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Abstract Bladder cancer (BC) is the second most common urologic cancer in western countries. New strategies for managing high-grade muscle-invasive bladder cancer (MIBC) are urgently required because MIBC has a high risk of recurrence and poor survival. A growing body of evidence indicates that microRNA has potent antitumorigenic properties in various cancers, and thus, therapeutic strategies based on microRNA may show promising results in cancer therapy. Analysis of The Cancer Genome Atlas (TCGA) database indicated that hsa-miR-30a-3p is downregulated in human BC. Our in vitro investigation demonstrated that hsa-miR-30a-3p suppresses the expression of matrix metalloproteinase-2 (MMP-2) and MMP-9 and reduces the cell invasive potential of BC cells. Furthermore, hsa-miR-30a-3p directly targets ATG5, ATG12, and Beclin 1; this in turn improves the chemosensitivity of BC cells to cisplatin through the repression of protective autophagy. In a tumor-xenograft mice model, hsa-miR-30a-3p suppressed muscle invasion. Cotreatment with hsa-miR-30a-3p enhanced the antitumor effect of cisplatin in reducing tumor growth in BC. The current study provides a novel strategy of using hsa-miR-30a-3p as an adjuvant or replacement therapy in future BC treatment.

Objective: Quercetin, a natural dietary compound, has been demonstrated with antitumor activities against several types of cancers by disrupting cell cycle and inducing apoptotic cell death. However, human bladder cancer cells such as 5637 and T24 cells expressing mutant p53 are resistant to a 24 hrs quercetin treatment. In this study, the anticancer effect of quercetin was evaluated in these bladder cancer cells. Materials and Methods: The bladder cancer cells treated with quercetin were subjected to evaluated cell apoptosis by caspase activity, TUNEL assay and cell viability assay. The cell autophagy was assessed by detecting procession of LC3-II autophagic marker protein. Results: After 48 and 72 hrs of incubation, quercetin was found to be significantly effective in inhibiting proliferation of 5637 and T24 cells in a dose-dependent manner. Quercetin treatment increased the caspase 3/7 activities, percentage of subG0/G1 cells, and DNA fragmentation, indicating an induced apoptotic cell death. Pretreatment of a pan-caspase inhibitor, Z-VAD-FMK, attenuated the quercetin-decreased cell viability, suggesting that the cytotoxicity caused by quercetin mainly via apoptotic cell death. We also found that quercetin induced autophagy, as evidenced by the increased processing of LC3-II, a specific marker of autophagy. The disruption of autophagic flux by using bafilomycin A1, an autophagy inhibitor, caused significant accumulation of cellular p62 and LC3-II. In addition, the pretreatment of autophagy inhibitors, Baf A1 and chloroquine, strongly augmented apoptosis in 5637 and T24 cells, indicating the suppression of quercetin-induced autophagy enhanced apoptosis. Furthermore, the decreased cell viability and increased LC3-II processing were attenuated in quercetin-treated cells which pretreated with a reactive oxygen species (ROS) scavenger, N-acetyl cystine (NAC) suggested that quercetin-induced cytotoxicity and autophagy were initiated by the generation of ROS. Conclusion: This study proposes that combined treatment of autophagy inhibitor which sensitizes cells to quercetin treatment may be a better therapeutic approach to reduce bladder cancer cells proliferation.

Background: Chloroquine (CQ) is recognized as a potent adjuvant when combined with other chemotherapies to treat cancers. However, the effects of a single treatment of CQ on bladder cancer (BC) cells have not been investigated. Methods: The growth and viability of CQ-treated BC cells were examined. The lysosomal morphology was detected using LysoTracker. The induction of lysosomal membrane permeability (LMP) was detected by acridine orange (AO) translocation, and cathepsin B and D release. The expression of the bid, caspase-3, and cytosolic cytochrome C (Cyto. C) in CQ-treated cells was detected by the Western blot. The pepstatin A and E64d were used to attenuate CQ-induced LMP. Results: A single dose of CQ treatment induced BC cell death, and attenuated by pepstatin A and E64d. The diminishing of fluorescent in CQ-treated cells stained with LysoTracker, suggesting that CQ targets lysosomal functions. This was further supported by increased AO translocation and the releasing of CatB and CatD into the cytosol. The increased level of cleavage bid and cytosolic Cyto. C indicated mitochondrial outer membrane permeabilization and subsequently leading to apoptosis induction judged by the increased level of activated caspase 3. Conclusion: CQ-induced LMP that enhances apoptosis and ultimately leading to BC cell death. The study results demonstrated for the first time that single CQ treatment against BC cells by inducing LMP and subsequent mitochondria membrane permeability that trigger apoptosis, making it a potential treatment for BC therapy in the future.

Background: Prostate cancer (PCa) is a leading cause of cancer-related death in men, which emphasizes the need for novel therapeutic approaches. Targeting microRNA (miRNA) has been considered as a therapeutic strategy against cancers. Human miR-204-5p potentially targeting BCL2 has been reported to be downregulated in various cancers. We hypothesized that miR-204-5p overexpression induces cancer cell apoptosis by repressing BCL2 expression. Methods: A vector harboring mature miR-204-5p was constructed and delivered into human PCa cells. The expression level of miR-204-5p was determined by miRNA quantitative polymerase chain reaction (QPCR). Luciferase reporter assays were performed to verify the function of mature miR-204-5p and its direct binding to BCL2 transcripts. The expression levels of BCL-2 messenger RNA (mRNA) and protein samples were measured by QPCR and Western blot, respectively. Cell viability was detected by WST-1 assays. Induction of apoptosis was determined by increased levels of cleavage caspase 3 and caspase 3/7 activity. Results: The expression levels of miR-204-5p were downregulated in PCa cells compared with normal prostate epithelial cells. Transfection of pSM-204 resulted in up to 6.2-fold higher expression of miR-204-5p when compared with pSM control. The mRNA levels of several potential target genes of miR-204-5p were decreased in pSM-204-transfected PC3 and Rv1 cells. BCL2 mRNA and protein expression decreased in miR-204-5p-transfected cells, which led to cytochrome C release from mitochondria. It subsequently increased cleaved caspase 3 and caspase 3/7 activities and reduced cell viability. Cotransfection of a reporter vector harboring the BCL2 3′-untranslated region to compete with endogenous transcripts partially rescued miR-204-5p-induced apoptosis. Conclusion: Human miR-204-5p targets BCL2 in PCa cells. Restoration of miR-204-5p in PCa could therefore be considered as a novel strategy by targeting antiapoptotic BCL2.

Chloroquine (CQ) and hydroxychloroquine (HCQ), two antimalarial drugs, are suggested to have potential anticancer properties. in the present study, we investigated the effects of CQ and HCQ on cell growth of bladder cancer with emphasis on autophagy inhibition and apoptosis induction in vitro. The results showed that CQ and HCQ inhibited the proliferation of multiple human bladder cell lines (including RT4, 5637, and T24) in a time- and dose-dependent fashion, especially in advanced bladder cancer cell lines (5637 and T24) compared to immortalized uroepithelial cells (SV-Huc-1) or other reference cancer cell lines (PC3 and MCF-7). We found that 24-hour treatment of CQ or HCQ significantly decreased the clonogenic formation in 5637 and T24 cells compared to SV-Huc-1. As human bladder cancer tumor exhibits high basal level of autophagic activities, we detected the autophagic flux in cells treated with CQ and HCQ, showing an alternation in LC3 flux in CQ- or HCQ-treated cells. Moreover, bladder cancer cells treated with CQ and HCQ underwent apoptosis, resulting in increased caspase 3/7 activities, increased level of cleaved poly(ADP-ribose) polymerase (PARP), caspase 3, and DNA fragmentation. Given these results, targeting autophagy with CQ and HCQ represents an effective cancer therapeutic strategy against human bladder cancer.

Background: Transrectal ultrasound-guided prostate (TRUS-P) biopsy is the standard diagnostic procedure for patients with suspected prostate cancer. However, infectious complications can occur. Purpose: To analyze the patients from a single institution who were hospitalized for infectious complications after TRUS-P biopsy. Materials and Methods: From 2003 to 2012, 985 TRUS-P biopsy procedures were performed in a medical center in Northern Taiwan; among these, 28 patients were admitted for infectious complications following the procedure. A retrospective review of the medical records was performed, and data regarding the demographics, details of infectious complications, and hospital course of these patients were collected. Results: The median age of the patients was 62 (27–82) years; the comorbidity rate was 57.1% (16/28); the admission rate was 2.8% (28/985). The most common causative pathogen was Escherichia coli. More than half of the cultured E. coli were not resistant to fluoroquinolones. The median hospital stay was 6 (3–16) days, and one patient was admitted to the intensive care unit. No mortality was observed in this study. Although the number of biopsies increased, no obvious increasing trend in the number of hospitalizations was noted in recent years. Conclusion: TRUS-P biopsy can be safely performed, and no obvious increasing trend in infectious complications and resistant strains has been observed in recent years.

Objective: Bladder cancer (BC) is one of the most common noncutaneous malignant cancers with high mortality and recurrence rates in the general population, particularly in men. We previously reported detecting dysregulated micro-RNAs (miRNAs) in human BC tissues. Using an miRNA targeting database, we found that miR-204, which is downregulated in BC, targets the B-cell lymphoma 2 gene (BCL2). We hypothesized that the overexpression of miR-204 induces apoptosis by repressing BCL2 expression and causing cancer cell death. Materials and methods: To overexpress miR-204 in cancer cells, a vector that expresses miR-204 was transfected into human BC, embryonic kidney, and cervical cancer cells. The expression of miR-204 in the transfected and mock cells was monitored through miRNA quantitative polymerase chain reactions (Q-PCRs). Luciferase reporter assays were performed to verify the direct binding of miR-204 to targeted sites on the BCL2 transcripts. The BCL2 mRNA and protein expression levels in the miR-204-transfected cells were measured using Q-PCRs and Western blotting, respectively. The cell viability of the miR-204 transfected cells was detected through WST-1 assays. The induction of apoptosis was determined by the increased release of cytochrome c, cleavage caspase 3, caspase 3/7 activity, and DNA fragmentation. Results: The expression of miR-204 in the transfected cells was elevated to 6.2-fold, compared with the controls, and the function of matured miR-204 was confirmed through the luciferase reporter assays. The expressions of BCL2 mRNA and protein were reduced in the miR-204-transfected cells, which generated increased DNA fragmentation, caspase 3 cleavage, caspase 3/7 activity, and reduced cell viability. Cotransfection of the reporter vector that harbored the BCL2 3′-untranslated region to compete with endogenous transcripts partially rescued the apoptosis that was induced by the overexpressed miR-204. This finding further supported that miR-204 targeting BCL2 induces apoptosis in BC cells. Conclusion: miR-204 targets BCL2 in human BC cells. The overexpression of miR-204 in tumors could thus be a novel strategy for inducing cell death by targeting antiapoptotic BCL2.

Objective: Urothelial carcinoma is one of the most common pathological types of bladder cancer. Several studies have shown that dysregulated microRNAs (miRNAs) play an important role in bladder cancer progression. We performed the present miRNA microarray analysis in samples of urothelial carcinoma of the bladder and adjacent normal bladder tissue from Taiwanese patients to investigate dysregulated miRNAs. Materials and methods: To detect dysregulated miRNAs in urothelial carcinoma of the bladder, samples of tumor and adjacent normal tissues were collected from 10 patients. Tissue samples from three patients were subjected to miRNA microarray analysis, and the remaining tissue samples from the other seven patients were used to validate the results obtained from the microarray data. Potential targets of these dysregulated miRNAs were identified using online databases, including MicroCosm and TargetScan. Results: A panel of 30 differentially expressed miRNAs with at least fourfold differences in expression compared with normal controls, including 19 upregulated and 11 downregulated miRNAs, was generated. The expression levels of miR-30a-5p, miR-30a-3p, miR-99a, miR-130b, miR-133b, miR-135b, miR-145, miR-195, miR-204, and miR-214 were experimentally verified using real-time RT-PCR analysis. Using an online miRNA target database, we discovered that these dysregulated miRNAs potentially control components of the insulin-like growth factor 1 receptor (IGF1R) signaling pathway. Conclusion: Our results indicate that dysregulated miRNAs may be involved in bladder cancer pathogenesis and are potential biomarkers.

Background/Objective: Standard laparoscopic adrenalectomy requires early control of the main adrenal vein; however, the small retroperitoneal working space is challenging for beginners to perform this maneuver. We report a technical modification of retroperitoneal laparoscopic adrenalectomy (RLA) for primary hyperaldosteronism (PHA) and the clinical outcomes. Methods: A total of 38 RLAs were performed for the patients with PHA. The patients were placed in true lateral position with mild bending to expand the surgical field. Instead of attempting to control the main adrenal vein initially, we adopted a technical modification that manipulating and freeing the gland first before controlling the main adrenal vein. Results: The RLAs were successfully performed in all but one case, which was converted to open surgery due to pancreatic injury. Mean operative time was 124 minutes and estimated blood loss was 74 ml. Mean maximal fluctuation of systolic blood pressure was 29 mmHg. For the right-side RLA, less operative time (113.5 vs. 137.9 minutes) and estimated blood loss (59.5 vs. 91.2 ml) were noted compared with the left-side procedure. Postoperative complications included cerebrovascular accident in one patient, one surgical site hematoma, and two patients had postoperative fever. Potassium level returned to normal in all patients and 70% of the patients reduced their antihypertensives. Conclusion: Technical modification RLA for PHA without initial control of the main adrenal vein is a safe and feasible procedure. No vigorous blood pressure fluctuation was intraoperatively noted. No vascular injury occurred. Moreover, the right-side procedure became easier.

Purpose: Although intravesical chemotherapy has several antitumoral benefits, it can also have severe side effects. The development of novel therapeutic approaches for bladder cancer (BC) is thus warranted. Hyperthermia (HT) is a widely applicable adjuvant therapy in various cancers. Therefore, this study investigated the effect of HT on improving the chemosensitivity of BC. Materials and Methods: The BC cell lines 5637 and T24 were cultured and treated with HT (43°C) for 24 h. Then, cell viability and survival were assessed using resazurin reagent and colony formation assay, respectively. Western blot assay was used to analyze the levels of Bax, Bcl-2, cleaved caspase-3, and cleaved poly (ADP-ribose) polymerase (PARP) protein expression. Mitochondria degradation was observed by MitoTracker Green staining. Results: In BC cells, HT co-administered with various concentrations of doxorubicin significantly inhibited cell viability and survival. Moreover, HT combined with doxorubicin promoted mitochondrial apoptosis, which caused Bax upregulation and Bcl-2 downregulation. Levels of cleaved caspase-3 and PARP protein expression were also elevated after co-treatment. Conclusion: Taken together, HT improved the chemosensitivity of BC cells to doxorubicin. HT combined with chemotherapy further activated mitochondrial apoptosis in BC cells. The findings suggested that HT may serve as a potential adjunctive treatment for BC that is ready to be applied clinically. [ABSTRACT FROM AUTHOR]