Androgen Regulated Genes in Human Prostate Xenografts in Mice: Relation to BPH and Prostate Cancer

Benign prostatic hyperplasia (BPH) and prostate carcinoma (CaP) are linked to aging and the presence of androgens, suggesting that androgen regulated genes play a major role in these common diseases. Androgen regulation of prostate growth and development depends on the presence of intact epithelial-stromal interactions. Further, the prostatic stroma is implicated in BPH. This suggests that epithelial cell lines are inadequate to identify androgen regulated genes that could contribute to BPH and CaP and which could serve as potential clinical biomarkers. In this study, we used a human prostate xenograft model to define a profile of genes regulated in vivo by androgens, with an emphasis on identifying candidate biomarkers. Benign transition zone (TZ) human prostate tissue from radical prostatectomies was grafted to the sub-renal capsule site of intact or castrated male immunodeficient mice, followed by the removal or addition of androgens, respectively. Microarray analysis of RNA from these tissues was used to identify genes that were; 1) highly expressed in prostate, 2) had significant expression changes in response to androgens, and, 3) encode extracellular proteins. A total of 95 genes meeting these criteria were selected for analysis and validation of expression in patient prostate tissues using quantitative real-time PCR. Expression levels of these genes were measured in pooled RNAs from human prostate tissues with varying severity of BPH pathologic changes and CaP of varying Gleason score. A number of androgen regulated genes were identified. Additionally, a subset of these genes were over-expressed in RNA from clinical BPH tissues, and the levels of many were found to correlate with disease status. Our results demonstrate the feasibility, and some of the problems, of using a mouse xenograft model to characterize the androgen regulated expression profiles of intact human prostate tissues.     

Differential protein expression in anatomical zones of the prostate

The prostate has three anatomical zones: the peripheral (PZ), the transition (TZ), and the central (CZ) zone. It is proposed that the CZ may be of mesodermal origin, whereas the other two are of endodermal origin. Proteome patterns in the zones were characterized to test for differences. Cells were scraped from macroscopically normal areas of PZ, TZ, and CZ in radical prostatectomy specimens. After exclusion of samples with cancer or prostatic intraepithelial neoplasia, 18 cases remained for analysis. Cells were collected in a medium with protease inhibitors, and the protein material was prepared for two-dimensional gel electrophoresis. The proteins in spots that differed quantitatively between regions were identified via mass spectrometric fingerprinting of tryptic fragments and selected tandem mass spectrometry sequence analysis. Ten proteins with significant zonal differential expression were identified, eight with underexpression in the CZ versus the PZ and the TZ (arginase II, ATP synthase, cytokeratin 8, lamin A/C, peroxiredoxin 4, protein disulfide isomerase A3, tropomyosin, and vimentin), and two with overexpression in the CZ (peroxiredoxin 2 and creatine kinase B). The PZ and TZ, although differing in terms of incidence of cancer and hyperplasia, have epithelium with highly similar major protein expression profiles. However, the protein profile of the CZ differs from that of the other regions, suggesting functional differences.     

Quantitative analysis of diffusion-weighted magnetic resonance images:differentiation between prostate cancer and normal tissue based on a computer-aided diagnosis system

Diffusion-weighted imaging(DWI) is considered to be one of the dominant modalities used in prostate cancer(PCa) detection and the assessment of lesion aggressiveness,especially for peripheral zone(PZ) PCa.Computer-aided diagnosis(CAD),which is capable of automatically extracting and evaluating image features,can integrate multiple parameters and improve the detection of PCa.In this study,13 quantitative image features were extracted from DWI by CAD,and diagnostic efficacy was analyzed in both the PZ and transition zone(TZ).The results demonstrated that there was a significant difference(P0.05) between PCa and non-PCa for nine of the 13 features in the PZ and five of the 13 features in the TZ.Besides,the prediction outcome of CAD had a strong correlation with the DWI scores that were graded by experienced radiologists according to the Prostate Imaging-Reporting and Data System Version 2(PI-RADS v2).     

Overexpression of Periostin in Stroma Positively Associated with Aggressive Prostate Cancer

Background

Periostin is an important extracellular matrix protein involved in cell development and adhesion. Previously, we identified periostin to be up-regulated in aggressive prostate cancer (CaP) using quantitative glycoproteomics and mass spectrometry. The expression of periostin was further evaluated in primary radical prostatectomy (RP) prostate tumors and adjacent non-tumorous prostate tissues using immunohistochemistry (IHC). Our IHC results revealed a low background periostin levels in the adjacent non-tumorous prostate tissues, but overexpressed periostin levels in the peritumoral stroma of primary CaP tumors.

Methods

In this study, periostin expression in CaP was further examined on multiple tissue microarrays (TMAs), which were conducted in four laboratories. To achieve consistent staining, all TMAs were stained with same protocol and scored by same image computation tool to determine the total periostin staining intensities. The TMAs were further scored by pathologists to characterize the stromal staining and epithelial staining.

Results

The periostin staining was observed mainly in peritumoral stromal cells and in some cases in tumor epithelial cells though the stronger staining was found in peritumoral stromal cells. Both periostin stromal staining and epithelial staining can differentiate BPH from CaP including low grade CaP (Gleason score ≤6), with significant p-value of 2.2e-16 and 0.001, respectively. Periostin epithelial staining differentiated PIN from low grade CaP (Gleason score ≤6) (p=0.001), while periostin stromal staining differentiated low grade Cap (Gleason score ≤6) from high grade Cap (Gleason score ≤6) (p=1.7e-05). In addition, a positive correlation between total periostin staining and Gleason score was observed (r=0.87, p=0.002).

Conclusions

The results showed that periostin staining was positively correlated with increasing Gleason score and the aggressiveness of prostate disease.     

Predicting Prostate Biopsy Outcomes: A Preliminary Investigation on Screening with Ultrahigh B-Value Diffusion-Weighted Imaging as an Innovative Diagnostic Biomarker

Background

Routine screening of prostate specific antigen (PSA) is no longer recommended because of a high rate of over-diagnosis of prostate cancer (PCa).

Objective

To evaluate the efficacy of diffusion-weighted magnetic resonance imaging (DW-MRI) for PCa detection, and to explore the clinical utility of ultrahigh b-value DW-MRI in predicting prostate biopsy outcomes.

Methodology

73 male patients were selected for the study. They underwent 3T MRI using T2WI conventional DW-MRI with b-value 1000 s/mm², and ultrahigh b-value DW-MRI with b-values of 2000 s/mm² and 3000 s/mm². Two radiologists evaluated individual prostate gland images on a 5-point rating scale using PI-RADS, for the purpose of region-specific comparisons among modalities. Sensitivity, specificity, accuracy, positive predictive value (PPV), negative predictive value (NPV) and likelihood ratios (LR) were investigated for each MRI modality. The area under the receiver operating characteristic (ROC) curve (AUC) was also calculated.

Results

Results showed the improved diagnostic value of ultrahigh b-value DWI-MRI for detection of PCa when compared to other b values and conventional MRI protocols. Sensitivity values for 3000 s/mm² in both peripheral zone (PZ) and transition zone (TZ) were significantly higher than those observed with conventional DW-MRI—Specificity values for 3000 s/mm² in the TZ were significantly higher than other b-value images, whereas specificity values using 3000 s/mm² in the PZ were not significantly higher than 2000 s/mm² images. PPV and NPV between 3000 s/mm² and the other three modalities were significantly higher for both PZ and TZ images. The PLRs and NLRs of b-value 3000 s/mm² DW-MRI in the PZ and TZ were also recorded. ROC analysis showed greater AUCs for the b value 3000 s/mm² DWI than for the other three modalities.

Conclusions

DW-MRI with a b-value of 3000 s/mm² was found to be the most accurate and reliable MRI modality for PCa tumor detection and localization, particularly for TZ lesion discrimination. It may be stated that the b-value of 3000 s/mm² is a novel, improved diagnostic biomarker with greater predictive accuracy for PCa prior to biopsy.     

不同年龄段前列腺外周带基质细胞超微结构差异

目的:研究不同年龄段前列腺外周带基质细胞超微结构并探讨其意义。方法:透射电镜观察3例年轻组(23-、26-、32-岁)和3例老年组(56-、64-、71-岁)正常前列腺外周带和3例前列腺癌组织的超微结构。Masson’s Trichrome染色法观察前列腺外周带基质胶原的分布情况。结果:年轻组、老年组前列腺外周带间质均以平滑肌细胞分布为主,间杂成纤维细胞,但老年组平滑肌细胞和成纤维细胞体积较大、形态不规则。与正常年轻组相比,老年组前列腺外周带成纤维细胞存在明显老化和肌化改变,部分细胞内与蛋白合成相关的细胞器粗面内质网发达,呈现肌成纤维细胞特征。与年轻组和老年组外周带相比,前列腺癌基质中功能更加活跃的肌成纤维细胞和胶原蛋白数量增加明显。Masson’s Trichrome染色支持超微观察到的基质胶原分布差异:年轻组<老年组<前列腺癌组。结论:不同年龄段前列腺外周带基质细胞超微结构间存在差异,老年组前列腺外周带和前列腺癌基质组织中具有活性的肌成纤维细胞的增加可能是造成老年前列腺癌高发和恶性进展的重要病理基础之一。     

Elution of cell-bound anti-prostatic epithelial antibodies after multiple cryotherapy of carcinoma of the prostate

Antibodies reactive with the cytoplasmic membrane or intercellular areas of autologous human and monkey prostatic secretory epithelial cells have been demonstrated by immunofluorescence in an eluate prepared from prostatic tissue obtained from a patient with metastatic adenocarcinoma of the prostate (CaP) after multiple cryotherapy of his primary prostatic tumour. While limited to the study of one patient, elution of anti-prostatic antibodies offers further documentation to previous studies pointing toward the participation of immunologic processes in CaP.     

Cellular distribution of Glut-1 and Glut-5 in benign and malignant human prostate tissue

Over-expression of hexose transporters (Gluts), specifically Glut-1, is a common event in human malignancies. In prostate cancer (CaP), however, expression of Gluts has been characterized poorly. In this study, expression and distribution of Glut-1 and Glut-5 proteins were characterized using immunohistochemistry in 76 specimens of benign prostate, 10 specimens of high-grade intraepithelial neoplasia (HGPIN), and 28 specimens of CaP. In addition, mRNA expression of Glut-2, Glut-7, Glut-9, and Glut-11 was analyzed in a set of five specimens of benign prostate and CaP. In benign prostate, Glut-1 localized to the basal cells and to the basolateral membrane of secretory/luminal epithelial cells. Glut-5, however, localized to the apical membrane of secretory/luminal epithelial cells. In HGPIN, Glut-1 was immunohistochemically undetectable. Glut-5, however, localized to the apical membrane of the neoplastic epithelial cells. In CaP, Glut-1 and Glut-5, were immunohistochemically undetectable. However, over-expression of GLUT1 was observed in some specimens of highly proliferative intraductal CaP. Glut-7, Glut-9, and Glut-11 mRNAs were detected in benign prostate and CaP, however, only Glut-11 mRNA was consistently up-regulated in CaP compared to benign prostate. Low levels of expression of Glut-1 protein in the majority of CaP could explain, at least in part, the limited clinical applicability of positron emission tomography using 2-[18F]-fluoro-2-deoxy-D-glucose for imaging CaP. Moreover, expression of Glut-5 in HGPIN suggested that fructose could be utilized as potential metabolic substrate in HGPIN. Understanding the molecular mechanisms involved in regulation/dysregulation of Gluts in CaP could provide insight in the understanding of hexose metabolism in CaP.     

Identifying variables responsible for clustering in discriminant analysis of data from infrared microspectroscopy of a biological sample.

In the biomedical field, infrared (IR) spectroscopic studies can involve the processing of data derived from many samples, divided into classes such as category of tissue (e.g., normal or cancerous) or patient identity. We require reliable methods to identify the class-specific information on which of the wavenumbers, representing various molecular groups, are responsible for observed class groupings. Employing a prostate tissue sample divided into three regions (transition zone, peripheral zone, and adjacent adenocarcinoma), and interrogated using synchrotron Fourier-transform IR microspectroscopy, we compared two statistical methods: (a) a new "cluster vector" version of principal component analysis (PCA) in which the dimensions of the dataset are reduced, followed by linear discriminant analysis (LDA) to reveal clusters, through each of which a vector is constructed that identifies the contributory wavenumbers; and (b) stepwise LDA, which exploits the fact that spectral peaks which identify certain chemical bonds extend over several wavenumbers, and which following classification via either one or two wavenumbers, checks whether the resulting predictions are stable across a range of nearby wavenumbers. Stepwise LDA is the simpler of the two methods; the cluster vector approach can indicate which of the different classes of spectra exhibit the significant differences in signal seen at the "prominent" wavenumbers identified. In situations where IR spectra are found to separate into classes, the excellent agreement between the two quite different methods points to what will prove to be a new and reliable approach to establishing which molecular groups are responsible for such separation.     

Electrochemically induced FTIR difference spectroscopy in the mid- to far infrared (200 microm) domain: a new setup for the analysis of metal-ligand interactions in redox proteins

We report the setup of an electrochemical cell with chemical-vapor deposition diamond windows and the use of a Bruker 66 SX FTIR spectrometer equipped with DTGS and Si-bolometer detectors and KBr and mylar beam splitters, to record on the same sample, FTIR difference spectra corresponding to the structural changes associated with the change in redox state of active sites in proteins in the whole 1800-50 cm(-1) region. With cytochrome c we show that reliable reduced-minus-oxidized FTIR difference spectra are obtained, which correspond to single molecular vibrations. Redox-sensitive IR modes of the cytochrome c are detected until 140 cm(-1) with a good signal to noise. This new setup is promising to analyze the infrared spectral region where metal-ligand vibrations are expected to contribute and to extend the analysis of vibrational properties to metal sites or redox states not accessible to (resonance) Raman spectroscopy.     

不同年龄段前列腺外周带基质细胞超微结构差异

目的：研究不同年龄段前列腺外周带基质细胞超微结构并探讨其意义。方法：透射电镜观察3例年轻组（23-、26-、32-岁）和3例老年组（56-、64-、71-岁）正常前列腺外周带和3例前列腺癌组织的超微结构。Masson＇s Trichrome染色法观察前列腺外周带基质胶原的分布情况。结果：年轻组、老年组前列腺外周带间质均以平滑肌细胞分布为主,间杂成纤维细胞,但老年组平滑肌细胞和成纤维细胞体积较大、形态不规则。与正常年轻组相比,老年组前列腺外周带成纤维细胞存在明显老化和肌化改变,部分细胞内与蛋白合成相关的细胞器粗面内质网发达,呈现肌成纤维细胞特征。与年轻组和老年组外周带相比,前列腺癌基质中功能更加活跃的肌成纤维细胞和胶原蛋白数量增加明显。Masson＇s Trichrome染色支持超微观察到的基质胶原分布差异：年轻组〈老年组〈前列腺癌组。结论：不同年龄段前列腺外周带基质细胞超微结构间存在差异,老年组前列腺外周带和前列腺癌基质组织中具有活性的肌成纤维细胞的增加可能是造成老年前列腺癌高发和恶性进展的重要病理基础之一。     

The morphological study on the adrenal gland of African ostrich chicks

The morphology of the adrenal gland has been studied for a number of animal species all over the world, yet the detailed data about ostrich chick has not been reported. In the present study, the morphological features of the adrenal gland in African ostrich chicks were investigated by means of gross anatomy, light and electron microscope. Differences between the left and right adrenal glands were found in shape, size and location. The interrenal tissue and chromaffin cell interdigitated irregularly. The interrenal tissue was divided into a peripheral zone (PZ) and a central inner zone (CZ), and the PZ was further distinguished into an outer area (subcapsular zone, SCZ) and an inner area (IZ). The cellular arrangement in these zones showed evident zonation that resembled the mammalian. This phenomenon had been previously described only for the pelicanus. The cytoplasm of interrenal cells in SCZ was stained lightly than in IZ and CZ, and contained several vacuoles. Additionally, unlike CZ cells, SCZ cells appeared to contain more mitochondria and less lipid droplets. Two types of chromaffin cells: epinephrine cells and norepinephrine cells could be detected. The type 1 granules possessed a central core and a variable distance between membrane and core; the type 2 granules had an eccentric core, which leant to one side of granule and sticked to the membrane, giving a lager lacouna appearance in another side of the granule.     

Infrared biomarkers of impaired cystic fibrosis transmembrane regulator protein biogenesis

The mid‐infrared (IR) spectra of human cystic fibrosis (CF) cells acquired by Fourier transform infrared microspectroscopy were compared with those of non‐CF cells. Within the 1700 to 1480 cm^?1 spectral domain of amides, unsupervised explorative principal component analysis identified a few variables reflecting quantitative and qualitative vibrations arising from protein secondary structures and amino acid side chains. Their pattern reflected α‐helix to β‐sheet transitions in bronchial epithelial cells and in immortalized peripheral blood mononuclear cells from patients with R1162X missense or in‐frame F508del mutations in the cystic fibrosis transmembrane regulator gene (Cftr). Similar transitions have been described in IR spectra of cells, tissues and body fluids of patients affected with some neurodegenerative diseases characterized by the accumulation of misfolded protein aggregates. The variables pattern was able to distinguish CF cells from non‐CF cells and was modified by molecular compounds used to rescue the unbalanced folding process of mutated cystic fibrosis transmembrane regulator (CFTR) anion channel. To our knowledge, this is the first experimental evidence of spectroscopic biomarkers of the impaired biogenesis of CFTR by IR microanalysis in the spectra of human CF bronchial epithelial and lymphoblastoid cells.     

Rapid intra‐operative diagnosis of kidney cancer by attenuated total reflection infrared spectroscopy of tissue smears

Herein, a technique to analyze air‐dried kidney tissue impression smears by means of attenuated total reflection infrared (ATR‐IR) spectroscopy is presented. Spectral tumor markers—absorption bands of glycogen—are identified in the ATR‐IR spectra of the kidney tissue smear samples. Thin kidney tissue cryo‐sections currently used for IR spectroscopic analysis lack such spectral markers as the sample preparation causes irreversible molecular changes in the tissue. In particular, freeze‐thaw cycle results in degradation of the glycogen and reduction or complete dissolution of its content. Supervised spectral classification was applied to the recorded spectra of the smears and the test spectra were classified with a high accuracy of 92% for normal tissue and 94% for tumor tissue, respectively. For further development, we propose that combination of the method with optical fiber ATR probes could potentially be used for rapid real‐time intra‐operative tissue analysis without interfering with either the established protocols of pathological examination or the ordinary workflow of operating surgeon. Such approach could ensure easier transition of the method to clinical applications where it may complement the results of gold standard histopathology examination and aid in more precise resection of kidney tumors.