GnRH antagonist monotherapy versus a GnRH agonist plus bicalutamide for advanced hormone-sensitive prostate cancer; KYUCOG-1401.

OBJECTIVES: To compare the effectiveness and safety of gonadotropin-releasing hormone (GnRH) antagonist monotherapy to combined androgen blockade (CAB) with a GnRH agonist and bicalutamide in patients with advanced hormone-sensitive prostate cancer (HSPC). METHODS: The study was conducted as KYUCOG-1401 trial (UMIN000014243) and enrolled 200 patients who were randomly assigned to either group A (GnRH antagonist monotherapy followed by the addition of bicalutamide) or group B (CAB by a GnRH agonist and bicalutamide). The primary endpoint was PSA progression-free survival. The secondary endpoints were the time to CAB treatment failure, radiographic progression-free survival, overall survival, changes in serum parameters, including PSA, hormones, and bone and lipid metabolic markers, and adverse events. RESULTS: PSA progression-free survival was significantly longer in group B (hazard ratio [HR], 95% confidence interval [CI]; 1.40, 1.01-1.95, p = 0.041). The time to CAB treatment failure was slightly longer in group A (HR, 95% CI; 0.80, 0.59-1.08, p = 0.146). No significant differences were observed in radiographic progression-free survival or overall survival. The percentage of patients with serum testosterone that did not reach the castration level was higher at 60 weeks (p = 0.046) in group A. No significant differences were noted in the serum levels of bone metabolic or lipid markers between the two groups. An injection site reaction was more frequent in group A. CONCLUSIONS: The present results support the potential of CAB using a GnRH agonist and bicalutamide as a more effective treatment for advanced HSPC than GnRH antagonist monotherapy.

The safety and efficacy of relebactam/imipenem/cilastatin in Japanese patients with complicated intra-abdominal infection or complicated urinary tract infection: A multicenter, open-label, noncomparative phase 3 study.

INTRODUCTION: Relebactam, a novel class A/C ß-lactamase inhibitor developed as a fixed-dose combination with imipenem/cilastatin, restores imipenem activity against imipenem-nonsusceptible gram-negative pathogens. METHODS: This phase 3, multicenter, open-label, noncomparative study (NCT03293485) evaluated relebactam/imipenem/cilastatin (250 mg/500 mg/500 mg) dosed every 6 h for 5-14 days in Japanese patients with complicated intra-abdominal infections (cIAIs) or complicated urinary tract infections (cUTIs), including those with secondary sepsis. Sepsis was defined as an infection-induced systemic inflammatory response syndrome, with a documented positive blood culture; patients meeting these protocol-defined criteria were evaluated for efficacy against sepsis. RESULTS: Of 83 patients enrolled, 81 patients (cIAI, n = 37; cUTI, n = 44) received ≥1 dose of study treatment. Escherichia coli was the most common baseline pathogen isolated in both patients with cIAI and cUTI. Adverse events (AEs) were reported in 74.1% (n = 60/81) of patients, and drug-related AEs occurred in 18.5% (n = 15/81). The most common AEs were diarrhea and nausea (8.6%). Serious AEs occurred in nine patients, including one death, but none were considered treatment related. The primary efficacy endpoint for patients with cIAI was clinical response at end of treatment (EOT) in the microbiologically evaluable (ME) population, and for patients with cUTI was microbiological response at EOT in the ME population. The proportion of cIAI and cUTI patients achieving favorable responses were 85.7% (n = 24/28) and 100.0% (n = 39/39), respectively. All patients with sepsis (cIAI, n = 1; cUTI, n = 5) achieved a favorable composite clinical and microbiological response at EOT. CONCLUSIONS: A favorable safety and efficacy profile for relebactam/imipenem/cilastatin was observed in Japanese patients with cIAI and cUTI.

The efficacy and safety of tazobactam/ceftolozane in Japanese patients with uncomplicated pyelonephritis and complicated urinary tract infection.

We report efficacy and safety results for a combination of a novel cephalosporin class antibiotic and a ß-Lactamase inhibitor, tazobactam/ceftolozane (1:2) at a dose of 1.5 g intravenously every 8 h in Japanese patients with uncomplicated pyelonephritis and complicated urinary tract infection. This study design was a nonrandomized, multicenter, open-label trial, and the treatment period was 7 days. Of 115 patients enrolled in this study, 114 received tazobactam/ceftolozane, and 90 were included in the efficacy analyses. Ninety-nine isolates (bacterial count ≥105 CFU/mL) were identified by urine culture. The main baseline uropathogens were Escherichia coli (80 isolates), Klebsiella pneumoniae (8 isolates), and Proteus mirabilis (3 isolates). Of these, 13 isolates were ESBL-producers. The favorable per-patient microbiological response rate at 7 days after the final administration of tazobactam/ceftolozane was 80.7% (71/88). The response rate in uncomplicated pyelonephritis was 90.0% (36/40), complicated pyelonephritis 63.6% (14/22), and complicated cystitis 80.8% (21/26). The favorable clinical response rate was 96.6% (86/89), and composite response rate (based on microbiological and clinical response) was 80.7% (71/88). The eradication rate by uropathogen was 83.5% (66/79) in E. coli, 42.9% (3/7) in K. pneumoniae, and 100% (3/3) in P. mirabilis. The incidence of drug-related adverse events was 17.5% (20/114 patients). The most common drug-related adverse events were diarrhea and alanine aminotransferase increased in 5.3% (6/114 patients each). Drug-related serious adverse events and deaths were not observed. These results support the safety and efficacy of tazobactam/ceftolozane and suggest it will be a useful treatment for uncomplicated pyelonephritis and complicated urinary tract infection.

Therapeutic effect of propiverine hydrochloride on mixed-type urinary incontinence in women: The Female Urgency and Stress Urinary Incontinence Study of Propiverine Hydrochloride trial.

OBJECTIVES: To show the efficacy of propiverine hydrochloride in the management of symptoms of stress urinary incontinence in female patients with mixed-type urinary incontinence. METHODS: The study was carried out as a multicenter single-arm clinical trial at 64 institutions in Japan. The participants were female patients aged ≥20 years with mixed-type urinary incontinence. The frequency of stress urinary incontinence and urgency urinary incontinence was evaluated at baseline and 4, 8 and 12 weeks after treatment with propiverine hydrochloride. Subjective symptoms were evaluated using the Overactive Bladder Symptom Score and the International Consultation on Incontinence Questionnaire-Short Form. Functional urethral length and maximum urethral closing pressure were also measured at baseline and 12 weeks after treatment at the institutions where the urethral pressure profile was taken. RESULTS: In total, 49 mixed-type urinary incontinence patients were enrolled in the present study. The number of cases of urgency urinary incontinence was reduced time-dependently, which showed statistically significant differences between baseline and 4, 8 and 12 weeks after treatment. A similar statistically different reduction was also observed for stress urinary incontinence. The mean reduction rates of urgency urinary incontinence and stress urinary incontinence at 12 weeks after treatment were 63.9% and 44.3%, respectively. The total scores of International Consultation on Incontinence Questionnaire-Short Form and Overactive Bladder Symptom Score were gradually reduced, and the differences were statistically significant. Functional urethral length and maximum urethral closing pressure at 12 weeks after treatment did not show any statistical differences compared with those at baseline. CONCLUSIONS: Propiverine hydrochloride can be an effective therapeutic option for stress urinary incontinence in patients with mixed-type urinary incontinence.

Stereoselective Synthesis of Highly Functionalized 2,3-Dihydro-4-pyranones Using Phosphine Oxide as Catalyst.

2,3-Dihydro-4-pyranones were synthesized stereoselectively using a chiral phosphine oxide as the catalyst. The phosphine oxide sequentially activated silicon tetrachloride and promoted the double aldol reaction of 4-methoxy-3-buten-2-one with aldehydes. Subsequent stereoselective cyclization afforded the corresponding highly functionalized 2,3-dihydro-4-pyranones bearing three contiguous chiral centers in good yields and with high diastereo- and enantioselectivities.

MiR-96 and miR-183 detection in urine serve as potential tumor markers of urothelial carcinoma: correlation with stage and grade, and comparison with urinary cytology.

A new diagnostic marker for urothelial carcinoma (UC) is needed to avoid painful cystoscopy during the initial diagnosis and follow-up period. However, the current urine markers are useless because of the low sensitivities and specificities for UC detection. MiR-96 and miR-183 were differentially upregulated microRNA in our previous microRNA screening for UC. The expression levels of miR-96 and miR-183 in the urine samples were significantly higher in 100 UC than in healthy controls (miR-96, P=0.0059; and miR-183, P=0.0044). The receiver-operating characteristic curve analyses demonstrated that each microRNA had good sensitivity and specificity for distinguishing UC patients from non-UC patients (miR-96, 71.0% and 89.2%; and miR-183, 74.0% and 77.3%). Our cohort included 78 UC patients who had undergone urinary cytology. MiR-96 was positively detected in 27 of 44 patients who had had a "negative" urinary cytology diagnosis. We combined the miR-96 detection data with the urinary cytology data, and diagnosed 61 of 78 cases as UC; sensitivity rose from 43.6% to 78.2%. We found significant stepwise increases in miR-96 and miR-183 expression with advancing tumor grade (miR-96, P=0.0057; and miR-183, P=0.0036) and pathological stage (miR-96, P=0.0332; and miR-183, P=0.0117). The expression levels of the microRNA were significantly lower in urine collected after surgery (miR-96, P=0.0241; and miR-183, P=0.0045). In conclusion, miR-96 and miR-183 in urine are promising tumor markers for UC. In particular, miR-96 may be a good diagnostic marker in combination with urinary cytology.

Identification of novel microRNA targets based on microRNA signatures in bladder cancer.

MicroRNAs (miRNAs) are small noncoding RNAs that negatively regulate protein-coding genes. To identify miRNAs that have a tumor suppressive function in bladder cancer (BC), 156 miRNAs were screened in 14 BCs, 5 normal bladder epithelium (NBE) samples and 3 BC cell lines. We identified a subset of 7 miRNAs (miR-145, miR-30a-3p, miR-133a, miR-133b, miR-195, miR-125b and miR-199a*) that were significantly downregulated in BCs. To confirm these results, 104 BCs and 31 NBEs were subjected to real-time RT-PCR-based experiments, and the expression levels of each miRNA were significantly downregulated in BCs (p < 0.0001 in all). Receiver-operating characteristic curve analysis revealed that the expression levels of these miRNAs had good sensitivity (>70%) and specificity (>75%) to distinguish BC from NBE. Our target search algorithm and gene-expression profiling in BCs (Kawakami et al., Oncol Rep 2006;16:521-31) revealed that Keratin7 (KRT7) mRNA was a common target of the downregulated miRNAs, and the mRNA expression levels of KRT7 were significantly higher in BCs than in NBEs (p = 0.0004). Spearman rank correlation analysis revealed significant inverse correlations between KRT7 mRNA expression and each downregulated miRNA (p < 0.0001 in all). Gain-of-function analysis revealed that KRT7 mRNA was significantly reduced by transfection of 3 miRNAs (miR-30-3p, miR-133a and miR-199a*) in the BC cell line (KK47). In addition, significant decreases in cell growth were observed after transfection of 3 miRNAs and si-KRT7 in KK47, suggesting that miR-30-3p, miR-133a and miR-199a* may have a tumor suppressive function through the mechanism underlying transcriptional repression of KRT7.

CpG hypermethylation of collagen type I alpha 2 contributes to proliferation and migration activity of human bladder cancer.

In our microarray screening of methylated genes in bladder cancer (BC), the collagen type 1 alpha 2 (COL1A2) gene was the most up-regulated among the 30,144 genes screened. We hypothesize that inactivation of the COL1A2 gene through CpG methylation contributes to proliferation and migration activity of human BC. We subjected a bladder cancer cell line (BOY) and 67 BC specimens and 10 normal bladder epitheliums (NBEs) to conventional or real-time methylation quantitative polymerase chain reaction (PCR) and to real-time reverse transcriptase (RT)-PCR. We also established a stable COL1A2 transfectant for evaluating cell proliferation and migration activity. After 5-aza-dC treatment, the expression levels of COL1A2 mRNA transcript markedly increased in BOY. Our cell proliferation assays consistently demonstrated growth inhibition in the COL1A2 transfectant compared with control and wild-type BOY cells (p<0.0001). Wound healing assays also showed significant wound healing inhibition in the COL1A2 transfectant compared to the counterparts (p=0.0016). We demonstrated by bisulfite DNA sequencing that the promoter hypermethylation of COL1A2 was a frequent event in clinical BCs. The methylation index of COL1A2 was significantly higher in the 67 BCs than in the 10 NBEs (p=0.0011). Conversely, COL1A2 mRNA transcript was significantly lower in the BCs than in the NBEs (p=0.0052). The mechanism of COL1A2 down-regulation in BC is through CpG hypermethylation of the promoter region. COL1A2 gene inactivation through CpG hypermethylation may contribute to proliferation and migration activity of BC.

MiR-133a induces apoptosis through direct regulation of GSTP1 in bladder cancer cell lines.

OBJECTIVE: We previously demonstrated that miR-133a is a tumor-suppressive microRNA (miRNA) and is commonly down-regulated in human bladder cancer (BC). The aim of this study is to determine a novel oncogenic gene targeted by miR-133a in BC. METHODS: To identify genes targeted by miR-133a, an oligo-microarray analysis was performed using the miR-133a-transfected BC cell lines. For gain/loss-of-function studies, miR-133a/si-glutathione S-transferase π1 (GSTP1)-transfectants were subjected to XTT assay and flow cytometry to evaluate their cell viability and apoptosis status. The luciferase reporter assay was used to confirm the actual binding sites between miR-133a and GSTP1 mRNA. The mRNA and protein expression of GSTP1 in BC cell lines and clinical samples were evaluated by real-time RT-PCR and Western blot, respectively. RESULTS: MiR-133a transfection induced cell viability inhibition and apoptosis in BC cell lines. We focused on the GSTP1 gene that was the top 7 down-regulated one in the gene profile from the miR-133a-transfectants. MiR-133a transfection repressed expression levels of mRNA and protein levels of GSTP1. A luciferase reporter assay suggested that the actual binding may occur between miR-133a and GSTP1 mRNA. Cell viability inhibition and apoptosis were induced in the si-GSTP1 transfectants compared with the controls (P < 0.005). GSTP1 mRNA expression levels in 43 clinical BCs were significantly higher than those in eight normal bladder epitheliums (P = 0.0277). CONCLUSION: Our data suggest that tumor suppressive miR-133a directly regulated oncogenic GSTP1 gene in BC, and that an anti-apoptotic effect mediated by GSTP1 is maintained by miR-133a down-regulation in human BC.

Functional role of LASP1 in cell viability and its regulation by microRNAs in bladder cancer.

OBJECTIVE: Our previous study demonstrated that fascin homolog 1 (FSCN1) might have an oncogenic function in bladder cancer (BC) and that its expression was regulated by specific microRNAs (miRNAs). Recently, LIM and SH3 protein 1 (LASP1) as well as FSCN1 have been reported as actin filament bundling proteins in the same complexes attached to the inner surfaces of cell membranes. We hypothesize that LASP1 as well as FSCN1 have an oncogenic function and that is regulated by miRNAs targeting LASP1 mRNA. METHODS: The expression levels of LASP1 mRNA in 86 clinical samples were evaluated by real-time RT-PCR. LASP1-knockdown BC cell lines were transfected by siRNA in order to examine cellular viability by XTT assay, wound healing assay, and matrigel invasion assay. We employed web-based software in order to search for candidate miRNAs targeting LASP1 mRNA, and we focused on miR-1, miR-133a, miR-145, and miR-218. The luciferase reporter assay was used to confirm the actual binding sites between the miRNAs and LASP1 mRNA. RESULTS: Real-time RT-PCR showed that LASP1 mRNA expression was higher in 76 clinical BC specimens than in 10 normal bladder epitheliums (P < 0.05). Loss-of-function studies using si-LASP1-transfected BC cell lines demonstrated significant cell viability inhibition (P < 0.0005), cell migration inhibition (P < 0.0001), and a decrease in the number of invading cells (P < 0.005) in the transfectants compared with the controls. Transient transfection of three miRNAs (miR-1, miR-133a, and miR-218), which were predicted as the miRNAs targeting LASP1 mRNA, repressed the expression levels of mRNA and protein levels of LASP1. The luciferase reporter assay demonstrated that the luminescence intensity was significantly decreased in miR-1, miR-133a, and miR-218 transfectants (P < 0.05), suggesting that these miRNAs have actual target sites in the 3' untranslated region of LASP1 mRNA. Furthermore, significant cell viability inhibitions occurred in miR-218, miR-1, and miR-133a transfectants (P < 0.001). CONCLUSION: Our data indicate that LASP1 may have an oncogenic function and that it might be regulated by miR-1, miR-133a, and miR-218, which may function as tumor suppressive miRNAs in BC.

miR-218 on the genomic loss region of chromosome 4p15.31 functions as a tumor suppressor in bladder cancer.

Growing evidence suggests that microRNAs (miRNAs) are aberrantly expressed in many human cancers, and that they play significant roles in carcinogenesis and cancer progression. The identification of tumor suppressive miRNAs and their target genes could provide new insights into the mechanism of carcinogenesis. However, the genetic or epigenetic regulations of these miRNAs have not yet been fully elucidated in bladder cancer (BC). Chromosomal alterations of cancer cells give us important information for the identification of tumor suppressor genes. Our miRNA array-comparative genomic hybridization (CGH) analysis showed several miRNAs to be candidate tumor suppressors of BC. Our array-CGH analysis revealed that chromosome 4 was lost in all BC cell lines. We selected 19 miRNAs located on chromosome 4 and evaluated their expression levels in cancer cell lines as well as clinical samples. Gain-of-function analysis revealed that miR-218 inhibited BC cell proliferation, migration and invasion. Furthermore, flow cytometry analysis showed that it induced BC cell apoptosis. Genome-wide gene expression analysis showed that it targeted multiple oncogenes in BC. Our study is the first to demonstrate that miR-218 located on chrosomosme 4p15.31 is a tumor suppressive miRNA in BC. The identification of tumor suppressive miRNAs and their target genes on the basis of array-CGH analysis could provide new insights into the mechanisms of BC carcinogenesis.

CpG hypermethylation of human four-and-a-half LIM domains 1 contributes to migration and invasion activity of human bladder cancer.

We previously reported a simple technique that combines microarray data from clinical bladder cancer (BC) specimens with those from a BC cell line (BOY) treated with a pharmacologic demethylating agent (5-aza-dC). We focused on the human four-and-a-half LIM domains 1 (FHL1) gene which was selected on the basis of previous microarray data analysis. Because LIM domains provide protein-protein binding interfaces, FHL genes play an important role in cellular events, such as focal adhesion and differentiation, by interacting with the target protein as either a repressor or activator. We hypothesized that inactivation of the FHL1 gene through CpG methylation contributes to cell viability including migration and invasion activity of human BC. After 5-aza-dC treatment, the expression levels of FHL1 mRNA transcript markedly increased in all cell lines tested, as shown by real-time reverse transcription-polymerase chain reaction (RT-PCR). The methylation index of FHL1 in our samples was significantly higher in 70 BC specimens than in 10 normal bladder epithelium (NBE) specimens (63.9+/-25.5 and 0.3+/-0.2, respectively; p=0.0066). Conversely, FHL1 mRNA expression was significantly lower in the BC specimens than in the NBE ones (0.331+/-0.12 and 2.498+/-0.61, respectively; p=0.0011). In addition, significant inhibitions of wound healing (45.78+/-6.2, and 100+/-0, respectively; p=0.009) and of cell invasion (18.5+/-2.3 and 95.2+/-2.4, respectively; p=0.02) were observed in stable FHL1-transfected cells than in the control BC cells. In conclusion, we found that the mechanism of FHL1 down-regulation in BC is through CpG hypermethylation of the promoter region. FHL1 gene inactivation by CpG hypermethylation may thus contribute to migration and invasion activity of BC.

CpG hypermethylation of cellular retinol-binding protein 1 contributes to cell proliferation and migration in bladder cancer.

We have previously reported a simple technique that combines microarray data from clinical bladder cancer (BC) specimens with those from a BC cell line (BOY) treated with a pharmacological demethylating agent [5-aza-2'-deoxycytidine (5-aza-dC)] to find candidate genes that have tumor suppressive functions. We focused on the cellular retinol-binding protein 1 (CRBP1) gene that was selected by using the microarray data. As CRBP1 regulates intracellular retinoic acid (vitamin A) homeostasis, which is involved in morphogenesis, and cellular proliferation and differentiation, the loss of CRBP1 could cause tumorigenesis in BC. We hypothesized that the inactivation of the CRBP1 gene through CpG methylation contributes to cell viability, including the migration and invasion activity of human BC cells. After the 5-aza-dC treatment, the mRNA and protein expression levels of CRBP1 markedly increased in all BOY and T24 BC cell lines. Combined bisulfite-restriction analysis and bisulfite DNA sequencing revealed that promoter CpG hypermethylation existed in 28 out of the 65 BCs (43%) and in none of the 16 normal bladder epithelia (NBEs). Conversely, CRBP1 mRNA expression in the BCs was significantly lower than that in the NBEs (0.63 ± 0.11 vs. 4.92 ± 0.80, p<0.0001). We found significant inhibition of cell growth (p<0.0001) and migration (p<0.0001) in the CRBP1 stable transfectants compared to the control cell line, in a cell proliferation and wound-healing assay, respectively. In conclusion, the aberrant CpG hypermethylation of the CRBP1 gene promoter could be involved in the development of BC. We demonstrate here for the first time that the CRBP1 gene could have a tumor suppressive function in BC.