Structural modeling and mRNA expression of epididymal ß-defensins in GnRH immunized boars: A model for secondary hypogonadism in man.

Human secondary hypogonadism is associated with impaired testicular function, however, little is known about its impact on sperm epididymal maturation. Endocrine disruption in the epididymis could impair the secretion of key proteins, such as ß-defensins, responsible for spermatozoa maturation during epididymal transit. This study evaluated the sequence and structural similarities between porcine epididymal ß-defensins porcine ß-defensins (pBD3), pBD4, pBD125, and pEP2C and their human homologs using bioinformatics integrated with information derived from protein databanks. We then verified whether the expression of pBD3, pBD4, pBD125, and pEP2C genes in the testis and epididymis are influenced by disruption of the hypothalamic-pituitary-testicular (HPT) axis in a pig model for male human secondary hypogonadism. Upon modeling porcine ß-defensins, structural and functional analysis confirmed the presence of motifs associated with ß-defensin function, validating the models generated in silico. pBD3 and pBD4 showed acceptable structural alignments with human ß-defensins BDEF103 and BDEF110, respectively. In addition, evaluation of hormonal regulation of ß-defensins was assessed by analyzing the expression of these four ß-defensins in adult boars immunized against gonadotropin-releasing hormone (GnRH). Our results indicate that HPT axis disruption modifies the expression of pBD3, pBD4, pBD125, and pEP2C in boar testis and epididymis, suggesting an endocrine-dependent regulation of ß-defensins in swine epididymis. In conclusion, sequence and structural homology between pBD3 and pBD4 and their human homologs provide a basis for using the pig as a model for the study of human secondary hypogonadism. Further investigation of the human homologs in hypogonadal men could elucidate the connections between fertility and epididymal expression of ß-defensins.

H4K12 and H3K18 Acetylation Associates With Poor Prognosis in Pancreatic Cancer.

BACKGROUND/OBJECTIVES: Epigenetic deregulation may be involved in tumor cell biology, including differentiation, tumor progression, and cell death, and histone acetylation is a major regulatory mechanism of gene transcription. Patterns of global histone modifications have been recently suggested as outcome predictors in cancer patients, but few studies have been conducted on pancreatic ductal adenocarcinomas (PDACs). This study was designed to investigate the predictive value of histone acetylation modifications on PDAC. MATERIALS AND METHODS: A retrospective clinicopathologic analysis was undertaken in 119 patients diagnosed with PDAC between 2005 and 2011, and immunohistochemistry performed with polyclonal antibodies against H4K12ac, H3K9ac, and H3K18ac. Positive nuclear staining for each histone was measured as the intensity and expression, being classified into low-staining or high-staining groups. Results were analyzed in relation to patients' clinicopathologic parameters. RESULTS: There was a positive relationship between tumor differentiation and H4K12ac high scores (P<0.05) and staining with the 3 markers correlated positively with tumor stage (P<0.01). Univariate analysis showed worse survival in patients with high detection levels of H4K12ac (P=0.038) and H3K18Ac (P=0.033). A backwards Cox proportional hazards model analysis revealed the independent prognostic effect of high H4K12ac and H3K18ac levels (hazard ratios of 1.6 and 1.7, respectively, P<0.05), especially for patients at early stages of disease. CONCLUSIONS: We propose that acetylation of H4K12 and H3K18 may be considered valuable prognostic factors for pancreatic cancer, although the mechanism involved needs further investigation. Increasing insights into histone acetylation modifications can ultimately generate new ideas for rational and molecularly based diagnostic and therapeutic approaches.

Ecto-5'-Nucleotidase Overexpression Reduces Tumor Growth in a Xenograph Medulloblastoma Model.

BACKGROUND: Ecto-5'-nucleotidase/CD73 (ecto-5'-NT) participates in extracellular ATP catabolism by converting adenosine monophosphate (AMP) into adenosine. This enzyme affects the progression and invasiveness of different tumors. Furthermore, the expression of ecto-5'-NT has also been suggested as a favorable prognostic marker, attributing to this enzyme contradictory functions in cancer. Medulloblastoma (MB) is the most common brain tumor of the cerebellum and affects mainly children. MATERIALS AND METHODS: The effects of ecto-5'-NT overexpression on human MB tumor growth were studied in an in vivo model. Balb/c immunodeficient (nude) 6 to 14-week-old mice were used for dorsal subcutaneous xenograph tumor implant. Tumor development was evaluated by pathophysiological analysis. In addition, the expression patterns of adenosine receptors were verified. RESULTS: The human MB cell line D283, transfected with ecto-5'-NT (D283hCD73), revealed reduced tumor growth compared to the original cell line transfected with an empty vector. D283hCD73 generated tumors with a reduced proliferative index, lower vascularization, the presence of differentiated cells and increased active caspase-3 expression. Prominent A1 adenosine receptor expression rates were detected in MB cells overexpressing ecto-5'-NT. CONCLUSION: This work suggests that ecto-5'-NT promotes reduced tumor growth to reduce cell proliferation and vascularization, promote higher differentiation rates and initiate apoptosis, supposedly by accumulating adenosine, which then acts through A1 adenosine receptors. Therefore, ecto-5'-NT might be considered an important prognostic marker, being associated with good prognosis and used as a potential target for therapy.

Carboplatin in the treatment of Ewing sarcoma: Results of the first Brazilian collaborative study group for Ewing sarcoma family tumors-EWING1.

BACKGROUND: Large cooperative group studies have shown the efficacy of risk-adapted treatment for Ewing sarcoma. However, validation and local adaptation by National cooperative groups is needed. A multicenter protocol to determine the efficacy and safety of a risk-adapted intensive regimen was developed by the Brazilian cooperative group. PROCEDURE: Patients <30 years old with Ewing sarcoma were eligible. Induction chemotherapy consisted of two cycles of ICE (ifosfamide, carboplatin, and etoposide) followed by two cycles of VDC (vincristine, doxorubicin, and cyclophosphamide), followed by local control. Patients with low risk (LR) disease (localized resectable with normal LDH) received 10 additional alternating courses of IE with VDC. For patients with high-risk (HR) disease (unresectable, pelvic, metastatic, or high LDH), two additional cycles of ICE were given. RESULTS: One-hundred seventy five patients (39% metastatic) were enrolled. Fifty-two patients (29.7%) were LR and 123 (70.3%) were HR. Overall response rate at end of induction was 27.4%. Five-year event-free survival (EFS) and overall survival (OS) estimates were 51.4% and 54.4%, respectively. Patients with localized disease had better outcomes than patients with metastases (5-year EFS 67.9% vs. 25.5%, and 5-year OS 70.3% vs. 29.1%, respectively). On multivariate analysis, the presence of metastatic disease was the only prognostic factor (P < 0.01). CONCLUSION: The VDC/ICE protocol was feasible, and considering the high tumor burden in our population, resulted in comparable results to those reported by cooperative groups in high-income countries. Further adaptation to maximize efficacy and minimize toxicity will be required.

Neutrality of miniSTR D22S1045 marker by Ewing's sarcoma phenotype.

Neutrality investigations of markers with forensic use are important to see if a phenotypic trait is being expressed in relation to the alleles of the marker. MiniSTR marker D22S1045 (locus 22q12.3) is localized near the breakpoint region of the EWS gene (22q12.2), which leads to the development of Ewing's Sarcoma. Analyzing allele frequencies and linkage disequilibrium in Ewing's sarcoma patients and non-affected populations, we found that the marker mD22S1045 was neutral when related to Ewing's Sarcoma.

Antiproliferative activity of the dimeric phloroglucinol and benzophenone derivatives of Hypericum spp. native to southern Brazil.

A large number of plants are known to possess strong antitumor properties. Previous studies have verified the antiproliferative activity of the extracts and fractions from six species of Hypericum spp. growing in southern Brazil. In the present study, the in-vitro antiproliferative effects of two dimeric phloroglucinols (japonicin A and uliginosin B, isolated from Hypericum myrianthum) and two benzophenones (cariphenone A and cariphenone B, isolated from H. carinatum) were investigated against three tumor cell lines (HT-29 - human colon carcinoma cells; U-251 - human glioma cell line, and OVCAR-3 - human ovarian carcinoma cells). In addition, different doses of these compounds were associated with cytotoxic drugs commonly used as chemotherapy in the clinic. Cariphenone A and cariphenone B showed moderate antiproliferative activity against all tumor cell lines at a dose of 100 µg/ml. Unlike benzophenones, japonicin A and uliginosin B exerted antiproliferative effects only in the OVCAR-3 cell line. Moreover, a very strong synergistic effect was demonstrated by the association of subeffective doses of japonicin A with the chemotherapeutic drug paclitaxel, decreasing cellular proliferation of the OVCAR-3 cell line. These preliminary results provide a scientific basis to further pursue these compounds as potential combined therapy for certain tumor types.

Influence of GRPR and BDNF/TrkB signaling on the viability of breast and gynecologic cancer cells.

Neuropeptide and neurotrophin receptors are increasingly important molecular targets in cancer. Scientific findings indicate that compounds blocking gastrin-releasing peptide receptors (GRPR) or tropomyosin receptor kinase (Trk) receptors are likely to have antiproliferative activities against cancer cells. The present study aimed to demonstrate that, in contrast to previous findings, GRPR activation reduces, whereas its blockade increases the viability of breast, ovarian and cervical cancer cell lines. However, consistent with previous studies, Trk inhibition was demonstrated to reduce the viability of these cells. MCF-7 (breast), OVCAR-3 (ovarian) and HeLa (cervical) human cancer cell lines were treated with GRP, the GRPR antagonists RC-3095 and RC-3940-II, brain-derived neurotrophic factor (BDNF) and the Trk antagonist K252α. Cell viability was measured by the MTT assay. Expression of GRPR and BDNF was confirmed with reverse transcription-polymerase chain reaction (RT-PCR). GRP reduced, whereas RC-3940-II enhanced the viability of the three cell lines. Treatment with K252α inhibited the viability of the cell lines, while BDNF increased the viability of OVCAR-3 cells. The results supported the hypothesis that GRPR and BDNF/TrkB signaling regulates cancer cell viability. Most importantly, these findings are the first to demonstrate that GRPR blockade can stimulate, rather than inhibits the viability of breast and gynecologic cancer cell lines.

Histone deacetylase inhibitors reverse CpG methylation by regulating DNMT1 through ERK signaling.

Methylation of CpG repeats in the upstream/promoter regions of genes is an established mechanism of gene silencing in many cell types. DNA methylation results in the recruitment of histone deacetylases (HDACs) to promoter regions, thereby repressing expression of genes. General inhibitors of class I and II HDACs (HDACi), such as sodium butyrate and suberoylanilide hydroxamic acid, suppress the growth of prostate cancer cells in vitro and in vivo. In this study, we investigated the mechanism of re-expression of silenced cell cycle inhibitors and retinoic acid receptor B2 (RARB2). HDACi inhibited cell cycle progression, and reversed promoter methylation and silencing of three tumor suppressor genes: RARB2 and the cell cycle regulating cyclin-dependent kinase inhibitors p16 and p21. HDACi repressed MAP kinase I (ERK) activation and down-regulated DNA (cytosine-5-)-methyltransferase 1 (DNMT1) levels. Direct inhibition of ERK activity similarly decreased DNMT1 protein levels and reversed the basal hypermethylation of the promoters and silencing of the RARB2, p21 and p16 tumor suppressor genes. Suppression of DNMT1 level by siRNA also reversed methylation of these tumor suppressor genes with similar kinetics. Collectively, these data demonstrate that HDACi, by inhibiting ERK activity, regulate DNMT1 and ultimately DNA methylation. These results demonstrate that HDACs regulate gene methylation, in addition to the established and reciprocal ability of CpG methylation to recruit HDACs to repress transcription.

Leukemia virus long terminal repeat activates NFkappaB pathway by a TLR3-dependent mechanism.

The long terminal repeat (LTR) region of leukemia viruses plays a critical role in tissue tropism and pathogenic potential of the viruses. We have previously reported that U3-LTR from Moloney murine and feline leukemia viruses (Mo-MuLV and FeLV) upregulates specific cellular genes in trans in an integration-independent way. The U3-LTR region necessary for this action does not encode a protein but instead makes a specific RNA transcript. Because several cellular genes transactivated by the U3-LTR can also be activated by NFkappaB, and because the antiapoptotic and growth promoting activities of NFkappaB have been implicated in leukemogenesis, we investigated whether FeLV U3-LTR can activate NFkappaB signaling. Here, we demonstrate that FeLV U3-LTR indeed upregulates the NFkappaB signaling pathway via activation of Ras-Raf-IkappaB kinase (IKK) and degradation of IkappaB. LTR-mediated transcriptional activation of genes did not require new protein synthesis suggesting an active role of the LTR transcript in the process. Using Toll-like receptor (TLR) deficient HEK293 cells and PKR(-/-) mouse embryo fibroblasts, we further demonstrate that although dsRNA-activated protein kinase R (PKR) is not necessary, TLR3 is required for the activation of NFkappaB by the LTR. Our study thus demonstrates involvement of a TLR3-dependent but PKR-independent dsRNA-mediated signaling pathway for NFkappaB activation and thus provides a new mechanistic explanation of LTR-mediated cellular gene transactivation.

Mutations that abrogate transactivational activity of the feline leukemia virus long terminal repeat do not affect virus replication.

The U3 region of the LTR of oncogenic Moloney murine leukemia virus (Mo-MuLV) and feline leukemia viruses (FeLV) have been previously reported to activate expression of specific cellular genes in trans, such as MHC class I, collagenase IV, and MCP-1, in an integration-independent manner. It has been suggested that transactivation of these specific cellular genes by leukemia virus U3-LTR may contribute to the multistage process of leukemogenesis. The U3-LTR region, necessary for gene transactivational activity, also contains multiple transcription factor-binding sites that are essential for normal virus replication. To dissect the promoter activity and the gene transactivational activity of the U3-LTR, we conducted mutational analysis of the U3-LTR region of FeLV-A molecular clone 61E. We identified minimal nucleotide substitution mutants on the U3 LTR that did not disturb transcription factor-binding sites but abrogated its ability to transactivate the collagenase gene promoter. To determine if these mutations actually have altered any uncharacterized important transcription factor-binding site, we introduced these U3-LTR mutations into the full-length infectious molecular clone 61E. We demonstrate that the mutant virus was replication competent but could not transactivate cellular gene expression. These results thus suggest that the gene transactivational activity is a distinct property of the LTR and possibly not related to its promoter activity. The cellular gene transactivational activity-deficient mutant FeLV generated in this study may also serve as a valuable reagent for testing the biological significance of LTR-mediated cellular gene activation in the tumorigenesis caused by leukemia viruses.