Teruo Inamoto

Sensitivity to Epidermal Growth Factor Receptor Inhibitor Requires E-Cadherin Expression in Urothelial Carcinoma Cells

Purpose: Epidermal growth factor receptor (EGFR) is an attractive target for the treatment of urothelial carcinoma, but a clinical response can be expected in only a small proportion of patients. The aim of this study was to define molecular markers of response to cetuximab therapy in a panel of urothelial carcinoma cell lines. Experimental Design: Eleven cell lines were investigated for antiproliferative response to cetuximab based on [3H]thymidine incorporation. A variety of markers, including EGFR expression, phosphorylation, and gene amplification, as well as the expression of other growth factor receptors, their ligands, and markers of epithelial-to-mesenchymal transition were investigated. Cohens κ statistic was used to estimate the agreement between response and expression of these markers. E-cadherin was silenced by small interfering RNA in two sensitive cell lines, and the effect on the response to cetuximab was measured. Results: We were able to identify a panel of relevant markers pertaining especially to alternate growth factor receptor expression and epithelial-to-mesenchymal transition that predicted response to cetuximab. The data suggested that expression of intact HER-4 (κ, 1.00; P = 0.008), E-cadherin (κ, 0.81; P = 0.015), and β-catenin (κ, 0.81; P = 0.015) and loss of expression of platelet-derived growth factor receptor β (κ, 0.57; P = 0.167) were associated with response to cetuximab therapy. Silencing E-cadherin in two sensitive cell lines reduced responsiveness to cetuximab in both (P < 0.001). Conclusions: A panel of predictive markers for cetuximab response has been established in vitro and is currently being evaluated in a prospective clinical trial of neoadjuvant EGFR-targeted therapy. Most importantly, E-cadherin seems to play a central role in modulation of EGFR response in urothelial carcinoma.

Curcumin Potentiates the Antitumor Effects of Gemcitabine in an Orthotopic Model of Human Bladder Cancer through Suppression of Proliferative and Angiogenic Biomarkers

Little progress has been made in the last three decades in the treatment of bladder cancer. Novel agents that are nontoxic and can improve the current standard of care of this disease are urgently needed. Curcumin, a component of Curcuma longa (also called turmeric), is one such agent that has been shown to suppress pathways linked to oncogenesis, including cell survival, proliferation, invasion and angiogenesis. We investigated whether curcumin has potential to improve the current therapy for bladder cancer, using an orthotopic mouse model. Curcumin potentiated the apoptotic effects of gemcitabine against human bladder cancer 253JBV cells in culture. Electrophoretic mobility shift assay revealed that curcumin also suppressed the gemcitabine-induced activation of the cell survival transcription factor NF-kappaB. In an orthotopic mouse model, bioluminescence imaging revealed that while curcumin alone significantly reduced the bladder tumor volume, maximum reduction was observed when curcumin was used in combination with gemcitabine (P<0.01 versus vehicle; P<0.01 versus gemcitabine alone). Curcumin also significantly decreased the proliferation marker Ki-67 and microvessel density (CD31) (P<0.01 versus vehicle; P<0.01 versus gemcitabine alone), but maximum reduction occurred when it was combined with gemcitabine (P<0.01 versus vehicle; P<0.01 versus gemcitabine alone). Curcumin abolished the constitutive activation of NF-kappaB in the tumor tissue; induced apoptosis, and decreased cyclin D1, VEGF, COX-2, c-myc and Bcl-2 expression in the bladder cancer tissue. Overall our results suggest that curcumin alone exhibits significant antitumor effects against human bladder cancer and it further potentiates the effects of gemictabine, possibly through the modulation of NF-kappaB signaling pathway.

1,1-Bis(3′-indolyl)-1-(p-chlorophenyl)methane activates the orphan nuclear receptor Nurr1 and inhibits bladder cancer growth

Nurr1 is an orphan nuclear receptor and a member of the nerve growth factor I-B subfamily of transcription factors with no known endogenous ligand or stimulator. We show, for the first time, evidence that Nurr1 is expressed in a panel of 11 human bladder cancer cell lines. A new class of methylene-substituted diindolylmethanes (C-DIM) were screened and 1,1-bis(3′-indolyl)-1-(p-chlorophenyl)methane (DIM-C-pPhCl) activated the ligand-binding domain of Nurr1. Treatment of bladder cancer cells with Nurr1-active C-DIM resulted in decreased cell survival (MTT assay) and induction of cell death pathways, resulting in poly(ADP-ribose) polymerase cleavage and DNA fragmentation. The specificity of the Nurr1-active compound was shown using RNA interference in 253J B-V cells, whereby small interfering RNA against Nurr1 attenuated ligand-dependent activation of Nurr1 and poly(ADP-ribose) polymerase cleavage. Furthermore, activation of Nurr1 resulted in stimulation of tumor necrosis factor-related apoptosis-inducing ligand and small interfering RNA experiments attenuated tumor necrosis factor-related apoptosis-inducing ligand production. In an orthotopic model of human bladder tumors established in nude mice, administration of a Nurr1-active C-DIM suppressed bladder cancer growth. These results identify Nurr1 as a potential target for bladder cancer therapy and also identify a novel agent for activating Nurr1. [Mol Cancer Ther 2008;7(12):3825–33]

Anti-CD26 Monoclonal Antibody–Mediated G1-S Arrest of Human Renal Clear Cell Carcinoma Caki-2 Is Associated with Retinoblastoma Substrate Dephosphorylation, Cyclin-Dependent Kinase 2 Reduction, p27kip1 Enhancement, and Disruption of Binding to the Extracellular Matrix

Purpose: CD26 is a 110-kDa cell surface glycoprotein with a role in tumor development through its association with key intracellular proteins. In this report, we show that binding of soluble anti-CD26 monoclonal antibody (mAb) inhibits the growth of the human renal carcinoma cells in both in vitro and in vivo experiments. Experimental Design: Growth inhibition by anti-CD26 mAb was assessed using proliferation assay and cell cycle analysis. Anti-CD26 mAb, chemical inhibitors, dominant-negative, or constitutively active forms of specific signaling molecules were used to evaluate CD26-associated pathways. The in vivo growth-inhibitory effect of anti-CD26 mAb was also assessed in a human renal carcinoma mouse xenograft model. Results:In vitro experiments show that anti-CD26 mAb induces G1-S cell cycle arrest associated with enhanced p27kip1 expression, down-regulation of cyclin-dependent kinase 2, and dephosphorylation of retinoblastoma substrate. Moreover, our data show that enhanced p27kip1 expression is dependent on the attenuation of Akt activity. Anti-CD26 mAb also internalizes cell surface CD26, leading to decreased binding to collagen and fibronectin. Experiments with a mouse xenograft model involving human renal carcinoma cells show that anti-CD26 mAb treatment drastically inhibits tumor growth in tumor-bearing mice, resulting in enhanced survival. Conclusions: Taken together, our data strongly suggest that anti-CD26 mAb treatment may have potential clinical use for CD26-positive renal cell carcinomas.

Cytoplasmic mislocalization of the orphan nuclear receptor Nurr1 is a prognostic factor in bladder cancer

Nurr1 belongs to a novel class of orphan nuclear receptors (the NR4A family). The authors have previously shown that Nurr1 is important in carcinogenesis. In the current study, they examined the clinicopathologic relevance of expression patterns of Nurr1 in bladder tumors.

Superagonistic CD28 Antibody Induces Donor-Specific Tolerance in Rat Renal Allografts

The ultimate goal of organ transplantation is to establish graft tolerance where CD4+CD25+FOXP3+ regulatory T (Treg) cells play an important role. We examined whether a superagonistic monoclonal antibody specific for CD28 (CD28 SA), which expands Treg cells in vivo, would prevent acute rejection and induce tolerance using our established rat acute renal allograft model (Wistar to Lewis). In the untreated or mouse IgG‐treated recipients, graft function significantly deteriorated with marked destruction of renal tissue, and all rats died by 13 days with severe azotemia. In contrast, 90% of recipients treated with CD28 SA survived over 100 days, and 70% survived with well‐preserved graft function until graft recovery at 180 days. Analysis by flow cytometry and immunohistochemistry demonstrated that CD28 SA induced marked infiltration of FOXP3+ Treg cells into the allografts. Furthermore, these long‐surviving recipients showed donor‐specific tolerance, accepting secondary (donor‐matched) Wistar cardiac allografts, but acutely rejecting third‐party BN allografts. We further demonstrated that adoptive transfer of CD4+CD25+ Treg cells, purified from CD28 SA‐treated Lewis rats, significantly prolonged allograft survival and succeeded in inducing donor‐specific tolerance. In conclusion, CD28 SA treatment successfully induces donor‐specific tolerance with the involvement of Treg cells, and thus the therapeutic value of this approach warrants further investigation and preclinical studies.

Metastases to the penis from carcinoma of the prostate

Abstract A 58‐year‐old man presented with dysuria at the Osaka Medical College Hospital in November 1996. Laboratory examination revealed elevated serum prostate‐specific antigen (PSA) to > 100 ng/mL. Adenocarcinoma of the prostate with metastasis to the bone was diagnosed after a biopsy of the prostate and bone scintigraphy; hormonal therapy was administered. Although bone metastasis was well controlled and the serum PSA level declined to within normal levels (2.0 ng/mL), several painless nodules were found on the penile glans. Biopsy of the nodules showed that the penile tumor was a metastasis from the prostate cancer. The patient underwent partial penectomy for relief from penile pain. The serum PSA level showed no elevation 3 months after the partial penectomy, suggesting that careful observation of prostate cancer patients is necessary, even when oseous metastasis is well controlled and serum PSA levels are kept within normal ranges by hormonal therapy. The case also indicates that urologists should consider the possibility of metastasis to the penis from prostate cancer.

Adipose-derived stromal cells inhibit prostate cancer cell proliferation inducing apoptosis.

Mesenchymal stem cells (MSCs) have generated a great deal of interest in the field of regenerative medicine. Adipose-derived stromal cells (AdSCs) are known to exhibit extensive proliferation potential and can undergo multilineage differentiation, sharing similar characteristics to bone marrow-derived MSCs. However, as the effect of AdSCs on tumor growth has not been studied sufficiently, we assessed the degree to which AdSCs affect the proliferation of prostate cancer (PCa) cell. Human AdSCs exerted an inhibitory effect on the proliferation of androgen-responsive (LNCaP) and androgen-nonresponsive (PC3) human PCa cells, while normal human dermal fibroblasts (NHDFs) did not, and in fact promoted PCa cell proliferation to a degree. Moreover, AdSCs induced apoptosis of LNCaP cells and PC3 cells, activating the caspase3/7 signaling pathway. cDNA microarray analysis suggested that AdSC-induced apoptosis in both LNCaP and PC3 cells was related to the TGF-β signaling pathway. Consistent with our in vitro observations, local transplantation of AdSCs delayed the growth of tumors derived from both LNCaP- and PC3-xenografts in immunodeficient mice. This is the first preclinical study to have directly demonstrated that AdSC-induced PCa cell apoptosis may occur via the TGF-β signaling pathway, irrespective of androgen-responsiveness. Since autologous AdSCs can be easily isolated from adipose tissue without any ethical concerns, we suggest that therapy with these cells could be a novel approach for patients with PCa.

Invasive ability of human renal cell carcinoma cell line Caki-2 is accelerated by gamma-aminobutyric acid, via sustained activation of ERK1/2 inducible matrix metalloproteinases.

Gamma-aminobutyric acid (GABA) was first discovered as an inhibitory neurotransmitter in the central nervous system (CNS) and has been reported to have a variety of functions, including regulation of cell division, cell differentiation and maturation, and to be involved in the development of certain cancers outside the CNS. In the present study, using the human renal cell carcinoma cell line Caki-2, we demonstrated that GABA stimulation significantly increased the expression of MMP-2 and -9 and subsequently increased the invasive activity of the cancer cells. Because MAPK signaling is one of the key regulators of MMP expression, we further evaluated MAPK signaling after stimulation with GABA. It was found that GABA stimulation promoted the phosphorylation of MAPKs, including ERK1/2, JNK, and p38. ERK1/2 phosphorylation was sustained for up to 12 h, while phosphorylation of JNK and p38 returned to the endogenous level by 30 min. It was noteworthy that the ras/raf/MEK/ERK pathway inhibitor PD98059 attenuated GABA-induced MMP-9 expression and that both PD98059 and MMP inhibitors attenuated the GABA-induced invasive activity of Caki-2 cells. Moreover, data obtained by depletion of the MEK/ERK pathway using interfering RNA transfection of Caki-2 cells clearly corroborated the above results, as both MMP-9 expression and GABA-induced invasive ability were decreased significantly. We also demonstrated that the GABA-induced increase in invasive ability via ERK1/2 up-regulation was mediated mainly through the GABA-B receptor. These results indicate that GABA stimulation promotes cancer cell invasion and that the effect is partly due to ERK1/2-dependent up-regulation of MMPs.

Incidence of urethral stricture after bipolar transurethral resection of the prostate using TURis: results from a randomised trial

To assess whether bipolar transurethral resection of the prostate (B‐TURP) using the TURis® system has a similar level of efficacy and safety to that of the traditional monopolar transurethral resection of the prostate (M‐TURP), and to evaluate the impact of the TURis system on postoperative urethral stricture rates over a 36‐month follow‐up period.