Metabolite profiling of blood plasma of patients with prostate cancer

Prostate cancer is one of the most common types of cancer in men. It is though extremely important to search for specific markers including metabolites, which concentration in blood could be a diagnostic measure. In this regard, the metabolite profiling of blood plasma was performed with two groups of people: healthy volunteers (n = 30) and patients with prostate cancer, second stage (n = 40). The profiling protocol included proteins removal from blood plasma with methanol and direct analysis of metabolite fractions by mass spectrometry. Identification of the most abundant metabolites in samples was performed using an accurate mass tag and an isotope pattern methods. Cancer-specific metabolites were revealed by statistical analysis of metabolite intensities in the mass spectra. Six different metabolites were found to be cancer-specific. Two metabolites, acylcarnitine and arachidonoyl amine, have the AUC 0.97 and 0.86, respectively, which are higher than those from PSA test, 0.59.     

The expression and functional activities of smooth muscle myosin and non‐muscle myosin isoforms in rat prostate

Benign prostatic hyperplasia (BPH) is mainly caused by increased prostatic smooth muscle (SM) tone and volume. SM myosin (SMM) and non‐muscle myosin (NMM) play important roles in mediating SM tone and cell proliferation, but these molecules have been less studied in the prostate. Rat prostate and cultured primary human prostate SM and epithelial cells were utilized. In vitro organ bath studies were performed to explore contractility of rat prostate. SMM isoforms, including SM myosin heavy chain (MHC) isoforms (SM1/2 and SM‐A/B) and myosin light chain 17 isoforms (LC_17a/b), and isoform ratios were determined via competitive RT‐PCR. SM MHC and NM MHC isoforms (NMMHC‐A, NMMHC‐B and NMMHC‐C) were further analysed via Western blotting and immunofluorescence microscopy. Prostatic SM generated significant force induced by phenylephrine with an intermediate tonicity between phasic bladder and tonic aorta type contractility. Correlating with this kind of intermediate tonicity, rat prostate mainly expressed LC_17a and SM1 but with relatively equal expression of SM‐A/SM‐B at the mRNA level. Meanwhile, isoforms of NMMHC‐A, B, C were also abundantly present in rat prostate with SMM present only in the stroma, while NMMHC‐A, B, C were present both in the stroma and endothelial. Additionally, the SMM selective inhibitor blebbistatin could potently relax phenylephrine pre‐contracted prostate SM. In conclusion, our novel data demonstrated the expression and functional activities of SMM and NMM isoforms in the rat prostate. It is suggested that the isoforms of SMM and NMM could play important roles in BPH development and bladder outlet obstruction.     

Regulation of the expression of protein kinase C isoenzymes in rat ventral prostate: effects of age,castration and flutamide treatment

Protein kinase C (PKC) isoenzymes are involved in cell function, growth, apoptosis and neoplastic transformation in the prostate gland. We detected by means of Western blot the expression of the classical alpha and beta1, the novel epsilon and the atypical zeta isoforms of PKC in ventral prostates from rats with different extents of plasma testosterone levels and/or androgen imprinting on the gland. The expression of the four isoforms decreased in 5-day castrated rats showing apoptotical regression of the gland and a drastic reduction of circulating testosterone. However, the expression of PKC isoenzymes (alpha, beta1, epsilon ) increased in prostates from pubertal (35-days old) rats that are characterized by relatively low but extremely bioactive testosterone plasma levels. Treatment of adult rats for 14 days with flutamide (daily s.c. injection of 15 mg/Kg B.W.) resulted in increased expression of the four isoenzymes; it occurred in the presence of increased (normal rats) or drastically reduced (rats castrated after 9 days of flutamide administration) levels of plasma testosterone conceivably through a direct effect of this nonsteroidal antiandrogen on prostate cells. Measurements of PKC(alpha) activity were in agreement with the observations on protein expression and showed that flutamide (that is extensively used in the treatment of advanced prostate cancer) elicits some impairment in the mechanisms of translocation of this isoform from the cytosol to the membrane. Thus, in addition to the possibility of direct effects of flutamide upon the rat prostate, we present evidence that the levels of circulating androgens and/or their bioactivity in the gland regulate the expression of various important PKC isoforms.     

Biomarkers for prostate cancer

The detection of prostate cancer using a blood test has by many standards changed the face of the disease. Despite this tremendous success, there are limitations attributed to the use of prostate specific antigen (PSA) as a means to screen and detect prostate cancer. PSA, as its name implies, is not specific for prostate cancer and as such is often found elevated in other prostatic diseases/symptoms associated with the aging male. Clearly, more specific marker(s) that could identify which individuals actually have prostate cancer and differentiate them from those without the disease would be of tremendous value. The search for more accurate and clinically useful biomarkers of prostate cancer has been extensive. This has focused on individual markers, as well as groups of markers. Included among these are PSA isoforms, pathological indicators and stains, nucleic acids and others. This article highlights the discovery of PSA as a first blood‐based biomarker for prostate cancer detection, as well as other molecular biomarkers and their potential application in detection of the disease. J. Cell. Biochem. 108: 3–9, 2009. © 2009 Wiley‐Liss, Inc.     

Knockdown of Akt isoforms by RNA silencing suppresses the growth of human prostate cancer cells in vitro and in vivo

The serine/threonine kinase Akt has three highly homologous isoforms in mammals: Akt1, Akt2, and Akt3. Recent studies indicate that Akt is often constitutively active in many types of human malignancy. Here we investigated the expression and function of Akt isoforms in human prostatic carcinoma cells. Initially, we used Western blotting to examine Akt expression in four human prostate cancer cell lines. Next, small-interfering RNAs (siRNAs) specific for Akt isoforms were used to elucidate their role on the in vitro and in vivo growth of prostate cancer cells. Expression of Akt1 and Akt2 was detected in all cells tested, but Akt3 was expressed only in cancer cells that did not express androgen receptors. All synthetic siRNAs against Akt isoforms suppressed their expression and inhibited the growth of cancer cells in vitro. Furthermore, atelocollagen-mediated systemic administration of siRNAs significantly reduced the growth of tumors that had been subcutaneously xenografted. These results suggest that targeting Akt isoforms could be an effective treatment for prostate cancers.     

Plasma netrin-1 is a diagnostic biomarker of human cancers

Context:?The axon guidance cues netrin-1 is a secreted protein overexpressed in many different cancer tissues.

Objectives:?To determine whether plasma netrin-1 can be used as a diagnostic biomarker of human cancer.

Materials and Methods:?A total of 300 cancer plasma samples from breast, renal, prostate, liver, meningioma, pituitary adenoma, glioblastoma, lung, pancreatic and colon cancer patients were compared against 138 control plasma samples. Netrin-1 levels were quantified by ELISA and immunohistochemistry.

Results:?Plasma netrin-1 levels were significantly increased in breast, renal, prostate, liver, meningioma, pituitary adenoma, and glioblastoma cancers as compared to control samples.

Discussion and Conclusion:?Our results suggest that plasma netrin-1 can be used as a diagnostic biomarker for many human cancers.     

Proteomic analysis of N‐glycosylation of human seminal plasma

Seminal plasma is a mixture of secretions from several male accessory glands. The seminal plasma contains many secreted proteins which are important for sperm function and male fertility. In this study, we employed N‐linked glycosylated peptide enrichment, combined with LC–MS/MS analysis, and establish the first large scale N‐linked glycoproteome of human seminal plasma. Combined with the results of five biological replicates, a total of 720 N‐glycosylated sites on 372 proteins were identified. Analysis of variations among five individuals revealed similar compositions of N‐glycosylated proteins in seminal plasma. The N‐linked glycoproteome could help us understanding the biological functions of human seminal plasma. The data set could also be a resource for further screening of biomarkers for male diseases including cancer and infertility at the level of N‐glycosylation. For example, N‐glycosylated prostate‐specific antigen is known to be an efficient biomarker that can distinguish benign prostate hyperplasia from prostate cancer. All MS data have been deposited in the ProteomeXchange with identifier PXD000959 ( http://proteomecentral.proteomexchange.org/dataset/PXD000959 ).     

Effect of Influenza Virus Proteins Modulate Hemostasis in vitro and in vivo

We studied the effect of influenza virus proteins—hemagglutinin, neuraminidase, nucleoprotein, and membrane protein—on hemostasis in vitro and in vivo. The results demonstrated that envelope proteins hemagglutinin and neuraminidase enhanced the fibrinolytic and anticoagulant activities of blood plasma and the activity of human tissue plasminogen activator. The membrane protein proved to have the highest activity among the core proteins of influenza virus; in contrast to hemagglutinin and neuraminidase, it inhibited fibrinolysis, increased the coagulant activity of blood plasma, and decreased the activity of human tissue plasminogen activator. The combined action of hemagglutinin and neuraminidase increased the plasma fibrinolytic and anticoagulant activities to a greater extent than the individual action of each agent. The combined action of hemagglutinin and membrane protein also increased the plasma fibrinolytic and anticoagulant activities, although to a lesser extent than the action of hemagglutinin alone. These data indicate that viral proteins are physiologically active and can cause influenza-specific disturbances of hemostasis.     

A comparative study of glycoproteomes in androgen-sensitive and -independent prostate cancer cell lines

Prostate cancer is one of the most common malignancies in men and is predicted to be the second leading cause of cancer-related deaths. After 6–18 months, hormone ablation treatment results in androgen-independent growth of cancer cells, metastasis and progression. The mechanism of androgen-independent growth of prostatic carcinoma cells is still unknown. Identification of factors that facilitate the transition from androgen-dependent to independent states is crucial in designing future diagnostics and medication strategies. To understand the biochemical meaning of hormone dependency deprivation, glycoproteins enriched profiles were compared between DU145 (hormone non-responding) and LNCaP (hormone responding) prostate cancer cells. These results allow for anticipation on the important role of glycosylation in malignant transformation. Both Tn antigen and complex antennary N-oligosaccharides were recognized. Their occurrence might be involved in the development and progression of tumor, and failure of hormone ablation therapy. Among identified proteins in androgen-sensitive cells nucleolin (P19338) was found that is widely described as apoptosis inhibitor, and also transporter of molecules from the membrane to the cytoplasm or nucleus. In addition, 14-3-3 protein family (P27348, P31946, P61981, P63104, P62258, Q04917, and P31947) was investigated across available databases as it forms stable complexes with glycoproteins. Our studies indicate that isoforms: sigma and eta were found in androgen-dependent prostate cancer cells, while other isoforms were present in androgen non-responding cells. 14-3-3 binding partners are involved in cancer pathogenesis. These findings may contribute to a better understanding of prostate cancer tumorigenesis and to a more efficient prognosis and individual therapy in a future. However, it still remains to be revealed how important those changes are for androgen dependency loss in prostate cancer patients carried out on clinically relevant populations.     

Quercetin can be a more reliable treatment for metastatic prostate cancer than the localized disease: An in vitro study

Quercetin is a plant flavonoid that has been recognized to have anti-inflammatory, antioxidant and anti-proliferative activities. This study aims to evaluate the inhibitory effects of quercetin against prostate malignancy in vitro and the underlying resistance mechanism. IC₅₀ values of quercetin were determined by MTT assay. Annexin-V/PI staining was used to measure the rate of apoptosis. DNA cell cycle was analysed by PI staining method. Real-time PCR was performed to assess mRNA levels of OPN isoforms, VEGF isoforms, P53 and KLK2. Migration potential, proliferative capability and nucleus morphology of cells were evaluated by the scratch-wound assay, colony-forming assay and Hoechst staining, respectively. Quercetin significantly increased the apoptosis rate of PC-3 and LNCaP cell lines, arrested the cell cycle at the sub-G1/G1 phase, and reduced the migration potential and colony-forming capability. Moreover, upregulation of apoptosis-related genes and downregulation of genes involved in proliferation and angiogenesis was also observed. Although our results elucidated that quercetin has antitumor effects on PC-3 and LNCaP, for the first time, we showed that quercetin treatment causes alterations in the expression of OPN and VEGF isoforms, which are cancer-promoting modulators through various processes such as angiogenesis and drug-resistance. Prostate malignant cells can dodge the anti-carcinogenic properties of quercetin via modulation of OPN and VEGF isoforms in vitro. Therefore, quercetin acts as a double-edged sword in prostate cancer treatment.     

Detection of aggressive prostate cancer associated glycoproteins in urine using glycoproteomics and mass spectrometry

Clinical management of prostate cancer remains a significant challenge due to the lack of available tests for guiding treatment decisions. The blood prostate‐specific antigen test has facilitated early detection and intervention of prostate cancer. However, blood prostate‐specific antigen levels are less effective in distinguishing aggressive from indolent prostate cancers and other benign prostatic diseases. Thus, the development of novel approaches specific for prostate cancer that can differentiate aggressive from indolent disease remains an urgent medical need. In the current study, we evaluated urine specimens from prostate cancer patients using LC‐MS/MS, with the aim of identifying effective urinary prostate cancer biomarkers. Glycoproteins from urine samples of prostate cancer patients with different Gleason scores were characterized via solid phase extraction of N‐linked glycosite‐containing peptides and LC‐MS/MS. A total of 2923 unique glycosite‐containing peptides were identified. Glycoproteomic comparison on urine and tissues from aggressive and non‐aggressive prostate cancers as well as sera from prostate cancer patients revealed that the majority of AG prostate cancer associated glycoproteins were more readily detected in patient's urine than serum samples. Our data collectively indicate that urine provides a potential source for biomarker testing in patients with AG prostate cancer.     

Fatty acid regulation of protein kinase C isoforms in prostate cancer cells

Polyunsaturated fatty acids influence the aetiology of prostate cancer. Their effects on cellular mechanisms regulating prostate tumorigenesis are unclear. Using prostate cancer cells (LNCaP), we determined effects of n-9-OA, n-6-LA, and n-3-EPA on total PKC and its isoforms in relation to cell proliferation and PSA production. PKC-alpha, delta, gamma, iota, mu, and zeta were present in LNCaP cells; PKC-beta, epsilon, eta, and theta isoforms were not. PKC-alpha was detected only in cytosol; PKC-delta, iota, gamma, and mu were present in cytosol and in membranes. Fatty acids increased cell proliferation, total PKC activity and elicited pro-proliferative effects on specific PKC isoforms (PKC-delta and -iota). EPA and LA increased total PKC activity and reduced membrane-abundance of PKC-delta. OA reduced cytosolic and membrane PKC-delta. Only EPA reduced PKC-gamma membrane abundance. Fatty acids enhanced cytosolic PKC-iota abundance but only EPA and to a lesser extent LA increased its membrane content. Changes in PKC-delta, -iota, and -gamma did not affect PSA production.     

Establishment of a three-dimensional human prostate organoid coculture under microgravity-simulated conditions: Evaluation of androgen-induced growth and psa expression

Summary A novel in vitro human prostate cancer model was established by using a coculture technique in which isolated human prostate fibroblasts were observed to grow as a mixed culture with isolated human prostate cancer cells (LNCaP) on microcarrier beads under microgravity-simulated conditions. This model appears to be promising and deserves further exploration because: (a) cocultured human prostate fibroblasts and cancer epithelial cells appear to undergo patterns of histogenesis similar to those observed in human prostate tumors and (b) unlike the conventional cell culture on plastic dishes, cocultured human prostate fibroblasts and LNCaP cells in microgravity-simulated conditions responded to the inductive signals of growth and differentiation from dihydrotestosterone in a manner similar to that observed in the in vivo condition. These results offer an opportunity to examine molecular mechanisms of cellular signaling in response to androgen stimulation during normal and aberrant human prostate development. The microgravity-simulated three-dimensional prostate epithelial cell culture with prostate fibroblasts can be further explored as an ideal in vitro model for the study of normal and neoplastic prostate development. This model could also be adopted as a drug screening program for the discovery of novel therapeutic agents in the treatment of human prostate cancer and benign hyperplastic growth.     

Levels of plasma glycan-binding auto-IgG biomarkers improve the accuracy of prostate cancer diagnosis

Strategies to improve the early diagnosis of prostate cancer will provide opportunities for earlier intervention. The blood-based prostate-specific antigen (PSA) assay is widely used for prostate cancer diagnosis but specificity of the assay is not satisfactory. An algorithm based on serum levels of PSA combined with other serum biomarkers may significantly improve prostate cancer diagnosis. Plasma glycan-binding IgG/IgM studies suggested that glycan patterns differ between normal and tumor cells. We hypothesize that in prostate cancer glycoproteins or glycolipids are secreted from tumor tissues into the blood and induce auto-immunoglobulin (Ig) production. A 24-glycan microarray and a 5-glycan subarray were developed using plasma samples obtained from 35 prostate cancer patients and 54 healthy subjects to identify glycan-binding auto-IgGs. Neu5Acα2-8Neu5Acα2-8Neu5Acα (G81)-binding auto-IgG was higher in prostate cancer samples and, when levels of G81-binding auto-IgG and growth differentiation factor-15 (GDF-15 or NAG-1) were combined with levels of PSA, the prediction rate of prostate cancer increased from 78.2% to 86.2% than with PSA levels alone. The G81 glycan-binding auto-IgG fraction was isolated from plasma samples using G81 glycan-affinity chromatography and identified by N-terminal sequencing of the 50 kDa heavy chain variable region of the IgG. G81 glycan-binding 25 kDa fibroblast growth factor-1 (FGF1) fragment was also identified by N-terminal sequencing. Our results demonstrated that a multiplex diagnostic combining G81 glycan-binding auto-IgG, GDF-15/NAG-1 and PSA (≥?2.1 ng PSA/ml for cancer) increased the specificity of prostate cancer diagnosis by 8%. The multiplex assessment could improve the early diagnosis of prostate cancer thereby allowing the prompt delivery of prostate cancer treatment.

     

Variations of components of the plasminogen activation system with the cell cycle in benign prostate tissue and prostate cancer.

Background: Components of the fibrinolytic system are involved in tumor cell invasion and metastasis. Previous investigations suggested a cell cycle-dependent expression of urokinase-type plasminogen activator (u-PA) in epithelial cells. In order to determine a correlation of cell cycle phases with the fibrinolytic system, we investigated the expression of u-PA, tissue-type plasminogen activator (t-PA), and plasminogen activator inhibitor type 1 (PAI-1) in normal and tumor-containing prostate extracts and analyzed a possible relationship with flow cytometry-determined proliferative activity of the samples. Cell cycle phases were correlated with fibrinolytic parameters in prostate tissue. Methods: Samples were obtained from patients undergoing radical prostatectomy for prostate cancer and separated into two portions for DNA analysis and the detection of u-PA, t-PA, and PAI-1. Flow cytometric analysis was performed according to the Vindelov technique. The concentrations of u-PA, t-PA, and PAI-1 were determined from tissue extracts after homogenization by an enzyme-linked immunosorbent assay (ELISA) technique. Results: Correlations of u-PA and t-PA expression with the frequency of G0/G1, S, G2M, S-phase fraction (SPF), and proliferation index (PI) for normal prostate and prostate cancer revealed no significant correlation. The only significant finding was observed in normal tissue revealing a positive correlation between PAI-1 expression and G0/G1 and a negative correlation with S-phase, SPF, and PI. No dependence of PAI-1 expression on different cell phases was found in prostate cancer. Furthermore, no significant correlation of u-PA, t-PA, and PAI-1 with cell cycles in organ-confined ( or = pT3a) tumors was found. No significant correlation in prostate cancer of components of the fibrinolytic system differentiated according to tumor grade or perineural tumor infiltration and cell cycle analysis was found. Only in highly differentiated G1 (Gleason 2-4) cancer, u-PA had a significant positive correlation with G2M-phase. Conclusion: Absence of a correlation between levels of components of the fibrinolytic system and cell cycle phases suggests that the reported association between increases of some of these components and aggressive biological behavior of prostate cancer is secondary to non-cell cycle-related mechanisms.     

Monitoring mouse prostate development by profiling and imaging mass spectrometry

Mass spectrometry-based tissue profiling and imaging are technologies that allow identification and visualization of protein signals directly on thin sections cut from fresh frozen tissue specimens. These technologies were utilized to evaluate protein expression profiles in the normal mouse prostate during development (1-5 weeks of age), at sexual maturation (6 weeks of age), and in adult prostate (at 10, 15, or 40 weeks of age). The evolution of protein expression during normal prostate development and maturation were subsequently compared with 15-week prostate tumors derived from genetically engineered mice carrying the Large T antigen gene under regulation of the prostate-specific probasin promoter (LPB-Tag mouse model for prostate cancer). This approach identified proteins differentially expressed at specific time points during prostate development. Furthermore expression of some of these proteins, for example probasin and spermine-binding protein, were associated with prostate maturation, and prostate tumor formation resulted in their loss of expression. Cyclophilin A, a protein found in other cancers, was differentially alpha-acetylated on the N terminus, and both isoforms appeared during normal prostate and prostate tumor development. Imaging mass spectrometry localized the protein signals to specific prostatic lobes or regions. Thus, tissue profiling and imaging can be utilized to analyze the ontogeny of protein expression during prostate morphogenesis and tumorigenesis and identify proteins that could potentially serve as biomarkers for prostate cancer.