Satoshi Anai

A Novel Human AlkB Homologue, ALKBH8, Contributes to Human Bladder Cancer Progression

We recently identified a novel human AlkB homologue, ALKBH8, which is expressed in various types of human cancers including human urothelial carcinomas. In examining the role and function of ALKBH8 in human bladder cancer development in vitro, we found that silencing of ALKBH8 through small interfering RNA transfection reduced reactive oxygen species (ROS) production via down-regulation of NAD(P)H oxidase-1 (NOX-1) and induced apoptosis through subsequent activation of c-jun NH(2)-terminal kinase (JNK) and p38. However, we also found that JNK and p38 activation resulted in phosphorylation of H2AX (gammaH2AX), a variant of mammalian histone H2A, which contributes to the apoptosis induced by silencing ALKBH8 and NOX-1. Silencing of ALKBH8 significantly suppressed invasion, angiogenesis, and growth of bladder cancers in vivo as assessed both in the chorioallantoic membrane assay and in an orthotopic mouse model using green fluorescent protein-labeled KU7 human urothelial carcinoma cells. Immunohistochemical examination showed high expression of ALKBH8 and NOX-1 proteins in high-grade, superficially and deeply invasive carcinomas (pT(1) and >pT(2)) as well as in carcinoma in situ, but not in low-grade and noninvasive phenotypes (pT(a)). These findings indicate an essential role for ALKBH8 in urothelial carcinoma cell survival mediated by NOX-1-dependent ROS signals, further suggesting new therapeutic strategies in human bladder cancer by inducing JNK/p38/gammaH2AX-mediated cell death by silencing of ALKBH8.

1-tert-Butyl-3-[6-(3,5-dimethoxy-phenyl)-2-(4-diethylamino-butylamino)-pyrido[2,3-d]pyrimidin-7-yl]-urea (PD173074), a Selective Tyrosine Kinase Inhibitor of Fibroblast Growth Factor Receptor-3 (FGFR3), Inhibits Cell Proliferation of Bladder Cancer Carrying the FGFR3 Gene Mutation along with Up-Regulation of p27/Kip1 and G1/G0 Arrest

Activating mutation of the fibroblast growth factor receptor-3 (FGFR3) gene is known as a key molecular event in both oncogenesis and cell proliferation of low-grade noninvasive human bladder urothelial carcinoma (UC), which is characterized by frequent intravesical recurrence. In this study, we investigated the antitumor potentiality of 1-tert-butyl-3-[6-(3,5-dimethoxy-phenyl)-2-(4-diethylamino-butylamino)-pyrido[2,3-d]pyrimidin-7-yl]-urea (PD173074), a small-molecule FGFR3-selective tyrosine kinase inhibitor (TKI), as a therapeutic modality using eight UC cell lines. In our in vitro cell proliferation assay, PD173074 suppressed cell proliferation remarkably in two cell lines, namely, UM-UC-14 and MGHU3, which expressed mutated FGFR3 protein. In contrast, the other six cell lines expressing wild-type FGFR3 or without FGFR3 expression were resistant to PD173074 treatment. Cell cycle analysis revealed the growth inhibitory effect of PD173074 was associated with arrest at G1-S transition in a dose-depending manner. Furthermore, we observed an inverse relationship between Ki-67 and p27/Kip1 expression after PD173074 treatment, suggesting that up-regulation of p27 recruited UC cells harboring activating FGFR3 mutations in G1 that was analogous with the other receptor TKIs acting on the epidermal growth factor receptors. In the mouse xenograft models using subcutaneously transplanted UM-UC-14 and MGHU3, orally administered PD173074 suppressed tumor growth and induced apoptotic changes comparable with the results of our in vitro assay. These findings elucidated the effectiveness of molecular targeted approach for bladder UC harboring FGFR3 mutations and the potential utility to decrease the intravesical recurrence of nonmuscle invasive bladder UC after transurethral surgical resection.

Heme oxygenase-1 promotes angiogenesis in urothelial carcinoma of the urinary bladder

Angiogenesis is necessary for the growth, invasion, and metastasis of solid tumors. Previous studies have shown that heme oxygenase-1 (HO-1) plays an important role in angiogenesis in both normal and cancerous cells, such as vascular endothelial cells and pancreatic cancer cells, respectively. In this study, we analyzed the role of HO-1 and other angiogenic factors in urothelial carcinoma of the bladder. Specifically, we used real-time reverse transcription polymerase chain reaction (PCR) and Western blotting to investigate the upregulation of 7 angiogenic factors, namely, HO-1, vascular endothelial growth factor (VEGF), hypoxia-inducible factor (HIF)-1α, HIF-2α, cyclooxygenase-2 (COX-2), interleukin-8 (IL-8), and basic fibroblast growth factor (bFGF) under hypoxic conditions in the T24 urothelial carcinoma cell line. We also used enzyme-linked immunosorbent assay (ELISA) to measure the amount of VEGF secreted into the growth media. In addition, we administered an HO-1 inhibitor, zinc protoporphyrin IX, to mice with subcutaneous T24 tumors to assess the modulation of angiogenesis in solid tumors in vivo. We also performed immunohistochemical analyses of 23 primary bladder cancer specimens with high-grade tumors infiltrating into the stroma (pT1) for expression of HO-1, VEGF, HIF-1α, HIF-2α, COX-2, and CD31. Image analysis of CD31 staining was performed to estimate microvessel density (MVD), a measure of angiogenesis. Hypoxic conditions induced upregulation of HO-1, VEGF, HIF-1α, HIF-2α, and COX-2 in T24 cells and increased VEGF secretion, which could be suppressed by zinc protoporphyrin IX. In vivo, inhibition of HO-1 decreased tumor growth and MVD by suppressing angiogenic factors, particularly VEGF and HIF-1α. In clinical specimens of bladder cancer, high expression of HO-1 was correlated with high expression of HIF-1α (P=0.027) and high MVD (P=0.005), but not with VEGF expression (P=0.19). In conclusion, since overexpression of HO-1 promotes angiogenesis in urothelial carcinoma cells, HO-1 inhibitors could be used as novel therapeutics for urothelial carcinoma of the urinary bladder.

p37 Induces tumor invasiveness.

Previous studies have shown a statistically significant correlation between human carcinomas and monoclonal antibody detection of a Mycoplasma hyorhinis–encoded protein known as p37. A potential mechanism of p37 is that it might promote invasion and metastasis. Recombinant p37 enhanced the invasiveness of two prostate carcinoma and two melanoma cell lines in a dose-dependent manner in vitro, but did not have a significant effect on tumor cell growth. Furthermore, the increased binding to cell surfaces and the enhanced invasive potential of cancer cells from exposure to p37 could be completely reversed by preincubation of the cancer cells with an anti-p37 monoclonal antibody. Sequence comparisons, followed by three-dimensional molecular modeling, revealed a region of similarity between p37 and influenza hemagglutinin A, a sialic acid–binding protein that plays a critical role in viral entry. Binding of p37 to prostate carcinoma cells was found to be at least partially sialic acid dependent because neuraminidase treatment decreased this binding. Taken together, these observations suggest that M. hyorhinis can infect humans and may facilitate tumor invasiveness via p37. These results further suggest that p37 may be a molecular target for cancer therapy.

Prognostic significance of CD45RO+ memory T cells in renal cell carcinoma

Background:Memory T cells are well known to have a critical role for host defense in humans. However, their role in actual human cancer remains largely unknown. In this study, we tried to reveal the clinical importance of tumour-infiltrating CD45RO+ memory T cells in renal cell carcinoma (RCC).Methods:We analysed 105 patients with RCC, who received radical or partial nephrectomy. Those were 65 in TNM stage I, 7 in stage II, 15 in stage III, and 18 in stage IV, respectively. CD45RO expression was evaluated by immunohistochemistry. CD4 and CD8 expressions were also systematically assessed in the same manner.Results:Patients with higher TNM stage or high nuclear grade were found to have higher densities of CD45RO. Furthermore, CD45RO status was positively correlated with preoperative C-reactive protein level. In prognostic analysis, CD45RO+lo patients had a significantly better prognosis than CD45RO+hi patients. There was also a significant difference between CD4+lo and CD4+hi groups, whereas no significant difference was observed in CD8 T-cell status. Finally, multivariate analysis revealed that CD45RO+ status was the independent prognostic factor for patient overall survival.Conclusion:CD45RO+ memory T-cell status has a significant independent prognostic value, indicating that the adaptive immune response is functionally critical in human RCC.

ROS generation via NOX4 and its utility in the cytological diagnosis of urothelial carcinoma of the urinary bladder

BackgroundReactive oxygen species (ROS) production via NADPH oxidase (NOX) contributes to various types of cancer progression. In the present research, we examined the pathobiological role of NADPH oxidase (NOX)4-mediated generation of reactive oxygen species (ROS) in urothelial carcinoma (UC) of the urinary bladder, and demonstrated the utility of ROS labeling in urine cytology.MethodsNOX4 gene was silenced in vivo and in vitro by NOX4 siRNA transfection with or without atlocollagen. Cell cycle and measurement of ROS were analyzed by flowcytometry. Orthotopic implantation animal model was used in vivo experiment. NOX4 expression in urothelial carcinoma cells was observed by immunohistochemical analysis using surgical specimens of human bladder cancer. Urine cytology was performed after treatment with ROS detection reagents in addition to Papanicolaou staining.ResultsNOX4 was overexpressed in several UC cell lines and the NOX inhibitor, diphenylene iodonium reduced intracellular ROS and induced p16-dependent cell cycle arrest at the G1 phase. Moreover, silencing of NOX4 by siRNA significantly reduced cancer cell growth in vivo as assessed in an orthotopic mouse model. Immunohistochemistry demonstrated high expression of NOX4 in low grade/non-invasive and high grade/invasive UC including precancerous lesions such as dysplasia but not in normal urothelium. Then, we assessed the usefulness of cytological analysis of ROS producing cells in urine (ROS-C). Urine samples obtained from UC cases and normal controls were treated with fluorescent reagents labeling the hydrogen peroxide/superoxide anion and cytological atypia of ROS positive cells were analyzed. As a result, the sensitivity for detection of low grade, non-invasive UC was greatly increased (35% in conventional cytology (C-C) vs. 75% in ROS-C), and the specificity was 95%. Through ROS-C, we observed robust improvement in the accuracy of follow-up urine cytology for cases with previously diagnosed UC, especially in those with low grade/non-invasive cancer recurrence (0% in C-C vs. 64% in ROS-C).ConclusionsThis is the first report demonstrating that ROS generation through NOX4 contributes to an early step of urothelial carcinogenesis and cancer cell survival. In addition, cytology using ROS labeling could be a useful diagnostic tool in human bladder cancer.

Inhibition of COX-2 expression by topical diclofenac enhanced radiation sensitivity via enhancement of TRAIL in human prostate adenocarcinoma xenograft model

BackgroundCOX-2 inhibitors have an antitumor potential and have been verified by many researchers. Treatment of cancer cells with external stressors such as irradiation can stimulate the over-expression of COX-2 and possibly confer radiation resistance. In this study, we tested if topical diclofenac, which inhibits both COX-1 and COX-2, administration rendered prostate tumor cells sensitize to the effects of radiation.MethodsLNCaP-COX-2 and LNCaP-Neo cells were treated with 0 to 1000 μM diclofenac. Next, a clonogenic assay was performed in which cells were subjected to irradiation (0 to 4 Gy) with or without diclofenac. COX-2 expression and other relevant molecules were measured by real-time PCR and immunohistochemistry after irradiation and diclofenac treatment. In addition, we assessed the tumor volumes of xenograft LNCaP-COX-2 cells treated with topical diclofenac with or without radiation therapy (RT).ResultsLNCaP-COX-2 and LNCaP-Neo cell lines experienced cytotoxic effects of diclofenac in a dose related manner. Clonogenic assays demonstrated that LNCaP-COX-2 cells were significantly more resistant to RT than LNCaP-Neo cells. Furthermore, the addition of diclofenac sensitized LNCaP-COX-2 not but LNCaP-Neo cells to the cytocidal effects of radiation. In LNCaP-COX-2 cells, diclofenac enhanced radiation-induced apoptosis compared with RT alone. This phenomenon might be attributed to enhancement of RT-induced TRAIL expression as demonstrated by real-time PCR analysis. Lastly, tumor volumes of LNCaP-COX-2 cells xenograft treated with diclofenac or RT alone was >4-fold higher than in mice treated with combined diclofenac and radiation (p<0.05).ConclusionsThese in vitro and in vivo findings suggest that conventional COX inhibitor, diclofenac enhances the effect of RT on prostate cancer cells that express COX-2. Thus, diclofenac may have potential as radiosensitizer for treatment of prostate cancer.

Delivery of PTEN via a novel gene microcapsule sensitizes prostate cancer cells to irradiation

The tumor suppressor gene MMAC/PTEN located on chromosome10q23.3 has dual phosphatase activity in the phosphoinositide-3-kinase signaling pathway and inhibits Akt activation, a serine-threonine kinase, which is involved in proliferative and antiapoptotic pathways. Furthermore, MMAC/PTEN is frequently inactivated in a variety of tumors including prostate cancer. In this study, we generated a new type of gene transfer drug, GelaTen, which is a microsphere of cationized gelatin hydrogels incorporating PTEN plasmid DNA. Using our previously reported radiation-resistant PC3-Bcl-2 human prostate cancer cells (PTEN deleted), we examined the efficacy of GelaTen to force the expression of PTEN in vivo to inhibit tumor growth after intratumoral injection alone or with irradiation. Combinational therapy with GelaTen and irradiation improved both the in vitro and in vivo efficacy of growth inhibition compared with GelaTen or irradiation alone. These data show that GelaTen gene therapy, enabling radiosensitization, can potentially treat prostate cancers that have MMAC/PTEN gene alterations associated with radioresistance. [Mol Cancer Ther 2008;7(7):1864–70]

Clinical Significance of Heme Oxygenase-1 Expression in Non-Muscle-Invasive Bladder Cancer

Introduction: In several malignant diseases, elevated heme oxygenase-1 (HO-1) is associated with progression or resistance to chemotherapy. We evaluated the clinical significance of HO-1 expression in non-muscle-invasive bladder cancer. Patients and Methods: We examined 109 patients with non-muscle-invasive bladder cancer. The immunoexpression of HO-1, p53, and Ki-67 was analyzed using paraffin-embedded tissue from transurethral resection in comparison with the clinicopathological variables. Results: Positive expression of HO-1 was found in 66 of 109 tumors (61%), and the positivity of HO-1 correlated significantly with high tumor grade and the altered expression patterns of p53 and Ki-67. In our analysis of 16 cases treated by intravesical administration of anthracyclines, the positive expression of HO-1 correlated with poor disease-free survival (p = 0.015). In in vitro experiments using urothelial cancer cell lines, HO-1 upregulation was observed by exposure to doxorubicin. Moreover, siRNA-mediated suppression of HO-1 upregulation sensitized the urothelial cancer cells to doxorubicin. Conclusions: Our findings suggested that resistance against anthracyclines correlated with HO-1 and expression analysis of HO-1 may be a useful predictive marker for intravesical administration of anthracyclines.

Syndecan‐1 (CD138) contributes to prostate cancer progression by stabilizing tumour‐initiating cells

Increasing evidence suggests that tumour‐initiating cells (TICs) contribute to the development of prostate cancer. Here, we identified syndecan‐1 as a key molecule maintaining the stability of prostate cancer TICs. Holoclones harbouring the biological properties of stemness were derived from single‐cell cultures of the PC3 human prostate cancer cell line. These holoclones over‐expressed syndecan‐1, but showed reduced expression of NADPH oxidase (NOX) and synthesis of hydrogen peroxide and oxygen radicals. Stable RNA‐mediated silencing of syndecan‐1 gene expression up‐regulated NOX‐dependent generation of reactive oxygen species and reduced the survival of holoclones in vitro. Syndecan‐1 down‐regulation also strongly reduced the number of CD133+/CD44+ primitive cancer cells and tumour growth in vivo. Interestingly, syndecan‐1 gene knockdown significantly enhanced the tumour‐suppressive effects of docetaxel by inhibiting the docetaxel‐induced increase in CD133+/CD44+ cells in vivo. In the transgenic adenocarcinoma of the mouse prostate (TRAMP) mouse model of prostate cancer, early intervention with a syndecan‐1 inhibitor (OGT2115) or syndecan‐1 RNAi reduced the incidence of adenocarcinoma and the number of c‐kit+/CD44+ cells in cancer foci. Finally, we found that syndecan‐1 immunopositivity in prostate cancer cells was significantly associated with biochemical recurrence after radical prostatectomy. Taken together, our results show that syndecan‐1 contributes to prostatic carcinogenesis by maintaining TICs and may be a target molecule for therapy. Copyright