Susumu Kageyama

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.

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.

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.

PRIMARY ADENOSQUAMOUS CELL CARCINOMA OF THE MALE DISTAL URETHRA: MAGNETIC RESONANCE IMAGING USING A CIRCULAR SURFACE COIL

A 78-year-old man presented with dysuria and penile induration. Physical examination demonstrated a 2.5 cm. nontender induration on the penile shaft. Urethroscopy revealed stricture of the fossa navicularis without tumor. Computerized tomography and ultrasonography did not show the extent of tumor due to poor tissue contrast. Although T1 (spin-echo V i 3 ) and T2weighted (spinecho 2,000/80) MRI images were obtained using a 1.5 Tesla superconducting magnet and body coil, they did not enable clear visualization of the tumor (fig. 1, A). Therefore, MRI was performed using a circular surface coil with an aperture of 100 mm., which offered better spatial resolution and a better signal-to-noise ratio. The lesion, which extended into the corpus spongiosum and corpora cavernma, was clearly shown on T2 but not T1-weighted images (fig. 1, B). Percutaneous needle biopsy specimens revealed adenocarcinoma. Further examination demonstrated no primary lesions other than those on the penis. The patient underwent partial penectomy (fig. 2). Pathological examination demonstrated adenosquamous cell carcinoma arising in the urethral mucosa (fig. 3). The low intensity region on T2-weighted images corresponded to the region with histological findings of tumor.

Multiple NF-Y-binding CCAAT boxes are essential for transcriptional regulation of the human C7orf24 gene, a novel tumor-associated gene

Human chromosome 7 ORF 24 (C7orf24) has been identified as a tumor‐related protein, and shown to be a γ‐glutamyl cyclotransferase. In the current study, we characterized the promoter region of the human C7orf24 gene to explore the transcriptional regulation of the gene. We revealed that the human C7orf24 promoter is a TATA‐less promoter, containing five CCAAT boxes aligned in a forward orientation. By performing a luciferase reporter assay with 5′‐deleted and site‐directed mutated constructs in HeLa, MCF‐7 and IMR‐90 cells, we found that three proximal CCAAT boxes are important for basal transcription. Electrophoretic mobility gel shift assay and chromatin immunoprecipitation assay demonstrated that NF‐Y specifically bound to all three CCAAT boxes. In addition, the mRNA and protein expression levels of C7orf24 were significantly reduced in HeLa cells depleted of NF‐YB, a subunit of NF‐Y. These results suggested that NF‐Ys bound to the three proximal CCAAT boxes play a central role in the transcription of the gene. Furthermore, as in the case of other genes transcribed under the control of multiple NF‐Ys, such as human E2f1 and cyclin b1, the C7orf24 gene expression profile oscillated during the cell cycle, implying that C7orf24 is a novel cell cycle‐associated gene.

Expression of platelet-derived endothelial cell growth factor/thymidine phosphorylase in human bladder cancer

We investigated the expression of platelet‐derived endothelial cell growth factor/thymidine phosphorylase (PD‐ECGF/TP) in primary bladder cancer, its association with clinicopathologic findings, and their prognostic value. mRNA was extracted from 20 bladder cancer specimens and 6 normal bladder mucosal tissues. Relative amounts of PD‐ECGF/TP mRNA were evaluated by reverse transcriptase‐polymerase chain reaction (RT‐PCR) and compared with the level of glycer‐aldehyde‐3‐phosphate dehydrogenase mRNA (used as an internal standard). PD‐ECGF/TP expression was examined by immunohistochemistry in 85 patients who underwent cystectomy for bladder cancer. Serum PD‐ECGF/TP levels were measured in 23 patients using a sandwich‐type enzyme‐linked immunosorbent assay. By RT‐PCR analysis, expression of PD‐ECGF/TP was found to be 7‐fold higher in invasive tumors than in superficial tumors (P<0.01) and 9‐fold higher than in normal bladder (P<0.01). Out of 85 transitional cell carcinoma tissue samples, 69 (81%) were evaluated as PD‐ECGF/TP‐positive by immunohistochemical staining. PD‐ECGF/TP expression correlated significantly with tumor grade (P=0.001), depth of invasion (P=0.012), and lymphatic invasion (P=0.01). No correlation was found between expression of PD‐ECGF/TP and the number of tumors, tumor configuration, lymph node involvement, venous invasion, c‐erbB‐2 expression, or overall survival. We could not detect a significant serum level of PD‐ECGF/TP in any patient. The results suggest that PD‐ECGF/TP might give valuable information for bladder cancer management, though it may not be a good new tumor marker for bladder cancer.

Pharmacokinetic/Pharmacodynamic Analysis of a Hemodialyzed Patient Treated with 25 mg of Sunitinib

Sunitinib has been approved for the treatment of advanced and/or metastatic renal cell carcinoma (RCC). Information on the dosage adjustment of sunitinib for patients undergoing hemodialysis is limited. Especially, efficacy and tolerance of sunitinib at a low dose in such patients are not fully understood. Thus, we examined the effect of hemodialysis on the pharmacokinetics, safety and efficacy of 25 mg of sunitinib. The patient was a 66-year-old man diagnosed with RCC and undergoing hemodialysis. He was treated with sunitinib at 25 mg daily for 4 weeks of a 6-week cycle. There were little differences in the AUC0–24 h of sunitinib and its major active metabolite SU12662 on day 17 (on hemodialysis) and day 18 (off hemodialysis) of the first cycle. The total sunitinib concentration (sunitinib and SU12662) was approximately 50 ng/ml at a steady state in every cycle. The patient’s genotype was wild type for ABCG2 421C>A, which is associated with increased sunitinib exposure. In the following two cycles of sunitinib, computed tomography scan showed a partial response of the lung metastasis. During the first cycle, the patient developed grade 2 thrombocytopenia and leukocytopenia. After four cycles of treatment, the patient developed grade 3 fatigue and the sunitinib treatment was discontinued. Our patient on hemodialysis could be safely and effectively treated with 25 mg of sunitinib, and a total sunitinib concentration of about 50 ng/ml was maintained. The pharmacokinetics of sunitinib and SU12662 were rarely affected by hemodialysis. Therapeutic drug monitoring could be helpful during sunitinib therapy, especially in a specific population.

Gamma-Glutamylcyclotransferase: A Novel Target Molecule for Cancer Diagnosis and Treatment

Gamma-glutamylcyclotransferase (GGCT) is one of the major enzymes involved in glutathione metabolism. However, its gene locus was unknown for many years. Recently, the gene for GGCT was found to be identical to C7orf24, which is registered as a hypothetical protein. Orthologs have been found in bacteria, plants, and nematodes as well as higher organisms, and the GGCT gene is highly preserved among a wide range of species. GGCT (C7orf24) was also reported as an upregulated protein in various cancers. Although the function of GGCT in cancer cells has not been determined, the following important activities have been reported: (1) high expression in various cancer tissues and cancer cell lines, (2) low expression in normal tissues, (3) inhibition of cancer cell proliferation via anti-GGCT RNAi, (4) inhibition of cancer cell invasion and migration via anti-GGCT RNAi, (5) an epigenetic transcriptional regulation in cancer cells, and (6) an antitumor effect in cancer-bearing xenograft mice. Therefore, GGCT is promising as a diagnostic marker and a therapeutic target for various cancers. This review summarizes these interesting findings.