Eddie Shu-Yin Chan

Activation of GPR30 inhibits the growth of prostate cancer cells through sustained activation of Erk1/2, c-jun/c-fos-dependent upregulation of p21, and induction of G 2 cell-cycle arrest

G-protein-coupled receptor-30 (GPR30) shows estrogen-binding affinity and mediates non-genomic signaling of estrogen to regulate cell growth. We here showed for the first time, in contrast to the reported promoting action of GPR30 on the growth of breast and ovarian cancer cells, that activation of GPR30 by the receptor-specific, non-estrogenic ligand G-1 inhibited the growth of androgen-dependent and androgen-independent prostate cancer (PCa) cells in vitro and PC-3 xenografts in vivo. However, G-1 elicited no growth or histological changes in the prostates of intact mice and did not inhibit growth in quiescent BPH-1, an immortalized benign prostatic epithelial cell line. Treatment of PC-3 cells with G-1 induced cell-cycle arrest at the G2 phase and reduced the expression of G2-checkpoint regulators (cyclin-A2, cyclin-B1, cdc25c, and cdc2) and phosphorylation of their common transcriptional regulator NF-YA in PC-3 cells. With extensive use of siRNA-knockdown experiments and the MEK inhibitor PD98059 in this study, we dissected the mechanism underlying G-1-induced inhibition of PC-3 cell growth, which was mediated through GPR30, followed by sustained activation of Erk1/2 and a c-jun/c-fos-dependent upregulation of p21, resulting in the arrest of PC-3 growth at the G2 phase. The discovery of this signaling pathway lays the foundation for future development of GPR30-based therapies for PCa.

Expression of microRNAs in the Urine of Patients With Bladder Cancer

UNLABELLED We quantified the urine sediment and supernatant levels of microRNA (miRNA) targets related to epithelial-mesenchymal transition in 51 patients with bladder cancer and in 24 controls. We found that patients with bladder cancer had depressed levels of the miR-200 family, miR-192, and miR-155 in urinary sediment. The urinary level of these miRNAs may be developed as noninvasive markers for bladder cancer. BACKGROUND MicroRNAs (miRNA) have been implicated to play an important role in the pathogenesis of a variety of cancers. We studied the levels of miRNAs related to epithelial-mesenchymal transition (EMT) in the urine of patients with bladder cancer. METHOD The expression of the miR-200 family, miR-205, miR-192, miR-155, and miR-146a in the urine sediment and supernatant of 51 patients with bladder cancer and in 24 controls was determined by real-time quantitative polymerase chain reaction. RESULTS Compared with controls, the patients with bladder cancer had a lower expression of the miR-200 family, miR-192, and miR-155 in the urinary sediment; lower expression of miR-192; and higher expression of miR-155 in the urinary supernatant. The expression of the miR-200 family, miR-205, and miR-192 in the urine sediment significantly correlated with urinary expression of EMT markers, including zinc finger E-box-binding homeobox 1, vimentin, transforming growth factor β1, and Ras homolog gene family, member A. Furthermore, the levels of miR-200c and miR-141 in the urine sediment became normalized after surgery. CONCLUSION We found that the urinary miR-200 family, miR-155, miR-192, and miR-205 levels are depressed in patients with bladder cancer. The level of these miRNA targets in urine has the potential to be developed as noninvasive markers for bladder cancer.

Development of a Scoring System From Noncontrast Computerized Tomography Measurements to Improve the Selection of Upper Ureteral Stone for Extracorporeal Shock Wave Lithotripsy

PURPOSE We investigated the role of noncontrast computerized tomography in predicting the treatment outcome of shock wave lithotripsy on upper ureteral stones to formulate a clinical algorithm to facilitate clinical management. MATERIALS AND METHODS Adult patients with upper ureteral stones confirmed by noncontrast computerized tomography and scheduled for primary in situ shock wave lithotripsy were prospectively recruited. Standardized treatment was performed on each patient. The primary end point was stone-free status at 3 months. Pretreatment noncontrast computerized tomography was assessed by a single radiologist blinded to the clinical parameters. Predictive values of computerized tomography measurements on the treatment outcome were then assessed. RESULTS Between October 2004 and July 2007 a total of 94 patients (60 male and 34 female) were recruited for the study. Logistic regression showed that stone volume, mean stone density and skin-to-stone distance were potential predictors of successful treatment. From ROC curves the optimum cutoff for predicting treatment outcomes for stone volume, mean stone density and skin-to-stone distance was 0.2 cc, 593 HU and 9.2 cm, respectively. A simple scoring system was constructed based on the 3 factors of stone volume less than 0.2 cc, mean stone density less than 593 HU or skin-to-stone distance less than 9.2 cm. The stone-free rate for patients having 0, 1, 2 and 3 factors was 17.9%, 48.4%, 73.3% and 100%, respectively (linear-by-linear association test 22.83, p <0.001). CONCLUSIONS Stone volume, mean stone density and skin-to-stone distance were potential predictors of the successful treatment of upper ureteral stones with shock wave lithotripsy. A scoring system based on these 3 factors helps separate patients into outcome groups and facilitates treatment planning.

Mouse orthotopic models for bladder cancer research

Bladder cancer is a common malignancy in the urinary tract. Despite different therapeutic options, recurrence and progression of urothelial carcinoma after treatment is not uncommon. Novel therapeutic options of bladder cancer are urgently needed. The preclinical evaluation of new treatments requires an animal tumour model that mimics the human counterpart. To date, various animal orthotopic bladder cancer models have been described, but the reported rate of tumour ‘take’ is 30–100%. The establishment of reliable and reproducible animal models remains an ongoing challenge. We review different kinds of mouse models of orthotopic bladder cancer used in urothelial cancer studies, the methods of implantation, and the reported rate of tumour take. Significant progress has been made recently in noninvasive small animal‐imaging in tumour models. It is now possible for researchers to investigate the effects of studied agents by monitoring of in vivo tumour growth directly and noninvasively, as well as measuring a wide range of tumour‐related variables in small animals. We summarize the recent development in small‐animal imaging for tumour detection and quantification.

Cell-Free Urinary MicroRNA-99a and MicroRNA-125b Are Diagnostic Markers for the Non-Invasive Screening of Bladder Cancer

Background Evidence implicated the diagnostic significance of microRNAs in whole urine/urine sediments in urothelial carcinoma of the bladder (UCB). However, the contaminated blood cells in patients with haematouria significantly altered the expression profiles of urinary microRNA, influencing the test accuracy. Methods MicroRNA profiles of the urine supernatants of UCB patients and controls without any malignancy and profiles of malignant and corresponding normal mucosa tissues from the patients were determined by microRNA microarray and compared to identify differentially expressed microRNAs. The differential expression was verified in the tissues of an independent patient cohort by RT-qPCR. The diagnostic significance of selected microRNAs as biomarkers in the urine supernatant was investigated in the expanded cohorts. Results MicroRNA-99a and microRNA-125b were down-regulated in the urine supernatants of UCB patients. The degree of down-regulation was associated with the tumor grade. A diagnostic model was developed using a combined index of the levels of microRNA-99a and microRNA-125b in the urine supernatant with a sensitivity of 86.7%, a specificity of 81.1% and a positive predicted value (PPV) of 91.8%. Discriminating between high- and low-grade UCB, the model using the level of microRNA-125b alone exhibited a sensitivity of 81.4%, a specificity of 87.0% and a PPV of 93.4%. Conclusions The results revealed a unique microRNA expression signature in the urine supernatants of UCB patients for the development of molecular diagnostic tests. An effective cell-free urinary microRNA-based model was developed using a combined index of the levels of microRNA-99a and microRNA-125b to detect UCB with good discriminating power, high sensitivity and high specificity.

Optimizing Orthotopic Bladder Tumor Implantation in a Syngeneic Mouse Model

PURPOSE We established a reliable technique for orthotopically implanting bladder tumor cells in a syngeneic mouse model. MATERIALS AND METHODS MBT-2 murine bladder cancer cells were transurethrally implanted in the bladder of syngeneic C3H/He mice (Jackson Laboratory, Bar Harbor, Maine). Different chemical pretreatments were used before tumor implantation, including phosphate buffered saline (control), HCl, trypsin and poly-L-lysine. MBT-2 cells (1 x 10(6) or 2 x 10(6)) were instilled into the intravesical space after chemical pretreatment. Tumor take and bladder tumor volume were determined by micro ultrasound. Bladders were harvested at the end of the study to measure bladder weight and for histopathological examination. RESULTS Bladder pretreatment with HCl in 5 preparations was discontinued due to significant adverse reactions, resulting in death in 1 mouse, and severe bladder inflammation and hematuria 3 days after pretreatment in 2. Pretreatment with phosphate buffered saline, trypsin and poly-L-lysine in 6 animals each was tolerated well without significant adverse reactions or mortality. The tumor take rate in the control, trypsin and poly-L-lysine pretreatment groups was 33%, 83% and 83%, respectively. The take rate was higher in mice instilled with 2 x 10(6) cells than in those with 1 x 10(6) cells (93% vs 73%, p <0.05). CONCLUSIONS We report a reliable, feasible method of orthotopically implanting bladder tumor cells into a syngeneic mouse model. Poly-L-lysine and trypsin are useful adjunctive pretreatment agents to improve bladder tumor uptake. This model may be suitable to evaluate treatment paradigms for bladder cancer.

A MicroRNA-7 Binding Site Polymorphism in HOXB5 Leads to Differential Gene Expression in Bladder Cancer

Purpose To investigate the biological function of HOXB5 in human bladder cancer and explore whether the HOXB5 3′-UTR SNP (1010A/G), which is located within the microRNA-7 binding site, was correlated with clinical features of bladder cancer. Methods Expression of HOXB5 in 35 human bladder cancer tissues and 8 cell lines were examined using real-time PCR and immunohistochemistry. Next, we explored the biological function of HOXB5 in vitro using cell proliferation, migration and colony formation assays. Using bioinformatics, a SNP (1010A/G) was found located within the microRNA-7 binding site in the 3′-UTR of HOXB5. Real-time PCR was used to test HOXB5 expression affected by different alleles. Finally, multivariate logistic regression analysis was used to determine the relationship between SNP (1010A/G) frequency and clinical features in 391 cases. Results HOXB5 was frequently over-expressed both in bladder cancer tissues and cell lines. Inhibition of HOXB5 suppressed the oncogenic function of cancer cells. Next, we demonstrated that a SNP (1010A/G), located within the microRNA-7 binding site in the 3′-UTR of HOXB5, could affect HOXB5 expression in bladder cancer mainly by differential binding activity of microRNA-7 and SNP-related mRNA stability. Finally, we also showed the frequency of 1010G genotype was higher in cancer group compared to normal controls and correlated with the risk of high grade and high stage. Conclusion HOXB5 is overexpressed in bladder cancer. A miRNA-binding SNP (1010A/G) located within 3′-UTR of HOXB5 is associated with gene expression and may be a promising prognostic factor for bladder cancer.

Effect of Vardenafil on Blood Pressure Profile of Patients With Erectile Dysfunction Concomitantly Treated With Doxazosin Gastrointestinal Therapeutic System for Benign Prostatic Hyperplasia

PURPOSE We investigated the effect of the combination of the doxazosin gastrointestinal therapeutic system and 10 mg vardenafil on the hemodynamic status of patients with benign prostatic hyperplasia and erectile dysfunction. MATERIALS AND METHODS This was a double-blinded, randomized, placebo controlled crossover trial. Patients with benign prostatic hyperplasia and erectile dysfunction treated with the doxazosin gastrointestinal therapeutic system on a regular basis, with no other antihypertensive events, were recruited. Subjects took 10 mg vardenafil or placebo in a randomized crossover fashion with a washout period of at least 7 days between each treatment. The supine and standing blood pressure of the subjects was recorded from 1 hour before to 6 hours after the administration of vardenafil or placebo. The primary outcome of the study was the maximal change in standing systolic blood pressure of the subjects from 1 half hour before to 6 hours after the administration of drugs. RESULTS A total of 37 patients, 25 (67.6%) and 12 (32.4%) on the doxazosin gastrointestinal therapeutic system at 4 mg and 8 mg, respectively, completed the trial. The combination drug therapy resulted in a maximal decrease in standing systolic blood pressure of 6.18 mm Hg (95% CI -12.02, -0.33; p = 0.039). Only 1 patient had an asymptomatic standing systolic blood pressure of less than 85 mm Hg. Otherwise no symptomatic hypotension or clinically significant adverse cardiovascular event was observed during the study. CONCLUSIONS In patients on the doxazosin gastrointestinal therapeutic system for benign prostatic hyperplasia a single 10 mg dose of vardenafil had no symptomatic hemodynamic effects.

Sunitinib Malate Provides Activity Against Murine Bladder Tumor Growth and Invasion in a Preclinical Orthotopic Model

OBJECTIVE To evaluate the effects of sunitinib on localized bladder cancer in a mouse orthotopic bladder tumor model. METHODS We used an established orthotopic mouse bladder cancer model in syngeneic C3H/He mice. Treatment doses of 40 mg/kg of sunitinib or placebo sterile saline were administrated daily by oral gavage. Tumor volume, intratumoral perfusion, and in vivo vascular endothelial growth factor receptor-2 expression were measured using a targeted contrast-enhanced micro-ultrasound imaging system. The findings were correlated with the total bladder weight, tumor stage, and survival. The effects of sunitinib malate on angiogenesis and cellular proliferation were measured by immunostaining of CD31 and Ki-67. RESULTS Significant inhibition of tumor growth was seen after sunitinib treatment compared with the control. The incidence of extravesical extension of the bladder tumor and hydroureter in the sunitinib-treated group (30% and 20%, respectively) was lower than the incidence in the control group (66.7% and 55.6%, respectively). Sunitinib therapy prolonged the survival in mice, with statistical significance (log-rank test, P = .03). On targeted contrast-enhanced micro-ultrasound imaging, in vivo vascular endothelial growth factor receptor-2 expression was reduced in the sunitinib group and correlated with a decrease in microvessel density. CONCLUSION The results of our study have demonstrated the antitumor effects of sunitinib in the mouse localized bladder cancer model. Sunitinib inhibited the growth of bladder tumors and prolonged survival. Given that almost 30% of cases in our treatment arm developed extravesical disease, sunitinib might be suited as a part of a multimodal treatment regimen for bladder cancer.

Transabdominal Micro-ultrasound Imaging of Bladder Cancer in a Mouse Model: A Validation Study

OBJECTIVES To validate the use of transabdominal micro-ultrasound imaging (MUI) in an orthotopic murine bladder cancer model. The current in vivo imaging systems for murine bladder cancer include magnetic resonance imaging, bioluminescent and fluorescent imaging, and intravesical ultrasound. METHODS We implanted murine bladder tumor-2 tumor cells into C3H/He female mice. Mice underwent MUI before, and every 3 days after instillation of tumor cells. Three mice were killed at every MUI session. Bladder tumors were measured and tumor volumes were calculated during MUI and gross stereomicroscopy. Bladders were harvested and examined under gross stereomicroscopy to confirm the presence, location, and size of bladder tumors, and were prepared for histology review. RESULTS Overall, 15 of 33 (45%) mice were confirmed to have tumors, using MUI, gross stereomicroscopy, and histology. Measurements of tumor size by MUI and gross microscopy had a high correlation coefficient (r = 0.97). MUI identified all tumors that were present on final histology. The smallest confirmed tumor on MUI was detected at 0.52 mm(3), and mean tumor volume was 0.95 mm(3). No tumors that were not detected first using MUI were found on final histology. CONCLUSIONS Transabdominal MUI is a valuable tool to use for translational studies involving orthotopic mouse bladder cancer models. MUI provides real-time, high resolution in vivo images of bladder tumors. Tumor presence can be confirmed with a high degree of accuracy pertaining to tumor volume before initiation of treatment. In addition, tumor growth or regression can be followed up in vivo longitudinally.