Induction of apoptotic cell death in human bladder cancer cells by ethanol extract of Zanthoxylum schinifolium leaf, through ROS-dependent inactivation of the PI3K/Akt signaling pathway

BACKGROUND/OBJECTIVES@#Zanthoxylum schinifolium is traditionally used as a spice for cooking in East Asian countries. This study was undertaken to evaluate the anti-proliferative potential of ethanol extracts of Z. schinifolium leaves (EEZS) against human bladder cancer T24 cells.MATERIALS/METHODS: Subsequent to measuring the cytotoxicity of EEZS, the anti-cancer activity was measured by assessing apoptosis induction, reactive oxygen species (ROS) generation, and mitochondrial membrane potential (MMP). In addition, we determined the underlying mechanism of EEZS-induced apoptosis through various assays, including Western blot analysis. @*RESULTS@#EEZS treatment concentration-dependently inhibited T24 cell survival, which is associated with apoptosis induction. Exposure to EEZS induced the expression of Fas and Fas-ligand, activated caspases, and subsequently resulted to cleavage of poly (ADPribose) polymerase. EEZS also enhanced the expression of cytochrome c in the cytoplasm by suppressing MMP, following increase in the ratio of Bax:Bcl-2 expression and truncation of Bid. However, EEZS-mediated growth inhibition and apoptosis were significantly diminished by a pan-caspase inhibitor. Moreover, EEZS inhibited activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway, and the apoptosis-inducing potential of EEZS was promoted in the presence of PI3K/Akt inhibitor. In addition, EEZS enhanced the production of ROS, whereas N-acetyl cysteine (NAC), a ROS scavenger, markedly suppressed growth inhibition and inactivation of the PI3K/Akt signaling pathway induced by EEZS. Furthermore, NAC significantly attenuated the EEZS-induced apoptosis and reduction of cell viability. @*CONCLUSIONS@#Taken together, our results indicate that exposure to EEZS exhibits anticancer activity in T24 bladder cancer cells through ROS-dependent induction of apoptosis and inactivation of the PI3K/Akt signaling pathway.

CDC6 mRNA Expression Is Associated with the Aggressiveness of Prostate Cancer

BACKGROUND: Cell division cycle 6 (CDC6) is an essential regulator of DNA replication and plays important roles in the activation and maintenance of the checkpoint mechanisms in the cell cycle. CDC6 has been associated with oncogenic activities in human cancers; however, the clinical significance of CDC6 in prostate cancer (PCa) remains unclear. Therefore, we investigated whether the CDC6 mRNA expression level is a diagnostic and prognostic marker in PCa. METHODS: The study subjects included 121 PCa patients and 66 age-matched benign prostatic hyperplasia (BPH) patients. CDC6 expression was evaluated using real-time polymerase chain reaction and immunohistochemical (IH) staining, and then compared according to the clinicopathological characteristics of PCa. RESULTS: CDC6 mRNA expression was significantly higher in PCa tissues than in BPH control tissues (P = 0.005). In addition, CDC6 expression was significantly higher in patients with elevated prostate-specific antigen (PSA) levels (> 20 ng/mL), a high Gleason score, and advanced stage than in those with low PSA levels, a low Gleason score, and earlier stage, respectively. Multivariate logistic regression analysis showed that high expression of CDC6 was significantly associated with advanced stage (≥ T3b) (odds ratio [OR], 3.005; confidence interval [CI], 1.212–7.450; P = 0.018) and metastasis (OR, 4.192; CI, 1.079–16.286; P = 0.038). Intense IH staining for CDC6 was significantly associated with a high Gleason score and advanced tumor stage including lymph node metastasis stage (linear-by-linear association, P = 0.044 and P = 0.003, respectively). CONCLUSION: CDC6 expression is associated with aggressive clinicopathological characteristics in PCa. CDC6 may be a potential diagnostic and prognostic marker in PCa patients.

Baicalein Inhibits the Migration and Invasion of B16F10 Mouse Melanoma Cells through Inactivation of the PI3K/Akt Signaling Pathway

Baicalein, a natural flavonoid obtained from the rhizome of Scutellaria baicalensis Georgi, has been reported to have anticancer activities in several human cancer cell lines. However, its antimetastatic effects and associated mechanisms in melanoma cells have not been extensively studied. The current study examined the effects of baicalein on cell motility and anti-invasive activity using mouse melanoma B16F10 cells. Within the noncytotoxic concentration range, baicalein significantly inhibited the cell motility and invasiveness of B16F10 cells in a concentration-dependent manner. Baicalein also reduced the activity and expression of matrix metalloproteinase (MMP)-2 and -9; however, the levels of tissue inhibitor of metalloproteinase-1 and -2 were concomitantly increased. The inhibitory effects of baicalein on cell motility and invasiveness were found to be associated with its tightening of tight junction (TJ), which was demonstrated by an increase in transepithelial electrical resistance and downregulation of the claudin family of proteins. Additionally, treatment with baicalein markedly reduced the expression levels of lipopolysaccharide-induced phosphorylated Akt and the invasive activity in B16F10 cells. Taken together, these results suggest that baicalein inhibits B16F10 melanoma cell migration and invasion by reducing the expression of MMPs and tightening TJ through the suppression of claudin expression, possibly in association with a suppression of the phosphoinositide 3-kinase/Akt signaling pathway.

Erratum: Increased Expression of Herpes Virus-Encoded hsv1-miR-H18 and hsv2-miR-H9-5p in Cancer-Containing Prostate Tissue Compared to That in Benign Prostate Hyperplasia Tissue / 대한배뇨장애요실금학회지

In this article, a part of fund and grant supports was omitted unintentionally.

Metabolic Pathway Signatures Associated with Urinary Metabolite Biomarkers Differentiate Bladder Cancer Patients from Healthy Controls

PURPOSE: Our previous high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry study identified bladder cancer (BCA)-specific urine metabolites, including carnitine, acylcarnitines, and melatonin. The objective of the current study was to determine which metabolic pathways are perturbed in BCA, based on our previously identified urinary metabolome. MATERIALS AND METHODS: A total of 135 primary BCA samples and 26 control tissue samples from healthy volunteers were analyzed. The association between specific urinary metabolites and their related encoding genes was analyzed. RESULTS: Significant alterations in the carnitine-acylcarnitine and tryptophan metabolic pathways were detected in urine specimens from BCA patients compared to those of healthy controls. The expression of eight genes involved in the carnitine-acylcarnitine metabolic pathway (CPT1A, CPT1B, CPT1C, CPT2, SLC25A20, and CRAT) or tryptophan metabolism (TPH1 and IDO1) was assessed by RT-PCR in our BCA cohort (n=135). CPT1B, CPT1C, SLC25A20, CRAT, TPH1, and IOD1 were significantly downregulated in tumor tissues compared to normal bladder tissues (p<0.05 all) of patients with non-muscle invasive BCA, whereas CPT1B, CPT1C, CRAT, and TPH1 were downregulated in those with muscle invasive BCA (p<0.05), with no changes in IDO1 expression. CONCLUSION: Alterations in the expression of genes associated with the carnitine-acylcarnitine and tryptophan metabolic pathways, which were the most perturbed pathways in BCA, were determined.

Increased Expression of Herpes Virus-Encoded hsv1-miR-H18 and hsv2-miR-H9-5p in Cancer-Containing Prostate Tissue Compared to That in Benign Prostate Hyperplasia Tissue / 대한배뇨장애요실금학회지

PURPOSE: Previously, we reported the presence of virus-encoded microRNAs (miRNAs) in the urine of prostate cancer (CaP) patients. In this study, we investigated the expression of two herpes virus-encoded miRNAs in prostate tissue. METHODS: A total of 175 tissue samples from noncancerous benign prostatic hyperplasia (BPH), 248 tissue samples from patients with CaP and BPH, and 50 samples from noncancerous surrounding tissues from these same patients were analyzed for the expression of two herpes virus-encoded miRNAs by real-time polymerase chain reaction (PCR) and immunocytochemistry using nanoparticles as molecular beacons. RESULTS: Real-time reverse transcription-PCR results revealed significantly higher expression of hsv1-miR-H18 and hsv2-miRH9- 5p in surrounding noncancerous and CaP tissues than that in BPH tissue (each comparison, P<0.001). Of note, these miRNA were expressed equivalently in the CaP tissues and surrounding noncancerous tissues. Moreover, immunocytochemistry clearly demonstrated a significant enrichment of both hsv1-miR-H18 and hsv2-miR-H9 beacon-labeled cells in CaP and surrounding noncancerous tissue compared to that in BPH tissue (each comparison, P<0.05 for hsv1-miR-H18 and hsv2- miR-H9). CONCLUSIONS: These results suggest that increased expression of hsv1-miR-H18 and hsv2-miR-H95p might be associated with tumorigenesis in the prostate. Further studies will be required to elucidate the role of these miRNAs with respect to CaP and herpes viral infections.

Urinary Nucleic Acid TSPAN13-to-S100A9 Ratio as a Diagnostic Marker in Prostate Cancer

The potential use of urinary nucleic acids as diagnostic markers in prostate cancer (PCa) was evaluated. Ninety-five urine samples and 234 prostate tissue samples from patients with PCa and benign prostatic hyperplasia (BPH) were analyzed. Micro-array analysis was used to identify candidate genes, which were verified by the two-gene expression ratio and validated in tissue mRNA and urinary nucleic acid cohorts. Real-time quantitative polymerase chain reaction (qPCR) was used to measure urinary nucleic acid levels and tissue mRNA expression. The TSPAN13-to-S100A9 ratio was selected to determine the diagnostic value of urinary nucleic acids in PCa (P = 0.037) and shown to be significantly higher in PCa than in BPH in the mRNA and nucleic acid cohort analyses (P < 0.001 and P = 0.013, respectively). Receiver operating characteristic (ROC) analysis showed that the area under the ROC curve was 0.898 and 0.676 in tissue mRNA cohort and urinary nucleic acid cohort, respectively. The TSPAN13-to-S100A9 ratio showed a strong potential as a diagnostic marker for PCa. The present results suggest that the analysis of urine supernatant can be used as a simple diagnostic method for PCa that can be adapted to the clinical setting in the future.

Urinary MicroRNAs of Prostate Cancer: Virus-Encoded hsv1-miRH18 and hsv2-miR-H9-5p Could Be Valuable Diagnostic Markers / 대한배뇨장애요실금학회지

PURPOSE: MicroRNAs (miRNAs) in biological fluids are potential biomarkers for the diagnosis and assessment of urological diseases such as benign prostatic hyperplasia (BPH) and prostate cancer (PCa). The aim of the study was to identify and validate urinary cell-free miRNAs that can segregate patients with PCa from those with BPH. METHODS: In total, 1,052 urine, 150 serum, and 150 prostate tissue samples from patients with PCa or BPH were used in the study. A urine-based miRNA microarray analysis suggested the presence of differentially expressed urinary miRNAs in patients with PCa, and these were further validated in three independent PCa cohorts, using a quantitative reverse transcriptionpolymerase chain reaction analysis. RESULTS: The expression levels of hsa-miR-615-3p, hsv1-miR-H18, hsv2-miR-H9-5p, and hsa-miR-4316 were significantly higher in urine samples of patients with PCa than in those of BPH controls. In particular, herpes simplex virus (hsv)-derived hsv1-miR-H18 and hsv2-miR-H9-5p showed better diagnostic performance than did the serum prostate-specific antigen (PSA) test for patients in the PSA gray zone. Furthermore, a combination of urinary hsv2-miR-H9-5p with serum PSA showed high sensitivity and specificity, providing a potential clinical benefit by reducing unnecessary biopsies. CONCLUSIONS: Our findings showed that hsv-encoded hsv1-miR-H18 and hsv2-miR-H9-5p are significantly associated with PCa and can facilitate early diagnosis of PCa for patients within the serum PSA gray zone.

Lower Levels of Human MOB3B Are Associated with Prostate Cancer Susceptibility and Aggressive Clinicopathological Characteristics

Mps one binder (MOB) proteins are integral components of signaling pathways that control important cellular processes, such as mitotic exit, centrosome duplication, apoptosis, and cell proliferation. However, the biochemical and cellular functions of the human MOB (hMOB) protein family remain largely unknown. The present study investigated the association between hMOB3B expression and clinicopathological characteristics of prostate cancer (PCa).Study subjects included 137 PCa patients and 137 age-matched benign prostatic hyperplasia (BPH) patients. hMOB3B expression was estimated using real-time PCR and compared with clinicopathological parameters of PCa. hMOB3B mRNA expression was significantly lower in PCa tissues than in BPH control tissues (P or =10 ng/mL), a Gleason score> or =8, and metastatic disease (any T, N+/M+) than in those with low PSA levels, a low Gleason score, and non-metastatic disease (each P<0.05). In conclusion, low levels of hMOB3B are closely associated with aggressive clinicopathologic features in patients with PCa. Our results suggest that hMOB3B may act as a tumor suppressor in human PCa.

Novel Combination Markers for Predicting Survival in Patients with Muscle Invasive Bladder Cancer: USP18 and DGCR2

We performed gene expression profiling in bladder cancer patients to identify cancer-specific survival-related genes in muscle invasive bladder cancer (MIBC) patients. Sixty-two patients with MIBC were selected as the original cohort and another 118 MIBC patients were chosen as a validation cohort. The expression of USP18, DGCR2, and ZNF699 genes were measured and we analyzed the association between gene signatures and survival. USP18 and DGCR2, were significantly correlated to cancer-specific death (P=0.020, P=0.007, respectively). Cancer-specific survival in the low USP18 or DGCR2 expression group was significantly longer than the high expression group (P=0.018, P=0.006, respectively). In multivariate Cox regression analysis, a combination of USP18 and DGCR2 mRNA expression levels were significant risk factors for cancer-specific death (HR, 2.106; CI, 1.043-4.254, P=0.038). Overall survival and cancer-specific survival rates in the low-combination group were significantly longer than those in the high-expression group (P=0.001, both). In conclusion, decreased expressions of USP18 and DGCR2 were significantly associated with longer cancer-specific survival, and also the combination of two genes was correlated to a longer survival for MIBC patients. Thus, the combination of USP18 and DGCR2 expression was shown to be a reliable prognostic marker for cancer-specific survival in MIBC.

Decreased DBC1 Expression Is Associated With Poor Prognosis in Patients With Non-Muscle-Invasive Bladder Cancer

PURPOSE: The deleted in bladder cancer 1 (DBC1) gene is located within chromosome 9 (9q32-33), a chromosomal region that frequently shows loss of heterozygosity in bladder cancer (BC). It is suspected that it acts as a tumor suppressor gene, but its prognostic value remains unclear. The aim of the present study was to investigate the value of DBC1 as a prognostic marker in BC. MATERIALS AND METHODS: The expression of DBC1 was determined by real-time polymerase chain reaction analysis in 344 patients with BC (220 non-muscle-invasive BC [NMIBC] and 124 muscle-invasive BC [MIBC]) and in 34 patients with normal bladder mucosa. The results were compared with clinicopathologic parameters, and the prognostic value of DBC1 was evaluated by Kaplan-Meier analysis and a multivariate Cox regression model. RESULTS: DBC1 expression was significantly decreased in patients with MIBC compared with those diagnosed with NMIBC (p=0.010). Patients with aggressive tumor characteristics had lower DBC1 expression levels in NMIBC (each, p<0.05). By multivariate Cox regression analysis, low DBC1 expression was a predictor of progression to MIBC (hazard ratio, 7.104; p=0.013). Kaplan-Meier estimates revealed a significant difference in tumor recurrence, progression to MIBC, and cancer-specific survival depending on the level of DBC1 expression in NMIBC (log-rank test, each, p<0.05). CONCLUSIONS: The expression of DBC1 was associated with tumor aggressiveness, progression to MIBC, and survival in NMIBC. Our results suggest that DBC1 expression can be a useful prognostic marker for patients with NMIBC.

Clinical Flow and Outcomes in Patients with Malignant Spinal Cord Compression in Korea / 대한내과학회지

BACKGROUND/AIMS: We investigated the process from the development of symptoms to treatment and analyzed the clinical characteristics, treatment outcomes, and prognostic factors related to the treatment response and survival of patients with malignant spinal cord compression (SCC). METHODS: This study retrospectively reviewed the medical records of 56 patients diagnosed with metastatic SCC using magnetic resonance imaging (MRI) from January 2002 to December 2011. RESULTS: The median age of the patients was 59.5 years, and the most common origin of metastatic SCC was lung cancer. The median interval from symptom development to visiting the hospital was 7 days, and the median interval from admission to the date of clinical diagnosis was 0 days. The median interval from clinical diagnosis to the date of MRI or therapy was 1 or 4 days, respectively. Twenty-six patients (46.4%) had ambulation dysfunction at initial presentation, and 33 patients (61.1%) had ambulation dysfunction after radiotherapy or surgery. The rate of patients regaining walking ability was 17.6% with radiotherapy and 25% with surgery. In univariate analysis, good performance status, ambulatory function, and autonomic function before therapy were favorable predictors of ambulatory function after treatment in all patients. No significant factor was found in multivariate analysis. Median overall survival (OS) was 67 days, and the significant factors for survival by multivariate analysis were performance status and the presence of prostate cancer. CONCLUSIONS: The therapeutic response of ambulatory function and OS in malignant SCC is very poor. Multidisciplinary communication is required for the prompt and optimal management of patients with malignant SCC.

GSTM1 Tissue Genotype as a Recurrence Predictor in Non-muscle Invasive Bladder Cancer

Tissue genotyping is more useful approach than using blood genomic DNA, which can reflect the effects of the somatic mutations in cancer. Although polymorphisms in glutathione S-transferase (GST) have been associated with the risk of bladder cancer (BC) development, few reports provide information about the prognosis of BC. We investigated glutathione S-transferase mu (GSTM1) and glutathione S-transferase theta (GSTT1) genotypes using genomic DNA from primary 165 BC tissue samples to assess the association with disease prognosis. DNA samples from tumor were analyzed by multiplex polymerase chain reaction (PCR). The results were compared with clinicopathological parameters. The prognostic significance of the GSTs was evaluated by Kaplan-Meier and multivariate Cox regression model. Kaplan-Meier estimates revealed significant differences in time to tumor recurrence according to the GSTM1 tissue genotype (P = 0.038) in non-muscle invasive bladder cancer (NMIBC). Multivariate Cox regression analysis also revealed that the tissue GSTM1 genotype (hazards ratio [HR]: 0.377, P = 0.031) was an independent predictor of bladder tumor recurrence in NMIBC. This identification of GSTM1 tissue genotype as a prognosticator for determining recurrence in NMIBC should prove highly useful in a clinical setting.

Prostate Size Correlates with Fasting Blood Glucose in Non-Diabetic Benign Prostatic Hyperplasia Patients with Normal Testosterone Levels

We evaluated the correlations between BMI, fasting glucose, insulin, testosterone level, insulin resistance, and prostate size in non-diabetic benign prostatic hyperplasia (BPH) patients with normal testosterone levels. Data from 212 non-diabetic BPH patients with normal testosterone levels, who underwent transurethral resection of the prostate (TURP) due to medical treatment failure, were evaluated retrospectively. Patients with prostate specific antigen (PSA) levels of > or = 3 ng/mL underwent multicore transrectal prostate biopsy before TURP to rule out prostate cancer. Patients with diabetes mellitus (DM) or serum testosterone levels of 0.05). Testosterone level inversely correlated with BMI (r = -0.327, P 0.05). Upon multiple adjusted linear regression analysis, prostate size correlated with elevated PSA (P < 0.001) and increased fasting glucose levels (P = 0.023). In non-DM BPH patients with normal testosterone levels, fasting glucose level is an independent risk factor for prostate hyperplasia.

A Comprehensive Dosimetric Analysis of Inverse Planned Intensity Modulated Radiation Therapy and Multistatic Fields Technique for Left Breast Radiotherapy / 대한방사선종양학회지

PURPOSE: This aim of this study is to analyze the dosimetric difference between intensity-modulated radiation therapy (IMRT) using 3 or 5 beams and MSF in the radiotherapy of the left breast. MATERIALS AND METHODS: We performed a comparative analysis of two radiotherapy modalities that can achieve improved dose homogeneity. First is the multistatic fields technique that simultaneously uses both major and minor irradiation fields. The other is IMRT, which employs 3 or 5 beams using a fixed multileaf collimator. We designed treatment plans for 16 early left breast cancer patients who had taken breast conservation surgery and radiotherapy, and analyzed them from a dosimetric standpoint. RESULTS: For the mean values of V95 and dose homogeneity index, no statistically significant difference was observed among the three therapies. Extreme hot spots receiving over 110% of the prescribed dose were not found in any of the three methods. A Tukey test performed on IMRT showed a significantly larger increase in exposure dose to the ipsilateral lung and heart than multistatic fields technique (MSF) in the low-dose area, but in the high-dose area, MSF showed a slight increase. CONCLUSION: In order to improve dose homogeneity, the application of MSF, which can be easily planned and applied more widely, is considered an optimal alternative to IMRT for radiotherapy of early left breast cancer.

Furfural from Pine Needle Extract Inhibits the Growth of a Plant Pathogenic Fungus, Alternaria mali

The antifungal effect of pine needle extract prepared by a distinguishable extraction method and the dry distillation method, was examined. The effect of this extract itself was insignificant. The chemical components of pine needle extract were then investigated by gas chromatographic analysis, and four chemical components, acetol, furfural, 5-methyl furfural, and terpine-4-ol, were identified. The antifungal effects of those four chemical components against Alternaria mali (A. mali), an agent of Alternaria blotch of apple, were then examined. It was observed that the minimum inhibitory concentrations (MICs) were 6.25, 0.78, 0.78, and 12.5 (mg/ml) of acetol, furfural, 5-methyl furfural, and terpine-4-ol, respectively. MICs of furfural and 5-methyl furfural had the same order of magnitude as that of an antifungal agrochemical, chlorothalonil. Although furfural itself can not be completely substituted for an antifungal agrochemical, a partial mixture of furfural and antifungal agrochemical may be used as a substitute. The use of agrochemicals for the prevention of plant disease caused by pathogenic fungus such as A. mali could be partially reduced by the application of this mixture.