Cheng-Huang Shen

Distinct DNA methylation epigenotypes in bladder cancer from different Chinese sub-populations and its implication in cancer detection using voided urine.

BackgroundBladder cancer is the sixth most common cancer in the world and the incidence is particularly high in southwestern Taiwan. Previous studies have identified several tumor-related genes that are hypermethylated in bladder cancer; however the DNA methylation profile of bladder cancer in Taiwan is not fully understood.MethodsIn this study, we compared the DNA methylation profile of multiple tumor suppressor genes (APC, DAPK, E-cadherin, hMLH1, IRF8, p14, p15, RASSF1A, SFRP1 and SOCS-1) in bladder cancer patients from different Chinese sub-populations including Taiwan (104 cases), Hong Kong (82 cases) and China (24 cases) by MSP. Two normal human urothelium were also included as control. To investigate the diagnostic potential of using DNA methylation in non-invasive detection of bladder cancer, degree of methylation of DAPK, IRF8, p14, RASSF1A and SFRP1 was also accessed by quantitative MSP in urine samples from thirty bladder cancer patients and nineteen non-cancer controls.ResultsThere were distinct DNA methylation epigenotypes among the different sub-populations. Further, samples from Taiwan and China demonstrated a bimodal distribution suggesting that CpG island methylator phentotype (CIMP) is presented in bladder cancer. Moreover, the number of methylated genes in samples from Taiwan and Hong Kong were significantly correlated with histological grade (P < 0.01) and pathological stage (P < 0.01). Regarding the samples from Taiwan, methylation of SFRP1, IRF8, APC and RASSF1A were significantly associated with increased tumor grade, stage. Methylation of RASSF1A was associated with tumor recurrence. Patients with methylation of APC or RASSF1A were also significantly associated with shorter recurrence-free survival. For methylation detection in voided urine samples of cancer patients, the sensitivity and specificity of using any of the methylated genes (IRF8, p14 or sFRP1) by qMSP was 86.7% and 94.7%.ConclusionsOur results indicate that there are distinct methylation epigenotypes among different Chinese sub-populations. These profiles demonstrate gradual increases with cancer progression. Finally, detection of gene methylation in voided urine with these distinct DNA methylation markers is more sensitive than urine cytology.

Global analysis of modifications of the human BK virus structural proteins by LC-MS/MS.

BK virus, a human polyomavirus, may cause nephritis and urological disorders in patients who have undergone renal transplantation. Little is known about the characteristics of the BK viral proteins. In the current study, BK viral proteins were characterized by immunoblotting and LC-MS/MS. The results revealed that BK virus is composed of three structural proteins, VP1, VP2, and VP3 and four cellular histones, H2A, H2B, H3, and H4. The major structural protein, VP1, can be divided into 16 subspecies by two-dimensional gel electrophoresis. Modifications of VP1, VP2, and VP3 were comprehensively identified by LC-MS/MS. The presence of acetylation, cysteinylation, carboxymethylation, carboxyethylation, formylation, methylation, methylthiolation, oxidation, dioxidation, and phosphorylation could be identified. This is the first report providing an analysis of the global modifications present on polyomavirus structural proteins. The identification of these modifications of VP1, VP2, and VP3 should facilitate an understanding of the physiology of BKV during its life cycle.

Human JC virus-like particles as a gene delivery vector

Introduction: As a viral gene delivery vector, the recombinant JC virus-like particles (VLPs) can be easily generated in large quantities and at low cost. Exogenous genes of interest can be packaged by the VLP without the involvement of viral genetic material and then delivered into any tissue susceptible to JC virus (JCV) to allow gene transduction. Therefore, it should be possible in the future to develop a gene delivery vector using the human JC VLPs that will allow gene therapy. Areas covered: Development of a gene delivery vector using the polyomavirus VLPs is reviewed in this article. The advantages and disadvantages of using JC VLP for gene delivery are discussed. Expert opinion: Human JC VLPs are readily produced and can be engineered with ease; they allow specific targeting without the presence of any viral genetic material. For therapeutic purposes, gene(s) of interest or other compounds can be packaged into the VLP and delivered to JCV-susceptible cells at high efficiency.

Analysis of the size of DNA packaged by the human JC virus-like particle

Previously, it has been demonstrated that the JC virus-like particle (VLP) is able to package DNA in E. coli and deliver the DNA into human colon cancer cells for gene expression. In this study, the maximum size of DNA packaged by the VLP was determined further. Plasmid DNAs with various sizes were packaged by the VLP in E. coli. Human neuroblastoma cells were then infected with the VLPs containing the various sizes of DNA to allow gene expression. In addition, plasmid DNAs packaged in the VLPs were extracted and retransformed back into E. coli under selection to determine the size of the DNA packaged. The results showed that the JC VLP was able to package plasmid DNA in E. coli up to at least 9.4 kbp in size and this size of DNA could be delivered successfully into human neuroblastoma cells for gene expression. The JC VLP is able to package exogenous DNA up to at least 9.4 kbp in size for gene transduction. These findings will help with the development of gene delivery systems using the JC VLP as the gene delivery vector.

Methylomics analysis identifies ZNF671 as an epigenetically repressed novel tumor suppressor and a potential non-invasive biomarker for the detection of urothelial carcinoma.

The molecular mechanism underlying the lethal phenomenon of urothelial carcinoma (UC) tumor recurrence remains unresolved. Here, by methylation microarray, we identified promoter methylation of the zinc-finger protein gene, ZNF671 in bladder UC tumor tissue samples, a finding that was independently validated by bisulphite pyrosequencing in cell lines and tissue samples. Subsequent assays including treatment with epigenetic depressive agents and in vitro methylation showed ZNF671 methylation to result in its transcriptional repression. ZNF671 re-expression in UC cell lines, via ectopic expression, inhibited tumor growth and invasion, in possible conjunction with downregulation of cancer stem cell markers (c-KIT, NANOG, OCT4). Clinically, high ZNF671 methylation in UC tumor tissues (n=96; 63 bladder, 33 upper urinary tract) associated with tumor grade and poor locoregional disease-free survival. Quantitative MSP analysis in a training (n=97) and test (n=61) sets of voided urine samples from bladder UC patients revealed a sensitivity and specificity of 42%-48% and 89%-92.8%, respectively, for UC cancer detection. Moreover, combining DNA methylation of ZNF671 and 2 other genes (IRF8 and sFRP1) further increased the sensitivity to 96.2%, suggesting a possible three-gene UC biomarker. In summary, ZNF671, an epigenetically silenced novel tumor suppressor, represents a potential predictor for UC relapse and non-invasive biomarker that could assist in UC clinical decision-making.

Tubeless Percutaneous Nephrolithotomy for Geriatric Patients

Aim: To evaluate the safety of tubeless percutaneous nephrolithotomy (PCNL) in geriatric patients. Materials and Method: This is a retrospective review of 401 patients who received tubeless PCNL in a single institute. Among these, 50 were performed in patients aged older than 70 years (group 1), while 351 were performed in the remaining younger patients (group 2). Results: There was no significant difference in stone size between the 2 groups (3.6 ± 1.9 vs. 3.5 ± 2.0 cm). The average operative time was similar in both groups (92.8 ± 34.5 vs. 86.6 ± 32.0 min). The stone-free rate in groups 1 and 2 was 68.0% (34/50) and 83.8% (294/351), respectively, which was statistically significantly different. The average postoperative hospital stay was longer in group 1 (4.6 ± 3.4 days) than in group 2 (3.9 ± 2.5 days), but the difference was not statistically significant. There was no significant difference in postoperative urinary tract infection rate and blood transfusion rate in both groups (urinary tract infection: 18.0 vs. 8.8%; blood transfusion: 4 vs. 2.6%). Two patients in group 1 and 3 patients in group 2 experienced pulmonary complications. There was no other severe complication. Conclusion: Tubeless PCNL is a safe procedure for the treatment of geriatric patients with urolithiasis.

Proteomic Study of Renal Uric Acid Stone

OBJECTIVE To analyze urinary uric acid stone matrix proteins (SMP) with mass spectrometry (MS) to evaluate the mechanisms of uric acid stone formation. SMP plays an important role in urinary stone formation. Several proteomic studies apply to calcium-containing stones have been reported; however no proteomic study for urinary uric acid stone has been reported. METHODS Pure kidney uric acid stones from 5 individuals were demineralized, and SMPs were isolated. The obtained proteins were analyzed with reverse-phase liquid chromatography-tandem MS. The acquired data were searched against a Swiss Prot human protein database using Matrix Science, Mascot. The identified proteins were submitted to the AmiGO Web site for gene ontology analysis. They were also sumitted to Metacore software and Kyoto Encyclopedia of Genes and Genomes website (KEGG) for pathway analysis. MS-determined protein expressions were verified by immunoblot. RESULTS MS analysis identified 242 proteins from 5 proteomic results and the number of the identified protein of each result ranged from 52 to 156. Metacore software analysis suggested that inflammation may play an important role for kidney uric acid stone formation. Endogenous metabolic pathways were also analyzed and submitted to KEGG Web site, which revealed that these proteins may participate in fat metabolism. Five identified proteins were selected for immunoblot validation, and 3 proteins were confirmed. CONCLUSION Our results suggest that inflammatory process may play a role in kidney uric acid stone formation. Our endogenous metabolic pathway analysis data revealed that these proteins may participate in lipid metabolism. Whether this finding implies a relation between lipotoxicity and kidney uric acid stone former requires further investigation.

Contributing Factors for Fever After Tubeless Percutaneous Nephrolithotomy

OBJECTIVE To evaluate the contributing factors for fever after tubeless percutaneous nephrolithotomy (PCNL). METHODS Between May 2009 and December 2013, 395 tubeless PCNLs were performed at our hospital. After stone extraction, the bleeding points were cauterized for hemostasis to enable tubeless modification. In patients with troublesome bleeding after cauterization, oxidized regenerated cellulose (Surgicel) strips were used to tamponade the access tract to facilitate bleeding control. The contributory factors for fever were evaluated by a retrospective chart review. RESULTS Forty-four patients (11.7%) developed fever after tubeless PCNL. There was no difference in gender, age, and body mass index in the development of fever. Episodes of febrile or septic urinary tract infection before PCNL were found to have occurred in 35 patients, but the incidence of postoperative fever was not significantly higher in these patients. There is no significant difference in the mean stone size in fever and nonfever patients. Complete staghorn stones were noted in 40 patients, and their fever rate was not significantly higher than patients with nonstaghorn stone. The operation time is not significantly higher in the group with urinary tract infection. Patients with postoperative fever had a high incidence of residual stones than the remaining patients (38.9% vs 20.4%). There was no significant difference in incidence of postoperative fever in patients with struvite stones than patients with nonstruvite stones. In patients who received Surgicel packing, the incidence of fever was not significantly higher. CONCLUSION Incomplete stone extraction is a major contributing factor for the development of fever after tubeless PCNL.

The high incidence of JC virus infection in urothelial carcinoma tissue in Taiwan.

Human polyomaviruses, JC virus (JCV) and BK virus (BKV), usually remain latent in kidney and urothelial tissue after primary infection. Infection with human polyomavirus has still not been correlated conclusively with malignancy of kidney and urothelial tissue. The present study investigated further the possible relationship between JCV/BKV infection and urothelial carcinoma. Tissue samples were examined from 33 urothelial carcinomas and 5 renal cell carcinomas for JCV/BKV infection, using nested PCR with primers common to both JCV and BKV. The viral genotypes were further verified by endonuclease digestion and DNA sequencing following the PCR. In addition, immunohistochemistry and Western blotting were also performed to detect viral large tumor protein (LT) and the late capsid protein (VP1) in the tissue samples. The results from nested PCR showed that 90.1% (30/33) of the urothelial carcinomas samples and all of the renal cell carcinomas samples (5/5) were JCV DNA positive. Both archetypal and re‐arranged JCV genotypes were detected. On the other hand, BKV DNA was detected in only one (3%) of the urothelial carcinoma tissue samples. The immunohistochemical results showed that 30% (10/33) of urothelial carcinoma tissues was stained positive for large tumor antigen (LT). However, the structural protein VP1 was not detectable in any of the tissue samples examined. The present study demonstrated that JCV is highly prevalent in urothelial carcinoma tissue as is the expression of large tumor antigen. Therefore, the findings support the hypothesis that JCV infection is associated with urothelial carcinoma. J. Med. Virol. 83:2191–2199, 2011.

The investigation of a traditional Chinese medicine, Guizhi Fuling Wan (GFW) as an intravesical therapeutic agent for urothelial carcinoma of the bladder

BackgroundThe high risk of recurrence faced by patients with bladder cancer has necessitated the administration of supplemental intravesical chemotherapy; however, such treatments often result in severe side effects. As a result, novel intravesical agents with enhanced efficacy and minimal toxicity are urgently required for the treatment of bladder cancer.MethodsGuizhi Fuling Wan (GFW) is a traditional Chinese medicine shown to inhibit the growth of hepatocellular carcinoma. This study evaluated the growth inhibition of GFW using normal human urothelial cells and bladder cancer cells; the efficacy of GFW treatment was further compared with mitomycin C, epirubicin, and cisplatin. We also examined the progression of cell cycle and apoptosis in bladder cancer cells in response to GFW treatment. CCK-8 was employed to analyze cell viability and flow cytometry was used to study the cell cycle and apoptosis. The mechanisms underlying GFW-induced cell cycle arrest were determined by Western blot analysis.ResultsOur data demonstrate the potent inhibitory effect of GFW in the proliferation of bladder cancer cell lines, BFTC 905 and TSGH 8301. GFW presented relatively high selectivity with regard to cancer cells and minimal toxicity to normal urothelial cells. Our results also demonstrate that GFW interferes with cell cycle progression through the activation of CHK2 and P21 and induces apoptosis in these bladder cancer cells.ConclusionsOur results provide experimental evidence to support GFW as a strong candidate for intravesicle chemotherapy against bladder cancer.