Fluvastatin potentiates anticancer activity of vorinostat in renal cancer cells.

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.

Effect of increased expression of both ras-related C3 botulinum toxin substrate 1 and p21-activated kinase 1 in patients with N0M0 upper urinary tract urothelial carcinoma and cancer-free surgical margins.

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.

Ritonavir and ixazomib kill bladder cancer cells by causing ubiquitinated protein accumulation.

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.

Increased nucleophosmin expression is a strong predictor of recurrence and prognosis in patients with N0M0 upper tract urothelial carcinoma undergoing radical nephroureterectomy.

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.

Clinical significance of p21-activated kinase 1 expression level in patients with upper urinary tract urothelial carcinoma.

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.

Cycloheximide inhibits starvation-induced autophagy through mTORC1 activation.

Protein synthesis inhibitors such as cycloheximide (CHX) are known to suppress protein degradation including autophagy. The fact that CHX inhibits autophagy has been generally interpreted to indicate that newly synthesized protein is indispensable for autophagy. However, CHX is also known to increase the intracellular level of amino acids and activate mTORC1 activity, a master negative regulator of autophagy. Accordingly, CHX can affect autophagic activity through inhibition of de novo protein synthesis and/or modulation of mTORC1 signaling. In this study, we investigated the effects of CHX on autophagy using specific autophagy markers. We found that CHX inhibited starvation-induced autophagy but not Torin1-induced autophagy. CHX also suppressed starvation-induced puncta formation of GFP-ULK1, an early-step marker of the autophagic process which is regulated by mTORC1. CHX activated mTORC1 even under autophagy-inducible starvation conditions. Finally, the inhibitory effect of CHX on starvation-induced autophagy was cancelled by the mTOR inhibitor Torin1. These results suggest that CHX inhibits starvation-induced autophagy through mTORC1 activation and also that autophagy does not require new protein synthesis at least in the acute phase of starvation.

Prognostic impact of fatty acid synthase expression in upper urinary tract urothelial carcinoma.

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 = 0.0273). Patients with high fatty acid synthase expression had significantly worse recurrence-free survival and extravesical-recurrence-free survival than those with low fatty acid synthase expression (P = 0.0171, P = 0.0228, respectively). In multivariate analysis, high fatty acid synthase expression was an independent predictor of shortened recurrence-free survival (P = 0.0220, hazard ratio (HR) = 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.

Panobinostat synergizes with bortezomib to induce endoplasmic reticulum stress and ubiquitinated protein accumulation in renal cancer cells.

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 <1). It also suppressed colony formation significantly (p <0.05). In a murine subcutaneous tumor model, a 10-day treatment was well tolerated and inhibited tumor growth significantly (p <0.05). Enhanced acetylation of the HDAC6 substrate alpha-tubulin was consistent with the suppression of HDAC6 activity by panobinostat, and the combination was shown to induce ER stress and ubiquitinated protein accumulation synergistically. CONCLUSIONS: Panobinostat inhibits renal cancer growth by synergizing with bortezomib to induce ER stress and ubiquitinated protein accumulation. The current study provides a basis for testing the combination in patients with advanced renal cancer.

Development of enzyme-linked immunosorbent assays for urinary thiazide-sensitive Na-Cl cotransporter measurement.

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.

Vorinostat and bortezomib synergistically cause ubiquitinated protein accumulation in prostate cancer cells.

PURPOSE: Protein ubiquitination is a novel strategy used to treat malignancies. We investigated whether the histone deacetylase inhibitor vorinostat (Cayman Chemical, Ann Arbor, Michigan) and the proteasome inhibitor bortezomib (LC Laboratories, Woburn, Massachusetts) would synergistically cause the accumulation of ubiquitinated proteins in prostate cancer cells. MATERIALS AND METHODS: LNCaP, PC-3 and DU 145 cells (ATCC™) were treated with vorinostat and/or bortezomib. Cell viability and induction of apoptosis were assessed. In vivo efficacy was evaluated in a murine subcutaneous tumor model using PC-3 cells. The influence of androgen receptor expression on bortezomib efficacy was examined using RNA interference. Changes in the expression of ubiquitinated proteins, cell cycle associated proteins and acetylated histone were evaluated. RESULTS: Androgen receptor expression seemed to decrease bortezomib activity. PC-3 and DU 145 cells were more susceptible to bortezomib than LNCaP cells and the silencing of androgen receptor expression in LNCaP cells enhanced bortezomib activity. Vorinostat and bortezomib synergistically induced apoptosis, inhibited prostate cancer cell growth and suppressed tumor growth in a murine xenograft model. The combination decreased cyclin D1 and cyclin-dependent kinase 4 expression, and increased p21 expression. The combination synergistically caused the accumulation of ubiquitinated proteins and histone acetylation. This histone acetylation was a consequence of the accumulation of ubiquitinated proteins. CONCLUSIONS: Vorinostat and bortezomib inhibit the growth of prostate cancer cells synergistically by causing ubiquitinated proteins to accumulate in cells. The current study provides a framework for testing the combination in patients with advanced prostate cancer.

Suberoylanilide hydroxamic acid (SAHA) combined with bortezomib inhibits renal cancer growth by enhancing histone acetylation and protein ubiquitination 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 <1) and colony formation significantly (P < 0.05). In s.c. tumour models a 10-day treatment with a combination of SAHA (50 mg/kg) and bortezomib (60 µg/kg) inhibited tumour growth significantly (P < 0.05). Mechanistically, SAHA combined with bortezomib enhanced protein ubiquitination synergistically and enhanced histone acetylation by inhibiting the expression of HDACs. CONCLUSION: SAHA combined with bortezomib inhibits the proliferation of renal cancer cells in vitro and in vivo, and the effectiveness of the combination is due to its synergistic enhancement of histone acetylation and protein ubiquitination.

Prediction of biochemical recurrence after radical prostatectomy using peritumoral lymphatic vessel density in biopsy specimens in patients with localized prostate cancer.

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.

Ataxia telangiectasia and Rad3-related inhibition by AZD6738 enhances gemcitabine-induced cytotoxic effects in bladder cancer cells.

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.

Antitumor effect of suberoylanilide hydroxamic acid and topotecan in renal cancer cells.

The treatment modality for advanced renal cancer is limited. The development of novel systemic therapies has long been waited for. Suberoylanilide hydroxamic acid (SAHA) is one of the most potent histone deacetylase (HDAC) inhibitors, which are promising novel anticancer agents. SAHA has already been tested in phase II clinical trials; however, its effectiveness has been found to be limited. Recently, the combination of SAHA and topoisomerase I inhibitor, topotecan, was shown to be effective, but this treatment strategy has not been tested in renal cancer cells. In the present study, we found that the combination of SAHA and topotecan effectively inhibited the growth of renal cancer cells by suppressing the expression of cyclin-dependent kinase (CDK) 4 and cyclin D1, and promoting retinoblastoma protein (Rb) dephosphorylation. Furthermore, the combination therapy was found to inhibit both the function and expression of HDACs, which may be one of the main mechanisms of the combination therapy. To the best of our knowledge, this is the first report that has revealed the combined beneficial effect of SAHA and topotecan on renal cancer cells. Combining SAHA and topotecan is thus a promising approach to the treatment of renal cancer.

Glucose-regulated protein 78 positivity as a predictor of poor survival in patients with renal cell carcinoma.

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 <0.0001), advanced T stage (≥pT3; p = 0.0002), lymphovascular invasion (positive; p <0.0001), regional nodal involvement (≥N1; p = 0.0086), and distant metastases at presentation (M1; p = 0.001). Positivity of GRP78 expression was significantly associated with shorter disease-specific survival and shorter progression-free survival. Cox proportional hazard model showed that strong GRP78 positivity was an independent predictor of shortened progression-free survival in N0M0 RCC patients. 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.

Inhibition of checkpoint kinase 1 potentiates anticancer activity of gemcitabine in bladder cancer cells.

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.

Simvastatin-romidepsin combination kills bladder cancer cells synergistically.

The HMG-CoA reductase inhibitor simvastatin activates AMP-activated protein kinase (AMPK) and thereby induces histone acetylation. We postulated that combining simvastatin with the histone deacetylase (HDAC) inhibitor romidepsin would kill bladder cancer cells by inducing histone acetylation cooperatively. The combination of romidepsin and simvastatin induced robust apoptosis and killed bladder cancer cells synergistically. In murine subcutaneous tumor models using MBT-2 cells, a 15-day treatment with 0.5 mg/kg romidepsin and 15 mg/kg simvastatin was well tolerated and inhibited tumor growth significantly. Mechanistically, the combination induced histone acetylation by activating AMPK. The combination also decreased the expression of HDACs, thus further promoting histone acetylation. This AMPK activation was essential for the combination's action because compound C, an AMPK inhibitor, suppressed the combination-induced histone acetylation and the combination's ability to induce apoptosis. We also found that the combination increased the expression of peroxisome proliferator-activated receptor (PPAR) γ, leading to reactive oxygen species production. Furthermore, the combination induced endoplasmic reticulum (ER) stress and this ER stress was shown to be associated with increased AMPK expression and histone acetylation, thus playing an important role in the combination's action. Our study also suggests there is a positive feedback cycle between ER stress induction and PPARγ expression.

The Dual Histone Deacetylase-Proteasome Inhibitor RTS-V5 Acts Synergistically With Ritonavir to Induce Endoplasmic Reticulum Stress in Bladder Cancer Cells.

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.

Expressions of P-Glycoprotein, Multidrug Resistance Protein 1 and Annexin A2 as Predictive Factors for Intravesical Recurrence of Bladder Cancer after the Initial Transurethral Resection and Immediate Single Intravesical Instillation of Adriamycin.

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.

Ubiquitin-proteasome System Is a Promising Target for Killing Cisplatin-resistant 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.