Minyong Kang

Tissue-engineered heart valve leaflets: an animal study.

Background Tissue-engineered heart valve leaflets are a promising way to overcome the inherent limitations of current prosthetic valves. The aim of this study was to compare the biological responses of an autologous cell seeded scaffold and an acellular scaffold implanted in the pulmonary valve leaflet in the same animal. Methods Myofibroblasts and endothelial cells were isolated and cultured from an ovine artery. A synthetic biodegradable scaffold consisting of polyglycolic acid and polylactic acid was initially seeded with the myofibroblasts, then coated with endothelial cells. Cells were seeded using a medium containing collagen and cultured. A tissue-engineered construct and a plain scaffold were implanted as double pulmonary valve leaflet replacement in the same animal in an ovine model (n=3). Additionally, the tissue-engineered construct (n=2) and the plain scaffold (n=2) were implanted as single valve leaflet replacements for long-term analysis. After sacrifice, the implanted valve leaflet tissues were retrieved, analyzed visually and using light microscopy. Results Three animals that underwent replacement of two valve leaflets with a tissue-engineered construct and a plain scaffold, survived only a short-time (12, 24, 36 hours). The death was attributed to heart failure caused by severe pulmonary insufficiency. Animals that underwent single valve leaflet replacement survived longer and were electively sacrificed at 6 and 9 weeks after operation. The analysis of the leaflets from the short-term survivors showed that the tissue-engineered constructs contained less fibrins and protein exudates than the plain scaffold. In contrast, leaflets obtained from animals surviving 6 and 9 weeks showed similar well organized granulation tissues in the tissue-engineered constructs and the plain scaffolds. Conclusion This animal experiment demonstrates that in the early phase of implantation, the tissue-engineered construct shows a better biological response in terms of antithrombogenicity than the plain scaffold, although both of them have similar results in the later reparative phase.

Inhibition of Autophagy Potentiates Atorvastatin-Induced Apoptotic Cell Death in Human Bladder Cancer Cells in Vitro

Statins are cholesterol reduction agents that exhibit anti-cancer activity in several human cancers. Because autophagy is a crucial survival mechanism for cancer cells under stress conditions, cooperative inhibition of autophagy acts synergistically with other anti-cancer drugs. Thus, this study investigates whether combined treatment of atorvastatin and autophagy inhibitors results in enhancing the cytotoxic effects of atorvastatin, upon human bladder cancer cells, T24 and J82, in vitro. To measure cell viability, we performed the EZ-Cytox cell viability assay. We examined apoptosis by flow cytometry using annexin-V/propidium iodide (PI and western blot using procaspase-3 and poly (ADP-ribose) polymerase (PARP) antibodies. To examine autophagy activation, we evaluated the co-localization of LC3 and LysoTracker by immunocytochemistry, as well as the expression of LC3 and p62/sequestosome-1 (SQSTM1) by western blot. In addition, we assessed the survival and proliferation of T24 and J82 cells by a clonogenic assay. We found that atorvastatin reduced the cell viability of T24 and J82 cells via apoptotic cell death and induced autophagy activation, shown by the co-localization of LC3 and LysoTracker. Moreover, pharmacologic inhibition of autophagy significantly enhanced atorvastatin-induced apoptosis in T24 and J82 cells. In sum, inhibition of autophagy potentiates atorvastatin-induced apoptotic cell death in human bladder cancer cells in vitro, providing a potential therapeutic approach to treat bladder cancer.

Generation of Bladder Urothelium from Human Pluripotent Stem Cells under Chemically Defined Serum- and Feeder-Free System

Human stem cells are promising sources for bladder regeneration. Among several possible sources, pluripotent stem cells are the most fascinating because they can differentiate into any cell type, and proliferate limitlessly in vitro. Here, we developed a protocol for differentiation of human pluripotent stem cells (hPSCs) into bladder urothelial cells (BUCs) under a chemically defined culture system. We first differentiated hPSCs into definitive endoderm (DE), and further specified DE cells into BUCs by treating retinoic acid under a keratinocyte-specific serum free medium. hPSC-derived DE cells showed significantly expressed DE-specific genes, but did not express mesodermal or ectodermal genes. After DE cells were specified into BUCs, they notably expressed urothelium-specific genes such as UPIb, UPII, UPIIIa, P63 and CK7. Immunocytochemistry showed that BUCs expressed UPII, CK8/18 and P63 as well as tight junction molecules, E-CADHERIN and ZO-1. Additionally, hPSCs-derived BUCs exhibited low permeability in a FITC-dextran permeability assay, indicating BUCs possessed the functional units of barrier on their surfaces. However, BUCs did not express the marker genes of other endodermal lineage cells (intestine and liver) as well as mesodermal or ectodermal lineage cells. In summary, we sequentially differentiated hPSCs into DE and BUCs in a serum- and feeder-free condition. Our differentiation protocol will be useful for producing cells for bladder regeneration and studying normal and pathological development of the human bladder urothelium in vitro.

Non-invasive Parameters Predicting Bladder Outlet Obstruction in Korean Men with Lower Urinary Tract Symptoms

The goal of this study was to evaluate the clinical and urodynamic features in Korean men with lower urinary tract symptoms (LUTS) and to determine non-invasive parameters for predicting bladder outlet obstruction (BOO). Four hundred twenty nine Korean men with LUTS over 50 yr of age underwent clinical evaluations for LUTS including urodynamic study. The patients were divided into two groups according to the presence of BOO. These two groups were compared with regard to age, the results of the uroflowmetry, serum prostate-specific antigen (PSA) level, prostate volume, International Prostate Symptom Score (I-PSS), and the results of the urodynamic study. Patients with BOO had a lower maximal flow rate (Qmax), lower voided volume, higher serum PSA level and larger prostate volume (P<0.05). BOO group had a significantly higher rate of involuntary detrusor contraction and poor compliance compared to the patients without BOO (P<0.05). The multivariate analysis showed that Qmax and poor compliance were significant factors for predicting BOO. Our results show that Qmax plays a significant role in predicting BOO in Korean men with LUTS. In addition, BOO is significantly associated with detrusor dysfunction, therefore, secondary bladder dysfunction must be emphasized in the management of male patients with LUTS.

Cytotoxic effects of escin on human castration-resistant prostate cancer cells through the induction of apoptosis and G2/M cell cycle arrest.

OBJECTIVE To evaluate the in vitro and in vivo effects of escin on human castration-resistant prostate cancer (CRPC) cells, PC-3 and DU-145. MATERIALS AND METHODS The inhibition of cell proliferation and its mechanism were assessed through a cytotoxicity assay, flow cytometry, and Western blot. The in vivo efficacy of escin in CRPC cells was assessed using a xenograft tumor model subcutaneously established in BALB/c nude mice. RESULTS The treatment with escin significantly reduced cell viability of CRPC cells in a dose- and time-dependent manner. Escin induced apoptosis in a time-dependent manner, which was accompanied by increases in pro-apoptotic (BCL-2 associated X protein, cleaved-caspase3, and cleaved-poly [adenosine diphosphate-ribose] polymerase) proteins and decreases in antiapoptotic (X-linked inhibitor of apoptosis protein, cellular inhibitor of apoptosis protein-1, cellular inhibitor of apoptosis protein-2B-cell leukemia/lymphoma-2, and B-cell lymphoma-extra large) proteins. Escin induced G2/M-phase cell cycle arrest and thus led to a significant decrease in the expression of cyclinB1 and its activating partner cyclin-dependent kinase 1, with the concomitant induction of p21. In addition, escin significantly inhibited the growth of CRPC cells in xenograft models. CONCLUSION The results show that escin induced cytotoxic effects on CRPC cells through the induction of apoptosis and G2/M cell cycle arrest, indicating it may be a novel therapeutic agent for CRPC.

S100A3 suppression inhibits in vitro and in vivo tumor growth and invasion of human castration-resistant prostate cancer cells.

OBJECTIVE To investigate the role of S100A3 and the effect of S100A3 inhibition on human castration-resistant prostate cancer (CRPC) cells by using in vitro and in vivo functional assays. MATERIALS AND METHODS Using human CRPC cells (PC3 and DU145), S100A3 expression levels were assessed by reverse transcription-polymerase chain reaction and Western blot analysis. After S100A3-specific small interfering ribonucleic acid (RNA) treatment, cell viability was determined by Cell Counting Kit-8 assay, and apoptotic cell fractions were evaluated by flow cytometry. The invasive properties of these cells and the expression pattern of matrix metalloproteinases (MMPs) were assessed using transwell migration assays, reverse transcription-polymerase chain reaction, and gelatin zymography. Finally, the in vivo efficacy of S100A3 inhibition on human CRPC cells was investigated using human tumor xenograft models in nude mice. RESULTS Human CRPC cells showed overexpression of S100A3, and its suppression reduced cell viability owing to apoptotic cell death. Additionally, S100A3 inhibition decreased the invasiveness of human CRPC cells. Moreover, MMP-2 and MMP-9 were downregulated in PC3, whereas only MMP-9 was downregulated in D145 after S100A3 inhibition. In human CRPC xenograft models, we noted a marked reduction in tumor growth in mice injected with S100A3 short hairpin RNA-transfected PC3 and DU145 cells. CONCLUSION Human CRPC cells showed upregulation of S100A3 expression, and S100A3 inhibition reduced tumor cell viability. S100A3 inhibition reduced invasion capability with downregulation of MMP expression. More importantly, S100A3 inhibition resulted in tumor growth suppression in human CRPC xenograft models, suggesting S100A3 could serve as a novel therapeutic target for the treatment of human CRPC.

Preoperative neutrophil-lymphocyte ratio can significantly predict mortality outcomes in patients with non-muscle invasive bladder cancer undergoing transurethral resection of bladder tumor

The prognostic role of systemic inflammatory response (SIR) markers is unclear in patients with non-muscle invasive bladder cancer (NMIBC). Here, we aimed to investigate the prognostic role of various SIR markers in the oncological outcomes in non-muscle invasive bladder cancer (NMIBC) patients at a single institution in Korea. Neutrophil-lymphocyte ratio (NLR), derived-NLR (dNLR), and platelet-lymphocyte ratio (PLR) were examined as SIR markers. We retrospectively collected data of 1,698 NMIBC patients who underwent transurethral resection of the bladder (TURB) between 1990 and 2013. After excluding 147 patients, the study population finally consisted of 1,551 individuals. Overall survival (OS), cancer-specific survival (CSS), recurrence-free survival (RFS), and progression-free survival (PFS) were analyzed by using Kaplan-Meier estimates. Multivariate Cox regression model was adopted to identify the predictors of oncological outcomes. Notably, elevated NLR (≥2.0), dNLR (≥1.5) and PLR (≥124) were associated with poor OS outcomes. Patients with increased NLR, but not dNLR and PLR, only had poor CSS estimates compared to those with lower NLR. However, no significant differences were found in RFS and PFS according to the SIR status. In the multivariate Cox regression analysis, elevated NLR was identified as a key predictor of OS [hazard ratio (HR)=1.52, 95% confidence interval (CI)=1.19-1.95], in addition to age (HR=1.07, 95% CI=1.05-1.08), hemoglobin (HR=0.83, 95% CI=0.78-0.88), and high grade tumor (HR=1.88, 95% CI=1.45-1.08). With respect to CSS, increased NLR was also identified as an independent predictor (HR=1.12, 95% CI=1.01-1.25). In summary, our results indicate that NLR can be a very reliable SIR marker for predicting the oncological outcomes, particularly mortality outcomes.

Prognostic factors for conditional survival in patients with muscle-invasive urothelial carcinoma of the bladder treated with radical cystectomy

Because only a few studies have evaluated conditional survival (CS) in bladder cancer patients, we examined conditional overall survival (OS) and cancer-specific survival (CSS) in these patients after radical cystectomy (RC), and determined which prognostic variables affect CS over time. We reviewed 487 patients treated with RC and pelvic lymph node dissection at our institution between 1991 and 2012. Cox regression models were used to identify the significant prognostic factors for CS depending on clinicopathological characteristics. As survival time increased after RC, conditional OS and CSS rates increased when compared with baseline survival probability. CS more significantly improved in the patients with unfavorable pathologic characteristics. While many variables were associated with survival at baseline, only age was found to be a significant prognostic factor for 5-year conditional OS in all given survivorships. In conclusion, conditional OS and CSS probabilities significantly improved over time, with greater improvements in the cases with unfavorable pathologic features. Moreover, age remained the key prognostic factor for conditional OS estimates from baseline to 5 years after surgery. Our results provide practical survival information to guide adjustments in our current follow-up strategy for bladder cancer patients after RC.

Hypertriglyceridemia is a potential preoperative predictor for biochemical recurrence after radical prostatectomy.

Objectives Many previous studies have suggested that the outcome of prostate cancer (PCa) may be closely related to abnormal lipid metabolism. Therefore, in this study, we evaluated the preoperative lipid profiles of patients with clinically localized prostate cancer (PCa) who underwent radical prostatectomy (RP), with particular emphasis on the relationship between these profiles and biochemical recurrence (BCR). Patients and Methods We evaluated 715 consecutive men with clinically localized PCa who underwent RP at our institution between January 2011 and December 2013. We defined hypertriglyceridemia as a fasting serum triglyceride (TG) level greater than 200 mg/dL. We used the Kaplan—Meier method to predict BCR-free survival and applied the log-rank test to determine the statistical significance between survival curves. Cox proportional hazard ratio (HR) models were used to identify the significant predictors of BCR according to clinicopathological variables. Results Of 663 patients who underwent RP for clinically localized PCa, 66 (10.0%) showed BCR during a median follow-up period of 21 months. Patients without BCR had higher levels of serum TG, and patients with hypertriglyceridemia were significantly more likely to achieve BCR-free survival in the Kaplan—Meier analysis (log-rank test, P = 0.009). In the multivariable analysis, the presence of hypertriglyceridemia (HR 0.22), pathologic Gleason score (≥8; HR 2.85), pathologic T stage (≥pT3; HR 3.44), and a positive surgical margin (HR, 2.39) were still significant BCR predictors. Conclusions We found that preoperative hypertriglyceridemia was associated with a lower risk of BCR after RP in patients with clinically localized PCa. Our results could help to clarify the currently conflicting evidence on the relationship between serum lipid profiles, particularly the presence of hypertriglyceridemia, and the risk of BCR in PC a patients after surgery.

Single, immediate postoperative instillation of chemotherapy in non-muscle invasive bladder cancer: a systematic review and network meta-analysis of randomized clinical trials using different drugs

We performed a network meta-analysis of randomized controlled trials (RCTs) to compare the efficacy of several intravesical chemotherapeutic (IVC) agents after transurethral resection of bladder tumor (TURB) in non-muscle invasive bladder cancer patients. The literature search was conducted using the Embase, Scopus and PubMed databases for RCTs, including patients with single or multiple, primary or recurrent stage Ta or T1 urothelial carcinoma of the bladder managed with a single, immediate instillation of IVC after TURB. Thirteen RCTs met the eligibility criteria. Pair-wise meta-analysis (direct comparison) showed that pirarubicin [hazard ratio (HR): 0.31], epirubicin (HR: 0.62), and MMC (HR: 0.40) were the most effective drugs for reducing tumor recurrence. Bayesian network meta-analysis (indirect comparison) revealed that treatment with pirarubicin (HR: 0.31), MMC (HR: 0.44), or epirubicin (HR: 0.60) was associated with prolonged recurrence-free survival. Among the drugs examined, only pirarubicin reduced disease progression compared to controls. These results suggest that a single, immediate administration of IVC with pirarubicin, MMC, or epirubicin is associated with prolonged recurrence-free survival following TURB in non-muscle invasive bladder cancer patients, though only pirarubicin also reduced disease progression.