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Background/aim Simultaneous inhibition of histone deacetylase and proteasomes induces endoplasmic reticulum (ER) stress efficiently. RTS-V5 is the first dual histone deacetylase-proteasome inhibitor, and we anticipated that combining it with the cytochrome P450 family 3 subfamily A member 4 inhibitor ritonavir would enhance its activity in bladder cancer cells. Materials and methods Using bladder cancer cells (human T-24, J-82, murine MBT-2), we evaluated the ability and mechanism by which the combination of RTS-V5 and ritonavir induced ER stress and killed cancer cells. Results The combination of RTS-V5 and ritonavir triggered robust apoptosis and inhibited bladder cancer growth effectively in vitro and in vivo. It caused ubiquitinated protein accumulation and induced ER stress synergistically. The combination inhibited the mammalian target of rapamycin pathway by increasing the expression of AMP-activated protein kinase. We also found that the combination caused histone and tubulin hyperacetylation. Conclusion Ritonavir enhances the ability of RTS-V5 to cause ER stress in bladder cancer cells.

Background/aim Activation of the ubiquitin-proteasome system (UPS) has been shown to be associated with drug resistance in cancer. Using bladder cancer cells, we investigated the association between UPS activation and cisplatin resistance and also the efficacy of UPS-targeting drugs. Materials and methods We established cisplatin-resistant bladder cancer cells (J82-cisR, T24-cisR) and examined the activation status of the UPS and the efficacy of MLN7243, oprozomib, ixazomib, and RTS-V5. Results The UPS in cisplatin-resistant bladder cancer cells was activated compared to that in their parental controls. All the UPS-targeting drugs induced apoptosis and inhibited growth more effectively in the cisplatin-resistant bladder cancer cells than they did in the parental controls. Furthermore, these UPS-targeting drugs induced endoplasmic reticulum stress by causing unfolded protein accumulation at lower concentrations in the cisplatin-resistant bladder cancer cells. Conclusion Targeting the UPS could be an effective strategy for treating cisplatin-resistant bladder cancer.

Objective Immediate single instillation of chemotherapy following transurethral resection of bladder tumor (TURBT) is suggested for non-muscle invasive bladder cancer (NMIBC) patients. However, no study has evaluated molecular marker that was involved in intravesical recurrence (IVR) after single instillation of chemotherapy. Therefore, this study aimed to evaluate whether P-glycoprotein, multidrug resistance protein 1 (MRP1), Annexin A2 (ANXA2) or nucleophosmin (NPM) expression predicts IVR after initial TURBT and immediate single intravesical adriamycin instillation. Methods We retrospectively reviewed consecutive 443 patients who underwent TURBT. Of these, 54 patients who underwent initial TURBT and single instillation of adriamycin for NMIBC were included. The expressions of P-glycoprotein, MRP1, ANXA2 and NPM were evaluated immunohistochemically and were divided into 2 groups (low or high) according to the staining intensity and/or proportion of positive cells. IVR was assessed by Kaplan-Meier method. Cox`s multivaritate analyses were performed to identify independent predictors for IVR. Results Nineteen patients (35.1%) had IVR. High P-glycoprotein expression was significantly correlated with multiplicity, pT stage and high grade. High ANXA2 expression was significantly correlated with high grade. MRP1 and NPM were not correlated with any clinicopathological variables. MRP1 expression and ANXA2 expression were significantly correlated with P-glycoprotein expression. Patients with high P-glycoprotein expression had significantly worse IVR-free survival (IVRFS) than those with low P-glycoprotein expression (P =0.015). The difference in IVRFS rates between patients with high ANXA2 expression and those with low ANXA2 expression was nearly significant (P =0.057). Univariate analyses indicated multiplicity, high grade and high P-glycoprotein expression were significant predictors for IVR. Multivariate analysis indicated high grade was an independent predictor for IVR. Conclusions High P-glycoprotein expression was associated with IVR. Further study was needed to determine significance of P-glycoprotein expression in IVR after single intravesical adriamycin instillation.

Drug repositioning is an emerging approach to developing novel cancer treatments. Vorinostat is a histone deacetylase inhibitor approved for cancer treatment, but it could attenuate its anticancer activity by activating the mTOR pathway. The HMG‐CoA reductase inhibitor fluvastatin reportedly activates the mTOR inhibitor AMP‐activated protein kinase (AMPK), and we thought that it would potentiate vorinostat's anticancer activity in renal cancer cells. The combination of vorinostat and fluvastatin induced robust apoptosis and inhibited renal cancer growth effectively both in vitro and in vivo. Vorinostat activated the mTOR pathway, as evidenced by the phosphorylation of ribosomal protein S6, and fluvastatin inhibited this phosphorylation by activating AMPK. Fluvastatin also enhanced vorinostat‐induced histone acetylation. Furthermore, the combination induced endoplasmic reticulum (ER) stress that was accompanied by aggresome formation. We also found that there was a positive feedback cycle among AMPK activation, histone acetylation, and ER stress induction. This is the first study to report the beneficial combined effect of vorinostat and fluvastatin in cancer cells., Fluvastatin potentiates anticancer activity of vorinostat in renal cancer cells via AMP‐activated protein kinase activation in concert with histone acetylation and endoplasmic reticulum stress induction.

BACKGROUND/AIM Induction of endoplasmic reticulum (ER) stress is a novel approach to cancer treatment. This study investigated the ability of the clinically feasible combination of the human immunodeficiency virus protease inhibitors lopinavir and ritonavir to induce ER stress killing urological cancer cells. MATERIALS AND METHODS Renal cancer cells (769-P, 786-O) and bladder cancer cells (UMUC-3, T-24) were used to investigate the ability of the combination to induce ER stress and its mechanism of action. RESULTS The combination inhibited the growth of both renal and bladder cancer cells synergistically by inducing ER stress. The combination-induced ER stress increased the expression of AMP-activated protein kinase and suppressed the mammalian target of rapamycin pathway. It also increased the expression of a tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor and thereby sensitized the cancer cells to TRAIL. CONCLUSION The combination of lopinavir and ritonavir acts against urological cancer cells by inducing ER stress synergistically.

The ataxia telangiectasia and rad3-related-checkpoint kinase 1 (ATR-CHK1) pathway is involved in DNA damage responses in many cancer cells. ATR inhibitors have been used in clinical trials in combination with radiation or chemotherapeutics; however, their effects against bladder cancer remain unclear. Here, the efficacy of combining gemcitabine with the novel ATR inhibitor AZD6738 was investigated in vitro in three bladder cancer cell lines (J82, T24, and UM-UC-3 cells). The effects of gemcitabine and AZD6738 on cell viability, clonogenicity, cell cycle, and apoptosis were examined. The combined use of gemcitabine and AZD6738 inhibited the viability and colony formation of bladder cancer cells compared to either treatment alone. Gemcitabine (5 nM) and AZD6738 (1 μM) inhibited cell cycle progression, causing cell accumulation in the S phase. Moreover, combined treatment enhanced cleaved poly[ADP-ribose]-polymerase expression alongside the number of annexin V-positive cells, indicating apoptosis induction. Mechanistic investigations showed that AZD6738 treatment inhibited the repair of gemcitabine-induced double-strand breaks by interfering with CHK1. Combining AZD6738 with gemcitabine could therefore be useful for bladder cancer therapy.

INTRODUCTION AND OBJECTIVE:Inducing endoplasmic reticulum (ER) stress is a novel strategy used to treat malignancies. The human immunodeficiency virus protease inhibitor ritonavir inhibits molecula...

Background/aim In the present study, the antineoplastic activity and mechanism of action of phenoxodiol, a novel isoflavone analog, was investigated in renal cancer cells. Materials and methods A panel of renal cancer cells (769-P, 786-O, Caki-2) was treated with phenoxodiol in vitro, and the efficacy of treatment was evaluated. Results MTS assay results showed that phenoxodiol decreased renal cancer viability in a dose-dependent manner. In addition, it inhibited colony formation significantly and perturbed the cell cycle. Treatment with phenoxodiol increased the number of annexin-V-positive cells as well as the expression of cleaved poly ADP ribose polymerase, demonstrating that phenoxodiol induced apoptosis in renal cancer cells. Phenoxodiol also inhibited Akt pathway via dephosphorylation of Akt. Conclusion Phenoxodiol inhibited Akt pathway and induced apoptosis of renal cancer cells. The present study provides a theoretical basis for future development of a novel therapy effective against renal cancer.

Docosahexaenoic acid (DHA) has a variety of anti-tumor activities. The present study examined the anti-tumor activity of DHA in renal cancer cells and its underlying mechanisms of action. The effects of DHA on the viability and proliferation of the human renal cancer cell lines Caki-1 and 786-O were examined by an MTS assay and cell counting. In addition, cell cycle distribution and cell apoptosis were analyzed by flow cytometry and Annexin V staining, and modulation of cell mobility and invasiveness was assessed by wound healing and Matrigel invasion assays. Effects of DHA on intracellular signaling pathways were also analyzed by western blotting. It was observed that DHA significantly reduced the viability and proliferation of Caki-1 and 786-O cells (P

There is no curative treatment for advanced bladder cancer. Causing ubiquitinated protein accumulation and endoplasmic reticulum stress is a novel approach to cancer treatment. The HIV protease inhibitor ritonavir has been reported to suppress heat shock protein 90 and increase the amount of unfolded proteins in the cell. If the proteasome functions normally, however, they are rapidly degraded. We postulated that the novel proteasome inhibitor ixazomib combined with ritonavir would kill bladder cancer cells effectively by inhibiting degradation of these unfolded proteins and thereby causing ubiquitinated proteins to accumulate. The combination of ritonavir and ixazomib induced drastic apoptosis and inhibited the growth of bladder cancer cells synergistically. The combination decreased the expression of cyclin D1 and cyclin-dependent kinase 4, and increased the sub-G1 fraction significantly. Mechanistically, the combination caused ubiquitinated protein accumulation and endoplasmic reticulum stress. The combination-induced apoptosis was markedly attenuated by the protein synthesis inhibitor cycloheximide, suggesting that the accumulation of ubiquitinated proteins played an important role in the combination's antineoplastic activity. Furthermore, the combination induced histone acetylation cooperatively and the decreased expression of histone deacetylases was thought to be one mechanism of this histone acetylation. The present study provides a theoretical basis for future development of novel ubiquitinated-protein-accumulation-based therapies effective against bladder cancer.

Background As a member of the Rho small guanosine triphosphatase family, ras-related C3 botulinum toxin substrate 1 (RAC1) interacts with various specific effectors, and p21-activated kinase 1 (PAK1), which has a role in both carcinogenesis and cellular invasion, binds to RAC1, after which activated PAK1 regulates cellular functions. There have been few reports about the simultaneous analysis of RAC1 and its downstream effector PAK1 in upper urinary tract urothelial carcinoma (UTUC). We assessed the expressions of both RAC1 and PAK1 and evaluated their association with clinicopathological parameters. Methods Immunohistochemical studies of RAC1 or PAK1 were performed with specimens from 104 patients with N0M0 UTUC and cancer-free surgical margins. Correlation of the positive expression of RAC1 or PAK1 or both with clinicopathological parameters was evaluated. Results A hazard model showed that the presence of mixed histologic features and moderate or strong positive expression of both RAC1 and PAK1 were independent factors for shortened disease-specific survival time (Ps = 0.041 and 0.016, respectively), and another hazard model revealed that only moderate or strong positive expression of both RAC1 and PAK1 was an independent factor for shortened recurrence-free survival time in the multivariate analysis (P = 0.036). Neither moderate or strong positive expression of RAC1 alone nor moderate or strong positive expression of PAK1 alone was an independent factor for a worse rate of disease-specific or recurrence-free survival in multivariate analysis. Conclusions Patients with N0M0 UTUC, cancer-free surgical margins and moderate or strong positive expression of both RAC1 and PAK1 should be carefully monitored after surgery.

INTRODUCTION AND OBJECTIVES:Inducing endoplasmic reticulum (ER) stress is a novel approach to cancer treatment. The next-generation proteasome inhibitor oprozomib acts against cancer by increasing ...

INTRODUCTION AND OBJECTIVES:The histone deacetylase (HDAC) inhibitor vorinostat acts against malignancies by inducing histone acetylation but attenuates its anticancer activity by activating the ma...

PURPOSE We aimed to evaluate whether increased nucleophosmin expression predicts recurrence and survival in upper tract urothelial carcinoma (UTUC). METHODS Specimens from 101 patients with N0M0 UTUC undergoing radical nephroureterectomy were evaluated. Nucleophosmin expression was determined immunohistochemically and categorized into two groups according to nucleophosmin staining intensity. The association between nucleophosmin expression and various clinicopathological factors including Ki-67 expression was analyzed. Multivariate analyses were performed to identify the independent predictors of extraurothelial recurrence and cancer-specific survival. RESULTS High nucleophosmin expression was significantly correlated with tumor location, pT ≥3, lymphovascular invasion, lymph node metastasis, and high Ki-67 expression. Patients whose tumors demonstrated high nucleophosmin expression had a significantly higher rate of extraurothelial recurrence and a lower survival rate than those with low nucleophosmin expression. Multivariate analysis showed that pT ≥3, lymph node metastasis, high nucleophosmin expression, and high Ki-67 expression were independent predictors of extraurothelial recurrence. When patients were stratified into three groups according to the number of risk factors, the 2-year extraurothelial recurrence-free survival rates were 92.9% in patients with 0 or 1 risk factor, 76.5% in patients with 2 risk factors, and 9.1% in patients with 3 or 4 risk factors. Regarding cancer-specific survival, lymphovascular invasion and high nucleophosmin expression were independent predictors. CONCLUSIONS Increased nucleophosmin expression was a strong predictor of extraurothelial recurrence and cancer-specific survival in patients with N0M0 UTUC undergoing radical nephroureterectomy. Our risk stratification models integrating nucleophosmin expression may provide valuable information on disease recurrence and prognosis.

The histone deacetylase (HDAC) inhibitor belinostat increases the amount of unfolded proteins in cells by promoting the acetylation of heat shock protein 90 (HSP90), thereby disrupting its chaperone function. The human immunodeficiency virus protease inhibitor ritonavir, on the other hand, not only increases unfolded proteins by suppressing HSP90 but also acts as a proteasome inhibitor. We thought that belinostat and ritonavir together would induce endoplasmic reticulum (ER) stress and kill renal cancer cells effectively. The combination of belinostat and ritonavir induced drastic apoptosis and inhibited the growth of renal cancer cells synergistically. Mechanistically, the combination caused ER stress (evidenced by the increased expression of the ER stress markers) and also enhanced histone acetylation by decreasing the expression of HDACs. To our knowledge, this is the first study that showed a beneficial combined effect of belinostat and ritonavir in renal cancer cells, providing a framework for testing the combination in renal cancer patients.

The recent discovery and clinical development of targeted agents have expanded treatment options in metastatic renal cell carcinoma (RCC). However, metastatic RCC remains a lethal disease. Complete response is rare and treatment with targeted agents eventually fails in the majority of the patients. Therefore, there is a need for developing a prognostic tool and a novel therapeutic agent for RCC to improve the follow-up strategy after surgical treatment. Clinical data, including patient characteristics, serum fatty acid profile, clinicopathological parameters and clinical outcome, were obtained from 112 patients with RCC prior to surgical treatment. Preoperative fatty acid levels were grouped according to patient characteristics, such as performance status, body mass index or pathological parameters, and were analyzed using the Mann-Whitney U test. Cancer-specific survival in the high and low docosahexaenoic acid (DHA) level groups were compared using the Kaplan-Meier method. Cox proportional hazards models were applied to determine the independent prognostic factors associated with shortened cancer-specific survival. The serum DHA level in patients with metastasis was significantly lower compared with that in patients without metastasis (P=0.047). Low serum DHA level, presence of metastasis and cachexia were independent predictors of shortened cancer-specific survival in a multivariate Cox proportional hazard model (P=0.033, hazard ratio = 4.43). Patients with a serum DHA level below the median value exhibited significantly shorter cancer-specific survival compared with those with a higher serum DHA level (P=0.008). Thus, according to our results, the preoperative serum DHA level may be able to predict the surgical outcome of RCC. However, this finding requires validation by large-scale prospective studies.

Background/aim There is no curative treatment for patients with advanced renal cancer. We believed that the combination of the histone deacetylase inhibitor panobinostat and the human immunodeficiency virus protease inhibitor nelfinavir would kill renal cancer cells by inducing endoplasmic reticulum (ER) stress. Materials and methods Using renal cancer cells (769-P, 786-O, Caki-2), the ability of this combination to induce ER stress and its mechanism of action were investigated. Results The combination of drugs induced apoptosis and inhibited cancer growth effectively both in vitro and in vivo. Mechanistically, the combination induced ER stress and histone acetylation cooperatively. ER stress induction was shown to play a pivotal role in the anticancer effect of the combination because the protein synthesis inhibitor cycloheximide significantly attenuated combination-induced apoptosis. Nelfinavir was also found to increase the expression of the mammalian target of rapamycin (mTOR) inhibitor AMP-activated protein kinase (AMPK) and inhibited the panobinostat-activated mTOR pathway. Conclusion Panobinostat and nelfinavir inhibit renal cancer growth by inducing ER stress.

There is no curative treatment for advanced renal cancer, and a novel treatment approach is urgently required. Inducing ubiquitinated protein accumulation and endoplasmic reticulum (ER) stress has recently emerged as a new approach in the treatment of malignancies. In the present study, we hypothesized that the histone deacetylase inhibitor belinostat would increase the amount of unfolded proteins in cells by inhibiting heat-shock protein (HSP) 90, and that the proteasome inhibitor bortezomib would inhibit their degradation by inhibiting the proteasome, thus causing ubiquitinated protein accumulation and ER stress synergistically. The combination of bortezomib and belinostat induced significant increases in apoptosis and inhibited renal cancer growth synergistically (combination indexes

Background/aim To investigate the efficacy against renal cancer cells of combining the HIV protease inhibitor ritonavir with the novel proteasome inhibitor delanzomib. Materials and methods Renal cancer cell lines 769-P, 786-O, Caki-2 and Renca were treated with ritonavir and delanzomib in vitro and in vivo, and the efficacy of combination was evaluated. Results The combination of ritonavir and delanzomib synergistically inhibited renal cancer growth and suppressed colony formation. It induced robust apoptosis evidenced by increased cell population in the sub-G1 fraction and increased number of annexin-V-positive cells. A 13-day treatment with the combination was well tolerated in the mouse model and inhibited tumor growth significantly. Mechanistically, the combination synergistically induced endoplasmic reticulum stress and inhibited the mammalian target of rapamycin (mTOR) pathway. Conclusion The effectiveness of combination of ritonavir and delanzomib appears to be due to the induction of ER stress and inhibition of the mTOR pathway.

Signal transducer and activator of transcription 3 (STAT3) regulates the expression of genes mediating cell survival, proliferation and angiogenesis and is aberrantly activated in various types of malignancies, including bladder cancer. We examined whether it could be a novel therapeutic target for bladder cancer using the STAT3 inhibitor WP1066. In T24 and UMUC-3 bladder cancer cells, 5 µM WP1066 prevented the phosphorylation of STAT3 and 2.5 µM WP1066 decreased cell survival and proliferation significantly (P

Objective: The p21-activated kinase serine/threonine kinases have been outlined as the main cytoskeletal remolding regulators. The same holds true for cell proliferation and motility. They additionally have a part in cellular invasion and carcinogenesis, but the effect of p21-activated kinase 1 expression on the progression of upper urinary tract urothelial carcinoma remains unclear. Therefore, we assessed the relation of p21-activated kinase 1 positivity level to clinicopathological features in patients with upper urinary tract urothelial carcinoma. Methods: Immunohistochemical staining was performed using formalin-fixed and paraffin-embedded specimens, which were all from 124 patients with upper urinary tract urothelial carcinoma. The determination of staining level was based on the intensity of the staining along with portion of cells stained. Correlation of p21-activated kinase 1 positivity with clinicopathological parameters, including disease-specific or extravesical-recurrence-free survival, was evaluated. Results: Statistically significant association was observed between moderate or more than moderate p21-activated kinase 1 positivity and higher tumor grade, pathological T stage, lymphovascular invasion, history of adjuvant chemotherapy and extravesical recurrence. Positivity for p21-activated kinase 1 had a significant association with shortened disease-specific survival in a multivariate analysis among clinicopathological parameters. Strongly positive p21-activated kinase 1 expression was also one of the independent factors for shortened extravesical-recurrence-free survival time in N0M0 upper urinary tract urothelial carcinoma patients in another multivariate analysis as well as histology and lymphovascular invasion (P= 0.0304, hazard ratio = 4.425). Conclusions: We conclude that our findings can help us continue a careful follow-up for upper urinary tract urothelial carcinoma patients with high p21-activated kinase 1 expression in surgical specimens.

In patients undergoing chronic hemodialysis (HD), erythropoietin (EPO) production from the kidney generally decreases and renal anemia develops. Patients without anemia, but with high serum EPO (sEPO) levels are rare among HD patients. The current study presents the case of a 67-year-old female HD patient with autosomal dominant polycystic kidney disease (ADPKD) and renal cell carcinoma (RCC), manifesting polycythemia with elevated sEPO levels. A radical nephrectomy was performed, which diminished the polycythemia, but the sEPO levels remained high. To determine the origin of the EPO production, immunohistochemistry was performed to detect EPO in the RCC and the renal cysts of the surgically resected kidney. In addition, the sEPO and EPO levels in a renal cyst were determined by enzyme immunoassay. EPO expression was demonstrated in RCC and cyst epithelial cells using immunohistochemistry, revealing extremely high EPO levels in the cyst fluid. Due to the remission of polycythemia following the nephrectomy, EPO production from the resected kidney appeared to have been the cause of the polycythemia. Positive EPO staining of the renal cysts in the resected polycystic kidney and sustained sEPO elevation following nephrectomy led to the hypothesis of EPO production in the renal cysts of the contralateral polycystic kidney. Although the postoperative EPO level was higher than the normal range, the hematocrit (Hct) level gradually decreased and recombinant human EPO was required again three months following the nephrectomy. Eight months after the nephrectomy, the Hct level was 30.2% with the use of rHuEPO. In conclusion, EPO production from RCC and renal cysts in ADPKD appeared to cause polycythemia in the HD patient.

Objective: Fatty acid synthase has been shown to be highly expressed in various types of cancers with increased tumour aggressiveness. In this study we examined the level of fatty acid synthase expression in surgically resected upper urinary tract urothelial carcinoma specimens and evaluated the relations between fatty acid synthase expression and the patients’ pathological features and clinical outcomes. Methods: Sections of paraffin-embedded tumour specimens from 113 patients who underwent surgical treatment for upper urinary tract urothelial carcinoma were immunostained with a polyclonal fatty acid synthase antibody, and a tumour was considered to have high fatty acid synthase expression if .50% of the cancer cells stained with moderate-to-strong intensity. Associations between fatty acid synthase expression and the patients’ pathological parameters and survival were analyzed statistically. Results: During the follow-up time (median: 46.8 months), 61 patients (54.0%) had recurrence and 17 (15.0%) died of upper urinary tract urothelial carcinoma. High fatty acid synthase expression was significantly associated with high tumour grade (P ¼ 0.0273). Patients with high fatty acid synthase expression had significantly worse recurrence-free survival and extravesicalrecurrence-free survival than those with low fatty acid synthase expression (P ¼ 0.0171, P ¼ 0.0228, respectively). In multivariate analysis, high fatty acid synthase expression was an independent predictor of shortened recurrence-free survival (P ¼ 0.0220, hazard ratio (HR) ¼ 1.970). Conclusions: Fatty acid synthase expression in upper urinary tract urothelial carcinoma is an independent predictor for tumour recurrence. Patients with high fatty acid synthase expression in upper urinary tract urothelial carcinoma should be followed carefully and adjuvant therapy for them should be considered.Keywords: fatty acid synthase, upper urinary tract urothelial carcinoma, prognosis

The Na-Cl cotransporter (NCC) in the distal convoluted tubules in kidney is known to be excreted in urine. However, its clinical significance has not been established because of the lack of quantitative data on urinary NCC. We developed highly sensitive enzyme-linked immunosorbent assays (ELISAs) for urinary total NCC (tNCC) and its active form, phosphorylated NCC (pNCC). We first measured the excretion of tNCC and pT55-NCC in urinary exosomes in pseudohypoaldosteronism type II (PHAII) patients since PHAII is caused by NCC activation. Highly increased excretion of tNCC and pNCC was observed in PHAII patients. In contrast, the levels of tNCC and pNCC in the urine of patients with Gitelman's syndrome were not detectable or very low, indicating that both assays could specifically detect the changes in urinary NCC excretion caused by the changes of NCC activity in the kidney. Then, to test whether these assays could be feasible for a more general patient population, we measured tNCC and pNCC in the urine of outpatients with different clinical backgrounds. Although urinary protein levels >30 mg/dl interfered with our ELISA, we could measure urinary pNCC in all patients without proteinuria. Thus we established highly sensitive and quantitative assays for urinary NCC, which could be valuable tools for estimating NCC activity in vivo.

In the present study, the association between clinicopathological parameters and α-actinin-4 (ACTN4) expression in bladder cancer specimens was evaluated, and the functional role of ACTN4 in bladder cancer cells was investigated. Immunohistochemistry using anti-ACTN4 antibody was performed in bladder cancer specimens (53 superficial and 42 muscle-invasive cases) from 95 patients who underwent radical cystectomy (n=46) or transurethral resection (TUR) only (n=49). We divided the levels of ACTN4 expression into 2 groups (low or high) by comparing the staining intensity in each specimen with that of the vascular endothelial cells in the same specimen, and we evaluated the correlations between these levels and pathological parameters, recurrence and prognosis. We also investigated the effects of ACTN4 suppression by siRNA on the invasive ability and proliferation of T24 and KU19-19 cells. High ACTN4 expression was significantly associated with higher tumor grade and higher pT stage. In patients with superficial bladder cancer treated only by TUR, the rate of intravesical recurrence did not differ significantly between patients with high ACTN4 expression and patients with low ACTN4 expression. In patients who had muscle‑invasive tumors and underwent radical cystectomy, high ACTN4 expression was associated with neither recurrence nor poor prognosis. Nonetheless, high ACTN4 expression was shown by a large percentage (81%) of patients with muscle-invasive bladder cancer and by a small percentage (17%) of patients with superficial bladder cancer. Furthermore, the leading edges of the invasive bladder cancer showed increased ACTN4 expression. ACTN4 suppression significantly reduced the number of invading bladder cancer cells but unexpectedly increased the proliferation of bladder cancer cells. ACTN4 suppression increased the phosphorylation of ERKs but not AKT or STAT3, suggesting that the increased proliferation due to ACTN4 suppression was mediated in part by the ERK pathway. ACTN4 expression may suppress the proliferation of bladder cancer cells and may produce conditions which facilitate cancer cell invasion.

INTRODUCTION AND OBJECTIVES: Induction of endoplasmic reticulum (ER) stress is a novel strategy for treating malignancies. The human immunodeficiency virus (HIV) protease inhibitor ritonavir increases the amount of unfolded proteins by suppressing the heat shock protein 90. We thought that combining ritonavir with the proteasome inhibitor delanzomib would kill renal cancer cells effectively by inhibiting the degradation of these unfolded proteins and thereby inducing ER stress. METHODS: Renal cancer cells (769-P, 786-O, Caki-2, Renca) were treated with ritonavir (25-50 mM) and/or delanzomib (25-50 nM). Cell viability and clonogenicity were assessed by MTS assay and colony formation assay. In vivo efficacy was evaluated using murine subcutaneous tumor models. Flow cytometry was used for cell cycle analysis and annexin-V assay. Western blotting was used to evaluate the induction of ER stress (expression of glucose-regulated protein 78, endoplasmic reticulum resident protein 44, and endoplasmic oxidoreductin-1-like protein) and histone acetylation, as well as the expression of Akt, cyclin D1, cyclin-dependent kinase (CDK) 4, sestrin 2, AMPactivated protein kinase (AMPK), and mammalian target of rapamycin (mTOR). RESULTS: The combination of ritonavir and delanzomib inhibited renal cancer growth synergistically (combination indexes

INTRODUCTION AND OBJECTIVES: Induction of endoplasmic reticulum (ER) stress is a novel strategy for treating malignancies. The human immunodeficiency virus (HIV) protease inhibitor ritonavir increases the amount of unfolded proteins by suppressing the heat shock protein 90. We thought that combining ritonavir with the proteasome inhibitor delanzomib would kill renal cancer cells effectively by inhibiting the degradation of these unfolded proteins and thereby inducing ER stress. METHODS: Renal cancer cells (769-P, 786-O, Caki-2, Renca) were treated with ritonavir (25-50 mM) and/or delanzomib (25-50 nM). Cell viability and clonogenicity were assessed by MTS assay and colony formation assay. In vivo efficacy was evaluated using murine subcutaneous tumor models. Flow cytometry was used for cell cycle analysis and annexin-V assay. Western blotting was used to evaluate the induction of ER stress (expression of glucose-regulated protein 78, endoplasmic reticulum resident protein 44, and endoplasmic oxidoreductin-1-like protein) and histone acetylation, as well as the expression of Akt, cyclin D1, cyclin-dependent kinase (CDK) 4, sestrin 2, AMPactivated protein kinase (AMPK), and mammalian target of rapamycin (mTOR). RESULTS: The combination of ritonavir and delanzomib inhibited renal cancer growth synergistically (combination indexes

Introduction: Lymphatic invasion has been associated with biochemical recurrence (BCR), and many patients with postoperative elevation of prostate-specific antigen (PSA) develop distant metastases within several years. We previously found peritumoral lymphatic vessel density (PTLD) in biopsy cores to be an independent predictor of lymphatic invasion in radical prostatectomy specimens, so we speculate that PTLD parameters in biopsy specimens could also be independent predictors of BCR after surgery. Patients and Methods: We obtained positive biopsy cores from 110 patients who underwent radical prostatectomy at our institution. Biopsy cores were immunostained with the D2-40 monoclonal antibody, which specifically and selectively detects lymphatic endothelium. We evaluated differences between the BCR-free survival rates and used univariate and multivariate analyses to detect independent predictors of BCR. Results: The results of a Cox proportional hazards model showed that lymphatic invasion in prostatectomy specimens was one of the independent postoperative prognostic factors for BCR (p = 0.0338). An additional model showed that one PTLD parameter, maximal PTLD, was among the independent preoperative predictors of lower BCR-free survival rates (p = 0.0200). Conclusions: Information about PTLD in prostate biopsy specimens could be helpful for selecting patients as radical prostatectomy candidates, and patients with high PTLD values should be carefully monitored after surgery.

What's known on the subject? and What does the study add? The treatment modality for advanced renal cancer is limited and new treatment approaches are urgently needed. Beneficial effects of bortezomib combined with SAHA have recently been reported. However, there are no previous reports of this combination being tested against renal cancer and its further mechanisms of action should be clarified. This study examined the combined effects of these two clinically feasible drugs and showed that the combination inhibits renal cancer cell proliferation by enhancing both protein ubiquitination and histone acetylation synergistically. OBJECTIVE • To investigate the combined effect of two clinically feasible drugs, the proteasome inhibitor bortezomib and the histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA), on human renal cancer cells in vitro and in vivo. MATERIALS AND METHODS • The effectiveness of the combination of bortezomib (10–20 nm) and SAHA (1–5 µm) on renal cancer cells (Caki-1, ACHN, A-498, 786-O, 769-P) was assessed by MTS assay, colony formation assay, cell cycle analysis, and apoptosis assay. • In vivo efficacy was evaluated using murine subcutaneous (s.c.) tumour models. • Protein ubiquitination, unfolded protein response, histone acetylation, and changes in the expression of HDAC were evaluated by western blotting. RESULTS • The combination of SAHA and bortezomib induced apoptosis and inhibited cancer cell proliferation synergistically (combination indices

Introduction: Glucose-regulated protein 78 (GRP78), a chaperone for newly formed proteins during folding and glycosylation, is associated with resistance to apoptosis in some forms of cancer. We assessed GRP78 expression and its correlation with clinicopathological parameters and survival. Patients and Methods: Immunohistochemistry was performed using formalin-fixed, paraffin-embedded specimens: 128 primary renal cell carcinoma (RCC) specimens (120 conventional and 8 other cell types) and 9 metastatic specimens. GRP78 positivity was determined based on intensity of staining and percentage of cells stained. Correlation of GRP78 positivity with clinicopathological parameters including patients’ survival was evaluated. Results: A statistically significant association was found between GRP78 positivity and higher tumor grade (G3; p = 0.0002), lymphovascular invasion (positive; p Conclusions: There was a significant relationship between GRP78 expression levels and aggressiveness of RCC. Increased expression of GRP78 might be a useful parameter to predict shortened survival in patients with RCC.

In the present study, we tested the hypothesis that the renoprotective effect of an angiotensin receptor blocker depends on the angiotensin II type 1 (AT 1 ) receptor on podocytes. For this purpose, we generated podocyte-specific knockout mice for the AT 1 gene ( Agtr1a ) and crossed with NEP25, in which selective podocyte injury can be induced by immunotoxin, anti-Tac(Fv)-PE38. Four weeks after the addition of anti-Tac(Fv)-PE38, urinary albumin:creatinine ratio was not attenuated in Agtr1a knockout / NEP25 mice (n=18) compared with that in control NEP25 mice (n=13; 8.08±2.41 in knockout versus 4.84±0.73 in control). Both strains of mice showed similar degrees of sclerosis (0.66±0.17 versus 0.82±0.27 on a 0 to 4 scale) and downregulation of nephrin (5.78±0.45 versus 5.65±0.58 on a 0 to 8 scale). In contrast, AT 1 antagonist or an angiotensin I–converting enzyme inhibitor, but not hydralazine, remarkably attenuated proteinuria and sclerosis in NEP25 mice. Moreover, continuous angiotensin II infusion induced microalbuminuria similarly in both Agtr1a knockout and wild-type mice. Thus, angiotensin inhibition can protect podocytes and prevent the development of glomerulosclerosis independent of podocyte AT 1 . Possible mechanisms include inhibitory effects on AT 1 of other cells or through mechanisms independent of AT 1 . Our study further demonstrates that measures that directly affect only nonpodocyte cells can have beneficial effects even when sclerosis is triggered by podocyte-specific injury.