Xiaopeng Liu

Profiling protein markers associated with lymph node metastasis in prostate cancer by DIGE-based proteomics analysis.

Current predictive tools and imaging modalities are not accurate enough for preoperative diagnosis of lymph node metastatic prostate cancer (LNM PCa). Proteomic analysis is introduced to screen potential biomarkers for early detection of LNM PCa. In our initial study, protein samples from localized and LNM PCa as well as benign prostatic hyperplasia tissues were analyzed using two-dimensional fluorescence difference in gel electrophoresis (2-D DIGE) coupled with MALDI-TOF/TOF MS. We identified 58 proteins that were differentially expressed in the LNM PCa group relative to the localized PCa group. Six of these proteins, e-FABP5, MCCC2, PPA2, Ezrin, SLP2, and SM22, are functionally relevant to cancer metastasis. Expression of these proteins was therefore further validated in tissue samples from the original cohort and also from a larger, independent cohort of patients using real time PCR, Western blotting, and immunohistochemistry staining. In addition, the serum levels of e-FABP5 were also examined by ELISA. Relative to localized PCa tissues, LNM PCa tissues had increased expression of e-FABP5, MCCC2, PPA2, Ezrin, and SLP2 and decreased expression of SM22. Patients with LNM PCa had significantly higher levels of serum e-FABP5. This study presents evidence that increased expression of e-FABP5, MCCC2, PPA2, Ezrin, and SLP2 and decreased expression of SM22 are useful diagnostic markers for the existence of LNM PCa.

Comparative analysis of whole saliva proteomes for the screening of biomarkers for oral lichen planus

Abstract.ObjectiveTo screen possible biomarkers in whole saliva from oral lichen planus (OLP) patients by proteomic techniques.MethodsTwo-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were applied to whole saliva of OLP patients and controls, and the differential protein components were analyzed with peptide mass fingerprinting and database searching.ResultsA total of 31 protein spots representing 14 proteins with at least two-fold difference in abundance between OLP and controls were identified. Among these, the expression of urinary prokallikrein was increased while short palate, lung and nasal epithelium carcinoma associated protein (PLUNC) was decreased in all samples of OLP.ConclusionsUrinary prokallikrein and PLUNC may be the new biomarkers which play a role in inflammation and immune response of OLP.

Toll-like receptor 9 agonists promote IL-8 and TGF-β1production via activation of nuclear factor κB in PC-3 cells

Chronic infection and resulting inflammation promote tumor development and progression, and Toll-like receptors (TLRs) may play an important role in this process. The aim of this study was to determine whether CpG oligonucleotides (CpG-ODN), which are Toll-like receptor 9 (TLR9) agonists, can promote inflammatory cytokines release from the prostate cancer PC-3 cells through activation of nuclear factor-kappaB (NF-kappaB). Flow cytometry, semiquantitative real-time reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, and immunofluorescence analysis were used to detect the transforming growth factor-beta1 (TGF-beta1) and interleukin-8 (IL-8) release and NF-kappaB activation in PC-3 cells after CpG-ODN stimulation. CpG-ODN promoted the expression and secretion of immunosuppressive cytokines TGF-beta1 and IL-8 from PC-3 cells. In addition, after CpG-ODN stimulation, NF-kappaB nuclear translocation was also observed in PC-3 cells, contributing to CpG-induced upregulation of IL-8 and TGF-beta1. Thus, TLR9 agonists may promote IL-8 and TGF-beta1 production in human prostate cancer cells through NF-kappaB activation.

Dendritic cells transduced with a PSMA-encoding adenovirus and cocultured with autologous cytokine-induced lymphocytes induce a specific and strong immune response against prostate cancer cells

OBJECTIVE The lack of curative therapies for advanced prostate cancer (PCa) has prompted a search for novel treatments such as immunotherapy. In this study, we analyzed whether dendritic cells (DCs) from healthy donors transduced with a PSMA-encoding adenovirus (Ad-PSMA) and cocultured with autologous cytokine-induced killer cells (CIKs) can induce a strong specific immune response against PCa cells in vitro. MATERIALS AND METHODS Ad-PSMA was constructed by DNA recombination. DCs and CIKs were prepared by cytokines induction from peripheral blood mononuclear cells, and flow cytometry was used to measure the phenotypes of DCs and CIKs. DCs were transduced with Ad-PSMA and then cocultured with autologous CIKs. The cytotoxicity of the cocultured cells against specific target LNCaP cells and control targets DU145 and PC3 cells was analyzed by a 4-h LDH release assay. RESULTS DCs were transduced with Ad-PSMA with transfection efficiency of 70% and the transduction did not alter typical morphology of mature DCs. The PSMA protein was effectively expressed in DCs, which were transfected with Ad-PSMA. Ad-PSMA-transduced DCs stimulated CIKs strongly to lyse about 75% of PSMA-expressing PCa cells. Furthermore, the cocultivation of Ad-PSMA-transduced DCs with CIKs could significantly increase the production of interferon-gamma after restimulated with PSMA peptide mixtures. CONCLUSIONS The data demonstrate that DCs, which were transduced with a PSMA-expressing adenovirus and cocultured with autologous CIKs, induce a PSMA-specific, strong immune response against PCa cells. Therefore, this approach may have a potential for an adoptive immunotherapy for patients with advanced PCa.

Proteomic analysis of rat penile tissue in a model of erectile dysfunction after radical prostatectomy

To investigate the effect of the vitamin D receptor (VDR) FokI polymorphism on the clinical presentation of calcium urolithiasis, as a FokI polymorphism in the VDR gene was recently reported to be associated with calcium metabolism disorders.OBJECTIVE To identify differential protein expression in penile tissue in a rat model of erectile dysfunction (ED) at an early stage after bilateral cavernosal nerve (CN) neurectomy, using proteomic techniques. MATERIALS AND METHODS Twelve male adult Sprague-Dawley rats were randomly divided into two equal groups, one having bilateral CN resection and one a control group. The penises were harvested 7 days after CN resection. Total protein was separated into >1250 protein spots by two-dimensional electrophoresis using pH 3-10 nonlinear immobilized pH gradient strips. Differential expression of proteins was analysed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and database searching. RESULTS Thirty-two proteins were significantly changed in the denervated penis, of which 25 (including nine up-regulated and 16 down-regulated) with cytoskeletal functions, and pathophysiological functions related to energy metabolism and oxidative stress, were identified. Examples include transgelin, creatine kinase B, annexin-1 and galactin-7. CONCLUSIONS The expression of several important proteins participating in pathophysiological processes of penile tissue are changed early after bilateral CN neurectomy. These changes might give new insights into the cellular and molecular mechanisms involved in neurogenic ED development, and indicate potential therapeutic targets.

A new experimental inbred Wistar rat varicocele model: anatomy of the left spermatic vein and the effect on histology

Because of venous anatomical differences between rats and humans and the personal interpretation of these differences, there is neither consistent animal prototype nor consistent results in the study of varicocele. We established a new substrain of Wistar inbred rats, of which the left testis vein has no significant branches to the common iliac vein up pampiniform plexus, but instead enters the left renal vein directly (similar to humans) and used them to create experimental varicocele model by partial ligation of the left renal vein. One month later, the predominant lesion of the left testis in induced group was spermatogenic arrest at the spermatid and preliminary spermatocyte phases, and considerable interstitial and Sertoli cell vacuolation. The right testis also showed spermatogenic arrest. Most important, the characteristics of the lesions differed in both testes, with the left testis having more severe lesions. Allowing for the unique anatomy of the left spermatic vein, the standard of the surgical procedure, the high rate of varicocele induction, and identical histological alteration as occurs in humans, we believe that this inbred Wistar rat substrain is suitable for the creation of an experimental varicocele model, which has promise for practical application in humans.

Loupe-assisted versus microscopic varicocelectomy: is there an intraoperative anatomic difference?

The aim of this study was to compare the intraoperative difference in anatomic details between loupe-assisted and microscopic varicocelectomy within the same spermatic cord. Between April 2011 and August 2011, 26 men with 33 sides containing grade 2-3 varicocele were enrolled in this study. First, one surgeon performed the open inguinal varicocelectomy under × 3.5 loupe magnification. The presumed vascular channels and lymphatics were isolated and marked without ligation. Another surgeon then microsurgically dissected and checked the same spermatic cord using an operating microscope to judge the results in terms of the ligation of the internal spermatic veins and the preservation of the arteries and lymphatics. There were significant differences in the average number of internal spermatic arteries (1.51 vs 0.97), internal spermatic veins (5.70 vs 4.39) and lymphatics (3.52 vs 1.61) between the microscope and loupe-assisted procedures (P < 0.001, P < 0.001, P < 0.001, respectively). Meanwhile, in varicocele repair with loupe magnification, an average of 1.30 ± 1.07 (43/33) internal spermatic veins per side were missed, among the overlooked veins, 1.12 ± 0.93 (37/33) were adhered to the preserved testicular artery, as well as 0.55 ± 0.79 lymphatics and 0.36 ± 0.55 arteries that were to be ligated. In conclusion, microscopic varicocelectomy could preserve more internal spermatic arteries and lymphatics and could ligate more veins than the loupe-assisted procedure. To some degree, loupe magnification is inadequate for the reliable identification and dissection of the tiny vessels of the spermatic cord, as most of the overlooked veins were adhered to the preserved testicular artery.

Enhanced antitumor effects by the coculture of allotumor RNA-pulsed dendritic cells with autologous cytokine-induced killer cells on hormone-refractory prostate cancer.

In this study, we evaluated antitumor effects of allotumour RNA-transfected dendritic cells (DCs) cocultured with autologous cytokine-induced killer cells (CIKs) on hormone-refractory prostate cancer. The cocultured cells enhanced prostate cancer cytolysis from 26% (CIKs-induced cytolysis) to 80.8%. They also increased the productions of CD4+ Th1 (IFN-γ+IL-4−, 55.52%) and CD8+ T (IFN-γ+, 69.59%) cells determined by intracellular cytokines IFN-γ /IL-4 staining and reduced the rate of CD4+ CD25+ cells from 18.72% (in CIKs) to 9.72%. The cocultured cells significantly inhibited tumor growth in SCID mouse and induced cancer cells necrosis and apoptosis. Our study indicates that tumor RNA-pulsed DCs cocultured with autologous CIKs significantly enhance antitumor immunity, which can be induced by increased CD4+ Th1 and CD8+ T cells and decreased CD4+CD25+ regulatory T (Treg) cells. This provides a potential immunotherapy strategy for HRPC.

MP66-13 14-3-3 EPSILON MAY PLAY AN IMPORTANT ROLE IN TESTICULAR GERM CELL APOPTOSIS: A FUNCTIONAL PROTEOMIC STUDIES OF EXPERIMENTAL VARICOCELE

INTRODUCTION AND OBJECTIVES: Small RNA molecules, including small interfering RNAs (siRNAs), microRNAs (miRNAs), and piwi-interacting RNAs (piRNAs) have been found to be exclusively expressed in certain stages of spermatogenesis and have been detected as free circulated nucleic acids in seminal plasma of ejaculate. Vasectomy enables distinguishing between miRNAs originated form testis and epididymis, because after vasectomy seminal plasma contains only secretions of prostate and seminal vesicles. The aim of this study was to identify germcells andSertoli cells specificmiRNA in seminal plasmaasa noninvasive diagnostic parameter predicting fertility and successful sperm retrieval of infertile patients. METHODS: miRNA fraction has been enriched from seminal plasma of healthy men before and 6 weeks after vasectomy as well as from patient with Sertoli Cell Only Syndrom (SCO) using HighPure miRNA Isolation Kit (Roche, USA). The expression profile of 84 miRNATargets from each sample applying (miRNA Finder;Qiagen) have been investigated. cDNA synthesis and preamplification was performed using universal primer mix PreAmp PCR-Mix (Qiagen) and quantitative real time PCR was carried out. The calculation of relative miRNA expression was performed using SABioscience web tool http://pcrdataanalysis. sabiosciences.com.mirna. RESULTS: Differential expression of 84 known target miRNAs has been found comparing seminal plasma content prior and after vasectomy. We observed significant overexpression of 56 miRNAs (4-443 fold) 6 weeks after vasectomy, while 7 miRNAs were downregulated(0.85-2fold). Thehighest expressionof hsa-miR-141-3pand the lowest level of hsa-miR-181a-5p has been indicated after vasectomy. Furthermore, we found 26 overexpressed miRNAs in patient with SCO (5-50 fold) and 3 miRNAs that were significantly downregulated (5-14 fold); hsa-miR-210; hsa-miR-210; hsa-miR-22-3p). The comparative analysis allowed selection of 19 testicular/epidydimal miRNAs. CONCLUSIONS: In this pilot project, we successfully identified miRNAs circulating in seminal plasma and demonstrated that vasectomy modulates the expression level of 84 miRNAs. Genome wide Next Generation Sequencing and alignment of miRNAs to their target sequence will possibly unravel complex mechanism of mRNA interference for silencing of gene key factors of spermatogenesis. From clinical point of view, epigenetic biomarker in semen might facilitate diagnosis of infertility and constitute a noninvasive method predicting the outcome for surgical sperm retrieval.

GW24-e3520 Association of cardiovascular disease and erectile dysfunction: share the same risk factors?

Objectives To evaluate the association between the risk factors for cardiovascular disease (CVD) and erectile dysfunction (ED). Methods Relevant studies published for the time range 1992-2012 were searched thoroughly in Medline. Abstracts regarding the main epidemiological evidences on the association between the risk factors for CVD and ED were conducted to briefly review. Results A growing number of consistent epidemiological evidences suggest CVD and ED share substantially plenty of the same risk factors (OR: 1.93; 95%CI: 1.48-3.27, P < 0.05), which include age, hyperlipidemia, obesity, hypertension, diabetes mellitus, depression and smoking. The pathogenesis of both CVD and ED has been shown to be common conditions of endothelial dysfunction. They were linked through decreased expression and activation of endothelial nitric oxide synthase (eNOS) occurring with ageing. ED may be an early symptom of underlying systemic vascular disease especially coronary artery disease (CAD) rather than itself. Approximately 50-70% of men with CAD have ED. ED can arise before CAD is symptomatic with a time window of 3–5 years. Conclusions These reviews reveal that ED is beginning to be considered as an early manifestation of underlying CVD. It is a potential risk predictor for the subsequent development of CVD.