The Effect of Cyclooxygenase-2 Inhibitor on the Gene Expression Profile of N-butyl-N-(4-hydroxybutyl) nitrosamine-induced Rat Urinary Bladder Cancer / 대한비뇨기과학회지

PURPOSE: Cyclooxygenase (COX)-2 plays an important role in promoting cancer cell proliferation and angiogenesis in human bladder cancer. In this study, we investigated the antitumor or antiangiogenic effects of selective COX-2 inhibitor on N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN)-induced rat bladder tumorigenesis. MATERIALS AND METHODS: Forty male Fischer 344 rats (control) were given only 0.05% BBN, while 40 rats (experimental) were administered 1,500mg/ kg celecoxib once daily and this treatment started from 1 week before their BBN treatment. Ten rats from the control groups and the experimental groups were then sacrificed at 4, 12, 16 and 24 weeks after BBN treatment. We observed all the bladders macroscopically as well as microscopically, and we measured the COX-2 expression in the bladder tissues. Utilizing a cDNA microarray, we analyzed the significant differences of gene expression between the 12 week-control group and the 12 week-experimental group. RESULTS: The incidence of tumor was lower in the experimental group than in the control group from week 12 to week 24. The COX-2 expressions were more significantly decreased via the BBN induction (p<0.05) in the experimental groups than in the control groups after 4 weeks. For the 12 week-experimental group, there were 15 genes altered by the administration of selective COX-2 inhibitor, and the selective COX-2 inhibitor especially regulated transgelin, membrane metallo endopeptidase and apolipoprotein E of these 15 genes to prevent the incidence of bladder tumor. CONCLUSIONS: Selective COX-2 inhibitor has an inhibitory effect on BBN-induced rat bladder tumorigenesis. In the pre-neoplastic phase, selective COX-2 inhibitor regulates transgelin, membrane metallo endopeptidase and apolipoprotein E to prevent the incidence of bladder tumor.

Prognostic Value of Cyclooxygenase-2 and p53 Expression in Stage T1 Grade III Bladder Cancer / 대한비뇨기과학회지

Purpose: The prognostic value of p53 remains controversial in stage T1, grade III (T1GIII) transitional cell carcinomas (TCC) of the bladder. Recent studies have reported increased expression of cyclooxygenase-2 (COX-2) in bladder cancer. The prognostic values of p53 and COX-2 were compared in T1GIII TCC of the bladder. Materials and Methods: Among 57 consecutive patients, diagnosed with T1GIII TCC of the bladder by transurethral resection (TURB), 37 were eligible for this study. Exclusion criteria included; the first TURB performed elsewhere, no or inadequate (less than 6 weeks) bacillus Calmette- Guerin treatment and postoperative follow-up of less than 1 year. The expressions of p53 and COX-2 were evaluated by immunohistochemical staining of TURB tissues. Possible correlations of the p53 and COX-2 expressions with the clinicopathological features, such as age, shape and multiplicity of tumor, recurrence and progression, were examined. Results: During a mean follow-up of 27 months, the disease recurred in 43.2% and progressed in 16.2%. Of the 37 specimens, 31 (83.8%) and 16 (43.2%) stained positive for COX-2 and p53 expressions, respectively. There were no significant differences in age, shape and multiplicity of the tumors, recurrence-free survival and progression-free survival between the p53 positive and negative groups. However, the recurrence-free and progression-free survivals were significantly lower in the COX-2 positive than in the COX-2 negative group (p=0.049 and p=0.027, respectively). When combined, p53 and COX-2 more accurately predicted recurrence than COX-2 alone (p=0.036), but not the progression (p=0.776). Conclusions: In the pathologically homogeneous group of T1GIII TCC of the bladder, COX-2 was superior to p53 in predicting the prognosis.

Anti-angiogenic Effect of Selective Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor(ZD1839) against Murine Renal Cell Carcinoma Orthotopic Model / 대한비뇨기과학회지

PURPOSE: ZD1839(IressaTM) is a selective epidermal growth factor receptor tyrosine kinase inhibitor(EGFR-TKI) known to block the cell signaling pathway. However, the effect of ZD1839 in relation to renal cell carcinoma, which is highly angiogenic, has not been reported. Using an orthotopic model of murine renal cell carcinoma(Renca), we evaluated the inhibitory effect of ZD1839 on tumor growth and angiogenesis. MATERIALS AND METHODS: Renca cells (1x10(4)cells/10microliter) were first adsorbed in Gelfoam and were implanted into the balb/cj mouse kidney followed by obturation with the agarose bar. Then, tumor formation was assessed every week for 4 weeks. IC50 was obtained for ZD1839 and genistein in vitro. 7 days after the implantation, the mice were divided into three groups, and normal saline, ZD1839(40mg/kg/day), and genistein (80mg/kg/day) were subcutaneously injected for 14 days. 21 days after the implantation, the mice were sacrificed, and tumor volume measurement and analysis of microvessel density(MVD) were performed using the factor VIII-related antigen and vascular endothelial growth factor(VEGF). RESULTS: Renca tumors, which formed in the renal parenchyme and had a circular shape, reached the peak growth velocity between 14 and 21 days. MVD was the highest at 14 days of implantation. IC50 for ZD1839 and genistein were 4.68microM and 5.43microM, respectively. Tumor growth after the treatment with ZD1839 and genstein was inhibited by 86%(p<0.01) and 49%(p<0.05), respectively, compared to the control. MVD of ZD1839 and genistein-treated groups were 50%(p<0.01) and 29%(p<0.05) lower, respectively, and VEGF levels were 24%(p<0.05) and 27%(p<0.05) lower, respectively, compared to the control. CONCLUSIONS: This orthotopic implantation method of the Renca cell is an effective model for demonstrating the effect of an angiogenesis inhibitor. Our results suggest that the anti-angiogenesis effect of ZD1839 in the Renca orthotopic implantation model partially contributes to the tumor growth inhibition, and that ZD1839 may be more effective than genistein in the tumor growth regulation through the inhibition of angiogenesis.

Cyclooxygenase-2 Inhibitor Delayed the Tumorigenesis of N-Butyl-N-(4-hydroxybutyl)nitrosamine-induced Rat Urinary Bladder Cancer Model / 대한비뇨기과학회지

PURPOSE: Cyclooxygenase-2 (COX-2) plays an important role in promoting cancer cell proliferation and angiogenesis in human bladder cancer. The selective COX-2 inhibitor has antitumor activities in vivo and in vitro in a variety of tumor types. In this study, the antitumor or antiangiogenic effects of selective COX-2 inhibitor on N-butyl-N-(4-hydroxybutyl)nitrosamine-induced rat bladder tumorigenesis were investigated. MATERIALS AND METHODS: Forty male Fischer 344 rats (Control group) were given only 0.05% BBN in water ad libitum, while 40 others (Experimental group) were administered 1,500mug/kg celecoxib once daily through the gavage tube, which started 1 week before the BBN treatment. Ten rats were used as the normal bladder. Ten rats from the control and experimental group were sacrificed 4, 12, 16, and 24 weeks after the start of the BBN treatment. All bladders were evaluated both macroscopically and microscopically. We also measured COX-2 expression, microvessel density (MVD), and vascular endothelial growth factor (VEGF) protein concentrations in the bladder tissues. RESULTS: Macroscopically and microscopically, the incidence of tumor was lower in the experimental group than in the control group from the 12th week to the 24th week. Each incidence of tumor in week 12, week 16, and week 24 was 20%, 50%, and 80% in the control group and 0%, 20%, and 40% in the experimental group, respectively. In both the control and experimental groups, COX-2 expression had a tendency to be concentrated in the cytoplasm of the epithelial cells of the papillary tumor and the endothelial cells adjacent to the vessel the basal layer of bladder. COX-2 and VEGF expression were significantly more decreased in the experimental groups than in the control groups after 4 weeks from the BBN induction (p<0.05). MVD was significantly decreased in the experimental group at week 16 (p<0.05). CONCLUSIONS: The selective COX-2 inhibitor has an inhibitory effect on BBN-induced rat bladder tumorigenesis because of its partially antiangiogenic properties. In the future, the selective COX-2 inhibitor could be expected to play an important role as a chemo-preventive agent and as therapeutic aids in bladder cancer if these inhibitory effects can be reproduced in human bladder tumorigenesis.

Expression of Heme Oxygenase-2 and Smooth Muscle Relaxation by Carbon Monoxide in Rat Prostate: Effects of Age and Castration / 대한비뇨기과학회지

PURPOSE: Carbon monoxide (CO), as well as nitric oxide (NO), have been proposed as potential effectors in the non-adrenergic, non-cholinergic (NANC), nerve-mediated relaxation of the prostate. Attempts were made to determine the localization and expression of heme oxygenase-2 (HO-2), and to observe the change in the relaxation of the smooth muscle induced by CO, depending on age and castration, in rat prostate glands. MATERIALS AND METHODS: The prostate smooth muscles, isolated from young (125+/-4.5g, n=18), adult (321+/-17.8g, n=18), old (413+/-6.4g, n=18) and castrated adult (318+/-15.2g, n=18) rats, were used. The HO-2 immunohistochemistry was observed using the rabbit anti-HO-2 polyclonal antibody. The expressions of HO-2 were measured by a reverse transcriptase-polymerase chain reaction (RT-PCR) and western blotting. Polygraphy, connecting the force displacement transducer, was used to observe the relaxation effect of CO. To investigate the relaxation action of the CO mediated, to the soluble guanylate cyclase (sGC) enzyme inhibitors, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and methylene blue were added to the reaction solutions. RESULTS: The HO-2 was located in the nerve fibers of the rat prostates. A quantitative analysis of PCR products revealed greater decreased levels of HO-2 mRNA and protein expression in the young (p<0.05) and castrated adults (p<0.05) than in the normal adult rats. The relaxation effect of CO was greater in the adult than in the young (p<0.05), old and castrated adult rats (p<0.05), but the effect was inhibited by the addition of ODQ and methylene blue in all groups (p<0.05). CONCLUSIONS: CO has an effect on the relaxation of rat prostate smooth muscle. The expression of HO-2 in the prostate became higher with increasing age, so it is estimated the relaxation effect of CO in the older adults will be higher than with the other ages. Androgen deprivation decreases relaxation effect of CO in prostate smooth muscle.

Expression of Neuroendocrine Cells in Benign Prostatic Hyperplasia and the Effect of Dihydrotestosterone / 대한비뇨기과학회지

PURPOSE: Neuroendocrine (NE) cells of the prostate are considered to be involved in the pathogenesis of benign prostate hyperplasia (BPH). By a comparative analysis of NE cell density in BPH tissue of men who were either exposed to or not exposed to 5alpha-reductase inhibitor, we investigated the relationship between NE cells and BPH, and the effect of androgen deprivation on NE cells. MATERIALS AND METHODS: Prostate tissue specimens, obtained from 30 men by transurethral resection of the prostate or radical cystoprostatectomy, were used. Of the 30 patients, 10 had a prostate smaller than 25 ml (normal control), the other 20 had a prostate larger than 40ml, 10 of who had taken 5alpha-reductase inhibitor (finasteride) for 3 months before surgery (androgen blockade group), and 10 who had not (BPH group). The distribution of NE cells in the prostate was examined using the anti-chromogranin A (CgA) antibody, and the density of the CgA-positive cells was compared by an optical dissector method. Immunoblotting was performed using the neuron specific enolase (NSE) antibody. A Mann-Whitney U test was used in a statistical analysis. RESULTS: Most of the CgA-positive NE cells were localized between the acinar epithelial cells. The mean numbers of CgA-positive NE cells per acinus in the normal controls and the BPH groups were 1.67+/-0.78 and 4.45+/-2.54, respectively, and the difference was statistically significant (p<0.05). However, the mean number of CgA-positive NE cells in the androgen blockade group, was 4.93+/-2.17, which was similar to the BPH group. In a NSE immunoblotting study, a distinct band was observed in the BPH and androgen blockade groups, but the density of the band was higher in the androgen blockade group. CONCLUSIONS: Our results suggest that NE cells may be involved in the hyperplastic process of BPH. Inhibition of dihydrotestosterone, caused by the oral administration of the 5alpha-reductase inhibitor, failed to induce any significant change in the NE cells, probably due to the incomplete androgen blockade.

Comparative Study of Concentration of Isoflavones and Lignans in Plasma and Prostatic Tissues of Normal Control and Benign Prostatic Hyperplasia

Objective: Isoflavones and lignans are phytoestrogens that have recently gained interest as dietary factors related to prostatic diseases. However, no data on the concentrations in prostate tissue in humans is available. Therefore, the concentrations of isoflavones and lignans in plasma and prostatic tissues according to the prostate volume were compared to determine their possible effect on the benign prostatic growth. Methods: Fasting plasma and prostatic tissue specimens were acquired from 25 men over 50 years of age with similar normal dietary habits and no previous history of drug intake that could affect the isoflavones and lignans levels. The tissue was acquired either during a transurethral resection of the prostate in 15 patients with benign prostatic hyperplasia (BPH) with prostate volume over 40 ml or during a radical cystoprostatectomy in 10 patients with bladder cancer with a prostate volume < 25 ml, who were used as the controls. Quantitative analysis of the isoflavones, specifically equol, daidzein and genistein and lignans, particularly enterodiol and enterolactone, was performed by gas chromatography-mass spectrometry. Results: The mean prostatic concentrations of enterodiol, enterolactone, equol and daidzein in the BPH and the control groups were similar. However, the mean prostatic concentration of genistein was significantly lower in the BPH group than in the control group (65.43 +/- 17.05 vs 86.96 +/- 37.75 ng/ ml, respectively, p=0.032). The plasma concentration of isoflavones and lignans in the two groups were comparable. Conclusion: Isoflavones, but not lignans, have some influence the benign prostatic growth, and the prostatic concentration of genistein possibly has the closest association among them. More studies to further clarify the roles and mechanisms of isoflavone action on BPH including pharmacokinetic studies are recommended.

Changes in the Expression of Bone Morphogenetic Protein Receptor Type IA, IB and II in Human Prostate by the Suppression of Dihydrotestosterone / 대한비뇨기과학회지

PURPOSE: To investigate whether the expression of type IA, IB and II bone morphogenetic protein receptors (hBMPRs) are affected by the suppression of dihydrotestosterone (DHT) in the prostate tissues of patients with benign prostatic hyperplasia (BPH), we determined mRNA levels and protein expression patterns of the hBMPRs in human prostate tissues. MATERIALS AND METHODS: Frozen tissues were obtained during the transurethral resection of the prostate (TURP) in BPH patients who had taken the 5alpha-reductase inhibitor (finasteride), for 3 months prior to surgery, for the reduction of the prostate volume. Tissues from patients who had not taken finasteride prior to the TURP were used as controls. Patients over 50 years old, and with a prostate volume over 50ml, were included. Semiquantitative polymerase chain reaction (PCR) and immunoblotting were used to compare the expressions of the human bone morphogenetic protein receptors (hBMPRs) between the experimental group and the controls. RESULTS: All of the BMPRs proteins were expressed in the human benign prostate tissues, with various concentrations. The semiquantitative PCR analysis showed that the mRNA level of the hBMPRs tended to decrease following 5alpha-reductase inhibition, and the magnitude of this decrease was the greatest for the hBMPR-IB. CONCLUSIONS: In the human prostate, only the expression of hBMPR-IB was significantly reduced by the suppression of DHT. Further studies on the possible role of the hBMP-hBMPR-IB complex, in the abnormal proliferation of the prostate under physiological conditions, are warranted.

Development of Rat Prostatitis Model by Oral Administration of Isoflavone and Its Characteristics

Inflammation of the prostate can be induced experimentally in rats by the subcutaneous administration of estrogen. However, it is usually achieved at the price of some alteration in the sex steroid hormone balance and morphological changes in the prostate. In this study, a soy-extracted isoflavone mixture with weak estrogenic activity was administered orally in an attempt to induce prostatitis in a more physiologic way and to characterize the inflammation induced. A total of 36 male Sprague-Dawley rats, 8 weeks old, were divided into 2 groups. The control group was fed with only an AIN-76A diet containing no detectable phytoestrogen and the experimental group was fed with AIN-76A and a soy- extracted isoflavone mixture (genistein 60.0% and daidzein 19.6%), 300mg/kg body weight for 9 weeks. The sequential body weight and prostate weight at necropsy were measured. A histologic examination and histomorphometry assessed the changes in the prostate. The serum concentrations of testosterone and dihydrotestosterone were measured to estimate the effects on the androgen level. Intraprostatic concentrations of genistein and daidzein were measured by gas chromatography/ mass spectroscopy (GC/MS). While no sign of prostate inflammation was apparent in the control group, severe inflammatory changes in the stroma, increased epithelial detachment and inflammatory exudates within the glandular lumen of the dorsolateral prostate were observed in more than 80%(15/18) of the experimental group. However, there was no significant difference in the ventral prostate between the two groups. The daidzein and genistein concentrations in both the lateral and ventral prostates were significantly higher in the experimental group than in the control group where no isoflavone was detectable. In addition, the concentrations were much higher in the dorsolateral than in the ventral prostate. Although the body weight gain was not consistent in the experimental group, there were no significant differences in the prostate weight and serum androgen level between groups. In summary, when a soy-extracted genistein and daidzein-rich isoflavone mixture was administered orally into rats, prostatic inflammation with characteristic lobe specificity developed. The present method of inducing prostatitis seems to be a more physiologic than an estrogen-induced experimental model, and sequential pharmacokinetic studies might help in establishing this model as a more valuable tool in assisting future research in this field.