotations of differentially expressed miRNA targets revealed that they were enriched in several signaling pathways important for scar wound healing. In conclusion, we demonstrate that the miRNA expression profile is altered in KFs compared with in fetal and adult dermal fibroblasts, and the expression profile may provide a useful clue for exploring the pathogenesis of keloids. miRNAs might partially contribute to the etiology of keloids by affecting several signaling pathways relevant to scar wound healing.

Transcriptomic analysis reveals shared gene signatures and molecular mechanisms between obesity and periodontitis.

Background: Both obesity (OB) and periodontitis (PD) are chronic non-communicable diseases, and numerous epidemiological studies have demonstrated the association between these two diseases. However, the molecular mechanisms that could explain the association between OB and PD are largely unclear. This study aims to investigate the common gene signatures and biological pathways in OB and PD through bioinformatics analysis of publicly available transcriptome datasets. Methods: The RNA expression profile datasets of OB (GSE104815) and PD (GSE106090) were used as training data, and GSE152991 and GSE16134 as validation data. After screening for differentially expressed genes (DEGs) shared by OB and PD, gene enrichment analysis, protein-protein interaction (PPI) network construction, GeneMANIA analysis, immune infiltration analysis and gene set enrichment analysis (GSEA) were performed. In addition, receiver operating characteristic (ROC) curves were used to assess the predictive accuracy of the hub gene. Finally, we constructed the hub gene-associated TF-miRNA-mRNA regulatory network. Results: We identified a total of 147 DEGs shared by OB and PD (38 down-regulated and 109 up-regulated). Functional analysis showed that these genes were mainly enriched in immune-related pathways such as B cell receptor signalling, leukocyte migration and cellular defence responses. 14 hub genes (FGR, MNDA, NCF2, FYB1, EVI2B, LY86, IGSF6, CTSS, CXCR4, LCK, FCN1, CXCL2, P2RY13, MMP7) showed high sensitivity and specificity in the ROC curve analysis. The results of immune infiltration analysis showed that immune cells such as macrophages, activated CD4 T cells and immune B cells were present at high infiltration levels in both OB and PD samples.The results of GeneMANIA analysis and GSEA analysis suggested that five key genes (FGR, LCK, FYB1, LY86 and P2RY13) may be strongly associated with macrophages. Finally, we constructed a TF-miRNA-mRNA regulatory network consisting of 233 transcription factors (TFs), 8 miRNAs and 14 mRNAs based on the validated information obtained from the database. Conclusions: Five key genes (FGR, LCK, FYB1, LY86, P2RY13) may be important biomarkers of OB and PD. These genes may play an important role in the pathogenesis of OB and PD by affecting macrophage activity and participating in immune regulation and inflammatory responses.

Enzalutamide-Resistant Progression of Castration-Resistant Prostate Cancer Is Driven via the JAK2/STAT1-Dependent Pathway.

Previous studies showed that CXCR7 expression was upregulated after enzalutamide (ENZ) treatment, and an increased level of CXCR7 could increase the invasion, migration, and angiogenesis of castration-resistant prostate cancer (CRPC) cells. This study demonstrated that the levels of p-JAK2, p-STAT1, C-Myc, and VEGFR2 were significantly reduced after CCX771, a specific CXCR7 inhibitor, treatment. This effect further increased after the combination treatment of ENZ and CCX771. Then, we verified that targeting the inhibition of JAK2 or STAT1 could remarkably increase apoptosis and DNA damage and decrease the migration of CRPC cells. More importantly, the combination treatment of ENZ + JAK2/STAT1 led to much greater suppression than the single-agent treatment of JAK2 or STAT1. Subcutaneous CRPC xenograft tumor growth was also reduced by single-agent ENZ treatment and single-agent FLUD, a specific STAT1 antagonist, treatment; but much superior effect was elicited by the combination treatment of ENZ + FLUD. The proliferative indices significantly decreased following combination treatment in tumor tissues compared with control-treatment tissues and single-agent-treatment tissues. Our results demonstrated that CXCR7, which signifies an androgen receptor (AR)-independent signaling pathway, caused CRPC progression via the downstream JAK2/STAT1 signal transduction cascade. Combined inhibition targeting both the AR and JAK2/STAT1 resulted in substantial tumor suppression due to the reduction in DNA damage repair ability and increment in apoptosis.

ß­catenin nuclear translocation induced by HIF­1α overexpression leads to the radioresistance of prostate cancer.

Hypoxia-inducible factor­1α (HIF­1α) is known to play crucial roles in tumor radioresistance; however, the molecular mechanisms responsible for the promotion of tumor radioresistance by HIF­1α remain unclear. ß­catenin is known to be involved in the metastatic potential of prostate cancer (PCa). In this study, to investigate the role of HIF­1α and ß­catenin in the radioresistance of PCa, two PCa cell lines, LNCaP and C4­2B, were grouped as follows: Negative control (no treatment), HIF­1α overexpression group (transfected with HIF­1α overexpression plasmid) and ß­catenin silenced group (transfected with HIF­1α plasmids and ß­catenin-shRNA). Cell proliferation, cell cycle, cell invasion and radiosensitivity were examined under normal or hypoxic conditions. In addition, radiosensitivity was examined in two mouse PCa models (the LNCaP orthotopic BALB/c-nu mice model and the C4­2B subcutaneous SCID mice model). Our results revealed that in both the LNCaP and C4­2B cells, transfection with HIF­1α overexpression plasmid led to an enhanced ß­catenin nuclear translocation, while ß­catenin silencing inhibited ß­catenin nuclear translocation. The enhanced ß­catenin nuclear translocation induced by HIF­1α overexpression resulted in an enhanced cell proliferation and cell invasion, an altered cell cycle distribution, decreased apoptosis, and improved non­homologous end joining (NHEJ) repair under normal and irradiation conditions. Similar results were observed in the animal models. HIF­1α overexpression enhanced ß­catenin nuclear translocation, which led to the activation of the ß­catenin/NHEJ signaling pathway and increased cell proliferation, cell invasion and DNA repair. These results thus suggest that HIF­1α overexpression promotes the radioresistance of PCa cells.

ATRA increases iodine uptake and inhibits the proliferation and invasiveness of human anaplastic thyroid carcinoma SW1736 cells: Involvement of ß-catenin phosphorylation inhibition.

All-trans-retinoic acid (ATRA) can enhance iodine uptake capability of thyroid tumors, but the mechanisms remain poorly understood. The aim of the present study was to investigate the effects of ATRA on isotope susceptibility, proliferation and invasion of anaplastic thyroid carcinoma (ATC) and potential mechanisms. SW1736 cells were treated with 1 µmol/l ATRA or 1% ethanol for 5 days. A cell line stably expressing ß-catenin-shRNA was established. An iodine uptake assay was performed using 125I. Proliferation and invasiveness were tested using MTT and Transwell assays, respectively. Western blotting was used to assess the expression of ß-catenin, glycogen synthase kinase-3ß (GSK-3ß), sodium/iodine symporter (NIS) and proteins involved in epithelial-mesenchymal transition. Cells pretreated with ATRA were injected subcutaneously into SCID mice. Mice were intraperitoneally injected with 131I once on the first day of treatment, and tumor growth was then assessed. After 35 days of 131I treatment, ATRA-pretreated tumor volume and weight were decreased compared with the 131I alone group (163.32±19.57 vs. 332.06±21.37 mm3; 0.35±0.14 vs. 0.67±0.23 g, both P<0.05). Similar results were observed in the ß-catenin shRNA-pretreated tumors. ATRA also increased the uptake of iodine by SW1736 cells (P<0.01), and similar results were observed in ß-catenin shRNA cells. ATRA treatment decreased the cell proliferation and invasion compared with control cells (all P<0.05), similar to ß-catenin shRNA. ATRA treatment decreased the expression of phosphorylated (p-)ß-catenin, p-GSK-3ß, vimentin, and fibronectin, and increased the expression of NIS and E-cadherin, compared with the control. ATRA increased the iodine uptake and inhibited the proliferation and invasion of SW1736 cells, involving ß-catenin phosphorylation. In conclusion, ATRA could be used to improve the isotope sensitivity of ATC.

Inhibiting ß-catenin expression promotes efficiency of radioiodine treatment in aggressive follicular thyroid cancer cells probably through mediating NIS localization.

The present study investigated whether the efficacy of radioiodine therapy towards aggressive thyroid cancer cells was affected by ß-catenin activity and associated with sodium/iodine symporter (NIS) localization. Human thyroid cancer cell line follicular thyroid carcinoma (FTC) 133 was endowed with aggressiveness by HIF-1α or ß-catenin overexpression. The protein amount and subcellular localization of NIS, and the radioiodine uptake capacity were detected in the cells, as well as in cells subsequently undergoing ß-catenin knockdown. Xenograft experiments were conducted to compare the tumor growth ability and responsiveness to radioactive treatment among HIF-1α and ß-catenin overexpressing FTC cells, respectively with or without ß-catenin knockdown. ß-catenin increased upon HIF-1α overexpression, but not vice versa. This signal axis would prompt metastatic propensity in FTC cells, and translocate NIS from cytomembrane to cytoplasm. Consistently the radioiodine uptake capacity in the cells decreased obviously. Knockdown of ß-catenin reversed all these changes. Furthermore, the xenograft experiments showed that radioiodine treatment could thoroughly suppress tumor growth ability of aggressive FTC cells only if the HIF-1α-induced ß-catenin activation was disrupted by ß-catenin knockdown. ß-catenin nuclear translocation in tumor cells was accompanied by abnormal subcellular localization of NIS. Moreover, we found that only after inhibiting ß-catenin expression, can the radioiodine treatment promote apoptosis other than repress proliferation and survival in xenograft tumor cells. In conclusion, aggressive FTC cells overexpressing HIF-1α will be fully cracked down by radioiodine therapy once ß-catenin expression is inhibited, and regulated localization of NIS may account for underlying mechanisms.

Targeting DNA Damage Response in Prostate Cancer by Inhibiting Androgen Receptor-CDC6-ATR-Chk1 Signaling.

Cell division cycle 6 (CDC6), an androgen receptor (AR) target gene, is implicated in regulating DNA replication and checkpoint mechanisms. CDC6 expression is increased during prostate cancer (PCa) progression and positively correlates with AR in PCa tissues. AR or CDC6 knockdown, together with AZD7762, a Chk1/2 inhibitor, results in decreased TopBP1-ATR-Chk1 signaling and markedly increased ataxia-telangiectasia-mutated (ATM) phosphorylation, a biomarker of DNA damage, and synergistically increases treatment efficacy. Combination treatment with the AR signaling inhibitor enzalutamide (ENZ) and the Chk1/2 inhibitor AZD7762 demonstrates synergy with regard to inhibition of AR-CDC6-ATR-Chk1 signaling, ATM phosphorylation induction, and apoptosis in VCaP (mutant p53) and LNCaP-C4-2b (wild-type p53) cells. CDC6 overexpression significantly reduced ENZ- and AZD7762-induced apoptosis. Additive or synergistic therapeutic activities are demonstrated in AR-positive animal xenograft models. These findings have important clinical implications, since they introduce a therapeutic strategy for AR-positive, metastatic, castration-resistant PCa, regardless of p53 status, through targeting AR-CDC6-ATR-Chk1 signaling.

Androgen receptor inhibitor-induced "BRCAness" and PARP inhibition are synthetically lethal for castration-resistant prostate cancer.

Cancers with loss-of-function mutations in BRCA1 or BRCA2 are deficient in the DNA damage repair pathway called homologous recombination (HR), rendering these cancers exquisitely vulnerable to poly(ADP-ribose) polymerase (PARP) inhibitors. This functional state and therapeutic sensitivity is referred to as "BRCAness" and is most commonly associated with some breast cancer types. Pharmaceutical induction of BRCAness could expand the use of PARP inhibitors to other tumor types. For example, BRCA mutations are present in only ~20% of prostate cancer patients. We found that castration-resistant prostate cancer (CRPC) cells showed increased expression of a set of HR-associated genes, including BRCA1, RAD54L, and RMI2 Although androgen-targeted therapy is typically not effective in CRPC patients, the androgen receptor inhibitor enzalutamide suppressed the expression of those HR genes in CRPC cells, thus creating HR deficiency and BRCAness. A "lead-in" treatment strategy, in which enzalutamide was followed by the PARP inhibitor olaparib, promoted DNA damage-induced cell death and inhibited clonal proliferation of prostate cancer cells in culture and suppressed the growth of prostate cancer xenografts in mice. Thus, antiandrogen and PARP inhibitor combination therapy may be effective for CRPC patients and suggests that pharmaceutically inducing BRCAness may expand the clinical use of PARP inhibitors.

Monoclonal antibodies directed against the outer membrane protein of Bordetella avium.

Bordetella avium is the etiologic agent of coryza and rhinotracheitis in poultry. This respiratory disease is responsible for substantial economic losses in the poultry industry. Monoclonal antibodies (MAbs) were produced against the outer membrane proteins (OMPs) of B. avium isolated from diseased chickens. BALB/c mice were immunized with the extracted B. avium OMPs. Then the splenocytes from immunized mice and SP2/0 myeloma cells were fused using PEG 4000. Three stable hybridoma clones (designated as 3G10, 4A3, and 4E8) were produced via indirect ELISA and three rounds of subcloning. The MAbs were classified as IgG1, and can recognize the 58 kDa OMP band by Western blot assays. No MAb cross-reactivity with chicken Proteus mirabilis, Escherichia coli, and Salmonella was observed. A double antibody sandwich ELISA (DAS-ELISA) was developed using the rabbit polyclonal antibodies as the capture antibody and MAb 4A3 as the detection antibody. Under the DAS-ELISA, the minimum detectable concentration of B. avium was 1 × 10(4) CFU/mL, and no cross-reactivity occurred with chicken Proteus mirabilis, Escherichia coli, and Salmonella. Results showed that the DAS-ELISA has good sensitivity and specificity. Clinical application showed the DAS-ELISA was more sensitive than the plate agglutination test. This study may be used to develop a quick and specific diagnostic kit, analyze epitopes, and establish systems for typing B. avium.

Effects of Taishan Pinus massoniana pollen polysaccharide on the subunit vaccine of Proteus mirabilis in birds.

Three adjuvants, namely, Taishan Pinus massoniana pollen polysaccharide (TPPPS), white mineral oil (WO) and propolis (PP), were added to the outer membrane protein (OMP) of Proteus mirabilis (P. mirabilis) and their effects were compared. Three hundred 1-day-old chicks were randomly divided into five groups (I-V), with 60 chicks per group, and injected subcutaneously with WO-OMP vaccine (I), PP-OMP vaccine (II), TPPPS-OMP vaccine (III), OMP-only vaccine (IV) and physiological saline (V) at 3, 7 and 12 days old. On days 3, 7, 14, 21, 28, 35, 42 and 49 after the first vaccination, the antibody titers, interleukin-2 levels (IL-2) and T-lymphocyte proliferation rates in the peripheral blood as well as the secreting-type IgA levels (SIgA) in the duodenum were measured. On day 7 after the third vaccination, the chicks were challenged with P. mirabilis strain Q1 and the protective effects of each group were observed. The highest protective rate was observed in group III. Moreover, the antibody titers as well as IL-2, SIgA and T-lymphocyte proliferation rates in this group significantly increased and were significantly higher than those in the other groups at most time points. The results indicate that TPPPS could significantly enhance the effects of the subunit vaccine of P. mirabilis; induced stronger humoral, cellular and mucosal immunity as compared with WO and PP; and should be developed as a vaccine adjuvant.

Development and characterization of simple sequence repeat (SSR) markers for the mushroom Flammulina velutipes.

Ten polymorphic SSR markers of Flammulina velutipes were developed and characterized with FIASCO methods. The polymorphism information content (PIC) ranged from 0.13 to 0.69. This is the first report on development of SSR markers in F. velutipe.

Strain-typing of Lentinula edodes in China with inter simple sequence repeat markers.

To validate strain typing by inter simple sequence repeat (ISSR) analysis in Lentinula edodes cultivars, 17 Chinese L. edodes strains including 15 cultivated strains cultivated on a large scale and two wild strains were analyzed with the ISSR technique. With the use of two ISSR primers, a total of 32 DNA products were detected, of which, 31 DNA products (96.9% of the detected products) were polymorphic between two or more strains. The profiles of those two primers could be employed to differentiate all of the tested strains. A cluster analysis based on ISSR data revealed that the 17 strains could be classified into two distinct groups. One group consisted of eight strains in which the cultivated strains were H (high-temperature)-type or B (broad-temperature)-type, and the other group comprised cultivated strains that were of the L (low-temperature)-type or M (medium-temperature)-type. In contrast to the two wild strains, the genetic diversity of 15 cultivated strains was very rich based on a similarity coefficient analysis.