Hiroyuki Konaka

Molecular Features of Hormone-Refractory Prostate Cancer Cells by Genome-Wide Gene Expression Profiles

One of the most critical issues in prostate cancer clinic is emerging hormone-refractory prostate cancers (HRPCs) and their management. Prostate cancer is usually androgen dependent and responds well to androgen ablation therapy. However, at a certain stage, they eventually acquire androgen-independent and more aggressive phenotype and show poor response to any anticancer therapies. To characterize the molecular features of clinical HRPCs, we analyzed gene expression profiles of 25 clinical HRPCs and 10 hormone-sensitive prostate cancers (HSPCs) by genome-wide cDNA microarrays combining with laser microbeam microdissection. An unsupervised hierarchical clustering analysis clearly distinguished expression patterns of HRPC cells from those of HSPC cells. In addition, primary and metastatic HRPCs from three patients were closely clustered regardless of metastatic organs. A supervised analysis and permutation test identified 36 up-regulated genes and 70 down-regulated genes in HRPCs compared with HSPCs (average fold difference > 1.5; P < 0.0001). We observed overexpression of AR, ANLN, and SNRPE and down-regulation of NR4A1, CYP27A1, and HLA-A antigen in HRPC progression. AR overexpression is likely to play a central role of hormone-refractory phenotype, and other genes we identified were considered to be related to more aggressive phenotype of clinical HRPCs, and in fact, knockdown of these overexpressing genes by small interfering RNA resulted in drastic attenuation of prostate cancer cell viability. Our microarray analysis of HRPC cells should provide useful information to understand the molecular mechanism of HRPC progression and to identify molecular targets for development of HRPC treatment.

Human Osteocalcin and Bone Sialoprotein Mediating Osteomimicry of Prostate Cancer Cells: Role of cAMP-Dependent Protein Kinase A Signaling Pathway

Osteocalcin and bone sialoprotein are the most abundant noncollagenous bone matrix proteins expressed by osteoblasts. Surprisingly, osteocalcin and bone sialoprotein are also expressed by malignant but not normal prostate epithelial cells. The purpose of this study is to investigate how osteocalcin and bone sialoprotein expression is regulated in prostate cancer cells. Our investigation revealed that (a) human osteocalcin and bone sialoprotein promoter activities in an androgen-independent prostate cancer cell line of LNCaP lineage, C4-2B, were markedly enhanced 7- to 12-fold in a concentration-dependent manner by conditioned medium collected from prostate cancer and bone stromal cells. (b) Deletion analysis of human osteocalcin and bone sialoprotein promoter regions identified cyclic AMP (cAMP)-responsive elements (CRE) as the critical determinants for conditioned medium-mediated osteocalcin and bone sialoprotein gene expression in prostate cancer cells. Consistent with these results, the protein kinase A (PKA) pathway activators forskolin and dibutyryl cAMP and the PKA pathway inhibitor H-89, respectively, increased or repressed human osteocalcin and bone sialoprotein promoter activities. (c) Electrophoretic mobility shift assay showed that conditioned medium-mediated stimulation of human osteocalcin and bone sialoprotein promoter activities occurs through increased interaction between CRE and CRE-binding protein. (d) Conditioned medium was found to induce human osteocalcin and bone sialoprotein promoter activities via increased CRE/CRE-binding protein interaction in a cell background-dependent manner, with marked stimulation in selected prostate cancer but not bone stromal cells. Collectively, these results suggest that osteocalcin and bone sialoprotein expression is coordinated and regulated through cAMP-dependent PKA signaling, which may define the molecular basis of the osteomimicry exhibited by prostate cancer cells.

Androgen replacement therapy contributes to improving lower urinary tract symptoms in patients with hypogonadism and benign prostate hypertrophy: a randomised controlled study.

Purpose. We performed a randomised controlled study regarding the effects of androgen replacement therapy (ART) on lower urinary tract symptoms (LUTS) in hypogonadal men with benign prostate hypertrophy (BPH). Methods. Fifty-two patients with hypogonadism and BPH were randomly assigned to receive testosterone (ART group) as 250 mg of testosterone enanthate every 4 weeks or to the untreated control group. We compared International Prostate Symptom Score (IPSS), uroflowmetry data, post-voiding residual volume (PVR) and systemic muscle volume at baseline and 12 months after treatment. Results. Forty-six patients (ART group, n = 23; control, n = 23) were included in the analysis. At the 12-month visit, IPSS showed a significant decrease compared with baseline in the ART group (15.7 ± 8.7 vs. 12.5 ± 9.5; p < 0.05). No significant changes were observed in the control group. The ART group also showed improvement in maximum flow rate and voided volume (p < 0.05), whereas no significant improvements were observed in the controls. PVR showed no significant changes in either group. In addition, the ART group showed significant enhancement of mean muscle volume (p < 0.05), whereas no significant changes were seen in the controls. Conclusion. ART improved LUTS in hypogonadal men with mild BPH.

Simultaneous determination of salivary testosterone and dehydroepiandrosterone using LC–MS/MS: Method development and evaluation of applicability for diagnosis and medication for late-onset hypogonadism

Late-onset hypogonadism (LOH) is a male-specific disorder caused by the age-related decline in androgens, such as testosterone (T). A sensitive liquid chromatography-electrospray ionization-tandem mass spectrometric (LC-ESI-MS/MS) method for the simultaneous quantification of T and its precursor, dehydroepiandrosterone (DHEA), in human saliva has been developed and validated. The saliva was deprotenized with acetonitrile, purified using a Strata-X cartridge, derivatized with 2-hydrazino-1-methylpyridine, and subjected to LC-MS/MS. The recovery rates of the steroids during the pretreatment were about 90%. Quantification was based on selected reaction monitoring using characteristic transitions, and deuterated T and DHEA were used as internal standards. This method allowed the reproducible (inter- and intra-assay precisions, <2.9%) and accurate (accuracy, 98.5-101.8%) quantification of the salivary androgens using a 500-microl sample and the limits of quantification for both androgens were 10 pg/ml. As preliminary steps in the practical application of the developed method in diagnosis and medication for LOH, the diurnal rhythms, inter-day alternations and age differences in the salivary T and DHEA were examined; the method found that the salivary T and DHEA show specific diurnal rhythms, significant alternations in early morning and pronouncedly decline with age. The method also enabled the determination of the changes in the individual T and DHEA levels after the DHEA supplementation, which is expected to be a new and easy medication for LOH. Thus, the developed method has satisfactory applicability in the diagnosis and medication for LOH.

Gene therapy for prostate cancer using the cytosine deaminase/uracil phosphoribosyltransferase suicide system

Cytosine deaminase (CD) activates prodrug 5‐FC to 5‐FU and is used for suicide gene therapy (the CD/5‐FC system). E. coli uracil phosphoribosyltransferase (UPRT) is a pyrimidine salvage enzyme that directly converts 5‐FU into 5‐fluorouridine monophosphate and improves the antitumoral effect of 5‐FU. This study demonstrates the effectiveness of transduction of the UPRT gene in addition to CD/5‐FC cancer suicide gene therapy.

Etiologic role of human papillomavirus infection in bladder carcinoma

The authors elucidated an etiologic role of human papillomavirus (HPV) infection in carcinoma of the bladder.

Tranilast inhibits hormone refractory prostate cancer cell proliferation and suppresses transforming growth factor β1-associated osteoblastic changes

Tranilast is a therapeutic agent used in treatment of allergic diseases, although it has been reported to show anti‐tumor effects on some cancer cells. To elucidate the effects of tranilast on prostate cancer, we investigated the mechanisms of its anti‐tumor effect on prostate cancer.

Tri-Modality therapy with I-125 brachytherapy, external beam radiation therapy, and short- or long-term hormone therapy for high-risk localized prostate cancer (TRIP): study protocol for a phase III, multicenter, randomized, controlled trial

BackgroundPatients with high Gleason score, elevated prostate specific antigen (PSA) level, and advanced clinical stage are at increased risk for both local and systemic relapse. Recent data suggests higher radiation doses decrease local recurrence and may ultimately benefit biochemical, metastasis-free and disease-specific survival. No randomized data is available on the benefits of long-term hormonal therapy (HT) in these patients. A prospective study on the efficacy and safety of trimodality treatment consisting of HT, external beam radiation therapy (EBRT), and brachytherapy (BT) for high-risk prostate cancer (PCa) is strongly required.Methods/DesignThis is a phase III, multicenter, randomized controlled trial (RCT) of trimodality with BT, EBRT, and HT for high-risk PCa (TRIP) that will investigate the impact of adjuvant HT following BT using iodine-125 (125I-BT) and supplemental EBRT with neoadjuvant and concurrent HT. Prior to the end of September 2012, a total of 340 patients with high-risk PCa will be enrolled and randomized to one of two treatment arms. These patients will be recruited from more than 41 institutions, all of which have broad experience with 125I-BT. Pathological slides will be centrally reviewed to confirm patient eligibility. The patients will commonly undergo 6-month HT with combined androgen blockade (CAB) before and during 125I-BT and supplemental EBRT. Those randomly assigned to the long-term HT group will subsequently undergo 2 years of adjuvant HT with luteinizing hormone-releasing hormone agonist. All participants will be assessed at baseline and every 3 months for the first 30 months, then every 6 months until 84 months from the beginning of CAB.The primary endpoint is biochemical progression-free survival. Secondary endpoints are overall survival, clinical progression-free survival, disease-specific survival, salvage therapy non-adaptive interval, and adverse events.DiscussionTo our knowledge, there have been no prospective studies documenting the efficacy and safety of trimodality therapy for high-risk PCa. The present RCT is expected to provide additional insight regarding the potency and limitations of the addition of 2 years of adjuvant HT to this trimodality approach, and to establish an appropriate treatment strategy for high-risk PCa.Trial registrationUMIN000003992

Gold nanoparticle based immunochromatography using a resin modified micropipette tip for rapid and simple detection of human chorionic gonadotropin hormone and prostate-specific antigen

Abstract A novel bioanalysis system based on immunochromatography was developed in connection with a nitrocellulose resin-modified micropipette tip, namely as ZipTip®. The sandwich-type immunoassay was applied to our bioananalysis system. The first antibodies that were aspirated by the micropipette were immobilized on the immunochromatographic resin at the edge of micropipette tip. The blocking solution was also aspirated in the same fashion. The measurement operation was performed by aspirating the sample solution, and then the gold colloidal nanoparticles (Au naps) conjugated secondary antibody solution. Since this bioanalysis system utilizes a micropipette, it is possible to increase the sample volume, which would enable the detection of antigen at low concentrations. In addition, the washing procedure can also be performed easily to reduce the background level. After the antigen-antibody reaction, the color intensity of Au naps could be observed by the naked eye. For analytical evaluation, the color intensity was captured by a scanner, and processed by analysis software. We have achieved the detection of human chorionic gonadotropin hormone (hCG) and total prostate-specific antigen (TPSA), which are well-known as fundamental indicators of pregnancy and cancer. The limit of detection (LOD) for hCG was 8 ng/ml (0.8 ng/tip), which is comparable to that of other conventional systems based on immunochromatography. Moreover, the LOD for TPSA was improved over the existing systems with the application of different sample volumes, such as 5 ng/μl (1 ng/tip) in 200 μl sample volume, and 2 ng/ml (0.6 ng/tip) in 300 ml sample volume. Since our bioanalysis system requires a small amount of immobilized antigen, it would be greatly useful in basic research for screening the antigen–antibody reactions. Besides, our bioanalysis system can be applied to on-field screening, since its operation is simple, and the visual results can be obtained rapidly.

Etiological role of human papillomavirus infection for inverted papilloma of the bladder

The status of human papillomavirus (HPV) infection in urothelial inverted papilloma was examined in the present study. Formalin‐fixed and paraffin‐embedded tissues from eight cases of inverted papilloma of the bladder were studied. The presence of HPV‐DNA was examined by modified GP5/6+PCR using archival tissue sections by microdissection. HPV genotype was determined with a Hybri‐Max HPV genotyping kit. Immunohistochemical analysis for p16‐INK4a, mcm7, HPV‐E4, and L1, and in situ hybridization for the HPV genome were performed. HPV was detected in seven of eight cases (87.5%) of inverted papilloma. Three cases were diagnosed as inverted papilloma with atypia, while the remaining five were typical cases. HPV‐18 was detected in two cases, including one inverted papilloma with atypia, and HPV‐16 was detected in four cases, including one inverted papilloma with atypia. Multiple HPV type infection was detected in one typical case and one atypical case. High‐risk HPV was present in all HPV‐positive cases. Cellular proteins, p16‐INK4a and mcm7, which are surrogate markers for HPV‐E7 expression, were detected in all HPV‐positive cases, and their levels were higher in inverted papilloma with atypia than in typical cases. In contrast, HPV‐E4 and L1, which are markers for HPV propagation, were observed in some parts of the typical inverted papilloma tissue. High‐risk HPV infection may be one of the causes of urothelial inverted papilloma, and inverted papilloma with atypia may have malignant potential. J. Med. Virol. 83:277–285, 2011.