Hiroyoshi Nakatsuji

Prostate stem cell antigen predicts tumour recurrence in superficial transitional cell carcinoma of the urinary bladder

To evaluate the relationship between prostate stem cell antigen (PSCA) expression level in transitional cell carcinoma (TCC) of the urinary bladder and various clinicopathological features, including stage and grade; and to determine whether PSCA mRNA expression predicts disease recurrence in superficial (not muscle‐invasive) TCC of the bladder.

Involvement of actinin-4 in the recruitment of JRAB/MICAL-L2 to cell-cell junctions and the formation of functional tight junctions.

ABSTRACT Tight junctions (TJs) are cell-cell adhesive structures that undergo continuous remodeling. We previously demonstrated that Rab13 and a junctional Rab13-binding protein (JRAB)/molecule interacting with CasL-like 2 (MICAL-L2) localized at TJs and mediated the endocytic recycling of the integral TJ protein occludin and the formation of functional TJs. Here, we investigated how JRAB/MICAL-L2 was targeted to TJs. Using a series of deletion mutants, we found the plasma membrane (PM)-targeting domain within JRAB/MICAL-L2. We then identified actinin-4, which was originally isolated as an actin-binding protein associated with cell motility and cancer invasion/metastasis, as a binding protein for the PM-targeting domain of JRAB/MICAL-L2, using a yeast two-hybrid system. Actinin-4 was colocalized with JRAB/MICAL-L2 at cell-cell junctions and linked JRAB/MICAL-L2 to F-actin. Although actinin-4 bound to JRAB/MICAL-L2 without Rab13, the actinin-4-JRAB/MICAL-L2 interaction was enhanced by Rab13 activation. Depletion of actinin-4 by using small interfering RNA inhibited the recruitment of occludin to TJs during the Ca2+ switch. During the epithelial polarization after replating, JRAB/MICAL-L2 was recruited from the cytosol to cell-cell junctions. This JRAB/MICAL-L2 recruitment as well as the formation of functional TJs was delayed in actinin-4-depleted cells. These results indicate that actinin-4 is involved in recruiting JRAB/MICAL-L2 to cell-cell junctions and forming functional TJs.

Effect of vascular endothelial growth factor and its receptor inhibitor on proliferation and invasion in bladder cancer.

Background: Vascular endothelial growth factor (VEGF) and its receptors are major regulators of cancer cell growth and metastases. We investigated the association between serum VEGF levels and clinicopathological parameters in bladder cancer patients. We also evaluated the effects of VEGF and its receptor inhibitor on proliferation and invasion in bladder cancer cell lines. Methods: Serum VEGF levels were measured in 52 patients with bladder cancer and 45 healthy controls. In highly invasive bladder cancer cell lines (T-24, UMUC-3 and J82), we assessed the effect of VEGF on proliferation and invasion of bladder cancer cell lines. The effect of VEGF receptor (VEGFR) tyrosine kinase inhibitor against bladder cancer cell lines was also measured. Results: Serum levels of VEGF were significantly higher in patients with muscular invasive bladder cancer than in patients with superficial bladder cancer (p < 0.005). VEGF increased tumor proliferation in a dose-dependent manner in all cell lines. VEGFR-2 tyrosine kinase inhibitor inhibited proliferation in all three cell lines, and inhibited invasion in T24. Conclusions: In bladder cancer, the serum VEGF level correlates significantly with muscular invasiveness. This study suggests that VEGF promotes tumor proliferation and invasion through VEGFR-2. VEGF-targeted therapy may be effective in treating invasive bladder cancers.

Role of phosphatidylinositol-3 kinase/Akt pathway in bladder cancer cell apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand

TRAIL/Apo2L is a pro‐apoptotic cytokine that is capable of inducing apoptosis in a wide variety of cancer cells but not in normal cells. Among various molecular strategies by which cancer cells evade apoptosis, PI3K/Akt signaling represents a dominant survival pathway. In this report, we investigated the role of PI3K/Akt pathway in TRAIL‐induced apoptotic death in human bladder cancer cells. We observed that RT4 cells had very low level of constitutively active Akt and were sensitive to TRAIL, whereas UM‐UC‐3 and T24 cells had higher levels of constitutively active Akt and were resistant to TRAIL. Downregulation of constitutively active Akt by PI3K inhibitors, wortmannin and LY294002, reversed cellular resistance to TRAIL. However, transfecting constitutively active Akt into RT4 cells increased Akt activity and inhibited TRAIL‐induced apoptosis. These results suggest that elevated Akt activity protects UM‐UC‐3 and T24 cells from TRAIL‐induced apoptosis, and the PI3K/Akt signaling might inhibit apoptotic signals. Thus, the modulation of Akt activity by combining pharmacological drugs or genetic alterations of the Akt expression could induce cellular responsiveness to TRAIL and PI3K/Akt signaling pathway could serve as a novel target for therapeutic intervention in bladder cancer. (Cancer Sci 2006; 97: 1093–1098)

Involvement of Rab13 and JRAB/MICAL-L2 in epithelial cell scattering

Epithelial cell scattering recapitulates the first steps of carcinoma invasion/metastasis. While the balance between cell–cell adhesive activity and cell motility ultimately determines this process, its molecular mechanisms remain unclear. Adherence junctions and tight junctions (TJs) are primarily responsible for cell–cell adhesive activity and subjected to dynamic remodeling. We previously showed that Rab13 and its effector protein JRAB/MICAL-L2 mediate the endocytic recycling of the integral TJ protein occludin and the assembly of functional TJs. In this study, we examined the role of Rab13 and JRAB/MICAL-L2 in the scattering of Madin-Darby canine kidney (MDCK) cells in response to 12-O-tetradecanoylphorbol-13-acetate (TPA). Knockdown of Rab13 in canine MDCK cells suppressed the TPA-induced scattering, and this phenotype was restored by re-expression of human Rab13. During TPA-induced MDCK cell scattering, Rab13 was transiently activated and returned to its basal level, and both Rab13 and JRAB/MICAL-L2 were colocalized with F-actin at cell–cell contact sites and then accumulated at emerging lamellipodial structures. TPA-induced MDCK cell scattering was also inhibited by knockdown of canine JRAB/MICAL-L2 and rescued by re-expression of mouse JRAB/MICAL-L2. These results indicate that Rab13 and JRAB/MICAL-L2 are involved in epithelial cell scattering.

The role of actinin-4 in bladder cancer invasion.

OBJECTIVES To examine actinin-4 expression levels in bladder cancer, in particular its levels during cellular growth and invasion. Actinin-4 is an actin-binding protein that is associated with cell motility and cancer metastasis. METHODS Relative messenger ribonucleic acid (mRNA) and protein expression of actinin-4 in normal bladder and bladder cancer cell lines was determined by quantitative real-time polymerase chain reaction and Western blot analysis. Actinin-4 expression was also localized in bladder cancer cells and tissues using immunohistochemistry. The growth and invasion activity of bladder cancer cells was evaluated using cell growth and in vitro cell invasion assays, and compared with that of bladder cancer cells treated with actinin-4 small interfering ribonucleic acids. RESULTS Actinin-4 mRNA and protein levels were elevated in bladder cancer cells that are known to exhibit increased growth and invasion activity. Protein expression was predominantly observed in the cytoplasm of the invasive bladder cancer cells and tissues. Treatment of bladder cancer cell lines with actinin-4 small interfering ribonucleic acids suppressed the invasive potential of the cells, but did not alter their growth. CONCLUSIONS The current study demonstrates that actinin-4 mRNA and protein levels are elevated in bladder cancer cells lines that exhibit increased growth and invasion activity. In addition, actinin-4 knockdown inhibited invasion of bladder cancer cells, but did not alter their growth. In conclusion, we hypothesize that the accumulation of actinin-4 in the cell cytoplasm is related to an increased susceptibility of tumor invasion and metastasis.

Up-regulation of plakophilin-2 and Down-regulation of plakophilin-3 are correlated with invasiveness in bladder cancer.

OBJECTIVE To examine plakophilin proteins (Pkp) and 3 expression levels in bladder cancer, in particular their levels during cellular growth and invasion. Pkp is associated with the binding of cadherin to intermediate filaments of the cytoskeleton. METHODS The relative mRNA and protein expression levels of Pkp2 and 3 in bladder cancer cell lines were determined using quantitative real-time polymerase chain reaction and Western blot analyses. The cellular localization of Pkp2 and 3 proteins in bladder cancer cells was also assayed using immunohistochemistry. The proliferation and invasive activities of bladder cancer cells were evaluated using cell growth and in vitro cell invasion assays, and were compared with those of bladder cancer cells treated with Pkp2 and 3 small interfering RNAs. RESULTS Pkp2 mRNA and protein levels were elevated, and those of Pkp3 were reduced, in bladder cancer cells that are known to exhibit increased proliferation and invasive activity. Pkp2/3 protein expression was predominantly observed in the cytoplasm of invasive bladder cancer cells and tissues. Pkp2 knockdown inhibited, and Pkp3 knockdown enhanced, invasion of bladder cancer cells, but these knockdowns did not alter cell proliferation. CONCLUSION We conclude that high Pkp2, and low Pkp3, expression is associated with bladder cancer cell invasion and that neither Pkp2 nor Pkp3 is associated with cell proliferation. We further hypothesize that accumulation of Pkp2 and 3 in the cell cytoplasm, rather than their recruitment to the cell membrane, is related to an increased ability of the tumor to invade and metastasize.

Laparoscopic Findings of Transverse Testicular Ectopia

Transverse testicular ectopia (TTE) is an extremely rare congenital anomaly in which both testes descend through the same inguinal canal. We report an 8-month-old male with TTE and hypospadias. To help manage the patient, we conducted laparoscopy to elucidate the anatomy of the spermatic cord of the ectopic testis. On laparoscopy, we clearly identified the spermatic cord of the right ectopic testis. In addition, the laparoscopic guide was helpful when doing the trans-septal orchidopexy, in that the ectopic testis could be precisely discriminated without confusion as to the laterality of the testes. Long-term follow-up at 32 months confirmed that both testes were properly positioned in the scrotum and had a good consistency.

Clinical research of renal vein control using Hem‐o‐lok clips in laparoscopic nephrectomy

Abstract  Control of the renal vein represents a crucial step in laparoscopic nephrectomy. Although endovascular gastrointestinal anastomosis (GIA) staplers have generally been used for renal vein control because of the large diameter of the vessel, Hem‐o‐lok clips have recently been used for renal artery control. GIA staplers are expensive and can malfunction on rare occasions, resulting in severe complications. We evaluated renal vein control using Hem‐o‐lok clips (adaptive vascular width 7–16 mm) in laparoscopic nephrectomy. Since April 2004, we have ligated renal arteries using Hem‐o‐lok clips. From June 2004, this method was applied for renal vein control in 40 laparoscopic nephrectomies. After renal pedicle dissection, renal pedicle ligation was accomplished using extra large (XL) Hem‐o‐lok clips on both the renal arteries and veins by placing two clips on the patient side and one clip on the specimen side. Ligation times for obtaining renal vein control were compared between XL Hem‐o‐lok clips and GIA staplers in 40 cases before June 2004. Vascular control using XL Hem‐o‐lok clips was successful in all 40 cases, without any slipping of clips or uncontrolled bleeding. After renal pedicle dissection, ligation time for achieving renal vein control was 167.0 ± 48 s (range: 122–295 s) using XL Hem‐o‐lok clips (mean, three clips) and 68 ± 24.0 s (range: 54–150 s) using a GIA stapler. XL Hem‐o‐lok clips allow safe and reliable control of renal veins in laparoscopic nephrectomy. Ligation time is only 100 s longer than using a GIA stapler. In addition, costs are reduced by more than 90% compared to GIA stapling.

Strategy for Repeat Prostate Biopsy: Predictors of Positive Biopsy and Additional Biopsy Location

Purpose: We analyzed patterns of tumor distribution in radical prostatectomy specimens from patients with repeat biopsies to determine additional appropriate biopsy locations for repeat biopsy. Methods: Between January 2000 and June 2005, a total of 382 patients underwent transrectal ultrasound-guided prostate biopsy. Of these, 47 patients underwent repeat biopsy. Radical prostatectomy was performed for 7 of 22 cancer-positive cases. The 7 specimens were superimposed to create an idealized prostate gland at 3 levels: apex, mid-prostate, and base. We compared these tumor maps with those from 35 initial biopsy positive patients. Results: Prostate cancer was detected in 22 of 47 patients who underwent repeat biopsy. Tumor mapping showed that tumors detected on repeat biopsy in comparison with tumor maps of initial biopsy were dense at the periurethral area of the apex in prostate. Conclusions: Additional biopsy cores taken from periurethral area of the apex on repeat biopsy might further enhance the detection of cancers.