Tatsuhiro Yoshiki

Intravesical administration of small interfering RNA targeting PLK-1 successfully prevents the growth of bladder cancer

The mainstay in the management of invasive bladder cancer continues to be radical cystectomy. With regard to improvement of quality of life, however, therapies that preserve the bladder are desirable. We investigated the use of intravesical PLK-1 small interfering RNA (siRNA) against bladder cancer. Patients with bladder cancers expressing high levels of PLK-1 have a poor prognosis compared with patients with low expression. Using siRNA/cationic liposomes, the expression of endogenous PLK-1 could be suppressed in bladder cancer cells in a time- and dose-dependent manner. As a consequence, PLK-1 functions were disrupted. Inhibition of bipolar spindle formation, accumulation of cyclin B1, reduced cell proliferation, and induction of apoptosis were observed. In order to determine the efficacy of the siRNA/liposomes in vivo, we established an orthotopic mouse model using a LUC-labeled bladder cancer cell line, UM-UC-3(LUC). PLK-1 siRNA was successfully transfected into the cells, reduced PLK-1 expression, and prevented the growth of bladder cancer in this mouse model. This is the first demonstration, to our knowledge, of inhibition of cancer growth in the murine bladder by intravesical siRNA/cationic liposomes. We believe intravesical siRNA instillation against bladder cancer will be useful as a therapeutic tool.

Diagnostic potential in bladder cancer of a panel of tumor markers (calreticulin, γ‐synuclein, and catechol‐o‐methyltransferase) identified by proteomic analysis

Using proteomic analysis, we previously identified calreticulin (CRT) as a potentially useful urinary marker for bladder cancer. Now, we have also identified γ‐synuclein (SNCG) and a soluble isoform of catechol‐o‐methyltransferase (s‐COMT) as novel candidates for tumor markers in bladder cancer, by means of proteomic analysis. In the process of establishing a superior tumor marker system, we investigated the diagnostic value of a combination assay of these three proteins. Voided urine samples were obtained from 112 bladder cancer and 230 control patients. Urinary CRT, SNCG, and s‐COMT were measured as a combined marker by quantitative western blot analysis. Relative concentration of each protein was calculated and the diagnostic value of a concomitant examination of these markers was evaluated by receiver operator characteristic analysis. With the best diagnostic cutoff, the overall sensitivity of the combined markers was 76.8% (95% confidence interval, 69–81%) with a specificity of 77.4% (72–80%), while those of a single use of CRT were 71.4% and 77.8%, respectively. When evaluated in relation to tumor characteristics, such as grade, stage, size, and outcome of urinary cytology, the diagnostic capacity of the combined markers was equal to or better than that of CRT in all categories. Concomitant use of CRT, SNCG, and s‐COMT had higher sensitivity for detection of bladder cancer than did single use of CRT. Our study suggests that use of this panel of markers will improve the diagnosis of bladder cancer and may allow the development of a protein microarray assay or multi‐channel enzyme‐linked immunosorbent assay.

c-erbB-2 oncoprotein : A potential biomarker of advanced prostate cancer

Overexpression of the c‐erbB‐2 oncogene has been implicated in the development and/or prognosis of several human carcinomas, including that of the prostate. Recently, c‐erbB‐2 protein was found to be released in the circulation. The present study was undertaken to study the significance of serum c‐erbB‐2 protein determination in men with prostate cancer.

Expression of transitional cell‐specific genes, uroplakin Ia and II, in bladder cancer: Detection of circulating cancer cells in the peripheral blood of metastatic patients

Background: Uroplakins (UP), urothelium‐specific transmembrane proteins, are present only in urothelia and may be good candidates as tumor markers specific for transitional cell carcinomas (TCC). We investigated the expression of UP‐Ia and UP‐II genes in the tissues and peripheral blood of patients with TCC.

Expression and Immunogenicity of a Tumor- Associated Antigen, 90K/Mac-2 Binding Protein, in Lung Carcinoma The Possibility of Its Clinical Use as a Tumor Marker and a Target Antigen in Cancer Immunotherapy

The authors attempted to obtain shared proteins among lung carcinoma cells by column chromatographies. A glycoprotein with approximately 500 kDa isolated from QG56 cells showed an identical amino acid sequence to 90K/Mac‐2 binding protein (M2BP). This protein has been reported to be highly expressed and to modulate the expression of surface molecules involved in immune responses on cultured cancer cells. Therefore, it would be beneficial for M2BP to be targeted in cancer immunotherapy.

A novel tumor-related protein, c7orf24, identified by proteome differential display of bladder urothelial carcinoma

Proteome analysis of bladder cancer with narrow‐range pH 2‐DE has identified a novel protein on chromosome 7 encoded by ORF 24 (C7orf24) as one of the highly expressed proteins in cancer cells. C7orf24 is currently registered in the protein database as a hypothetical protein with unknown function. The homologs of C7orf24 in other animals have also been registered as putative protein genes. Western blot analysis using a mAb against C7orf24 confirmed its higher expression in bladder cancer compared with normal tissue. Several other cancer cell lines were also found to express C7orf24. However, the introduction of C7orf24 into Rat‐1 or NIH3T3 cells did not cause malignant transformation. A stable transfectant of NIH3T3 cells with recombinant retrovirus vector was produced for a growth rate assay, and a higher growth rate was observed in C7orf24‐expressing cells compared with the controls. Six kinds of small interfering RNAs (siRNAs) were then produced, and C7orf24‐siRNA#5 showed a strong knockdown effect on protein expression and significant antiproliferative effects on cancer cell lines were demonstrated by the MTT assay. Therefore, C7orf24 may have an important role in cancer cell proliferation, and may be an appropriate therapeutic target molecule against cancer.

Urinary calreticulin in the diagnosis of bladder urothelial carcinoma

Objectives:  To evaluate the potential suitability of calreticulin (CRT) as a urinary marker for bladder cancer.

Expression of Uroplakin Ib and Uroplakin III Genes in Tissues and Peripheral Blood of Patients with Transitional Cell Carcinoma

ABSTRACT Uroplakins (UPs), urothelium‐specific transmembrane proteins, are present only in urothelial cells. We have determined the nucleotide sequences of human UP‐Ib and UP‐III and synthesized specific primer pairs. The two UP genes were expressed in both cancerous and noncancerous urothelia taken from all patients examined by reverse transcription‐polymerase chain reaction (RT‐PCR). These genes were also detected in the peripheral blood of 3 patients with metastatic transitional cell carcinoma (TCC), but not in that from 9 patients with non‐metastatic TCC or 3 healthy volunteers. The sensitivity of our assay was sufficient to detect one cancer cell in 5 ml of peripheral blood. Detection of UP gene‐expression in blood by RT‐PCR may provide helpful information for the diagnosis and management of TCC.

High Expression of Human Uroplakin Ia in Urinary Bladder Transitional Cell Carcinoma

Uroplakins (UPs) Ia, Ib, II, and III are tissue‐specific and differentiation‐dependent transmem‐brane proteins of the urothelium. We assessed the usefulness of human UP Ia as a histological marker by examining its expression in urinary bladder transitional cell carcinoma (TCC). A polyclonal antibody against human UP Ia was raised using a synthesized polypeptide. We applied our antibody to various organ tissues, including urothelium, and observed no crossreactivity. Analysis by RT‐PCR of normal urothelium, TCC and other organ tissues indicated that the human UP Ia gene expression is highly specialized to urothelium, and is conserved in TCC. Using immunohistochemistry, we investigated the expression of UP Ia in TCC from patients who had undergone radical cystectomy and from autopsy cases. Positive staining (10% or more positive cancer cells) was noted in primary lesions from 61 of 63 (96.8%) cystectomy patients. Depending on pathological grade, high expression (50% or more positive cancer cells) was observed in 17 of 18 (94.4%) moderately‐ to well‐differentiated TCC and in 36 of 45 (80.0%) poorly differentiated TCC. With regard to tumor invasion, high expression was noted in 20 of 22 (90.9%) superficial and 33 of 41 (80.5%) muscle‐invasive TCC. Cause‐specific survival rates were 68.6% and 75.0% in high‐ and low‐expression patients, respectively (log‐rank test, P=0.855, mean follow‐up; 65.0 months). In metastases, positive reactions were observed in 13 of 18 (72.2%) lesions. UP Ia may represent a specific histological marker judging from the stable expression, although its value as a prognostic factor remains undetermined.

Evaluation of a rapid qualitative prostate specific antigen assay, the One Step PSATM test

Recently, highly sensitive prostate specific antigen (PSA) kits have been developed and reported to be useful for the early identification of a chemical relapse. However, if the measurement time was short and the cost low, such an assay kit should be sufficient for cancer screening when dealing with a large number of samples. The One Step PSA test uses an immunochromatographic method to qualitatively, not quantitatively, judge a positive or negative result. We confirmed the sensitivity of the kit using purified PSA. Serum specimens from 147 men with or without prostate diseases were tested using the kit. PSA concentration of each serum specimen was independently measured by a quantitative ACS-PSA2 EIA kit (Chiron, cut-off: 2.1 ng/ml). The sensitivity of this kit was determined to be 4 ng/ml. All 33 samples with a value of greater than 4 ng/ml were clearly positive. Of the 94 samples with values less than 4 ng/ml, nine were judged as positive. The remaining 85 cases were judged as completely negative. These results indicate that the sensitivity of the One Step PSA test is 100% and the specificity is 90.4%. Tests using this kit can be easily performed at outpatient clinics or elsewhere. This kit is useful for initial cancer screening, because results can be obtained within 15 min and at a cost lower than that of ordinary PSA kits.