Simultaneous analyses of N-linked and O-linked glycans of ovarian cancer cells using solid-phase chemoenzymatic method

Glycans play critical roles in a number of biological activities. Two common types of glycans, N-linked and O-linked, have been extensively analyzed in the last decades. N-glycans are typically released from glycoproteins by enzymes, while O-glycans are released from glycoproteins by chemical methods. It is important to identify and quantify both N- and O-linked glycans of glycoproteins to determine the changes of glycans. The effort has been dedicated to study glycans from ovarian cancer cells treated with O-linked glycosylation inhibitor qualitatively and quantitatively. We used a solid-phase chemoenzymatic approach to systematically identify and quantify N-glycans and O-glycans in the ovarian cancer cells. It consists of three steps: (1) immobilization of proteins from cells and derivatization of glycans to protect sialic acids; (2) release of N-glycans by PNGase F and quantification of N-glycans by isobaric tags; (3) release and quantification of O-glycans by β-elimination in the presence of 1-phenyl-3-methyl-5-pyrazolone (PMP). We used ovarian cancer cell lines to study effect of O-linked glycosylation inhibitor on protein glycosylation. Results suggested that the inhibition of O-linked glycosylation reduced the levels of O-glycans. Interestingly, it appeared to increase N-glycan level in a lower dose of the O-linked glycosylation inhibitor. The sequential release and analyses of N-linked and O-linked glycans using chemoenzymatic approach are a platform for studying N-glycans and O-glycans in complex biological samples. The solid-phase chemoenzymatic method was used to analyze both N-linked and O-linked glycans sequentially released from the ovarian cancer cells. The biological studies on O-linked glycosylation inhibition indicate the effects of O-glycosylation inhibition to glycan changes in both O-linked and N-linked glycan expression.https://doi.org/10.1186/s12014-017-9137-

Downregulation of Homologous Recombination DNA Repair Genes by HDAC Inhibition in Prostate Cancer Is Mediated through the E2F1 Transcription Factor

Histone deacetylase inhibitors (HDACis) re-express silenced tumor suppressor genes and are currently undergoing clinical trials. Although HDACis have been known to induce gene expression, an equal number of genes are downregulated upon HDAC inhibition. The mechanism behind this downregulation remains unclear. Here we provide evidence that several DNA repair genes are downregulated by HDAC inhibition and provide a mechanism involving the E2F1 transcription factor in the process.Applying Analysis of Functional Annotation (AFA) on microarray data of prostate cancer cells treated with HDACis, we found a number of genes of the DNA damage response and repair pathways are downregulated by HDACis. AFA revealed enrichment of homologous recombination (HR) DNA repair genes of the BRCA1 pathway, as well as genes regulated by the E2F1 transcription factor. Prostate cancer cells demonstrated a decreased DNA repair capacity and an increased sensitization to chemical- and radio-DNA damaging agents upon HDAC inhibition. Recruitment of key HR repair proteins to the site of DNA damage, as well as HR repair capacity was compromised upon HDACi treatment. Based on our AFA data, we hypothesized that the E2F transcription factors may play a role in the downregulation of key repair genes upon HDAC inhibition in prostate cancer cells. ChIP analysis and luciferase assays reveal that the downregulation of key repair genes is mediated through decreased recruitment of the E2F1 transcription factor and not through active repression by repressive E2Fs.Our study indicates that several genes in the DNA repair pathway are affected upon HDAC inhibition. Downregulation of the repair genes is on account of a decrease in amount and promoter recruitment of the E2F1 transcription factor. Since HDAC inhibition affects several pathways that could potentially have an impact on DNA repair, compromised DNA repair upon HDAC inhibition could also be attributed to several other pathways besides the ones investigated in this study. However, our study does provide insights into the mechanism that governs downregulation of HR DNA repair genes upon HDAC inhibition, which can lead to rationale usage of HDACis in the clinics

A Comprehensive Analysis of FUT8 Overexpressing Prostate Cancer Cells Reveals the Role of EGFR in Castration Resistance

The emergence of castration-resistance is one of the major challenges in the management of patients with advanced prostate cancer. Although the spectrum of systemic therapies that are available for use alongside androgen deprivation for treatment of castration-resistant prostate cancer (CRPC) is expanding, none of these regimens are curative. Therefore, it is imperative to apply systems approaches to identify and understand the mechanisms that contribute to the development of CRPC. Using comprehensive proteomic approaches, we show that a glycosylation-related enzyme, alpha (1,6) fucosyltransferase (FUT8), which is upregulated in CRPC, might be responsible for resistance to androgen deprivation. Mechanistically, we demonstrated that overexpression of FUT8 resulted in upregulation of the cell surface epidermal growth factor receptor (EGFR) and corresponding downstream signaling, leading to increased cell survival in androgen-depleted conditions. We studied the coregulatory mechanisms of EGFR and FUT8 expression in CRPC xenograft models and found that castration induced FUT8 overexpression associated with increased expression of EGFR. Taken together, our findings suggest a crucial role played by FUT8 as a mediator in switching prostate cancer cells from nuclear receptor signaling (androgen receptor) to the cell surface receptor (EGFR) mechanisms in escaping castration-induced cell death. These findings have clinical implication in understanding the role of FUT8 as a master regulator of cell surface receptors in cancer-resistant phenotypes

Unusual presentation of parasitic leiomyoma; a tale of twists and turns

Uterine leiomyoma is the most common benign pelvic tumor of the myometrium, as the prevalence could be as high as 70%. Major risk factors include age between 40–60 years and African descent. It usually presents with abnormal uterine bleeding and/or pelvic pain or pressure. Extra-uterine cases of leiomyoma have been reported including Leiomyomatosis Peritonealis Disseminata (LPD), in which multiple nodules are found in the pelvis, peritoneum, or intestine. The term parasitic leiomyoma has been used in literature to describe a non-disseminating pattern . There is no clear explanation for pathogenesis; however, some reports linked it to previous uterine procedures. We are presenting here a case report of an unusual presentation of extra-uterine leiomyoma in a patient with a remote history of hysterectomy for uterine fibroids

Loss of Cyclin-Dependent Kinase Inhibitor Alters Oncolytic Adenovirus Replication and Promotes More Efficient Virus Production

We elucidate the role of p21/Waf-1, a cyclin-dependent kinase inhibitor, on the oncolytic infection and replication cycle of adenovirus by studying both mRNA and adenoviral proteins expression. We found that infection in the absence of p21 causes a significant increase in adenoviral genomes and late gene expression. Similarly, the oncolytic adenoviral infected p21−/− cells have earlier formation of replication foci and robust replication kinetics that were not observed in the wild type p21/Waf-1 intact cells. These findings suggest a culmination that the presence of intact p21 in host cells causes defects in the oncolytic viral life cycle which results in the production of immature and noninfectious particles

Giant Benign Prostatic Hyperplasia in a Pakistani Patient

“Giant hyperplasia” of the prostate is a rare pathology of the prostate gland. We report one such case, in which a successful retropubic prostatectomy was performed on an elderly male patient in Pakistan. The weight of the resected prostate was 700 g, which is the eighth largest prostate with benign prostatic hyperplasia reported

Development of a glycoproteomic strategy to detect more aggressive prostate cancer using lectin-immunoassays for serum fucosylated PSA

Abstract Background Prostate-specific antigen (PSA) is commonly used as a serum biomarker for the detection of prostate cancer. However, levels of PSA in serum do not reliably distinguish aggressive prostate cancer from non-aggressive disease. Therefore, there is an urgent need for biomarkers that can differentiate aggressive prostate cancers from non-aggressive phenotypes. Fucosylation is one of the glycosylation-based protein modifications. Previously we demonstrated increased levels of serum fucosylated PSA in patients with aggressive prostate cancer using lectin selection followed by PSA immunoassay. Methods We developed two lectin-immunoassays, Lens culinaris agglutinin (LCA) and Aleuria aurantia lectin (AAL) followed by clinical PSA immunoassay and investigated the levels of PSA and its fucosylated glycoforms in serum specimens from prostate cancer patients with different Gleason scores. First, we developed standard curves for lectins enrichment, which were applied to lectin-immunoassay for fucosylated PSA–LCA and PSA–AAL quantification in serum samples. Results Our results showed that both LCA- and AAL-immunoassays detected elevated fucosylated PSA and were correlated with higher Gleason scores but only AAL-immunoassay detected an increased percentage of fucosylated PSA in patient serum with higher Gleason scores. Conclusion We have developed quantitative lectin-immunoassays for serum fucosylated PSA. Our data demonstrated that fucosylated PSA–AAL, % fucosylated PSA–AAL and fucosylated PSA–LCA levels could be effective biomarkers to differentiate aggressive prostate cancer [especially Gleason 7 (4 + 3) or above] from non-aggressive disease. We believe that application of these lectin-immunoassays to a larger patient population is needed to evaluate the clinical utilities of fucosylated PSA using AAL–PSA and LCA–PSA for aggressive prostate cancer