OBJECTIVETo establish enzyme-linked immunosorbent assay (ELISA) for quantitative detection of Golgi protein73 (GP73) in serum.

METHODSA sandwich reaction was preformed with horseradish peroxidase labeled monoclonal antibody of GP73 as the catalytic enzyme. Several reactions liquid's concentration and reaction conditions were optimized. The method was evaluated in all aspects such as linear range, sensitivity, specificity, stability and so on.

RESULTSThe linear range was 25-500 ng/ml. The detection limit was 18.5 ng/ml. Inter-assay and intra-assay RSD were both less than 10%. The recoveries of three different spiked concentration samples were 95.3%, 92.6% and 103.7%. After stored at 4 degrees C and 37 degrees C for 3, 5, 7 days, the analysis showed correlation coefficient higher than 0.98 and RSD lower than 10%.

CONCLUSIONEstablished ELISA for quantity determination of serum GP73 has high accuracy, sensitivity and repeatability.

Antibodies, Monoclonal ; analysis ; Enzyme-Linked Immunosorbent Assay ; methods ; Humans ; Membrane Proteins ; blood ; Sensitivity and Specificity

This study was designed to investigate the different involvements of prostatic stromal cells from the normal transitional zone (TZ) or peripheral zone (PZ) in the carcinogenesis of prostate cancer (PCa) epithelial cells (PC-3) in vitro and in vivo co-culture models. Ultra-structures and gene expression profiles of primary cultures of human prostatic stromal cells from the normal TZ or PZ were analyzed by electron microscopy and microarray analysis. In vitro and in vivo co-culture models composed of normal TZ or PZ stromal cells and human PCa PC-3 cells were established. We assessed tumor growth and weight in the in vivo nude mice model. There are morphological and ultra-structural differences in stromal cells from TZ and PZ of the normal prostate. In all, 514 differentially expressed genes were selected by microarray analysis; 483 genes were more highly expressed in stromal cells from TZ and 31 were more highly expressed in those from PZ. Co-culture with PZ stromal cells and transforming growth factor-beta1 (TGF-beta1) increased the tumor growth of PC-3 cells in vitro and in vivo, as well as Bcl-2 expression. On the other hand, stromal cells of TZ suppressed PC-3 cell tumor growth in the mouse model. We conclude that ultra-structures and gene expression differ between the stromal cells from TZ or PZ of the normal prostate, and stroma-epithelium interactions from TZ or PZ might be responsible for the distinct zonal localization of prostate tumor formation.
Adenocarcinoma ; drug therapy ; genetics ; pathology ; Adult ; Animals ; Cell Line, Tumor ; Coculture Techniques ; Gene Expression ; drug effects ; Gene Expression Profiling ; Humans ; Male ; Mice ; Mice, Nude ; Oligonucleotide Array Sequence Analysis ; Prostate ; drug effects ; metabolism ; pathology ; Prostatic Neoplasms ; drug therapy ; genetics ; pathology ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Stromal Cells ; metabolism ; pathology ; Transforming Growth Factor beta ; pharmacology ; Young Adult

The androgen receptor (AR) plays an important role in the development and progression of prostate cancer (PCa). Androgen deprivation therapy is initially effective in blocking tumor growth, but it eventually leads to the hormone-refractory state. The detailed mechanisms of the conversion from androgen dependence to androgen independence remain unclear. Several PCa cell lines were established to study the role of AR in PCa, but the results were often inconsistent or contrasting in different cell lines, or in the same cell line grown under different conditions. The cellular and molecular alteration of epithelial cells and their microenvironments are complicated, and it is difficult to use a single cell line to address this important issue and also to study the pathophysiological effects of AR. In this paper, we summarize the different effects of AR on multiple cell lines and show the disadvantages of using a single human PCa cell line to study AR effects on PCa. We also discuss the advantages of widely used epithelium-stroma co-culture systems, xenograft mouse models, and genetically engineered PCa mouse models. The combination of in vitro cell line studies and in vivo mouse models might lead to more credible results and better strategies for the study of AR roles in PCa.
Animals ; Cell Line, Tumor ; Disease Models, Animal ; Epithelial Cells ; pathology ; Humans ; Male ; Mice ; Prostatic Neoplasms ; pathology ; physiopathology ; Receptors, Androgen ; physiology ; Stromal Cells ; pathology

OBJECTIVETo study the roles of rhoassociated coiled coil forming protein kinase 2 (Rock2) and transforming growth factor-β1 (TGF-β1) mRNA in acute asthma and the effect of glucocorticoid intervention on the Rock2 and TGF-β1 mRNA expression in rats.

METHODSForty-eight male rats were randomly divided into 4 groups (n=12 each): asthma, control, dexamethasone treated (DXM) and budesonide treated (BUD). Rat model of asthma was prepared by the ovalbumin (OVA) challenge. The animals were sacrificed 24 hrs after the last challenge. The total cell number and differentiation cell number were counted in bronchoalveolar lavage fluid (BALF). The protein expression of Rock2 was ascertained by immunohistochemistry and the mRNA expression of TGF-β1 was ascertained by hybridization in situ.

RESULTSThe pathological changes in the BUD and the DXM groups were alleviated when compared with the asthma group. The total cell number and the percentage of eosinophil (EOS), polymorphonuclear leukocytes (PMN) and lymphocytes (Lym) in BALF in the asthma group were significantly higher than those in the control group (P<0.01). The percentage of macrophage (Mф) in the asthma group was significantly lower than that in the control group (P<0.01). The total cell number and the percentage of EOS and Lym in BALF in the DXM and the BUD groups decreased, while the percentage of Mф increased significantly compared with those in the asthma group (P<0.01). The Rock2 and TGF-β1 mRNA expression in lung tissues in the asthma group increased significantly compared with those in the control, BUD and DXM groups, while there were no significant differences in the Rock2 expression and TGF-β1 mRNA expression between the DXM or BUD group and the control group.

CONCLUSIONSThe expression of Rock2 and TGF-β1 mRNA in lung tissues is increased in rats with acute asthma. Glucocorticoids can significantly decrease the expression of Rock2 and TGF-β1 in lung tissues, thus alleviates airway inflammation.

Animals ; Asthma ; drug therapy ; metabolism ; Budesonide ; therapeutic use ; Dexamethasone ; therapeutic use ; Glucocorticoids ; therapeutic use ; Lung ; metabolism ; pathology ; Male ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Transforming Growth Factor beta1 ; genetics ; rho-Associated Kinases ; genetics

OBJECTIVETo investigate plasma hydrogen sulfide (H₂S) levels and cystathionine-gamma- lyase (CSE) and cystathionine-beta-synthase (CBS) mRNA expression in the lung tissues in asthmatic rats and to explore the roles of endogenous H₂S, CSE and CBS system in the pathogenesis of asthma.

METHODSThirty male Sprague-Dawley rats (age 5 to 7 weeks) were randomly divided into three groups: control, asthma and budesonide treatment (n = 10 each). The asthma model was established by ovalbumin (OVA) sensitization and challenge. The budesonide treatment group received inhaled budesonide before challenge. The contents of plasma H₂S were measured by spectrophotometry. The levels of CSE and CBS mRNA in the lung tissues were examined by reverse transcriptase polymerase chain reaction (RT-PCR).

RESULTSThe contents of plasma H₂S in the asthma group (61 ± 16 μmol/L) were significantly lower than those in the control group (84 ± 15 μmol/L) (P<0.01). The contents of plasma H₂S in the budesonide treatment group (71 ± 14 μmol/L) were not statistically different from those in the control and asthma groups. CSE mRNA and CBE mRNA expression in the asthma group were significantly lower than those in the control group (P < 0.01). The budesonide treatment group had a decreased CSE mRNA expression and CBE mRNA expression compared with the control group, but had significantly increased CSE and CBE mRNA expression compared with the asthma group (P < 0.01). There was a significantly negative correlation between H₂S contents in plasma and total inflammatory cells in bronchoalveolar lavage fluid (n = 30, r = -0.549, P < 0.01).

CONCLUSIONSPlasma H₂S levels and CSE and CBS expression in the lung decrease in asthmatic rats, which possibly promotes inflammatory cell aggregation to the airway. Budesonide may alleviate airway inflammation in asthmatic rats possibly through the system of endogenous H₂S, CSE and CBS.

Animals ; Asthma ; drug therapy ; etiology ; metabolism ; Budesonide ; pharmacology ; Cystathionine beta-Synthase ; genetics ; physiology ; Cystathionine gamma-Lyase ; genetics ; physiology ; Hydrogen Sulfide ; metabolism ; Male ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo isolate and identify the cancer stem cells from primary human ovarian cancer tissues.

METHODSFresh tumor tissues from five cases of pathologically diagnosed ovarian cancers were taken, minced and then digested with collagenase and hyaluronidase to obtain single cell suspension. The erythrocytes were removed with ACK Lysis buffer. The suspensions were sorted by magnetic activated cell sorting (MACS) using CD133-binding microbeads. Then the sorted CD133(+) cells were verified by flow cytometry. The cells were cultured in serum-free medium supplemented with EGF, bFGF, insulin and BSA, and grew into spheroids. Immunofluorescence, differentiation and tumor formation tests of the cells were performed to characterize the properties of cancer stem cells.

RESULTSThe ovarian cancer stem cells were successfully isolated from primary human ovarian tumors, which formed typical spheroids in serum-free medium and had stronger ability of tumorigenesis. The results of related experiments verified that CD133 positive cells owned the properties of cancer stem cells.

CONCLUSIONSThe ovarian cancer stem cells presenting the characteristics of stemness in vitro and in vivo, have been successfully isolated from primary human ovarian tumor tissues by MACS. The isolated ovarian cancer stem cells could be used in future researches of their biological functions.

AC133 Antigen ; Animals ; Antigens, CD ; metabolism ; Cell Differentiation ; Cell Separation ; methods ; Female ; Flow Cytometry ; methods ; Glycoproteins ; metabolism ; Humans ; Immunomagnetic Separation ; methods ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Neoplasm Transplantation ; Neoplastic Stem Cells ; metabolism ; pathology ; Ovarian Neoplasms ; metabolism ; pathology ; Peptides ; metabolism

OBJECTIVETo compare different expression profiles of all known chemokine receptors in human hepatocellular carcinoma (HCC) cell lines with different metastasis potentials.

METHODSEighteen pairs of chemokine receptor primers were designed using Premier software. Expression profiles of the 18 chemokine receptors on four HCC cell lines of lower to higher potentials of metastasis (SMMC-7721, MHCC97-L, MHCC97-H and HCCLM6) were analyzed by RT-PCR. Expression of CXCR4 was detected by RT-PCR.

RESULTSExpression profiles of chemokine receptors on four HCC cell lines with different metastatic potentials had significant differences (P < 0.01), in which CCR10, CXCR4 and CXCR6 expressions decreased gradually as the metastatic potential of the cell lines increased. The expressions of CCR3, CCR4, CCR10, CCR12 and XCR1 on HCCLM6 were significantly reduced compared with SMMC-7721 (P < 0.01), whereas the expressions of CXCR1 (P = 0.006) and CXCR5 (P = 0.003) exceeded that of SMMC-7721. Except for CXCR2, CXCR6 and XCR1, most of chemokine receptors on MHCC97-H were expressed differently compared with MHCC97-L (P < 0.05), in which expressions of CCR1 (P = 0.002), CCR2 (P = 0.004) and CCR5 (P = 0.046) exceeded MHCC97-L. CXCR4 was detected only on the positive controls and SMMC-7721 when the template of total RNA was reduced one-half in RT-PCR.

CONCLUSIONChemokine receptors are expressed very differently at mRNA level on HCC cell lines with different metastatic potentials. The different profiles of chemokine receptors in tumor microenvironment and the function of CXCR4 in HCC should be further studied. Our findings have important implications in understanding the relationship between chemokine receptors and the metastatic potential of HCC.

Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line, Tumor ; Humans ; Liver Neoplasms ; metabolism ; pathology ; RNA, Messenger ; genetics ; Receptors, Chemokine ; metabolism

BACKGROUNDThe p22phox is a critical component of the superoxide-generating vascular nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Several polymorphisms in p22phox gene are studied for their association with cardiovascular diseases. However, no publication is available to assess the relation of 549C > T polymorphism in p22phox gene to coronary artery disease (CAD) risk. This study was to investigate the effect of the p22phox gene 549C > T polymorphism on CAD risk.

METHODSHospital-based case-control study was conducted with 297 CAD patients and 343 healthy persons as the control group. Polymerase chain reaction and pyrosequencing using PSQ 96 MA Pyrosequencer (Biotage AB) were used to detect the polymorphisms. Multiple Logistic regression model was used to adjust the potential confounders and to estimate odds ratio (OR) with 95% confidence intervals (CIs).

RESULTSThe observed genotype frequencies of this polymorphism obeyed the Hardy-Weinberg equilibrium in both cases (P = 0.439) and controls (P = 0.668). The frequency of mutant genotypes (TT + CT) in cases (41.08%) was higher than that in controls (36.73%) with an OR = 1.20 (95%CI = 0.87-1.65). After the adjustment of the potential confounders, there was a significant association of the mutant genotypes with increased risk of CAD (OR = 1.57, 95%CI = 1.01-2.46, P = 0.047).

CONCLUSIONSThe mutant genotypes of the p22phox gene 549C > T polymorphism had a significant effect on the increased risk of CAD in this studied population.

Case-Control Studies ; Coronary Artery Disease ; etiology ; genetics ; Female ; Genotype ; Humans ; Logistic Models ; Male ; Middle Aged ; NADPH Oxidases ; genetics ; Polymorphism, Single Nucleotide

Objective To assess the contribution of contrast-enhanced spectral mammography (CESM) in detecting breast carcinoma of dense breasts. Methods To retrospectively analyze the imaging and clinical data of 52 female patients with breast carcinoma which were confirmed by pathology in Tai'an Central Hospital of Shandong Province from April 2017 to April 2018.All cases classified as dense or uneven dense breasts by DM examination underwent Ultrasound (US), digital mammography (DM), CESM, dynamic contrast enhanced MRI (DCE-MRI).The breast imaging report and data system (BI-RADS) and breast density classification were both evaluated using the 5th edition of BI-RADS. The efficacy of US, DM, DM+CESM, DCE-MRI in detecting breast carcinoma (BI-RADS 5) was evaluated by χ2 test. Results Histopathology confirmed that 87 lesions were malignant and 35 lesions were benign. The sensitivity of US, DM, DM +CESM, DCE-MRI were 66.67％(58/87), 64.37％(56/87), 100.00％(87/87), 100.00％(87/87) and the specificity were 94.28％(33/35), 74.28％(26/35), 85.71％(30/35), 51.43％(18/35), respectively. There was statistically significant difference in specificity (χ2=9.545, P=0.002) and BI-RADS 5 category, detection 39.08％(34/87), 22.99％(20/87), respectively (χ2=5.263, P=0.022) between the DM + CESM group and DCE-MRI group. Conclusion In dense breasts, CESM has a high application value in breast carcinoma diagnosis.

Post-translational degradation of protein plays an important role in cell life. We employed chimeric molecules (dihydrotestosterone-based proteolysis-targeting chimeric molecule [DHT-PROTAC]) to facilitate androgen receptor (AR) degradation via the ubiquitin-proteasome pathway (UPP) and to investigate the role of AR in cell proliferation and viability in androgen-sensitive prostate cancer cells. Western blot analysis and immunohistochemistry were applied to analyse AR levels in LNCaP cells after DHT-PROTAC treatment. Cell counting and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) cell viability assay were used to evaluate cell proliferation and viability after AR elimination in both LNCaP and PC-3 cells. AR was tagged for elimination via the UPP by DHT-PROTAC, and this could be blocked by proteasome inhibitors. Degradation of AR depended on DHT-PROTAC concentration, and either DHT or an ALAPYIP-(arg)(8) peptide could compete with DHT-PROTAC. Inhibition of cell proliferation and decreased viability were observed in LNCaP cells, but not in PC-3 or 786-O cells after DHT-PROTAC treatment. These data indicate that AR elimination is facilitated via the UPP by DHT-PROTAC, and that the growth of LNCaP cells is repressed after AR degradation.
Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Dihydrotestosterone ; pharmacology ; Dose-Response Relationship, Drug ; Humans ; Male ; Prostatic Neoplasms ; drug therapy ; metabolism ; pathology ; Proteasome Endopeptidase Complex ; metabolism ; Receptors, Androgen ; metabolism ; Recombinant Fusion Proteins ; pharmacology ; therapeutic use ; Signal Transduction ; drug effects ; Ubiquitin ; metabolism