Takashi Kasahara

Role of XIAP in the malignant phenotype of transitional cell cancer (TCC) and therapeutic activity of XIAP antisense oligonucleotides against multidrug-resistant TCC in vitro.

XIAP directly inhibits executor caspases, making it the most downstream antiapoptotic molecule. Here, we examined the expression and function of XIAP in normal urothelium and TCC. We also examined the therapeutic effect of xiap AS PODN on the cell cycle and apoptosis of multidrug‐resistant T24 bladder cancer cells. XIAP was moderately expressed in normal transitional epithelium with prominent expression on the superficial layer cells. Seventy‐nine of 108 (73.15%) tumor samples were positive for XIAP protein, but XIAP positivity was not correlated with tumor stage or grade. Moreover, 4 bladder cancer cell lines (SCaBER, HT1376, T24 and RT4) expressed similar levels of XIAP. xiap AS PODN dose‐dependently reduced the XIAP protein level and induced apoptosis, leading to decreased cell viability by 87%. Combined administration with doxorubicin resulted in marked cytotoxicity due to escalation of apoptosis. Overexpression of XIAP in T24 cells resulted in a modest but statistically significant (p < 0.01) survival advantage compared to parental cells. Thus, XIAP expression may be critical for maintaining the viability and drug resistance of TCC, and endogenous XIAP levels are sufficient to protect cells from apoptosis. Our results suggest that XIAP may play an important role early in human TCC carcinogenesis. xiap AS may be a candidate for use as a cancer therapy for overcoming drug resistance in highly malignant TCC.

Prognostic impact of FAS/CD95/APO-1 in urothelial cancers: decreased expression of Fas is associated with disease progression.

The death receptor Fas (Apo1/CD95) and Fas ligand (FasL) system is recognised as a major pathway for the induction of apoptosis in vivo, and antiapoptosis via its blockade plays a critical role in carcinogenesis and progression in several malignancies. However, the function of Fas–FasL system in urothelial cancer (UC) has not been elucidated. We therefore investigated the expression of Fas, FasL and Decoy receptor 3 for FasL (DcR3) in UC specimens and cell lines, and examined the cytotoxic effect of an anti-Fas-activating monoclonal antibody (mAb) in vitro. Immunohistochemical examinations of Fas-related molecules were performed on 123 UC and 30 normal urothelium surgical specimens. Normal urothelium showed Fas staining in the cell membrane and cytoplasm. In UC, less frequent Fas expression was significantly associated with a higher pathological grade (P<0.0001), a more advanced stage (P=0.023) and poorer prognosis (P=0.010). Fas and the absence thereof were suggested to be crucial factors with which to select patients requiring more aggressive treatment. Moreover, low-dose anti-Fas-activating mAb sensitised resistant cells to adriamycin, and this synergistic effect could be applied in the development of new treatment strategy for UC patients with multidrug-resistant tumours.

Role of IRF-1 and caspase-7 in IFN-γ enhancement of fas-mediated apoptosis in ACHN renal cell carcinoma cells

Caspases exist as zymogens, and are activated by various extracellular stimuli, leading to apoptosis. One such stimulus is Fas/CD95, a member of the tumor necrosis factor receptor family, providing one means of cytotoxic T lymphocyte (CTL)‐mediated cell lysis. Clinical evidence has shown that administration of cytokine leads to regression in selected patients with renal cell carcinomas (RCCs). Interferon‐γ (IFN‐γ) indicates its contribution to anti‐tumor activity of immune cells. IFN‐γ elicits its effect through the transcription factor signal transducer and activator of transcription‐1 (STAT‐1), and through interferon regulatory factor‐1 (IRF‐1), one of the target genes of STAT‐1. Our previous study demonstrated an increase in the susceptibility of ACHN cells, established from RCC, to Fas‐mediated apoptosis by IFN‐γ, and the inhibition of this effect by the caspase‐3 and ‐7 inhibitor, DEVD‐CHO. We demonstrated the following phenomena in IFN‐γ‐treated ACHN cells: 1) enhanced transcription of caspase‐1, 3 and 7 mRNAs without any change in cleavage of their substrates; 2) increased cleavage DEVD (specific for caspase‐3 and 7), but not YVAD (for caspase‐1) or DMQD (for caspase‐3), after anti‐Fas/CD95 MAb treatment; 3) activation of the STAT‐1 and IRF‐1 pathway; and 4) partial abrogation of the IFN‐γ‐induced increase in Fas‐mediated apoptosis by antisense IRF‐1 oligodeoxynucleotide. These results suggest that IRF‐1 plays a pivotal role in the IFN‐γ‐mediated‐enhancement of Fas/CD95‐mediated apoptosis, through regulation of DEVD‐CHO‐sensitive caspases, most likely caspase‐7.

Caspase involved synergistic cytotoxicity of bcl-2 antisense oligonucleotides and adriamycin on transitional cell cancer cells.

We have previously shown that Bcl-2 expression was negative prognostic factor in transitional cell cancer (TCC), and that TCC cell lines expressing high levels of Bcl-2 are resistant to Adriamycin triggered apoptosis. Here we examined antisense oligonucleotide-mediated downregulation of Bcl-2 expression and its effect on sensitivity to Adriamycin (ADM) treatment in T24 cells. Treatment of T24 cells with 20 microM of bcl-2 antisense phosphorothioate oligodeoxynucleotide (PODN) reduced the Bcl-2 protein level. Combined administration with Adriamycin resulted in synergistic cytotoxicity, accompanied with a 2.4-fold increase in DEVD-specific caspase activity. The finding provides evidence that Bcl-2 expression may be critical for maintaining the drug resistance of TCC. bcl-2 antisense PODN might be useful means for overcoming drug resistance in highly malignant TCC.

Prognostic factors of patients with metastatic renal cell carcinoma with removed metastases: a multicenter study of 556 patients.

OBJECTIVE To investigate the prognosis and prognostic factors of patients with metastatic renal cell carcinoma who underwent metastasectomy. METHODS We sent questionnaires to Japanese hospitals. The questionnaires included data of patients with metastatic renal cell carcinoma who had their metastatic lesions removed between January 1988 and December 2009. We collected them and retrospectively analyzed these data and calculated the overall survival from the first metastasectomy until death or last follow-up. We also analyzed the relationship between survival and clinico-pathologic features and determined adverse prognostic factors. Furthermore, we identified a poor prognostic group by counting the number of prognostic factors. RESULTS A sample size of 556 patients from 48 institutions was studied. The median overall survival was 80 months. Four adverse prognostic factors were detected: incomplete resection by metastasectomy (hazard ratio [HR], 2.15), brain metastasis (HR, 3.73), >1.0 mg/dL C-reactive protein (HR, 2.45), and the highest histologic grade in Japanese classification (nuclei of tumor cells are larger than nuclei of normal tubular cells; HR, 1.88). The median overall survivals of patients with 3 or 4 prognostic factors, 2 factors, and 0 and 1 factors were 10 months, 42 months, and 105 months, respectively. CONCLUSION Four adverse prognostic factors for predicting the survival of patients with removed metastases were identified. Patients with 3 or 4 of these adverse prognostic factors had a worse prognosis.

Frequency of PSA-mRNA-bearing cells in the peripheral blood of patients after prostate biopsy

Transrectal ultrasound (TRUS) guided prostate biopsy is standard diagnostic procedure for prostate cancer (PCa). However, possibility of dissemination of cancer cells by biopsy is not negligible. To investigate this possibility, we examined prostate specific antigen (PSA)-bearing cells in peripheral blood of the 108 patients before and after prostate biopsy. Peripheral blood samples were obtained from 108 patients with elevated serum PSA (sPSA) levels, who had undergone sextant prostate biopsy using TRUS. The presence of PSA-mRNA bearing cells was examined using the nested RT-PCR method enabling detection of one LNCaP cell diluted in 1 ml of whole blood. Among 108 patients, 62 and 46 were diagnosed with benign prostatic hyperplasia (BPH) and PCa, respectively. PSA-mRNA was detected in 3 PCa cases but in no BPH patients before and after biopsy, and in 16 BPH (25.8%) and in 21 PCa (45.7%) patients only after biopsy (P < 0.01). The patients with positive mRNA before biopsy had higher sPSA (P < 0.001), and those after biopsy had higher sPSA and PSA density (PSAD) levels (P < 0.05). Positive PSA-mRNA cases had more cancer involved biopsy cores than the negative PSA-mRNA cases (P < 0.001). Although further investigations are needed, the present findings suggest that prostate biopsy might scatter prostate cells in the blood stream especially in cases with high sPSA and, thus, might contribute to tumour spreading in the cases of prostate cancer.

Laparoscopic adrenalectomy for pheochromocytoma: evaluation of experience and strategy at a single institute.

To assess the utility, safety and feasibility of laparoscopic adrenalectomy (LA) for pheochromocytoma. We reviewed our experience, focusing on surgical outcomes compared with our historical open adrenalectomy (OA) and modifications in surgical procedures.

Transforming growth factor-β1 regulates cell growth and causes downregulation of SMemb/non-muscle myosin heavy chain B mRNA in human prostate stromal cells

Objectives. SMemb/non-muscle myosin heavy chain B (SMemb/NMMHC-B) is most abundantly expressed in proliferating smooth muscle cells and correlates with phenotypic changes from a contractive to a proliferative type. The stromal cells of the prostate play a crucial role in the regulation of prostatic growth and function. The aim of this study was to investigate the effects of the multifunctional cytokine transforming growth factor-β1 (TGF-β1) on SMemb/NMMHC-B mRNA expression and stromal cell growth. The expression of the SM2 isoform of smooth muscle myosin heavy chain (SMMHC) mRNA was also examined. Material and methods. Primary cultures of prostate stromal cells were established by means of an explant method from eight normal prostates. The effects of TGF-β1 on stromal cell growth were determined by means of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide conversion assay. SMemb/NMMHC-B and SM2 mRNA expression were analyzed quantitatively by means of real-time polymerase chain reaction. Results. In the absence of TGF-β1, cells expressed α-smooth muscle actin and vimentin. After TGF-β1 treatment, the expression of α-smooth muscle actin increased and cells also expressed desmin. TGF-β1 at concentrations of 1.0, 5.0 and 10 ng/ml suppressed cell growth by 72%, 62% and 56%, respectively, downregulated SMemb/NMMHC-B mRNA expression by 71%, 52% and 38%, respectively and upregulated SM2 mRNA expression 2.1-, 3.0- and 5.3-fold, respectively. Conclusions. These results demonstrate that TGF-β1 modulates the smooth muscle cell phenotype from a proliferative to a contractile type and that the inhibitory effects of TGF-β1 on stromal cell growth correlate with downregulation of the SMemb/NMMHC-B gene.

Immunohistochemical studies of caveolin-3 in germ cell tumors of the testis.

Background/Aim: Caveolin, which is a major constructive component of the caveolar membranes, plays a key role in transcytosis of molecules into cells and regulation of several signal transductions. Caveolin has three isoforms, and recent studies suggest that in some malignant tumors an alteration of caveolin-1 expression correlates with oncogenetic changes. Caveolins have been reported to be negative regulators of inducible nitric oxide synthase (iNOS) which can provoke an antitumor response via infiltrating immune cells. The aim of this study was to examine the expressions of caveolin-1 and caveolin-3 (which is a caveolin-1 homologue localized predominantly in muscle tissue) in testicular cancer. Methods: We evaluated the expressions of caveolin-1, caveolin-3, and iNOS in 16 seminoma and 10 non-seminoma testicular cancer specimens as well as normal testicular tissue, using a streptavidin-biotin bridge technique on cryostat sections. The expression of caveolin-3 was confirmed by slot-blot analysis. Tumor-infiltrating immune cells were also studied immunohistochemically, and the correlation between the number of immune cells and caveolin-3 concentrations was calculated. Results: Immunohistochemistry revealed that caveolin-3, but not caveolin-1, was frequently expressed in seminomas (12 positive out of 16 specimens) without positive staining in their normal counterparts or in nonseminomatous germ cell tumors, except for muscle components in the teratoma. iNOS was not expressed in any tissues examined. Samples with high levels of caveolin-3 tended to have higher degrees of tumor-infiltrating immune cells, although this finding was not statistically significant. Conclusion: This is the first report demonstrating the involvement of caveolin-3 in germ cell tumors.

Retroperitoneoscopic donor nephrectomy with a gel-sealed hand-assist access device

BackgroundThe hand-assisted technique enables the rapid extraction of the graft, shortening the warm ischemia time (WIT), and the retroperitoneoscopic approach is potentially associated with a less incidence of postoperative ileus in donor nephrectomy for living kidney transplantation. The aim of this study was to assess the efficacy and safety of retroperitoneoscopic donor nephrectomy with a gel-sealed hand-assist access device (GelPort), which is a wound sealing device that permits the access of the hand to the surgical field, free trocar site choice within it, and rapid conversion to open surgery if necessary, while preserving the pneumoperitoneum/pneumoretroperitoneum.MethodsSeventy-five consecutive donors receiving this procedure were retrospectively studied. A 2-cm skin incision was made at the midpoint between the tip of the 12th rib and superior border of the iliac bone in the midaxillary line, through which retroperitoneal space was made. Preperitoneal wound with a 6 – 7-cm pararectal incision in the upper abdominal region was connected to the retroperitoneal space. A GelPort was put inside the pararectal surgical wound. The principle was pure retroperitoneoscopic surgery; hand-assist was applied for retraction of the kidney in the renal vessel control and graft extraction.ResultsThe mean operation time including waiting time for recipient preparation was 242.2±37.0 (range: 214.0–409.0) min, and the mean amount of blood loss was 164.3±146.6 (range: 10.0–1020.0) ml. The mean WIT was 2.8±1.0 (range: 1.0–6.0) min. The shortage of renal vessels or ureter was observed in none of the grafts. No donor experienced blood transfusion, open conversion, or injury of other organs. Blood loss was greater in patients with body mass index (BMI) of 25 kg/m2 or higher than in those with BMI of <25 kg/m2 (218.4±98.8 vs. 154.8±152.1 ml, P=0.031). No donor had postoperative ileus or reported wound pain leading to decreased activity of daily life or wound cosmetic problem.ConclusionsRetroperitoneoscopic hand-assisted donor nephrectomy with the mentioned approach was suggested to be a feasible option without compromising safety, although further improvement in surgical techniques is warranted.