Protein complexes/aggregates as potential cancer biomarkers revealed by a nanoparticle aggregation immunoassay

Protein–protein interactions and protein complex/aggregate formation play an essential role in almost all biological functions and activities. Through a nanoparticle aggregation immunoassay, we discovered that some proteins are substantially more complexed/aggregated in cancer tissues than normal tissues. This study examined four biomarkers proteins, CA125, CEA (carcinoembryonic antigen), CA19-9 and PAP (prostatic acid phosphatase) in ovarian, colon and prostate tissue lysates. The most exciting results were observed from the PAP assay of prostate tissues: prostate cancer can be clearly distinguished from normal prostate and prostate with benign conditions such as BPH (benign prostate hyperplasia) based on the complex/aggregation level of PAP in prostate tissue lysates. The complex/aggregate level of a protein can be potential biomarkers for cancer detection and diagnosis.     

Purification and determination of human prostate specific antigen in the serum

A human prostate specific antigen (PA) has been purified from an extract of prostatic tissue obtained during operation for benign prostatic hypertrophy (BPH). The antigen, which can be demonstrated a single component by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), has an apparent molecular weight of about 34,000 and has lower mobility for the positive pole than prostatic acid phosphatase (PAP). Double antibody radioimmunoassay (RIA) for PA in serum was developed with the antiserum raised in rabbit against partially purified PA. In normal serum of 30 controls the concentration were studied by the RIA. The normal upper limit of the serum PA levels in assay was set at 2.5 ng/ml. Elevated levels were observed in serum from 19 out of 21 untreated patients with prostatic carcinoma and 9 out of 23 patients with BPH, but latter less than 10 ng/ml. The results indicate that the PA is a potentially useful marker as well as PAP for prostatic cancer.     

Immobilization of Anti-Galectin-3 onto Polysiloxane–Polyvinyl Alcohol Disks for Tumor Prostatic Diseases Diagnosis

This work aimed to immobilize the antibody anti-galectin-3 onto polysiloxane–polyvinyl alcohol (POS-PVA) support, to evaluate its capacity to capture the serum antigen galectin-3 and to quantify by ELISA the antigen levels in sera from patients with prostatic adenocarcinoma (PA) and benign prostatic hyperplasia (BPH) and healthy individuals. Also, for comparative effect, the galectin-3 expression in the prostate tissue through immunohistochemistry was evaluated. The optical density (galectin-3 level) values established for the sera from PA and BPH patients were lower compared with those found for the healthy individuals. Galectin-3 immunohistochemically showed a significant increase and reduction of the cytoplasmatic protein expression in BPH and PA, respectively, compared with the normal prostate. These results showed that POS-PVA disks could be used as solid phase to immobilize serum galectins and in immunoassays procedures for the correspondent IgG anti-galectins detection in human sera.     

Identification of potential complementary serum biomarkers to differentiate prostate cancer from benign prostatic hyperplasia using gel- and lectin-based proteomics analyses

Diagnosis of prostate cancer (PCa) is currently much reliant on the invasive and time-consuming transrectal ultrasound-guided biopsy of the prostate gland, particularly in light of the inefficient use of prostate-specific antigen as its biomarker. In the present study, we have profiled the sera of patients with PCa and benign prostatic hyperplasia (BPH) using the gel- and lectin-based proteomics methods and demonstrated the significant differential expression of apolipoprotein AII, complement C3 beta chain fragment, inter-alpha-trypsin inhibitor heavy chain 4 fragment, transthyretin, alpha-1-antitrypsin, and high molecular weight kininogen (light chain) between the two groups of patients' samples. Our data are suggestive of the potential use of the serum proteins as complementary biomarkers to effectively discriminate PCa from BPH, although this requires further extensive validation on clinically representative populations.     

Comparative Proteomic Analysis of Human Benign Prostatic Hyperplastic Tissue Before and After Irradiation with Radioactive Nuclide

The expressed proteins were extracted from human benign prostatic hyperplastic tissues obtained with transurethral resection of the prostate before and after their irradiation with radioactive nuclide. The proteins were separated by two-dimensional gel electrophoresis and analyzed by mass spectrometry. Four proteins were differentially expressed and were identified with a database search. Three were associated with the regulation of cell motion and one was lactate dehydrogenase B, which plays an important r...     

高危良性前列腺增生的术式探讨

目的探讨经尿道前列腺剜除术与经尿道双极等离子前列腺电切术治疗高危良性前列腺增生的临床疗效。方法选取高危良性前列腺增生患者86例,随机分为实验组和对照组,两组患者分别应用经尿道前列腺剜除术、经尿道双极等离子前列腺电切术治疗,对比两组患者的手术效果。结果两组患者的临床疗效及不良反应均无明显差异(P>0.05),但实验组患者的手术时间、膀胱冲洗时间及住院时间均短于对照组,术中出血量少于对照组,差异有统计学意义(P<0.01)。结论经尿道前列腺剜除术与经尿道双极等离子前列腺电切术治疗高危良性前列腺增生均安全有效,但经尿道前列腺剜除术的手术时间、膀胱冲洗时间及住院时间更短,术中出血量更少。     

Effect of β Radiation on Bcl-2 and Bax Expressions in Benign Prostate Hyperplasia Tissues

The authors chose specimens from nine normal prostate tissues（NP group）, 15 benign prostate hyperplasia（BPH） prostates（BPH group）, and 35 BPH prostates that had been treated＇with ^90Sr/^90Y Prostatic Hyperplasia Applicator（exposure group）. The expressions of bcl-2 and bax in stroma and epithelia of prostate tissues were demonstrated by means of immunohistochemical staining, and the staining positive rate was semiquantatively determined, so as to observe the expression of bcl-2 and bax genes in the prostate tissues of normal individuals and BPH patients, before and after fl radiation, and to evaluate the influence of fl radiation on bcl-2 and bax expressions. The expressions of gene bcl-2 in the prostate epithelia of NP and BPH are significantly higher than those in the prostate stroma（P〈0.01）. However, the expressions of bcl-2 in the prostate epithelia and stroma of the BPH group are obviously higher than those in the NP group（P〈0.01）. The expression of gene bax in the prostate epithelia of the NP group is higher than that in the BPH group（P〈0.05）. However, bcl-2 expressions in the prostate epithelia and stroma of the BPH group are significantly higher than the bax expressions（P〈0.01）. Compared with those of the NP group, the expressions of bcl-2 in the prostate epithelia and stroma of the exposure group decrease remarkably, even as the expressions of the bax notably increase（P〈0.01）. Thus, the administration of β radiation can remarkably affect bcl-2 and bax gene expressions, to regulate cell apoptosis, in the prostate tissues of BPH.     

Differential diagnosis of prostate cancer and benign prostate hyperplasia using two-dimensional electrophoresis.

Prostate specific antigen (PSA) is a protease which is characteristic of the prostate. It is widely used as a serum marker for the early diagnosis of prostate cancer (PCa). Nevertheless, for concentrations between 4 and 10 ng/mL, PSA does not enable PCa to be distinguished from benign diseases, such as benign prostate hyperplasia (BPH). In sera, the use of a ratio between free PSA (PSA uncomplexed with protease inhibitor) and total PSA (free PSA and PSA bound to alpha-1 anti-chymotrypsin) enables the "gray zone" to be reduced, but an important proportion of patients are still wrongly classed. Using two-dimensional electrophoresis, we demonstrated using 52 PCa and 40 BPH well-documented clinical cases that BPH sera show a significantly greater percentage of low-molecular-weight free PSA elements (IwPSA) than PCa sera. In our study, the use of a ratio between IwPSA and standard free PSA enables the correct diagnosis of 100% of PCa and 82.5% of BPH cases as against when 73.1% and 42.5% respectively were correctly diagnozed using the total PSA and the free/total PSA ratio. This important finding may be related to differences in the mechanism secreting PSA from the prostate into the bloodstream. We have shown how a tissue marker may be turned into a powerful tumor marker by events probably unrelated to its expression.     

Zinc in human prostate gland: Normal, hyperplastic and cancerous

Zinc concentration in a prostate gland is much higher than that in other human tissues. Data about zinc changes for different prostate diseases are limited and greatly contradictory. Zinc content was determined for biopsy and resected materials of transrectal puncture tissues from benign prostate hyperplasia (BPH) and prostate cancer. There were 109 patients (50 BPH and 59 cancer) available for the present study. Control group consisted of 37 intact glands of men died an unexpected death (accident, murder, acute cardiac insufficiency, etc.). All materials studied were divided into two parts. One of them was morphologically examined, while another one was subjected to zinc analysis by INAA. Zinc contents (M±SE) of normal, benign hyperplastic and cancerous prostate glands were found to be 1018±124, 1142±77, and 146±10 g/g dry tissue, respectively. It was shown that zinc assessments in the materials of transrectal puncture biopsy of indurated prostate sites can be used as an additional test for differential diagnostics of BPH and cancer. Accuracy, sensitivity and specificity of the test are 98±2%.     

Discrimination of zone-specific spectral signatures in normal human prostate using Raman spectroscopy

The prostate gland is the most common site of pathology in human males. Using the urethra as an anatomical reference point, it can be divided into three distinct zones known as the transition zone (TZ), peripheral zone (PZ) and central zone (CZ). The pathological conditions of benign prostatic hypertrophy and/or prostate adenocarcinoma are highly prevalent in this gland. This preliminary study set out to determine whether biochemical intra-individual differences between normal prostate zones could be identified using Raman spectroscopy with subsequent exploratory analyses. A normal (benign) prostate transverse tissue section perpendicular to the rectal surface and above the verumontanum was obtained in a paraffin-embedded block. A 10-μm-thick slice was floated onto a gold substrate, de-waxed and analysed using Raman spectroscopy (200 epithelial-cell and 140 stromal spectra/zone). Raman spectra were subsequently processed in the 1800-367 cm(-1) spectral region employing principal component analysis (PCA) to determine whether wavenumber-intensity relationships expressed as single points in hyperspace might reveal biochemical differences associated with inter-zone pathological susceptibility. Visualisation of PCA scores plots and their corresponding loadings plots highlighted 781 cm(-1) (cytosine/uracil) and 787 cm(-1) (DNA) as the key discriminating factors segregating PZ from less susceptible TZ and CZ epithelia (P < 0.001). Conversely, 1459 cm(-1) (lipids and proteins) and 1003 cm(-1) (phenylalanine) were identified as the key biochemical factor distinguishing TZ from CZ epithelia (P < 0.05). All stromal zones were discriminated by the protein/lipid region (1459 cm(-1) and 1100 cm(-1)) with DNA/RNA region (781 cm(-1) and 787 cm(-1)) only highlighted between PZ and CZ (P < 0.05). This novel approach identifies biochemical markers that may have aetiological functional roles towards susceptibility of human prostate zones to specific pathological conditions.     

Proteomic analysis of laser capture microdissected human prostate cancer and in vitro prostate cell lines

Specific populations of normal and malignant epithelium from three radical prostatectomy tissue specimens were procured by laser capture microdissection (LCM) and analyzed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Six proteins that were only seen in malignant cells and two proteins that were only seen in benign epithelium were reproducibly observed in two of two cases examined. Furthermore, these proteins were not observed in the 2-D PAGE profiles from the patient-matched microdissected stromal cell populations, but were seen in the protein profiles from the undissected whole cryostat sections. One of these proteins was determined to be prostate-specific antigen (PSA) by Western blot analysis, and intriguingly the remaining protein candidates were found to be at least as abundant as the PSA protein. Comparison of 2-D PAGE profiles of microdissected cell with matched in vitro cell lines from the same patient, and metastatic prostate cancer cell lines (LnCaP and PC3) showed striking differences between prostate cells in vivo and in vitro with less than 20% shared proteins. The data demonstrate that 2-D PAGE analysis of LCM-derived cells can reliably detect alterations in protein expression associated with prostate cancer, and that these differentially expressed proteins are produced in high enough levels which could allow for their clinical utility as new targets for therapeutic intervention, serum markers, and/or imaging markers.     

Aberrant expression of acute-phase reactant proteins in sera and breast lesions of patients with malignant and benign breast tumors

We have analyzed unfractionated sera of newly diagnosed patients (n=10) with breast carcinoma (BC), prior to treatment, and patients (n=5) with fibrocystic disease of the breast (FDB) by two-dimensional gel electrophoresis (2-DE) and silver staining. The patients' 2-DE serum protein profiles obtained were then subjected to image analysis and compared to similar data generated from sera of normal healthy female controls (n=10) of the same range of age. The relative expression of alpha1-antichymotrypsin (ACT), clusterin, and complement factor B was significantly higher in all BC patients as compared to normal controls. However, the expression of alpha1-antitrypsin (AAT) in BC patients was apparently lower than that of the controls. Similar differential expression of ACT was detected in the FDB patients. The aberrant expression of the serum acute-phase proteins of patients with BC and FDB was confirmed by competitive enzyme-linked immunosorbent assay (ELISA). Similar altered proteins expression was also observed from immunohistochemical studies of malignant (n=5) and benign (n=5) breast lesions of the respective patients performed using antisera to the aberrantly expressed proteins. However, the malignant breast lesions were instead positively stained for AAT. The differential expression of the serum proteins was apparently abrogated when a six-month follow-up study was performed on nine of the BC patients subsequent to treatment.     

Micro and bulk analysis of prostate tissues classified as hyperplasia

BPH (Benign Prostatic Hyperplasia) is the most common benign neoplasm (non cancerous enlargement of the prostate gland), whose prevalence increases with age.The gland, when increased in size, exerts pressure on the urethra, causing obstruction to urine flow. The latter may result in severe urinary tract and kidney conditions.In this work prostate samples from patients diagnosed with BPH were analyzed using synchrotron radiation. Micro-analysis of the hyperplastic samples was carried out on the L-beam line at HASYLAB, DESY (Germany), while bulk analysis on selected samples was performed at the DRX2 beamline at LNF, Frascati (Italy).Microanalysis with a mono-energetic beam 15 μm in diameter confirmed that concentrations of certain elements, such as S, Mn, Cu, Fe and Zn, are good indicators of pathological disorders in prostate tissue that may be considered effective tracers of developing compliant. The concentrations of Mn, Cu, Fe and Zn are higher in hyperplastic tissues, as compared to normal ones, while for sulphur the opposite is observed.Additionally, Fe and S K-edge XANES (X-ray Absorption Near Edge Structure) spectroscopy experiments were carried out in order to determine the chemical speciation of these elements in our samples.     

Raman microscopy for the chemometric analysis of tumor cells

Raman spectroscopy is recognized as a tool for chemometric analysis of biological materials due to the high information content relating to specific physical and chemical qualities of the sample. Thirty cells belonging to two different prostatic cell lines, PNT1A (immortalized normal prostate cell line) and LNCaP (malignant cell line derived from prostate metastases), were mapped using Raman microscopy. A range of spectral preprocessing methods (partial least-squares discriminant analyses (PLSDAs), principal component analyses (PCAs), and adjacent band ratios (ABRs)) were compared for input into linear discriminant analysis to model and classify the two cell lines. PLSDA and ABR were able to correctly classify 100% of cells into benign and malignant groups, while PLSDA correctly classified a greater proportion of individual spectra. PCA was used to image the distribution of various biochemicals inside each cell and confirm differences in composition/distribution between benign and malignant cell lines. This study has demonstrated that PLSDAs and ABRs of Raman data can identify subtle differences between benign and malignant prostatic cells in vitro.     

Compatibility effects of herb pair Phellodendri chinensis cortex and Anemarrhenae rhizoma on benign prostatic hyperplasia using targeted metabolomics

Phellodendri chinensis cortex (P. C. cortex) and Anemarrhenae rhizoma (A. rhizoma) herb pair is a core component of traditional Chinese medicines used to treat inflammation and benign prostatic hyperplasia (BPH). The present study was designed to profile the arachidonic acid (AA) metabolomic characteristics in rat plasma and prostate after being treated with P. C. cortex and A. rhizoma as well as their combination. Plasma and prostate samples from sham group, BPH model group, herb pair group and two single herb groups were collected on days 7, 14, 21 and 28. Then, a systemic metabolomic analysis based on UFLC‐MS/MS was employed to quantify AA and its cyclooxygenase and lipoxygenase pathway metabolites (15‐HETE, 12‐HETE, 5‐HETE, AA, PGI₂, PGF_2α, 8‐HETE, PGD₂, PGE₂ and LTB₄). The results demonstrated that BPH led a significant increase of 10 biomarkers in plasma and tissue (p < 0.05). The clusters of herb pair group and single herb groups showed a tendency to return to the initial space, and the AA and its metabolites from those groups were differently downregulated to a healthier level, with the combination of single herbs most obvious. The present study demonstrated that P. C. cortex–A. rhizoma herb pair might produce synergistic or complementary compatibility effects on suppressing inflammatory processes occurring in BPH.     

New molecular markers for prostate tumor imaging: a study on 2-methylene substituted fatty acids as new AMACR inhibitors

The development of prostate carcinoma is associated with alterations in fatty acid metabolism. α‐Methylacyl‐CoA racemase (AMACR) is a peroxisomal and mitochondrial enzyme that catalyses interconversion between the (S)/(R)‐isomers of a range of α‐methylacyl‐CoA thioesters. AMACR is involved in the β‐oxidation of the dietary branched‐chain fatty acids and bile acid intermediates. It is highly expressed in prostate (more than 95 %), colon (92 %), and breast cancers (44 %) but not in the respective normal or hyperplastic tissues. Thus, targeting of AMACR could be a new strategy for molecular imaging and therapy of prostate and some other cancers. Unlabeled 2‐methylenacyl‐CoA thioesters ( 12 a – c ) were designed as AMACR binding ligands. The thioesters were tested for their ability to inhibit the AMACR‐mediated epimerization of (25R)‐THC‐CoA and were found to be strong AMACR inhibitors. Radioiodinated (E)‐¹³¹I‐13‐iodo‐2‐methylentridec‐12‐enoic acid (¹³¹I‐ 7 c ) demonstrated preferential retention in AMACR‐positive prostate tumor cells (LNCaP, LNCaP C4‐2wt and DU145) compared with both AMACR‐knockout LNCaP C4‐2 AMACR‐siRNA and benign BPH1 prostate cell lines. A significant protein‐bound radioactive fraction with main bands at 47 (sum of molecular weights of AMACR plus 12 c ), 70, and 75 kDa was detected in LNCaP C4‐2 wt cells. In contrast, only negligible amounts of protein‐bound radioactivity were found in LNCaP C4‐2 AMACR‐siRNA cells.     

Quantitative analysis of differentially expressed saliva proteins in human immunodeficiency virus type 1 (HIV-1) infected individuals

In the present study, we have established a new methodology to analyze saliva proteins from HIV-1-seropositive patients before highly active antiretroviral therapy (HAART) and seronegative controls. A total of 593 and 601 proteins were identified in the pooled saliva samples from 5 HIV-1 subjects and 5 controls, respectively. Forty-one proteins were found to be differentially expressed. Bioinformatic analysis of differentially expressed salivary proteins showed an increase of antimicrobial proteins and decrease of protease inhibitors upon HIV-1 infection. To validate some of these differentially expressed proteins, a high-throughput quantitation method was established to determine concentrations of 10 salivary proteins in 40 individual saliva samples from 20 seropositive patients before HAART and 20 seronegative subjects. This method was based on limited protein separation within the zone of the stacking gel of the 1D SDS PAGE and using isotope-coded synthetic peptides as internal standards. The results demonstrated that a combination of protein profiling and targeted quantitation is an efficient method to identify and validate differentially expressed salivary proteins. Expression levels of members of the calcium-binding S100 protein family and deleted in malignant brain tumors 1 protein (DMBT1) were up-regulated while that of Mucin 5B was down-regulated in HIV-1 seropositive saliva samples, which may provide new perspectives for monitoring HIV-infection and understanding the mechanism of HIV-1 infectivity.     

1-乙酰基-5-取代吲哚啉类化合物的合成及生物活性

根据拼合原理, 以1-乙酰基吲哚啉为起始原料, 设计合成了1-乙酰基-5-{2-[4-(取代苯氧基乙基)-1-哌嗪基]烷基}吲哚啉和1-乙酰基-5-[2-(4-取代苯基-1-哌嗪基)烷基]吲哚啉两类化合物, 所有目标化合物结构均经核磁共振氢谱、元素分析、红外光谱及质谱确证. 初步生物活性测试结果表明, 所有目标化合物均具有一定的α₁-AR拮抗活性.     

Proteomic approach for purification of seminal plasma proteins involved in tumor proliferation

Human seminal plasma contains a large array of proteins required for the normal physiology and metabolism of spermatozoa. These are mainly secreted from prostate epithelium, testes, and seminal vesicles. Fortunately, many of these are found to be present at elevated concentration in seminal plasma and act as a biomarker of different carcinomas as their levels are also enhanced in serum and are found to be involved in tumor progression and metastasis apart from fertility. The proteins which were overexpressed in the seminal plasma of prostate carcinoma patients were identified by 2-DE and MALDI-TOF/MS. We have designed a strategy to purify these four proteins prostate specific antigen (PSA), prostatic acid phosphatase (PAP), Zinc alpha2-glycoprotein (ZAG), and progastricsin (PG), together in homogeneity by using simple chromatographic techniques. Acidic and basic fractions of human seminal plasma were separated by ion exchange chromatography and further purified by gel permeation chromatography. Our results form a new and valuable resource for those attempting structure-based drug designing for prostate and other cancers where the amount of proteins is required in plenty and in native form.     

Prostate-specific antigen: update 1997.

Prostate-specific antigen (PSA) is the most important tumor marker for prostate cancer, although it is not a perfect marker as it is not cancer-specific. PSA, a member of the human kallikrein family, is present in two molecular forms in serum: free and complexed to protease inhibitors. PSA is now commonly measured on automated immunoassay systems employing monoclonal or polyclonal antibodies. Results from different assays can vary since some assays are not equimolar and react to the free and complexed forms differently. Utilization of the molecular forms of PSA is one approach to improve the sensitivity and specificity of the PSA assay. Patients with prostate cancer have a greater percentage of PSA bound to alpha1-antichymotripsin (ACT) than those without cancer. Measurement of the free to total PSA ratio in the diagnostic gray zone (usually 4-10 micrograms/liter of total PSA), where prostate cancer and benign prostatic hyperplasia (BPH) overlap, has been shown to eliminate between 16 and 79% of unnecessary biopsies. Free to total PSA cutoffs are influenced by the sensitivity and specificity values chosen, the reflex range for total PSA used, differences in free PSA assays, differences in populations studied, and factors such as total PSA concentrations, age, and prostate gland size. In addition to the molecular forms of PSA, age-specific reference ranges, rate of change of PSA concentrations (PSA velocity), ratio of serum PSA to prostate volume (PSA density), and neural network derived indices have been employed to improve the clinical utility of PSA measurements.