BackgroundOne key step in the development of prostate cancer (PCa) metastasis is the loss of E-cadherin expression associated with increased cellular motility and tumor invasion. This loss of E-cadherin expression is also required during normal embryogenesis and similar transcriptional repressors have been identified in both processes. We have previously reported the presence of one such transcription factor, WT1 in high Gleason grade prostate tumor tissues, and its absence in non-neoplastic or benign prostatic hyperplasia tissues.ResultsTo better understand the effect of WT1 on E-cadherin expression and migration of PCa cells we quantified WT1 and E-cadherin mRNA levels in normal prostate epithelial and PCa cell lines with varying migratory potential. In WT1 transfected cells E-cadherin transcript levels were decreased, while they were increased in siWT1-RNA transfected PCa cells, suggesting that elevated WT1 expression was sufficient to dampen E-cadherin levels and potentially enhance migratory ability. To delineate the mechanism of WT1-mediated repression of E-cadherin, potential WT1 binding sites were tested in vitro and in vivo binding of WT1 to the E-cadherin promoter in the chromatin of LNCaP and PC3 cells was assessed by Chromatin Immunoprecipitation. The effect of WT1 binding was measured in reporter assays; in PC3 and DU145 cells WT1 decreased the activity of the proximal E-cadherin promoter. Using site-directed mutagenesis, a newly identified WT1 binding site located 146 bp from the transcription start site was shown to be required for this repression by WT1. Transwell migration and wound healing assays revealed that in LNCaP cells with low migratory potential, over-expression of WT1 was sufficient to enhance migration, conversely, in the highly migratory PC3 cells silencing of WT1 decreased migration.ConclusionsThese findings suggested that WT1 expression in high grade prostate cancer may contribute to migration and metastasis. Thus, in prostate cancer WT1 may function as a novel oncogene facilitating development of the lethal metastatic phenotype.

Inflammation is a highly regulated biological response of the immune system that is triggered by assaulting pathogens or endogenous alarmins. It is now well established that some soluble extracellular matrix constituents, such as small leucine-rich proteoglycans (SLRPs), can act as danger signals and trigger aseptic inflammation by interacting with innate immune receptors. SLRP inflammatory signaling cascade goes far beyond its canonical function. By choosing specific innate immune receptors, coreceptors, and adaptor molecules, SLRPs promote a switch between pro- and anti-inflammatory signaling, thereby determining disease resolution or chronification. Moreover, by orchestrating signaling through various receptors, SLRPs fine-tune inflammation and, despite their structural homology, regulate inflammatory processes in a molecule-specific manner. Hence, the overarching theme of this review is to highlight the molecular and functional specificity of biglycan-, decorin-, lumican-, and fibromodulin-mediated signaling in inflammatory and autoimmune diseases

Tissue engineering shows great promise for the treatment of degenerative diseases, including bone repair. Polymer nanofibers provide a three-dimensional (3-D) scaffold for attachment and growth of mesenchymal stem cells. Increasing evidence supports that fiber alignment on scaffolds plays a major role in the viability and differentiation of stem cells. We compared the cell viability of canine adipose tissue-derived mesenchymal stem cells (cADMSCs) cultured in the aligned- (NanoAligned™) and random- (NanoECM™) oriented polycaprolactone (PCL) nanofiber-coated plates to control polystyrene tissue culture plates using a proliferation assay. Ability of the plates to induce differentiation of cADMSCs into osteocytes, adipocytes, and neurons was evaluated based on expression of the osteocyte markers, COL1A1 and osterix; adipocyte markers PPARγ2 and LPL; and neuronal marker nestin using RT-PCR. Proliferation results demonstrated that aligned-oriented PCL nanofiber-coated plates were more suitable substrate for cADMSCs after 7 days in culture compared to random-oriented PCL nanofiber-coated or control plates. Additionally, we demonstrated that both 3-D PCL nanofiber-coated plates were a better scaffold for cADMSCs differentiation into osteocytes compared to control plates. In conclusion, our results confirm that PCL nanofiber is a suitable tissue engineering material for use in regenerative medicine for canine patients in vivo. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1780-1788, 2018.

Background Reported efficacy of platelet-rich plasma (PRP) in regenerative medicine is contradictory. We validated the effects of PRP on proliferation of canine bone marrow-derived multipotent mesenchymal stromal cells (K9BMMSCs) in vitro. PRP was extracted from blood of six dogs with osteoarthritis. K9BMMSCs were established from bone marrow and characterized for CD90 and CD19 expression by immunocytochemistry. Effects of PRP concentrations on viability of matching autologous K9BMMSCs were validated using MTS assay. Results Positive CD90 and negative CD19 expression confirmed MSC origin. PRP at 40% volume/volume concentration increased, while PRP at 80 and 100% v/v concentrations suppressed viability of tested K9BMMSCs. Conclusion PRP concentration plays an important role in K9BMMSCs viability, which could affect tissue repairs in vivo. Electronic supplementary material The online version of this article (10.1186/s12917-019-2010-x) contains supplementary material, which is available to authorized users.

Due to doxorubicin (Dox) cardiotoxicity, the next generation of novel non-cardiotoxic anthracyclines, including AD 312 and AD 198, were synthesized and validated. In this study, we assessed the efficacy and mechanisms of anthracyclines-induced apoptosis and inhibition of cell viability in human bladder cancer cells expressing wild-type (wt) p53 (RT4 and SW780) and mutated (mt) p53 (UM-UC-3, 5637, T-24, J82, and TCCSUP) protein. Anthracyclines inhibited cell viability in tested TCC cells, but were less effective in mt-p53 TCC cells, especially in the drug-resistant J82 and TCCSUP cells. Anthracyclines upregulated the expression of wt p53 protein in RT4 and SW780 cells, but had no effect on expression of mt p53 protein in UM-UC-3, 5637, T-24, J82, and TCCSUP cells. The anthracyclines activated caspase 3/7 and cleavage of PARP in wt-p53 RT4 and SW780 cells, and mt-p53 5637, UM-UC-3, and T-24, but not in mt-p53 J82 and TCCSUP cells. The anthracyclines-induced cleavage of PARP was blocked by p53 siRNA in wt-p53 RT4 cells. Co-treatment of AD 198 with PRIMA-1 significantly inhibited cell viability of mt-p53 J82 cells, but had no effect in wt-p53 RT4 cells. AD 198 blocked c-myc expression in mt-p53 UM-UC-3, 5637, T-24, and J82 cells, however no expression of c-myc was detected in wt-p53 RT4 and SW780 cells. In conclusion, our results demonstrated that the anthracycline-induced resistance in bladder cancer cells positively correlated with TP53 mutations in the tetramerization domain in J82 and TCCSUP cells. Further, AD 312 and AD 198 are promising chemotherapeutic drugs for bladder cancer, especially in combination with PRIMA-1.

Among challenges of targeted therapies is the activation of alternative pro-survival signaling pathways in cancer cells, resulting in an acquired drug resistance. Cyclooxygenase-2 (COX-2) is overexpressed in bladder cancer cells, making it an attractive molecular target for the detection and treatment of cancer. Fluorocoxib A is an optical imaging agent that selectively targets COX-2. In this study, we evaluated the ability of fluorocoxib A to monitor the responses of bladder cancer to targeted therapies in vivo . The effects of several tyrosine kinase inhibitors (TKIs: axitinib, AB1010, toceranib, imatinib, erlotinib, gefitinib, imatinib, sorafenib, vandetanib, SP600125, UO126, and AZD 5438) on COX-2 expression were validated in ten human and canine bladder cancer cell lines (J82, RT4, T24, UM-UC-3, 5637, SW780, TCCSUP, K9TCC#1Lillie, K9TCC#2Dakota, K9TCC#5Lilly) in vitro . The effects of TKIs on bladder cancer in vivo were evaluated using the COX-2-expressing K9TCC#5Lilly xenograft mouse model and detected by fluorocoxib A. The increased COX-2 expression was detected by all tested TKIs in at least one of the tested COX-2-expressing bladder cancer cell lines (5637, SW780, TCCSUP, K9TCC#1Lillie, K9TCC#2Dakota, and K9TCC#5Lilly) in vitro . In addition, fluorocoxib A uptake correlated with the AB1010- and imatinib-induced COX-2 expression in the K9TCC#5Lilly xenografts in vivo . In conclusion, these results indicate that fluorocoxib A could be used for the monitoring the early responses to targeted therapies in COX-2-expressing bladder cancer.

The restricted expression of Wilms tumor 1 (WT1) and cyclin A1 (CCNA1) in normal tissues, as opposed to their abnormal expression in leukemia demonstrates the applicability of WT1 and CCNA1 as cancer antigens for immunotherapy, and as markers for prognosis and relapse. In this study, the WT1 and CCNA1 mRNA levels were found to be elevated in bone marrow samples from pediatric acute promyelocytic leukemia (APL or AML-M3) patients, and to be quite varied in pediatric acute lymphocytic leukemia (ALL) patients, compared to non-leukemic bone marrow controls. Consistent with the observed upregulation of both WT1 and CCNA1 in APL, WT1 overexpression elevated the CCNA1 mRNA levels in K562 leukemia cells. Treatment with curcumin decreased the WT1 levels in K562 cells, and also decreased CCNA1 protein expression. The examination of the CCNA1 promoter identified potential canonical WT1 binding sites within the 3-kb region upstream of the transcription start site. Chromatin immunoprecipitation and luciferase reporter assays confirmed WT1 binding and the activation of the CCNA1 promoter. Furthermore, the GC-rich core CCNA1 promoter region provided additional non-canonical WT1 activation sites, as revealed by promoter assays. The importance of the GC-rich core region of the CCNA1 promoter was confirmed by treating the K562 cells with mithramycin A, which blocks the binding of zinc finger transcription factors to GC-rich sequences. Mithramycin A subsequently suppressed both CCNA1 promoter activity and protein expression in the K562 cells. Taken together, the data from the WT1 overexpression, and curcumin and mithramycin A treatment experiments, as well as those from chromatin binding assays, along with inferences from patient RNA analyses, establish a plausible link between WT1 and CCNA1, and support the functional significance of an elevated WT1 expression in leukemia, which may also affect CCNA1 expression.

4645 Acute leukemias collectively comprise the most common group of malignancies in the pediatric age group. Increasingly, therapeutic approach and prognosis are influenced by leukemia-specific cytogenetic abnormalities and genetic alterations, thus highlighting the importance of identifying novel prognostic markers. The Wilms’ tumor suppressor gene WT1 is expressed in leukemic blasts and is found to be mutated in approximately 10 percent of leukemia cases. Although it is unclear whether WT1 acts as an oncogene or a tumor suppressor gene in leukemia, it is known to regulate genes involved in cancer progression, including the angiogenic and mitogenic factor, VEGF. Previous studies in kidney and prostate cell lines identified potential WT1 binding sites on the VEGF-A gene promoter and demonstrated that WT1 transcriptionally regulated VEGF expression. Thus, we hypothesized that WT1 transcriptionally regulates VEGF expression in leukemia. To examine WT1 and VEGF expression patterns in pediatric Acute Lymphocytic Leukemia (ALL), Acute Myeloid Leukemia (AML) and non-neoplastic bone marrow samples, we performed quantitative real time PCR. It was observed that WT1 and VEGF expression varied depending upon the type and sub-type of leukemia. Furthermore, to understand the significance of WT1 expression, we over-expressed GFP- WT1 in Molt-4 cells (T-ALL), HL-60 (AML) and K562 cells (CML) and then quantified mRNA levels of VEGF and the potential WT1 target genes CCNA1 and JAG. The results showed that WT1 levels induced variable expression of VEGF, CCNA1 and JAG in these different leukemic cell lines. Elevated expression of WT1 genes harboring mutations of the zinc finger (ZF) DNA binding domain has also been described in a subset of leukemias and has been associated with a poor prognosis. We therefore screened pediatric acute leukemia samples for novel ZF mutations that would abrogate its ability to regulate VEGF and other target genes. Conversely, a well described SNP rs16754 (in exon 7 of the WT1 gene) identified as a good prognostic marker in Cytogenetically Normal AML (CN-AML) was observed in our pediatric population as both homozygous and heterozygous variants of the WT1 gene. Our long term goal is to determine the molecular basis of the prognostic impact associated with variant WT1 expression in pediatric and adult leukemias. Disclosures: No relevant conflicts of interest to declare.