Kazuo Nishimura

Supervillin associates with androgen receptor and modulates its transcriptional activity

Activation of androgen receptor (AR) via androgen in muscle cells has been closely linked to their growth and differentiation. Here, we report the cloning and characterization of supervillin (SV), a 205-kDa actin-binding protein, as an AR coregulator from the skeletal muscle cDNA library. Mammalian two-hybrid and glutathione S-transferase pull-down assays indicate a domain within SV (amino acids 594-1268) can interact with AR N terminus and DNA-binding domain–ligand-binding domain in a ligand-enhanced manner. Subcellular colocalization studies with fluorescence staining indicate SV can colocalize with AR in the presence of 5α-dihydrotestosterone in COS-1 cells. The functional reporter assays showed full-length SV and the SV peptide (amino acids 831-1281) within the interaction domain can enhance AR transactivation. Furthermore, SV can enhance the endogenous AR target gene, p27KIP1, expression in prostate PC-3(AR2) cells. SV preferentially enhanced AR rather than other tested nuclear receptors and could be induced by natural androgens better than other steroids. SV can also cooperate with other AR coregulators, such as ARA55 or ARA70, to enhance AR transactivation further. Unlike SRC-1 that can enhance the interaction between AR N terminus and AR C terminus, SV shows a mild suppressive effect on N–C interactions, suggesting SV may go through a different mechanism to enhance AR transactivation. Together, our data demonstrate that SV is an AR coregulator that can enhance AR transactivation in muscle and other cells.

Sesquiterpene lactone parthenolide suppresses tumor growth in a xenograft model of renal cell carcinoma by inhibiting the activation of NF-κB

The transcription factor nuclear factor‐κB (NF‐κB) has been shown to be constitutively activated in various human malignancies, including leukemia, lymphoma and a number of solid tumors. NF‐κB regulates the transcriptional of genes important for tumor invasion, metastasis and chemoresistance. The sesquiterpene lactone parthenolide, an inhibition of NF‐κB, has been used conventionally to treat migraines and inflammation. In this study, renal cancer cell lines OUR‐10 and ACHN were used for in vitro experiments to evaluate growth‐inhibitory effects of parthenolide. An OUR‐10 xenograft model in nude mice was also used to investigate the in vivo growth‐inhibitory effects of parthenolide. Apoptosis in response to treatment of OUR‐10 cells with parthenolide was confirmed. Localization of NF‐κB in response to parthenolide treatment was examined of by immunofluorostaining of OUR‐10 cells with antibody against NF‐κB p65 and by Western blot analysis of OUR‐10 cell and tumor nuclear and cytosol fraction. Parthenolide effectively inhibited proliferation of cultured OUR‐10 cells and triggered apoptosis in vitro. Subcutaneous injection or oral administration of parthenolide showed significant tumor growth inhibition in the xenograft model via decreased production of interleukin‐8 (IL‐8) or vascular endothelial growth factor (VEGF). Immunohistochemistry and Western blot analysis showed decreased nuclear localization of NF‐κB and phosphorylated NF‐κB protein and subsequently expression of MMP‐9, Bcl‐xL and Cox‐2 in response to parthenolide treatment. These results indicate that parthenolide is a useful in the treatment of renal cell carcinoma and acts via inhibition of NF‐κB.

Differential induction of androgen receptor transactivation by different androgen receptor coactivators in human prostate cancer DU145 cells.

Recently identified androgen receptor (AR) coactivators were used in this study to determine whether the specificity of sex hormones and antiandrogens could be modulated at the coactivator level. We found that ARA 70 is the best coactivator to confer the androgenic activity on 17β-estradiol. Only ARA 70 and ARA 55 could increase significantly the androgenic activity of hydroxyflutamide, a widely used antiandrogen for the treatment of prostate cancer. None of the AR coactivators we tested could significantly confer androgenic activity on progesterone and glucocorticoid at their physiological concentrations (1–10 nM). We also found that ARA70, ARA55, and ARA 54, but not steroid receptor coactivator-1 (SRC-1) and Rb, could significantly enhance the Δ5-androstenediol-mediated AR transactivation. Furthermore, in comparing the relative specificity of these coactivators to AR in DU 145 cells, our results suggested that ARA70 has a relatively higher specificity and that SRC-1 can enhance almost equally well many other steroid receptors. Finally, our data demonstrated that AR itself and some select AR coactivators such as ARA70 or ARA54 could, respectively, interact with CBP and p300/CBP-associated factors that have histone acetyl-transferase activity for assisting chromatin remodeling. Together, our data suggest that the specificity of sex hormones and antiandrogens can be modulated by some selective AR coactivators. These findings may not only help us to better understand the specificity of the sex hormones and antiandrogens, but also facilitate the development of better antiandrogens to fight the androgen-related diseases, such as prostate cancer.

Increased Infiltration of Tumor Associated Macrophages is Associated With Poor Prognosis of Bladder Carcinoma In Situ After Intravesical Bacillus Calmette-Guerin Instillation

PURPOSE Tumor associated macrophages can regulate the growth of various cancers positively or negatively. Intravesical bacillus Calmette-Guerin instillation is now gold standard treatment for bladder carcinoma in situ. We investigated the correlation between tumor associated macrophages infiltrating bladder carcinoma in situ and the response to intravesical bacillus Calmette-Guerin therapy. MATERIALS AND METHODS We examined paraffin embedded tissues from 41 patients with bladder carcinoma in situ who received intravesical bacillus Calmette-Guerin therapy. Tumor associated macrophages were immunohistochemically stained by anti-CD68 monoclonal antibody. RESULTS The median number of tumor associated macrophages infiltrating among cancer cells and the number in the lamina propria were 4 and 24, respectively. Recurrent carcinoma in situ was found in 4.8% of cases with a lower cancer cell tumor associated macrophage count but in 47.6% of those with a higher cancer cell tumor associated macrophage count (less than 4 vs 4 or greater). Recurrence was found in 31.8% of patients with a lower lamina propria tumor associated macrophage count but in 21.1% of those with a higher lamina propria tumor associated macrophage count (less than 25 vs 25 or greater). The median ratio of tumor associated macrophages among cancer cells vs in the lamina propria was 0.2. Recurrence-free survival was significantly better in patients with a lower cancer cell tumor associated macrophage count (p = 0.0002). Those with a lower cancer cell-to-lamina propria tumor associated macrophage ratio had a higher recurrence-free rate (p <0.0001). Multivariate analysis revealed that the cancer cell tumor associated macrophage count and the cancer cell-to-lamina propria tumor associated macrophage ratio can be prognostic factors for bladder carcinoma in situ. CONCLUSIONS The count of tumor associated macrophages infiltrating the cancer area is useful for predicting the response of bladder carcinoma in situ to intravesical bacillus Calmette-Guerin instillation before treatment initiation.

Anticarcinogenic effect of FTY720 in human prostate carcinoma DU145 cells: Modulation of mitogenic signaling, FAK, cell‐cycle entry and apoptosis

Despite the high frequency of prostate cancer, therapeutic options for advanced disease are limited to chemotherapy, radiation or hormonal therapy and eventually fail in all patients. Therefore, alternative approaches need to be developed. We previously reported that FTY720, a metabolite from Isaria sinclarii, is a unique antitumor agent for an androgen‐independent prostate cancer cell line and requires caspase‐3 activation in apoptosis. In our study, we have evaluated the effect of FTY720 on a family of mitogen‐activated protein kinases (MAPKs), focal adhesion kinase (FAK), mitochondrial transmembrane potential, caspase‐9 and caspase‐8 and analyzed the expression of some cell‐cycle regulator proteins in DU145 cells in order to understand the various antitumor effects of FTY720. Apoptosis was quantified by phosphatidylserine exposure. Activation of MAPKs, cleavage of caspase‐9 and caspase‐8, status of cyclin‐dependent kinases (CDKs) and Cip1/p21, a cyclin‐dependent kinase inhibitor, were evaluated by Western blot analysis, in addition to FAK and phospho‐FAK immunoprecipitation and cell‐cycle analysis by FACScan. We found that in DU145 cells, 40 μM FTY720 caused activation of p38 MAPK and the upstream kinase MKK3/MKK6 but not SAPK/JNK. Mitochondrial transmembrane potential, FAK and ERK1/2 were reduced while caspase‐9 and caspase‐8 were cleaved. The p38‐specific inhibitor had no effect on apoptosis induced by FTY720, whereas z‐VAD.FMK, a broad‐spectrum caspase inhibitor, did not inhibit the p38 MAPK activation. An amount of 20 μM FTY720 resulted in G1 arrest and a decrease of CDK2 as well as CDK4, whereas it induced Cip1/p21. FTY720 may exert anticarcinogenic effects against prostate cancer cells possibly involving modulation of mitogenic signaling, cell‐cycle regulators, induction of G1 arrest and apoptotic death in DU145 cells.

Decreased number of mast cells infiltrating into needle biopsy specimens leads to a better prognosis of prostate cancer.

Mast cell infiltration is often observed around human tumours. Inflammatory cells such as macrophages, neutrophils and mast cells infiltrating around tumours are known to contribute to tumour growth; however, the clinical significance of mast cell invasion in prostate cancer (PCa) has not been investigated. Mast cell infiltration was evaluated in 104 patients (age range, 45–88 years; median, 72 years), who underwent needle biopsy of the prostate and were confirmed to have PCa. Needle biopsy specimens of prostate were sliced into 5-μm-thick sections and immunostained for mast cells with monoclonal antibody against mast cell-specific tryptase. Mast cells were counted systematically under a microscope (× 400 magnification), and the relations between mast cell numbers and clinicopathologic findings were evaluated. The mast cell count was evaluated for prognostic value by multivariate analysis. Mast cells were immunostained around the cancer foci. The median number of mast cells in each case was 16. The mast cell count was higher around cancer foci in patients with higher Gleason scores than in those with low Gleason scores. The mast cell number correlated well with clinical stage (P<0.001). Prostate-specific antigen-free survival of patients with higher mast cell counts was better than that in patients with lower mast cell counts (P<0.001). Multivariate analysis revealed that mast cell count was a significant prognostic factor (P<0.005). The number of mast cells infiltrating around cancer foci in prostate biopsy specimens can be a significant prognostic factor of PCa.

A dominant-negative mutant of androgen receptor coregulator ARA54 inhibits androgen receptor-mediated prostate cancer growth.

The ligand-bound androgen receptor (AR) regulates target genes via a mechanism involving coregulators such as androgen receptor-associated 54 (ARA54). We investigated whether the interruption of the AR coregulator function could lead to down-regulation of AR activity. Using in vitro mutagenesis and a yeast two-hybrid screening assay, we have isolated a mutant ARA54 (mt-ARA54) carrying a point mutation at amino acid 472 changing a glutamic acid to lysine, which acts as a dominant-negative inhibitor of AR transactivation. In transient transfection assays of prostate cancer cell lines, the mt-ARA54 suppressed endogenous mutated AR-mediated and exogenous wild-type AR-mediated transactivation in LNCaP and PC-3 cells, respectively. In DU145 cells, the mt-ARA54 suppressed exogenous ARA54 but not other coregulators, such as ARA55-enhanced or SRC-1-enhanced AR transactivation. In the LNCaP cells stably transfected with the plasmids encoding the mt-ARA54 under the doxycycline inducible system, the overexpression of the mt-ARA54 inhibited cell growth and endogenous expression of prostate-specific antigen. Mammalian two-hybrid assays further demonstrated that the mt-ARA54 can disrupt the interaction between wild-type ARA54 molecules, suggesting that ARA54 dimerization or oligomerization may play an essential role in the enhancement of AR transactivation. Together, our results demonstrate that a dominant-negative AR coregulator can suppress AR transactivation and cell proliferation in prostate cancer cells. Further studies may provide a new therapeutic approach for blocking AR-mediated prostate cancer growth.

Bacillus Calmette–Guérin Perfusion Therapy for the Treatment of Transitional Cell Carcinoma in situ of the Upper Urinary Tract

Objectives: The aim of this study is to evaluate the efficacy and safety of intrarenal bacillus Calmette–Guérin (BCG) instillation as a treatment for transitional cell carcinoma in situ (CIS) of the upper urinary tract.Methods: Diagnostic criteria of upper urinary tract CIS were (1) positive urinary cytology, (2) negative multiple random biopsy of the bladder and prostatic urethra, (3) negative radiographic findings in the upper urinary tract and (4) two serial positive cytologies in selective ipsilateral urine sampling from the pyeloureteral system. Eleven patients diagnosed as having upper urinary tract CIS were enrolled in this study. Thus, 11 renal units were treated with BCG instillation. After placing a 6–french Double–J stent, BCG (80 mg) in 40 ml saline was instilled into the bladder weekly, 6 times in total as one course.Results: At the end of one course, 9 cases showed negative urinary cytology. Among these 9 cases, 2 showed recurrence in the upper urinary tract after 4 months and 8 months of disease–free interval, respectively. These 2 cases have received an additional course of BCG instillation, but the urinary cytology did not normalize. Mean recurrence–free time was 19.6 months. Of the other 7 cases who responded to the first course of instillation, 6 cases were alive with no evidence of the disease. The remaining patient died of rectal cancer with no evidence of transitional cell carcinoma (TCC). Of the 2 cases who showed positive urinary cytology even after the first course, 1 underwent nephroureterectomy. The other case was diagnosed as having malignant lymphoma 3 months after the end of this instillation therapy, and he died of malignant lymphoma. As side effects, 8 cases (72.7%) showed bladder irritability, and 4 presented fever higher than 38°C. However, no patient needed antitubercular treatment.Conclusion: As for the short–term response, BCG instillation for the treatment of upper urinary tract CIS is considered to be effective and safe. Longer follow–up and further experience with this treatment are required.

Clinical Study of Brain Metastasis of Renal Cell Carcinoma

Objectives: To evaluate the natural history and the efficacy of treatments for renal cell carcinoma (RCC) with brain metastasis, we reviewed 18 patients with this disease. Methods: Out of 325 cases with RCC treated at Osaka University Hospital from 1957 to 1993, 18 (5.5%, male:female ratio 16:2) cases developed brain metastases. Median follow-up was 44 months after the initial treatment of the primary lesion. Twelve patients had surgical resection of brain metastases (surgical group), and 7 of them received adjuvant radiotherapy. Six patients with poor performance status were treated with supportive therapy alone (nonsurgical group). Results: Of 18 RCC patients with brain metastasis, 16 were male and 2 female. All brain metastases except for 1 case were symptomatic. Median interval between the initial treatment of the primary lesion and the diagnosis of brain metastasis was 19 months. The most frequent metastatic site prior to brain was the lung, which was detected in 7 cases (38.9%). Median survival of the entire group, measured from the onset of brain metastasis, was 9.5 months. One-year survival rate after the diagnosis of brain metastasis was 43.2% (64.8% in surgical group, 0% in nonsurgical group), 3-year 18.5% and 5-year 0%. Among 109 metastatic RCC, 14 patients were treated by lymphokine-activated killer (LAK) therapy. Out of 14 metastatic RCC patients treated by LAK therapy, 3 (21.4%) developed brain metastases. On the other hand, out of 95 metastatic RCC patients without LAK therapy, 15 (15.8%) had brain metastases. There was no significant difference in the rate of brain metastases between these two groups. Conclusion: There was a trend for prognosis of the surgical group to be better compared to that of the nonsurgical group, although it is not statistically significant. The optimum treatment for brain metastasis of RCC remains undefined, but our data suggested surgical resection in selected patients might contribute to prolonged survival of patients with brain metastasis. LAK therapy was not necessarily the risk factor of the brain metastasis.

Possible correlation between polymorphism in the tumor necrosis factor-beta gene and the clinicopathological features of bladder cancer in Japanese patients

Objectives:  In a variety of cancers, several polymorphisms of the tumor necrosis factor (TNF) genes have been reported to result in different clinical outcomes. We investigated whether a polymorphism of the TNF gene is associated with a susceptibility to bladder cancer and its disease status.