Association of oxidative stress with erectile dysfunction in community-dwelling men and men on dialysis.

OBJECTIVES: To investigate the association between oxidative stress and erectile dysfunction (ED) in community-dwelling men and men on dialysis. METHODS: This cross-sectional study included 398 community-dwelling men and 42 men on dialysis. Oxidative stress was assessed using 8-hydroxy-2'-deoxyguanosine (8-OHdG). Univariable and multivariable logistic regression analyses were performed to evaluate the association between oxidative stress and ED. RESULTS: Spearman's rank correlation test showed no significant correlation between urine 8-OHdG levels and the 5-Item International Index of Erectile Function scores in community-dwelling men (ρ = -0.005, p = 0.917) and between plasma 8-OHdG levels and the Sexual Health Inventory for Men scores in men on dialysis (ρ = 0.166, p = 0.295). In community-dwelling men, univariable and multivariable analyses revealed that urine 8-OHdG level was not significantly associated with ED (odds ratio [OR]: 1.005, 95% confidence interval [CI]: 0.884-1.144, p = 0.934; OR: 0.930, 95% CI: 0.798-1.084, p = 0.353; respectively). In men on dialysis, univariable analyses revealed that plasma 8-OHdG level was not significantly associated with severe ED (OR: 0.967, 95% CI: 0.876-1.066, p = 0.498). CONCLUSIONS: Oxidative stress was not significantly associated with ED prevalence and severity in community-dwelling men and men on dialysis.

Invadopodia are essential in transurothelial invasion during the muscle invasion of bladder cancer cells.

Muscle invasive bladder cancer is an aggressive type of epithelial tumor with a high rate of metastasis. For bladder cancer cells to reach the muscle layer, cells must invade through an urothelial cell monolayer (transurothelial invasion) and basement membrane. However, the process by which transurothelial invasion occurs has not been fully characterized. In this study we developed a novel method to evaluate the transurothelial invasion capacity and investigated its cellular and molecular processes using primary culture cells from bladder cancer patients. The analysis revealed that compared with the prognosis for patients with non­muscle invasive bladder cancer that of patients with muscle invasive bladder cancer was particularly poor due to metastatic recurrence. Cancer cells from patients with muscle invasive bladder cancer exhibited a higher invasive capacity through the urothelial cell monolayer compared with those from non­invasive bladder cancer patients. Furthermore, muscle invasive bladder cancer cells demonstrated a greater ability to form invadopodia, the filamentous actin­based membrane protrusions required for matrix degradation and invasion compared with non­invasive cells. Bladder cancer cell lines were established with reduced invadopodia formation by silencing the expression of cortactin, an essential component of invadopodia. The cortactin knockdown bladder cancer cells with reduced invadopodia formation demonstrated a markedly reduced ability to invade through the urothelial cell monolayer, indicating that invadopodia are essential for transurothelial invasion. The results indicate that invadopodia formation is required for muscle invasion of aggressive bladder cancer cells.

Extravasation during bladder cancer metastasis requires cortactin­mediated invadopodia formation.

Invasive cancer cells form the filamentous actin­based membrane protrusions known as invadopodia. Invadopodia are thought to play a critical role in cancer cell invasion and metastasis due to their ability to degrade the extracellular matrix. The present study assessed whether invadopodia formation is essential in extravasation of circulating bladder cancer cells and lung metastasis. To analyze the importance of invadopodia, bladder cancer cell lines with reduced invadopodia formation were established by silencing the expression of cortactin, an essential component of invadopodia, using cortactin short hairpin RNA. Bladder cancer cells with cortactin knockdown demonstrated a markedly decreased ability to form invadopodia, secrete matrix metalloproteinases and invade the extracellular matrix. In addition, the knockdown cells exhibited a reduced transendothelial invasion capacity and decreased formation of metastatic foci in the lungs. The present study demonstrated that bladder cancer cells with cortactin knockdown have a reduced capacity to extravasate into the lung from the circulation, due to the decreased invasive character of invadopodia. This suggests that invadopodia formation is a critical process for cancer cell extravasation.

In vivo selection of high-metastatic subline of bladder cancer cell and its characterization.

The majority of deaths associated with solid tumors are caused by tumor metastasis. To prevent metastasis, it is vital to understand its detailed process. In hematogenous metastasis of bladder cancer, some cancer cells disseminating into blood circulation extravasate into the lung tissues to form metastases. To study the molecular basis of the lung metastasis of bladder cancer, we employed an in vivo selection system that mimics hematogenous metastasis of bladder cancer on a low-metastatic bladder cancer cell line (KK-47). We have successfully isolated a high-metastatic bladder cancer subline, KK-47HM4, from KK-47 cells. We characterized KK-47HM4 in in vitro experimental systems. No significant difference in growth rate and susceptibility to NK cell attack between KK-47 and KK-47HM4 cells was observed. However, KK-47HM4 exhibited the higher capacities of Matrigel Matrix invasion and transendothelial invasion than KK-47. These results suggest that the extravasation of KK-47HM4 cells was enhanced among the multiple steps of the lung metastasis of bladder cancer. Our cDNA microarray analysis identified 67 genes whose expression was up- or downregulated in KK-47HM4 cells compared with KK-47 cells. This analysis data implied that one possible cause for enhanced extravasation of KK-47HM4 is its higher adhesion to extracellular matrix proteins. KK-47HM4 is the first bladder cancer subline with enhanced extravasation potential using the in vivo selection system. The information provided by our cDNA microarray analysis using KK-47HM4 will be useful for further investigation into the molecular basis of extravasation of cancer cells.

Core2 O-glycan-expressing prostate cancer cells are resistant to NK cell immunity.

Core2 ß-1,6-N-acetylglucosaminyltransferase (C2GnT) forms an N-acetylglucosamine branch in the O-glycans (core2 O-glycans) of cell surface glycoproteins. We previously revealed that the expression of C2GnT is positively correlated with poor prognosis in prostate cancer patients. However, the detailed mechanisms underlying their poor prognosis remain unclear. In the current study, we report that the core2 O-glycans carried by the surface MUC1 glycoproteins of prostate cancer cells play an important role in the evasion of NK cell immunity. In C2GnT­expressing prostate cancer cells, the MUC1 core2 O-glycans are modified with poly-N-acetyllactosamine. MUC1 glycoproteins carrying poly-N-acetyllactosamine attenuated the interaction of the cancer cells with NK cells, resulting in decreased secretion of granzyme B by the NK cells. Poly­N­acetyllactosamine also interfered with the ability of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to access the cancer cell surface. These effects of poly-N-acetyllactosamine on NK cells render C2GnT-expressing prostate cancer cells resistant to NK cell cytotoxicity. By contrast, C2GnT-deficient prostate cancer cells carrying a lower amount of poly-N-acetyllactosamine than the C2GnT-expressing prostate cancer cells were significantly more susceptible to NK cell cytotoxicity. Our results strongly suggest that C2GnT-expressing prostate cancer cells evade NK cell immunity and survive longer in the host blood circulation, thereby resulting in the promotion of prostate cancer metastasis.

Serum N-glycan profiling predicts prognosis in patients undergoing hemodialysis.

BACKGROUND: The aim of this study is to evaluate the usefulness of serum N-glycan profiling for prognosis in hemodialysis patients. METHODS: Serum N-glycan analysis was performed in 100 hemodialysis patients in June 2008 using the glycoblotting method, which allows high-throughput, comprehensive, and quantitative N-glycan analysis. All patients were longitudinally followed up for 5 years. To evaluate the independent predictors for prognosis, patients' background, blood biochemistry, and N-glycans intensity were analyzed using Cox regression multivariate analysis. Selected N-glycans and independent factors were evaluated using the log-rank test with the Kaplan-Meier method to identify the predictive indicators for prognosis. Each patient was categorized according to the number of risk factors to evaluate the predictive potential of the risk criteria for prognosis. RESULTS: In total, 56 N-glycan types were identified in the hemodialysis patients. Cox regression multivariate analysis showed cardiovascular events, body mass index, maximum intima media thickness, and the serum N-glycan intensity of peak number 49 were predictive indicators for overall survival. Risk classification according to the number of independent risk factors revealed significantly poor survival by increasing the number of risk factors. CONCLUSIONS: Serum N-glycan profiling may have a potential to predict prognosis in patients undergoing hemodialysis.

MUC1 carrying core 2 O-glycans functions as a molecular shield against NK cell attack, promoting bladder tumor metastasis.

Core 2 ß-1,6-N-acetylglucosaminyltransferase (C2GnT) forms an N-acetylglucosamine branch in O-glycans (core 2 O-glycans) of cell surface glycoproteins. C2GnT-expressing bladder tumors acquire highly metastatic phenotypes by surviving longer in host blood circulation. However, the detailed mechanisms underlying this increased survival remain unclear. In this study, we report that the expression of C2GnT in bladder tumors positively correlates with tumor progression and that bladder tumor cell-surface mucin 1 (MUC1) carrying core 2 O-glycans plays an important role in the evasion from natural killer (NK) cell attack. In C2GnT-expressing bladder tumor cells, heavily core 2 O-glycosylated MUC1 carries poly-N-acetyllactosamine in its O-glycans and galectin-3 binds to MUC1 through this poly-N-acetyllactosamine. The binding of galectin-3 to poly-N-acetyllactosamine in MUC1 core 2 O-glycans attenuates the interaction of the tumor cells with NK cells and interferes with the access of tumor necrosis factor-related apoptosis-inducing ligand to the tumor cell surface. These effects of MUC1 carrying core 2 O-glycans on NK cell attack facilitate C2GnT-expressing tumor cells to evade NK cell immunity and survive longer in host blood circulation. We reveal that MUC1 carrying core 2 O-glycans thus functions as a molecular shield against NK cell attack, thereby promoting bladder tumor metastasis.

A novel strategy for evasion of NK cell immunity by tumours expressing core2 O-glycans.

The O-glycan branching enzyme, core2 ß-1,6-N-acetylglucosaminyltransferase (C2GnT), forms O-glycans containing an N-acetylglucosamine branch connected to N-acetylgalactosamine (core2 O-glycans) on cell-surface glycoproteins. Here, we report that upregulation of C2GnT is closely correlated with progression of bladder tumours and that C2GnT-expressing bladder tumours use a novel strategy to increase their metastatic potential. Our results showed that C2GnT-expressing bladder tumour cells are highly metastatic due to their high ability to evade NK cell immunity and revealed the molecular mechanism of the immune evasion by C2GnT expression. Engagement of an NK-activating receptor, NKG2D, by its tumour-associated ligand, Major histocompatibility complex class I-related chain A (MICA), is critical to tumour rejection by NK cells. In C2GnT-expressing bladder tumour cells, poly-N-acetyllactosamine was present on core2 O-glycans on MICA, and galectin-3 bound the NKG2D-binding site of MICA through this poly-N-acetyllactosamine. Galectin-3 reduced the affinity of MICA for NKG2D, thereby severely impairing NK cell activation and silencing the NK cells. This new mode of NK cell silencing promotes immune evasion of C2GnT-expressing bladder tumour cells, resulting in tumour metastasis.

Requirement for FBP17 in invadopodia formation by invasive bladder tumor cells.

PURPOSE: Invadopodia (protrusions of the plasma membrane formed by invasive tumor cells) have an essential role in bladder tumor invasion. To understand the process of bladder tumor invasion it is crucial to investigate the molecular mechanisms of invadopodia formation. We found that invasive bladder tumor cells express FBP17. In this study we examined the role of FBP17 in bladder tumor cell invadopodia formation and invasion. MATERIALS AND METHODS: We used the 3 bladder tumor cell lines YTS-1, T24 and RT4 (ATCC®), and primary culture of bladder tumors from patients. Cells were stained with phalloidin for invadopodia formation. FBP17 knockdown cells were tested for invadopodia formation and subjected to invasion assay using a Transwell® cell culture chamber. We also examined the role of the extended FER-CIP4 homology and Src homology 3 domains of FBP17 in invadopodia formation in FBP17 mutant constructs. RESULTS: Invadopodia formation was observed in invasive bladder tumor cells and FBP17 was localized to invadopodia in invasive cells. FBP17 knockdown decreased invadopodia formation in invasive cells to 13% to 14% (p <0.0005) and decreased their invasive capacity to 14% to 16% (p <0.001). The extended FER-CIP4 homology and Src homology 3 domains of FBP17 were necessary for invadopodia formation and invasion. CONCLUSIONS: Invadopodia formation requires membrane deformation activity and recruitment of dynamin-2 mediated by FBP17. FBP17 has a critical role in the process of bladder tumor cell invasion by mediating invadopodia formation.

Invadopodia formation by bladder tumor cells.

A major cause of death in patients with bladder tumors is recurrence with metastasis. Bladder tumor metastasis is largely dependent upon the invasive capacity of tumor cells. Tumor cell invasion is mainly mediated by actin-rich protrusive membrane structures called invadopodia. The formation of invadopodia was observed in various types of invasive tumors such as breast cancer and melanomas. However, invadopodia formation so far has not been described in bladder tumor cells. We here report that human bladder tumor cells form functionally active invadopodia. By using a confocal laser scanning microscope, we demonstrated that invasive bladder tumor cell lines, YTS-1 and T24, with high Matrigel degradation activity form invadopodia but that noninvasive bladder tumor cell lines, RT4 and KK-47, form no detectable invadopodia. Invadopodia formed by YTS-1 cells had the ability to secrete matrix metalloproteases and degrade extracellular matrix to invade surrounding areas. Moreover, we observed that primary tumor cells obtained from patients with invasive bladder tumors also form invadopodia, validating the results from bladder tumor cell lines. Our results provide evidence that invasive human bladder tumor cells form invadopodia for tumor invasion.

FBP17 Mediates a Common Molecular Step in the Formation of Podosomes and Phagocytic Cups in Macrophages.

Macrophages act to protect the body against inflammation and infection by engaging in chemotaxis and phagocytosis. In chemotaxis, macrophages use an actin-based membrane structure, the podosome, to migrate to inflamed tissues. In phagocytosis, macrophages form another type of actin-based membrane structure, the phagocytic cup, to ingest foreign materials such as bacteria. The formation of these membrane structures is severely affected in macrophages from patients with Wiskott-Aldrich syndrome (WAS), an X chromosome-linked immunodeficiency disorder. WAS patients lack WAS protein (WASP), suggesting that WASP is required for the formation of podosomes and phagocytic cups. Here we have demonstrated that formin-binding protein 17 (FBP17) recruits WASP, WASP-interacting protein (WIP), and dynamin-2 to the plasma membrane and that this recruitment is necessary for the formation of podosomes and phagocytic cups. The N-terminal EFC (extended FER-CIP4 homology)/F-BAR (FER-CIP4 homology and Bin-amphiphysin-Rvs) domain of FBP17 was previously shown to have membrane binding and deformation activities. Our results suggest that FBP17 facilitates membrane deformation and actin polymerization to occur simultaneously at the same membrane sites, which mediates a common molecular step in the formation of podosomes and phagocytic cups. These results provide a potential mechanism underlying the recurrent infections in WAS patients.

Protein profiling of post-prostatic massage urine specimens by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry to discriminate between prostate cancer and benign lesions.

Post-prostatic massage urine specimens (PMUS) are expected to be rich in proteins originating from the prostatic acini. In this study, we created a PMUS bank consisting of 57 samples obtained from patients with biopsy-proven prostate cancer (PC) and 56 samples from subjects with biopsy-proven benign lesions to analyze protein profiles of PMUS by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). Strong anion-exchange (Q10), weak cation-exchange (CM10) and immobilized metal affinity capture (IMAC30) ProteinChip Arrays were used for protein profiling. In PC samples, single-marker analysis detected 49 mass peaks that were significantly up-regulated and 23 peaks that were significantly down-regulated, compared with peaks obtained from benign lesion samples. To confirm reproducibility we performed additional three rounds of assay using CM10 chip with pH 4.0 binding buffer. Among these significant peaks, a peak of m/z 10788 was significant throughout all 4 rounds of assays. For hierarchical clustering analysis (HCA), we used the 72 peaks which revealed significant differences in single-marker analysis. The heat map discriminated PC from benign lesions with a sensitivity of 91.7% and a specificity of 83.3%. Therefore, SELDI-TOF MS profiling of PMUS can be applied to differentiate patients with PC from cancer-free subjects. However, further investigation is required to verify the usefulness of this method in clinical practice.

Resistance training and reduction of treatment side effects in prostate cancer patients.

PURPOSE: To examine the effect of progressive resistance training on muscle function, functional performance, balance, body composition, and muscle thickness in men receiving androgen deprivation for prostate cancer. METHODS: Ten men aged 59-82 yr on androgen deprivation for localized prostate cancer undertook progressive resistance training for 20 wk at 6- to 12-repetition maximum (RM) for 12 upper- and lower-body exercises in a university exercise rehabilitation clinic. Outcome measures included muscle strength and muscle endurance for the upper and lower body, functional performance (repeated chair rise, usual and fast 6-m walk, 6-m backwards walk, stair climb, and 400-m walk time), and balance by sensory organization test. Body composition was measured by dual-energy x-ray absorptiometry and muscle thickness at four anatomical sites by B-mode ultrasound. Blood samples were assessed for prostate specific antigen (PSA), testosterone, growth hormone (GH), cortisol, and hemoglobin. RESULTS: Muscle strength (chest press, 40.5%; seated row, 41.9%; leg press, 96.3%; P < 0.001) and muscle endurance (chest press, 114.9%; leg press, 167.1%; P < 0.001) increased significantly after training. Significant improvement (P < 0.05) occurred in the 6-m usual walk (14.1%), 6-m backwards walk (22.3%), chair rise (26.8%), stair climbing (10.4%), 400-m walk (7.4%), and balance (7.8%). Muscle thickness increased (P < 0.05) by 15.7% at the quadriceps site. Whole-body lean mass was preserved with no change in fat mass. There were no significant changes in PSA, testosterone, GH, cortisol, or hemoglobin. CONCLUSIONS: Progressive resistance exercise has beneficial effects on muscle strength, functional performance and balance in older men receiving androgen deprivation for prostate cancer and should be considered to preserve body composition and reduce treatment side effects.

Changes in neutrophil surface receptor expression, degranulation, and respiratory burst activity after moderate- and high-intensity exercise.

Intense exercise stimulates the systemic release of a variety of factors that alter neutrophil surface receptor expression and functional activity. These alterations may influence resistance to infection after intense exercise. The aim of this study was to examine the influence of exercise intensity on neutrophil receptor expression, degranulation (measured by plasma and intracellular myeloperoxidase concentrations), and respiratory burst activity. Ten well-trained male runners ran on a treadmill for 60 min at 60% [moderate-intensity exercise (MI)] and 85% maximal oxygen consumption [high-intensity exercise (HI)]. Blood was drawn immediately before and after exercise and at 1 h postexercise. Immediately after HI, the expression of the neutrophil receptor CD16 was significantly below preexercise values (P < 0.01), whereas MI significantly reduced CD35 expression below preexercise values (P < 0.05). One hour after exercise at both intensities, there was a significant decline in CD11b expression (P < 0.05) and a further decrease in CD16 expression compared with preexercise values (P < 0.01). CD16 expression was lower 1 h after HI than 1 h after MI (P < 0.01). Immediately after HI, intracellular myeloperoxidase concentration was less than preexercise values (P < 0.01), whereas plasma myeloperoxidase concentration was greater (P < 0.01), indicating that HI stimulated neutrophil degranulation. Plasma myeloperoxidase concentration was higher immediately after HI than after MI (P < 0.01). Neutrophil respiratory burst activity increased after HI (P < 0.01). In summary, both MI and HI reduced neutrophil surface receptor expression. Although CD16 expression was reduced to a greater extent after HI, this reduction did not impair neutrophil degranulation and respiratory burst activity.