Combination treatment using percutaneous transluminal angioplasty and low-density lipoprotein apheresis in a patient with peripheral arterial disease and a history of chronic hemodialysis.

Peripheral arterial disease (PAD) is very common in dialysis patients, who tend to have diffuse calcification and severe peripheral arterial stenosis that make it difficult to treat limbs using only surgical or endovascular interventions. Better ways to treat this condition are therefore required and also follow-up studies to evaluate the effects of these treatments on the microcirculation. A 59-year-old man who had a cadaveric kidney transplant five years previously after 25 years of regular hemodialysis complained of pain at rest in his right lower limb and subsequently developed an intractable decubitus ulcer on his right fifth toe (Fontaine IV). Digital subtraction angiography revealed a severe obstruction of the right femoral artery and diffuse stenosis of the right superficial femoral artery. The patient underwent percutaneous transluminal angioplasty (PTA) and six sessions of low-density lipoprotein apheresis (LDL apheresis). At the end of these sessions his complaints were almost completely alleviated. The mean elevation in skin temperature after each session was (1.58 ± 0.99)°C [mean ± SD] over the right dorsalis pedis artery and (1.52 ± 0.88)°C at the tip of the right fifth toe. Ultrasound-measured blood flow rates in the right dorsalis pedis artery were 9.2 cm/s before PTA and 20.2 cm/s one month after PTA. Hemodialysis was resumed 3 days after the PTA due to contrast-induced nephropathy. The combination of PTA and LDL apheresis is useful for treating PAD in hemodialysis patients, with the changes in peripheral artery patency are able to be evaluated effectively by measuring skin temperature.

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.

The functional significance of miR-1 and miR-133a in renal cell carcinoma.

PURPOSE: The aim of this study was to find a novel molecular network involved in renal cell carcinoma (RCC) development through investigating the functions of miR-1 and miR-133a and their target genes. METHODS: We checked the expression levels of miR-1 and miR-133a in RCC cell lines and specimens (N=40) using real time RT-PCR. MiR-1 and miR-133a transfectants were subjected to a gain-of-function study to identify the functions of the miRNAs. To find the target genes of the miRNAs, we analysed the gene expression profile of their transfectants and performed a luciferase reporter assay. mRNA expression levels of the candidate target gene in the clinical specimens were examined, and loss-of-function studies were performed. RESULTS: The expression levels of miR-1 and miR-133a were significantly suppressed in RCC cell lines and specimens. Ectopic restoration of miR-1 and miR-133a showed significant inhibition of cell proliferation and invasion, and moreover, revealed induction of apoptosis and cell cycle arrest. The luciferase assay revealed transgelin-2 (TAGLN2), selected as a target gene for miR-1 and miR-133a on the basis of the gene expression profile, to be directly regulated by both miR-1 and miR-133a. The loss-of-function studies showed significant inhibitions of cell proliferation and invasion in the si-TAGLN2 transfectant. The expression level of TAGLN2 mRNA was significantly up-regulated in the RCC specimens; in addition, there was a statistically significant inverse correlation between TAGLN2 and miR-1 and miR-133a expression. CONCLUSIONS: Our data indicate that up-regulation of the oncogenic TAGLN2 was due to down-regulation of tumour-suppressive miR-1 and miR-133a in human RCC.

Unusual presentation of intraparenchymal renal artery aneurysm mimicking cystic renal cell carcinoma: a case report.

We describe a case of intraparenchymal renal artery aneurysm in a 58-year-old normotensive man with a history of distal ureterectomy. Imaging studies of the pre-existing right renal mass had been interpreted as being consistent with a simple renal cyst. Three years after ureterectomy, a small parietal nodule with contrast enhancement developed within the cyst. Partial nephrectomy was carried out based on a preoperative diagnosis of cystic kidney cancer. Surprisingly, pathological diagnosis was an aneurysm with a revascularized thrombus. Even though the present case represents an extremely rare clinical manifestation of intraparenchymal renal artery aneurysm, clinicians should be aware that imaging studies cannot distinguish all instances of renal vascular disease.

Restoration of miR-517a expression induces cell apoptosis in bladder cancer cell lines.

The aim of this study was to find novel tumor suppressor microRNAs through screening genes epigenetically silenced by methylation in bladder cancer (BC) cell lines using microRNA microarrays. Since miR-517a and miR-520g, both located on chromosome 19q13.42, were found to highly up-regulated genes after treatment with a demethylating agent, 5-aza-2'-deoxycytidine (5-Aza-dc), we hypothesized that they are tumor-suppressor microRNAs and performed a gain-of-function study using these mature microRNAs. The miR-517a restoration showed significant inhibition of cell proliferation in the transfectants compared to miR-control-transfected cells (p<0.0001 both in BOY and T24 cells). Furthermore, ectopic overexpression of miR-517a markedly induced apoptosis in the miR-517a-transfected BC cell lines. In addition, we carried out oligo microarray analysis using miR-517a transfectants and miR-control transfectants (BOY and T24), from which 35 down-regulated genes and 19 up-regulated genes were identified. These included amphiregulin (AREG) and BCL2-associated transcription factor 1, transcript variant 1 (BCLAF1), previously reported to be concerned with apoptosis, in both cell lines by miR-517a restoration. These data suggest that miR-517a functions as a tumor suppressor through inhibition of cell proliferation and induction of apoptosis under the regulation of AREG and/or BCLAF1 in BC cells. Anti-apoptotic effects may be maintained by down-regulation of miR-517a due to DNA hypermethylation in human BC cells, suggesting that restoration of miR-517a may be a novel therapeutic strategy for human 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.

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.

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.

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.

Skeletal-related events in urological cancer patients with bone metastasis: a multicenter study in Japan.

OBJECTIVE: To investigate the incidence of skeletal-related events (SRE) in urological cancer patients with bone metastases in Japan. METHODS: Five hundred eleven patients with urological cancer and documented bone metastases treated from January 2003 to April 2008 in ten Japanese institutions were included in a retrospective analysis. Type and incidence of SRE (fracture, radiotherapy, spinal cord compression, surgery, hypercalcemia, and bone pain) were determined from patient medical records. RESULTS: The overall incidence of SRE, including 'pain', was 61%. The most common event was radiotherapy for bone metastases, with an incidence of 31%. The overall incidence of events seemed to be similar among Japanese and Western patients with prostate cancer and renal cell carcinoma when comparing data with previously published reports. Nevertheless, a much lower incidence of fracture (19.1%) was observed in Japanese renal cell carcinoma patients. CONCLUSIONS: The overall incidence of SRE in Japanese urological cancer patients with bone metastasis was similar to that in Western patients, but the incidence of fracture was lower in Japanese renal cancer patients.

Prognosis of Japanese metastatic renal cell carcinoma patients in the cytokine era: a cooperative group report of 1463 patients.

BACKGROUND: Incidence rate of renal cell carcinoma (RCC) differs among countries. The rates of Asian countries are lower than those of countries in North America or Europe but are exceptionally high in Japanese males. Approximately 30% of patients with RCC have metastasis at initial diagnosis, and another 30% have metastasis after nephrectomy. Clinical studies of risk factors in patients with metastatic RCC (mRCC) are mainly based on data from non-Asian patients. OBJECTIVES: We aimed to investigate the prognosis of Japanese patients and their prognostic factors. DESIGN, SETTING, AND PARTICIPANTS: The subjects of this study were 1463 patients who were clinically diagnosed with RCC with metastasis in 40 Japanese hospitals between January 1988 and November 2002. MEASUREMENTS: The primary end point was overall survival calculated from first diagnosis of mRCC to death or last follow-up. We also investigated the relationship between survival and clinical features. RESULTS AND LIMITATIONS: The median overall survival time was 21.4 mo. The estimated survival rates at 1, 3, 5, and 10 yr were 64.2%, 35.2%, 22.5%, and 9.1%, respectively; they contrasted with data from the United States of 54%, 19%, 10%, and 6%, respectively for the same periods. A high percentage of patients had undergone nephrectomy (80.5%) and metastasectomy (20.8%), both of which were shown to prolong survival. CONCLUSIONS: The median survival time in the present study was approximately twice as long as that of previous studies from North America or Europe. Early diagnosis of metastasis, nephrectomy, metastasectomy, and cytokine-based therapy seemed to improve the prognosis of RCC patients in the present study.

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.

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 the UCHL1 gene promoter is associated with pathogenesis and poor prognosis in renal cell carcinoma.

PURPOSE: Aberrant DNA hypermethylation has been reported in renal cell carcinoma. We performed microarray analysis in the renal cancer cell line ACHN treated with the demethylating agent 5-aza-2'-deoxycytidine and investigated the UCHL1 gene involved in the regulation of cellular ubiquitin levels. MATERIALS AND METHODS: We subjected 131 renal cell carcinoma and 61 corresponding normal kidney tissue samples to real-time reverse transcriptase-polymerase chain reaction, quantitative methylation specific polymerase chain reaction and immunohistochemistry. We also established a stable UCHL1 transfectant to evaluate cell growth. RESULTS: We identified 10 genes that were up-regulated more than 2.5-fold in 5-aza-2'-deoxycytidine treated vs untreated ACHN cells. UCHL1 expression was increased 3.41-fold by 5-aza-2'-deoxycytidine treatment. In clinical samples the UCHL1 methylation index was significantly higher in renal cell carcinoma than in normal kidney tissue (p = 0.011). Conversely UCHL1 mRNA expression was significantly lower in renal cell carcinoma than in normal kidney tissue (p <0.0001). There was a negative correlation between mRNA expression and the UCHL1 methylation index (p = 0.017). The immunostaining score for UCHL1 was significantly higher in normal kidney tissue than in renal cell carcinoma (p <0.0001). Kaplan-Meier analysis showed that a positive UCHL1 methylation index had a significant adverse effect on prognosis (p = 0.048). Significant growth inhibition in UCHL1 transfectant compared to that in WT ACHN (p <0.0001) suggests that UCHL1 functions as a potential tumor suppressor gene in human renal cell carcinoma. CONCLUSIONS: To our knowledge we report the first study demonstrating that the mechanism of UCHL1 down-regulation in renal cell carcinoma is through CpG hypermethylation of the promoter region and methylation of the UCHL1 gene is associated with a poor prognosis in patients with renal cell carcinoma.

Isolation and characterization of arsenite-resistant human epidermoid carcinoma KB cells.

Arsenic trioxide (As2O3) has been used with success in the treatment of acute promyelocytic leukemia. However, resistance to arsenite agents reduces their efficacy. We have isolated arsenite-resistant human epidermoid carcinoma KB cells, termed KAS. KAS cells were resistant to sodium arsenite (22-fold) and showed a reduced accumulation of arsenite as a result of an active efflux mechanism. Further analysis indicated that resistance of KAS cells extended to other drugs including cisplatin (17-fold), antimony potassium tartrate (11-fold) and doxorubicin (27-fold). Although increased expression of multidrug resistance protein 1 (MRP1) in KAS cells was confirmed by quantitative RT-PCR and immunoblot analysis, specific inhibitors of MRP1 did not completely eliminate arsenite resistance. The level of glutathione (GSH) in KAS cells was 3-fold higher than that in KB-3-1 cells, and the inhibition of GSH synthesis by buthionine sulfoximine (BSO) considerably increased the cytotoxic effect of arsenite on KAS cells. A pyridine analog, 2-[4-(diphenylmethyl)-1-piperazinyl ethyl 5-(trans-4,6-dimethyl-1,3,2-dioxaphosphorinan-2-yl)-2,6-dimethyl-4-(3-nitrophenyl)-3-pyridine-carboxylate P oxide (PAK-104P), partially reversed the arsenite resistance and increased the arsenite accumulation in KAS cells. We suggest that the increased level of GSH is involved in arsenite resistance and an as yet unidentified arsenite transporter is expressed in the arsenite-resistant KAS cells.

Nuclear translocation of ADAM-10 contributes to the pathogenesis and progression of human prostate cancer.

A disintegrin and metalloproteases (ADAM) are cell membrane-anchored proteins with potential implications for the metastasis of human cancer cells via cell adhesion and protease activities. In prostate cancer (PC), the ADAM-10 protein showed a nuclear localization whereas in benign prostate hypertrophy (BPH) it was predominantly bound to the cell membrane. We hypothesized that the pathogenesis and progression of PC are attributable to the nuclear translocation of ADAM-10. Immunoblotting revealed that after 5alpha-dihydrotestosterone treatment, a 60-kDa active form of ADAM-10 was increased in the nuclear fraction but decreased in the cell membrane and cytoplasmic fractions of human androgen-dependent PC cells. Immunocytochemistry revealed that after 5alpha-dihydrotestosterone treatment, the ADAM-10 protein was translocated from the cell membrane to the nucleus. Coimmunoprecipitation of androgen receptor and ADAM-10 was detected in the nuclear fraction but not in the cell membrane and cytoplasmic fractions. Immunohistochemical study of 64 PC and 20 BPH samples showed that the intensity of ADAM-10 staining was significantly higher in the nuclei of PC cells than in the nuclei of BPH cells (P < 0.0001). It was also significantly lower in the cell membrane of PC cells than in the cell membrane of BPH cells (P = 0.0017). Nuclear staining intensity was significantly correlated with the clinical T-factor (P = 0.004), the Gleason score (P < 0.0001) and preoperative prostate-specific antigen levels (P = 0.0061). ADAM-10 small interfering RNA transfectants showed a significant decrease in cell growth compared to the controls. Our results suggest that in human PC, the nuclear translocation of ADAM-10 coupled with the androgen receptor is involved in tumor growth and progression.

Increased SKP2 and CKS1 gene expression contributes to the progression of human urothelial carcinoma.

PURPOSE: SKP2 and CKS1 promote aggressive tumor behavior via the regulation of p27 degradation. Our previous DNA microarray analysis of human urothelial carcinoma and normal urothelial epithelium showed that in urothelial carcinoma the 2 most highly up-regulated genes among SKP2-p27 interaction related genes are SKP2 (4.7-fold) and CKS1 (2.2-fold). We hypothesized that SKP2 and CKS1 gene expression is associated with urothelial carcinoma invasiveness and prognosis. MATERIALS AND METHODS: A total of 84 urothelial carcinoma specimens from patients with bladder (71) and upper urinary tract (13) cancer were examined by real-time reverse transcriptase-polymerase chain reaction and immunohistochemical study. RESULTS: Real-time reverse transcriptase-polymerase chain reaction showed that the average mRNA expression level of SKP2 and CKS1 significantly correlated with tumor stage, that is superficial vs invasive urothelial carcinoma (SKP2 and CKS1, p<0.001 and 0.006) and grade (p<0.001 and 0.009, respectively). Of the superficial urothelial carcinomas examined the SKP2 and CKS1 expression level was significantly higher in pT1 than in pTa samples (p=0.005 and 0.017, respectively). Immunohistochemical expression patterns of SKP2 and CKS1 also significantly correlated with tumor stage (p<0.001 and 0.048) and grade (p=0.003 and 0.025, respectively). In contrast, p27 expression inversely correlated with tumor stage and grade (p<0.001 and 0.011, respectively). Logistic regression analysis revealed that while SKP2 mRNA expression was a significant dependent predictor of p27 expression (p=0.021), there was no correlation between CKS1 mRNA expression and p27 (p=0.748). Kaplan-Meier curves and log rank tests revealed that the high mRNA expression levels of SKP2 and CKS1 had a significant adverse effect on prognosis (p=0.043 and 0.003, respectively). CONCLUSIONS: Our results suggest that SKP2 has a major role in the regulation of p27 degradation and CKS1 has a supporting role for SKP2 function in human urothelial carcinoma.

Inhibition of 5-lipoxygenase pathway suppresses the growth of bladder cancer cells.

AIM: Many stimuli, including growth factors and cytokines, activate arachidonic acid (AA) metabolic pathways, which are involved in cancer development and progression. We examined the effects of a series of pharmacological inhibitors of AA metabolic enzymes on bladder cancer cells to determine the role of AA pathway in this malignancy. METHODS: Human bladder cancer cell lines were treated with various AA metabolic enzymes inhibitors for lipoxygense (LOX) and cyclooxygenase (COX) pathways, and the growth suppression effects were examined. The enzyme expression in cancer cells was examined by immunoblot analyses. RESULTS: A 5-LOX-specific inhibitor, AA861, dose-dependently inhibited the growth of bladder cancer cells. The growth inhibitory effects were greatly abolished by 5-LOX product, 5-HETE, but not by other LOX products examined. They were observed in four cancer cells that expressed 5-LOX, but not in one that did not. Of a series of LOX and COX pathway inhibitors, AA861 was the strongest one to suppress the growth of cancer cells. CONCLUSIONS: Bladder cancer cells frequently expressed 5-LOX. A 5-LOX-specific inhibitor, AA861, revealed the strongest growth suppression of those cells compared to other LOX and COX pathway inhibitors, and the growth suppression effects were considered to be due to inhibition of the enzymatic activity. Therefore, 5-LOX may play a regulatory role in proliferation and/or survival of bladder cancer, and may be a therapeutic target for the disease.

Identification of differentially expressed genes in human bladder cancer through genome-wide gene expression profiling.

Large-scale gene expression profiling is an effective strategy for understanding the progression of bladder cancer (BC). The aim of this study was to identify genes that are expressed differently in the course of BC progression and to establish new biomarkers for BC. Specimens from 21 patients with pathologically confirmed superficial (n = 10) or invasive (n = 11) BC and 4 normal bladder samples were studied; samples from 14 of the 21 BC samples were subjected to microarray analysis. The validity of the microarray results was verified by real-time RT-PCR. Of the 136 up-regulated genes we detected, 21 were present in all 14 BCs examined (100%), 44 in 13 (92.9%), and the other 71 in 12 BCs (85.7%). Of 69 down-regulated genes, 25 were found in all 14 BCs (100%), 22 in 13 (92.9%), and the other 22 in 12 BCs (85.7%). Functional annotation revealed that of the up-regulated genes, 36% were involved in metabolism and 14% in transcription and processing; 25% of the down-regulated genes were linked to cell adhesion/surface and 21% to cytoskeleton/cell membrane. Real-time RT-PCR confirmed the microarray results obtained for the 6 most highly up- and the 2 most highly down-regulated genes. Among the 6 most highly up-regulated genes, CKS2 was the only gene with a significantly greater level of up-regulation in invasive than in superficial BC (p = 0.04). To confirm this result, we subjected all 21 BC samples to real-time PCR assay for CKS2. We found a considerable difference between superficial and invasive BC (p = 0.001). Interestingly, there was a considerable difference between the normal bladder and invasive BC (p = 0.001) and less difference between the normal bladder and superficial BC (p = 0.005). We identified several genes as promising candidates for diagnostic biomarkers of human BC and the CKS2 gene not only as a potential biomarker for diagnosing, but also for staging human BC. This is the first report demonstrating that CKS2 expression is strongly correlated with the progression of human BC.