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Bladder cancer is a major and fatal urological disease. Cisplatin is a key drug for the treatment of bladder cancer, especially in muscle-invasive cases. In most cases of bladder cancer, cisplatin is effective; however, resistance to cisplatin has a significant negative impact on prognosis. Thus, a treatment strategy for cisplatin-resistant bladder cancer is essential to improve the prognosis. In this study, we established a cisplatin-resistant (CR) bladder cancer cell line using a urothelial carcinoma cell line (UM-UC-3 cells). We screened for potential targets in CR cells and found that claspin (CLSPN) was overexpressed. CLSPNmRNA knockdown revealed that CLSPN had a role in cisplatin resistance in CR cells. In our previous study, we identified human leukocyte antigen (HLA)-A*02:01-restricted CLSPN peptide by HLA ligandome analysis. Thus, we generated a CLSPN peptide-specific cytotoxic T lymphocyte (CTL) clone that recognized CR cells at a higher level than wild-type UM-UC-3 cells. These findings indicate that CLSPN is a driver of cisplatin resistance and CLSPN peptide-specific immunotherapy may be effective for cisplatin-resistant cases.

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.

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.

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.

Panobinostat, a histone deacetylase inhibitor, induces histone acetylation and acts against cancer but attenuates its anticancer activity by activating the mammalian target of rapamycin (mTOR) pathway. AMP-activated protein kinase (AMPK) is a cellular energy sensor that reportedly inhibits the mTOR pathway. The antidiabetic drug metformin is also a potent AMPK activator and we investigated whether it augmented panobinostat's antineoplastic activity in bladder cancer cells (UMUC3, J82, T24 and MBT-2). Metformin enhanced panobinostat-induced apoptosis and the combination inhibited the growth of bladder cancer cells cooperatively in vitro and in vivo. As expected, metformin increased the phosphorylation of AMPK and decreased the panobinostat-caused phosphorylation of S6 ribosomal protein, thus inhibiting the panobinostat-activated mTOR pathway. The AMPK activation was shown to play a pivotal role in the combination's action because the AMPK inhibitor compound C attenuated the combination's anticancer activity. Furthermore, the AMPK activation by metformin enhanced panobinostat-induced histone and non-histone acetylation. This acetylation was especially remarkable in the proteins in the detergent-insoluble fraction, which would be expected if the combination also induced endoplasmic reticulum stress.

Checkpoint kinases (CHKs) are involved in the DNA damage response in many cancer cells. CHK inhibitors have been used in clinical trials in combination with chemotherapeutics; however, their effect against bladder cancer remains unclear. Here, we investigated the efficacy of combining gemcitabine with MK-8776, a novel CHK1 inhibitor, in four bladder cancer cell lines. The effects of gemcitabine and MK-8776 on cell viability, clonogenicity, cell cycle, and apoptosis were examined alongside in vivo efficacy using murine xenograft tumor models. Combined treatment inhibited the viability and colony formation of bladder cancer cells compared to either single treatment. Although gemcitabine (10 nM) alone increased the cell number in S-phase, it increased the cell number in sub-G1 phase when combined with MK-8776 (0.5 µM). Combined treatment enhanced cleaved poly[ADP-ribose]-polymerase expression alongside the number of annexin-V-positive cells, indicating the induction of apoptosis. In vivo, administration of gemcitabine and MK-8776 was well tolerated and suppressed tumor growth. Mechanistically, the combined treatment elevated γH2A.X and suppressed Rad51 expression. Our study demonstrates that MK-8776 and gemcitabine combined induces apoptosis and suppresses proliferation in bladder cancer cells by inhibiting CHKs and DNA repair. Therefore, CHK1 inhibition combined with gemcitabine may be a potential treatment for bladder cancer.

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.

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.

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...

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.

There is no established treatment for advanced rhabdomyosarcoma (RMS) with metastases at the time of diagnosis. A 17-year-old male was referred to our hospital because of a right scrotal mass. Computed tomography showed multiple lung metastases with pleural effusion and retroperitoneal lymph node metastasis, and bone scintigraphy revealed multiple bone metastases. Right high orchiectomy was performed and the tumor was diagnosed as paratesticular embryonal RMS. He was treated with a multimodal therapy including 17 cycles of combination chemotherapy consisting of cyclophosphamide, vincristine, doxorubicin and dacarbazine (CYVADIC) and achieved a long-term survival of 4 years.

_L-Carnitine is an essential component of mitochondrial fatty acid b-oxidation in the muscle and may control the acetyl moiety levels in the brain for acetylcholine synthesis. Carnitine transporter 1(CT1)is the high affinity _L-carnitine transporter whose localization was observed in the kidney, testis, liver, skeletal muscle and brain. To clarify the molecular mechanism of carnitine transport, we sought to find the interacting protein that may be related to the transport function of CT1. Using the intracellular C-terminal region of rat CT1 containing PDZ(PSD95/DLG/ZO-1)motif as bait, we performed the yeast two-hybrid screening against rat brain cDNA library. Thirty two positive clones were obtained from the 2.7×10^7 clones screened. One of them was PDZ domain-containing protein Lin-7. We found that Lin-7 interacts specifically with C-termini of CT1:deletion and mutation of the CT1 C-terminal PDZ-motif abolished the interaction with Lin-7 in the yeast two-hybrid assay. In addition, a PDZ domain within Lin-7 associates with the CT1 C-terminal. The association of CT1 with Lin-7 enhanced _L-carnitine transport activities in HEK293 cells although there is no statistical significance. Coexpression of Lin-7 and CT1 is identified in motor neurons of the spinal cord ventral horn together with Lin-2, a binding partner of Lin-7 known to assemble proteins involved in synaptic vesicle exocytosis and synaptic junctions. Therefore, Lin-7 interacts with CT1 and may regulate their subcellular distribution or function in central nervous system.

Primary lymphoma of the bladder is extremely rare. It represents less than 0.2% of all extranodal primary lymphomas and is usually extranodal marginal-zone B cell lymphoma of mucosa-associated lymphoid tissue (MALT).1 We herein report a case of primary MALT lymphoma of the bladder in a 77-year-old female patient who was successfully treated with radiotherapy. This lymphoma was not diagnosed earlier because it coincided with chronic cystitis. Case presentation A 77-year-old female had suffered from pollakisuria, voiding pain, and gross hematuria and had presented to several hospitals. As a result, she had been diagnosed with chronic cystitis and treated for 2 years with antibiotics and anticholinergics. She was referred to our hospital because her symptoms had not improved. At the initial presentation, the patient still had pyuria and urine culture was positive for levofloxacin-resistant Escherichia coli. She was given cefcapene, but pyuria and gross hematuria persisted. Cystoscopy revealed reddish edematous mucosa on the posterior wall of the bladder (Fig. 1A) and pelvic MRI showed mass lesions on the anterior and posterior walls (Fig. 1B). Cytological examination was negative for urothelial cancer. Open in a separate window Fig. 1 Cystoscopy and MRI at presentation. Cystoscopy showed reddish edematous mucosa on the posterior wall of the bladder (A). MRI showed mass lesions on the anterior and posterior walls (B, arrows). T2-weighted image.

A 74-year-old male patient with stage D1 prostate cancer with the initial prostate-specific antigen (PSA) level of 5570 ng/mL had received androgen deprivation therapy and the serum PSA level had decreased to 0.23 ng/mL when he developed macroscopic hematuria. MRI and cystoscopy suggested invasive urothelial cancer of the bladder, and transurethral resection was performed. The tumors were pathologically diagnosed as a Gleason score 9 prostate cancer with no PSA expression. Prostate cancer patients who develop novel symptoms should be screened for prostate cancer recurrence even if they have very low PSA levels.

Small cell carcinoma (SCC) of the urinary bladder is highly aggressive and portends a poor outcome. Herein, we report a patient with recurrent SCC of the urinary bladder who experienced an unusually long-term disease-free duration after radical cystectomy. The patient was a 60-year-old woman who had undergone transurethral resection followed by radical cystectomy for muscle-invasive bladder cancer (high-grade urothelial carcinoma with adenocarcinomatous differentiation) 6 years prior; the surgical specimen had a negative surgical margin. She was referred to our hospital because of continuous bleeding from her vagina. Magnetic resonance imaging showed a mass located at the anterior wall of her residual vagina, a biopsy of which confirmed a pathological diagnosis of adenocarcinoma. The vaginal tumor and a section of the sigmoid colon were resected en bloc and were pathologically diagnosed as adenocarcinoma and SCC. We reevaluated the initial transurethral resection specimen and found SCC with foci of adenocarcinoma concomitant with high-grade urothelial carcinoma. Local recurrence and metastasis at the pelvic bone occurred 4 months later; although radiation therapy was performed, she died of the progressive disease.

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.

(Objective) We investigated the efficacy and safety of percutaneous renal mass biopsy retrospectively. (Methods) A retrospective review was performed of 44 patients (46 renal masses) who received ultrasound and/or computed tomography guided percutaneous biopsy of a solid renal mass between April 2004 and December 2012 in National Defense Medical College Hospital. (Results) The median renal mass size was 45 (range 15-140) mm with a median of 2 (1-5) cores taken. Thirteen masses were biopsied for differential diagnosis between RCC and other malignancies (or benign renal tumors), 11 were biopsied for differential diagnosis between RCC and renal pelvic urothelial carcinoma, 10 unresectable masses were biopsied to confirm the diagnosis pathologically before starting medication, and 12 small masses were biopsied before radio-frequency ablation. Of the initial 46 biopsies, 38 (82.6%) were diagnostic. The median lesion sizes in the diagnostic and nondiagnostic biopsy specimens were 45 (15-140) mm and 43 (17-128) mm. The median numbers of diagnostic and nondiagnostic cores were 2 (1-5) and 1.5 (1-4). These size and core number differences between the diagnostic and nondiagnostic biopsy specimens are not statistically significant. Of initial nondiagnostic 8 masses, 3 masses that were performed repeat biopsy resulted in determined diagnosis finally. There were mild postprocedural hematomas not requiring blood transfusion. There was no tumor dissemination after renal mass biopsy. (Conclusions) Percutaneous biopsy of renal masses is a safe procedure that provides diagnostic information.

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.

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

A 64-year-old woman who has a history of congestive heart failure and atrial fibrillation was admitted to our hospital with the exacerbation of exertional dyspnea and urinary retention due to severe gross hematuria. Contrast-enhanced computed tomography showed a tumor involving the inferior and middle poles of the right kidney with no nodal involvement, or distant metastases, but that was accompanied by markedly proliferated blood vessels around the inferior vena cava and right renal vein, seemingly a result of an arteriovenous fistula. After embolization of the right renal artery, right radical nephrectomy was performed via a thoracoabdominal incision. The histological diagnosis of the tumor was clear cell renal cell carcinoma, G2 > G3, Fuhrman nuclear grade3, pT2a. Although the presence of an arteriovenous fistula was not confirmed histologically, the severely condensed proliferation of the blood vessels in the renal hilum is consistent with the diagnosis of an arteriovenous fistula accompanying renal cell carcinoma. Immediately after the operation, her symptoms of congestive heart failure, including dyspnea, subsided and her serum BNP levels and CTR value returned to normal levels. Two years after the operation, she shows no signs of recurrence or metastasis. To the best of our knowledge, there have been 25 cases of arteriovenous fistulas accompanied by renal cell carcinoma but only a few in which the symptoms were those of severe congestive heart failure. Clinicians should be aware that renal cell carcinoima could be a cause of heart failure.

Patient: Female, 78 Final Diagnosis: Adrenal pseudocyst Symptoms: None Medication: — Clinical Procedure: Operation Specialty: Urology Objective: Mistake in diagnosis Background: Adrenal pseudocysts are often discovered incidentally on imaging, but the diagnosis and treatment can be challenging. A case of adrenal pseudocyst with hemorrhage is presented that mimicked a solid tumor on imaging, resulting in adrenalectomy. Case Report: A 78-year-old woman was found to have a right adrenal lesion on abdominal imaging. Enhanced computed tomography (CT) showed a heterogeneously enhanced mass, and magnetic resonance imaging (MRI) showed a high-intensity T1-weighted and T2-weighed image, with an irregular enhanced margin. The imaging findings were suggestive of a solid tumor of the adrenal gland. Although full endocrine serological studies were negative, the lesion increased in size at two-year follow-up. Right laparoscopic adrenalectomy was performed, and a benign hemorrhagic adrenal pseudocyst was diagnosed histologically. Conclusions: Adrenal pseudocyst can be associated with acute intracystic hemorrhage, and imaging will show contrast enhancement, suggesting malignancy. In such cases, surgical excision is both diagnostic and curative.

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

Background More effective chemotherapies are urgently needed for bladder cancer, a major cause of morbidity and mortality worldwide. We therefore explored the efficacy of the combination of gemcitabine and AZD7762, a checkpoint kinase 1/2 (CHK1/2) inhibitor, for bladder cancer. Methods Viability, clonogenicity, cell cycle distribution and apoptosis were assessed in urothelial cancer cell lines and various non-malignant urothelial cells treated with gemcitabine and AZD7762. DNA damage was assessed by γH2A.X and 53-BP1 staining and checkpoint activation was followed by Western blotting. Pharmacological inhibition of CHK1 and CHK2 was compared to downregulation of either CHK1 or CHK2 using siRNAs. Results Combined use of gemcitabine and AZD7762 synergistically reduced urothelial carcinoma cell viability and colony formation relative to either single treatment. Non-malignant urothelial cells were substantially less sensitive to this drug combination. Gemcitabine plus AZD7762 inhibited cell cycle progression causing cell accumulation in S-phase. Moreover, the combination induced pronounced levels of apoptosis as indicated by an increase in the fraction of sub-G1 cells, in the levels of cleaved PARP, and in caspase 3/7 activity. Mechanistic investigations showed that AZD7762 treatment inhibited the repair of gemcitabine-induced double strand breaks by interference with CHK1, since siRNA-mediated depletion of CHK1 but not of CHK2 mimicked the effects of AZD7762. Conclusions AZD7762 enhanced sensitivity of urothelial carcinoma cells to gemcitabine by inhibiting DNA repair and disturbing checkpoints. Combining gemcitabine with CHK1 inhibition holds promise for urothelial cancer therapy. Electronic supplementary material The online version of this article (doi:10.1186/s13046-016-0473-1) contains supplementary material, which is available to authorized users.

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.

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

Background Inducing endoplasmic reticulum (ER) stress is a novel strategy used to treat malignancies. Inhibition of histone deacetylase (HDAC) 6 by the HDAC inhibitor panobinostat hinders the refolding of unfolded proteins by increasing the acetylation of heat shock protein 90. We investigated whether combining panobinostat with the proteasome inhibitor bortezomib would kill cancer cells effectively by inhibiting the degradation of these unfolded proteins, thereby causing ubiquitinated proteins to accumulate and induce ER stress. Methods Caki-1, ACHN, and 769-P cells were treated with panobinostat and/or bortezomib. Cell viability, clonogenicity, and induction of apoptosis were evaluated. The in vivo efficacy of the combination was evaluated using a murine subcutaneous xenograft model. The combination-induced ER stress and ubiquitinated protein accumulation were assessed. Results The combination of panobinostat and bortezomib induced apoptosis and inhibited renal cancer growth synergistically (combination indexes

There is currently no curative treatment for advanced renal cancer. Enhancing histone acetylation is a promising epigenetic-based therapy for cancer; however, in solid tumors, the efficacy of histone deacetylase (HDAC) inhibitors alone is limited. The human immunodeficiency virus protease inhibitor ritonavir is also a CYP3A4 inhibitor and we hypothesized that combining ritonavir with the HDAC inhibitor panobinostat, one of the substrates of CYP3A4, may effectively eliminate renal cancer cells by enhancing the activity of panobinostat. The combination of ritonavir and panobinostat synergistically inhibited cancer cell growth and cancer cell colony formation, while only slightly inhibiting the growth of renal proximal tubule epithelial cells. This combination significantly induced apoptosis in cancer cells and this apoptosis was considered to be caspase-dependent, since the pan-caspase inhibitor Z-VAD-FMK reduced the number of Annexin V-positive cells. In murine subcutaneous xenograft models using Caki-1 cells, a 10-day treatment with the combination of ritonavir and panobinostat significantly inhibited tumor growth. Panobinostat alone increased histone acetylation in a dose-dependent manner and the co-administration of ritonavir synergistically enhanced this acetylation. Furthermore, this combination inhibited the expression of HDACs, which may also play a role in the enhancement of histone acetylation. Thus, the present study may provide a basis for testing the combination of ritonavir and panobinostat for patients with advanced renal cancer.

Objective Direct vision internal urethrotomy (DVIU) has been considered to be a low invasive and widely used therapeutic modality for male urethral stricture. However, its efficacy is still controversial. We herein evaluated the efficacy of DVIU for male urethral stricture. Patients and methods Nineteen patients 27 to 78 years old (median age = 59) underwent DVIU for urethral strictures at our hospital were included in this study. Strictures were at bulbar urethra in 17 patients, membranous urethra in 1 patient, and pendulous urethra in 1 patient. The stricture lengths estimated on retrograde urethrography were less than 1 cm in 13 patients, 1-2 cm in 2 patients, and more than 2 cm in 4 patients. The etiology of stricture was straddle injury in 7 patients, post transurethral surgery in 7 patients, pelvic fracture in 1 patient, and unknown in 4 patients. The operation was done by cold knife incision using guidewire. The duration of postoperative urethral catheterization was 5 to 35 days (mean 12.8 days). Follow up duration ranged from 1 month to 139 months (mean 48.2 months). The definition of postoperative re-stricture was the confirmation of re-stricture on retrograde urethrography or deterioration of symptom. Results While no severe complication was observed, postoperative re-stricture was seen in 13 patients. Stricture-free rates at 3 months, 6 months, and 5 years after the first DVIU were 44.4%, 38.1%, 20.3% respectively. Although second DVIU was done for 7 patients with re-stricture, six patients resulted in failure. Stricture-free rates at 3 months, 6 months, and 5 years after the second DVIU were 42.2%, 28.6%, 14.3% respectively. Though the third DVIU was done for two of them, they were unable to void just immediately after the removal of urethral catheters. Stricture-free rate in stricture less than 1 cm was higher than that in 1 cm or longer, though it did not reach significant difference (p = 0.1813). Conclusion The efficacy of DVIU is lesser than we expected. DVIU seems to be excessively applied to male urethral strictures and should not be performed for long and recurrent urethral stricture.

Chitosanases 33 kDa and 40 kDa in size were detected in the culture supernatant of Bacillus circulans WL-12. One of the two chitosanases, chitosanse 40 (40-kDa chitosanase) has been shown to be identical to the enzyme which has been reported previously as a beta-1,3-1,4-glucanase by Bueno et al. The enzyme has been classified into family 8 glycosyl hydrolases together with the enzymes formally known as cellulase family D. This enzyme named chitosanase 40/beta-1,3-1,4-glucanase hydrolyzed both chitosan and beta-1,3-1,4-glucan with similar efficiency. However, the production of the enzyme was induced with chitosan but not by beta-1,3-1,4-glucan. Therefore, it seems possible that the major substrate of this enzyme is chitosan rather than beta-1,3-1,4-glucan. Analysis of degradation products generated from partially N-acetylated chitosan showed that chitosanase 40/beta-1,3-1,4-glucanse hydrolyzes GlcN-GlcN and GlcN-GlcNAc linkages but not GlcNAc-GlcNAc nor GlcNAc-GlcN. The specificity for hydrolyzing linkages of this enzyme is similar to that of the chitosanase from S. griseus HUT6037.