HOXA9, ISL1 and ALDH1A3 methylation patterns as prognostic markers for nonmuscle invasive bladder cancer: array-based DNA methylation and expression profiling.

DNA methylation patterns are associated with the development and prognosis of cancer. The aim of this study was to identify novel methylation markers for the prediction of patient outcomes using microarray analysis of DNA methylation and RNA expression patterns in samples from long-term follow-up patients with nonmuscle invasive bladder cancer (NMIBC). A total of 187 human bladder specimens were used for microarray array or pyrosequencing (PSQ) analyses: 6 normal controls (NC) and 181 NMIBC. Tumor-specific hypermethylated genes were selected from a data set comprising 24 matched microarray-based DNA methylation and gene expression profiles (6 controls and 18 NMIBC), and their clinical relevance was verified by quantitative PSQ analysis. The methylation status of Homeobox A9 (HOXA9), ISL LIM homeobox 1 (ISL1) and Aldehyde dehydrogenase 1 family, member A3 (ALDH1A3) was significantly associated with decreased gene expression levels and aggressive clinicopathological characteristics. Multivariate regression analyses showed that hypermethylation of these genes was an independent predictor of disease recurrence (HOXA9, ISL1 and ALDH1A3, either alone or in combination) and progression (ISL1 and ALDH1A3, either alone or in combination) (each p < 0.05). The results of this study suggest that these novel methylation markers are independent prognostic indicators in NMIBC patients, which may facilitate the assessment of disease recurrence and progression in NMIBC patients and inform clinical decision making regarding treatment.

Novel combination markers for predicting progression of nonmuscle invasive bladder cancer.

To identify prognostic markers in nonmuscle invasive bladder cancer (NMIBC), the combined effect of RUNX3 and MGC17624 for predicting NMIBC progression was assessed. RUNX3 promoter methylation was examined using methylation specific-polymerase chain reaction (MS-PCR). MGC17624 mRNA expression was evaluated by real-time PCR. Patients were divided into three groups according to the status of the two genes and the prognostic effects of these markers were evaluated. The median follow-up period was 57.8 months (range, 9.1-189.7). The mRNA expression level of MGC17624 was significantly lower in patients with positive RUNX3 methylation than in those with negative methylation (p = 0.047). Kaplan-Meier estimates showed significant differences in time-to-progression between the genetic combination predictors (log-rank test; p < 0.001). Patients with a poor predictive combination were at a significantly higher risk for progression [Hazard ratio (HR), 22.579] than patients with a good predictive combination in multivariate Cox regression analysis. In the subgroup analysis, a poor predictive combination accurately estimated progression in patients with intravesical therapy (HR, 20.081) and in those who experienced recurrence (HR, 54.233). Assessment of the status of RUNX3 and MGC17624 in combination was identified as a reliable method for predicting NMIBC progression.

Optimization of Accelerated Solvent Extraction of Ginsenosides from Cultivated Wild Ginseng Using Response Surface Methodology.

This study's aim is to apply response surface methodology (RSM) to model and optimize the accelerated solvent extraction (ASE) technique for extracting the sum of ginsenosides (Rg1, Rb1, and Rg3) and total ginsenosides from cultivated wild ginseng. To extract ginsenosides from cultivated wild ginseng, a new ASE-based method, combined with RSM modeling and optimization, was developed. The RSM method, which was based on a five-level, three-factor central composite design, was used to obtain the optimal combination of extraction conditions. Briefly, the optimal extraction conditions for the sum of ginsenosides (Rg1, Rb1, and Rg3) and total ginsenoside were as follows: 88.64% ethanol for each extraction solvent, 105.98°C and 129.66°C of extraction temperature, 28.77 and 15.92 min of extraction time, extraction pressure of 1,500 psi, nitrogen purge of 60 s, flush volume of 60%, and one extraction cycle. A 3D response surface plot and contour plot derived from the mathematical models were applied to obtain the optimal conditions. Under the above conditions, the experimental extraction yields of the sum of ginsenosides (Rg1, Rb1, and Rg3) and total ginsenoside content were 7.45 and 32.82 mg/g, respectively, which closely agrees with the model's prediction values.

Synthesis and Characterization of Phenolic Acid/Hydroxypropyl-ß-Cyclodextrin Inclusion Complexes.

The objective of this study was to synthesize and characterize inclusion complexes of phenolic acids with hydroxypropyl-ß-cyclodextrin (HP-ß-CD). The inclusion complexes were prepared by the freeze-drying method and characterized using a variety of analytical techniques, including ultraviolet-visible spectroscopy, Fourier transform infrared spectroscopy, differential scanning calorimetry, and X-ray diffractometry. The results of all these approaches indicated that phenolic acids were able to form an inclusion complex with HP-ß-CD, and the phenolic acids/HP-ß-CD inclusion compounds exhibited different spectroscopic features and properties based on the phenolic acids employed. The use of the HP-ß-CD matrix allowed for higher encapsulation efficiency and afforded capsules with distinct shapes.

Study of Flavonoid/Hydroxypropyl-ß-Cyclodextrin Inclusion Complexes by UV-Vis, FT-IR, DSC, and X-Ray Diffraction Analysis.

The objective of this study was to investigate characterization of inclusion complexes of flavonoids with hydroxypropyl-ß-cyclodextrin (HP-ß-CD). The inclusion complexes of flavonoids with HP-ß-CD was prepared by the freeze-drying method and its characterization was investigated by different analytical techniques including ultraviolet-visible spectroscopy, Fourier transform infrared spectroscopy (FT-IR), differential scanning calorimetry, and X-ray diffractometry. The catechin/HP-ß-CD complex exhibited the highest encapsulation efficiency (83.37%), followed by epicatechin/HP-ß-CD (81.51%), morin hydrate/HP-ß-CD (81.38%), and quercetin/HP-ß-CD (81.16%). The inclusion complexes of HP-ß-CD showed a decrease in the absorption of flavonoids with a small shift (≈2 nm) of the λmax, while similar to the characteristic absorption peak of flavonoids. However, the FT-IR spectra of the flavonoid/HP-ß-CD inclusion complexes did not display any features that were like the pure flavonoids, although the spectra were very similar to that of HP-ß-CD. The melting point of flavonoids disappeared, and the thermal properties of HP-ß-CD were altered following formation of the inclusion complex between flavonoids and HP-ß-CD, resulting in a shift in the melting peak.

Antioxidant Activity of Various Soluble Melanoidins Isolated from Black Garlic after Different Thermal Processing Steps.

To gain insight into the antioxidant activity of various soluble melanoidins isolated from black garlic after different thermal processing steps, the antioxidant activity was evaluated. Black garlic was produced in a ripening chamber using a programmed stepwise heating schedule as follows: Step 1, 90°C and 100% (RH) for 34 h; Step 2, 60°C and 60% RH for 6 h; Step 3, 75°C and 70% RH for 48 h; Step 4, 70°C and 60% RH for 60 h; Step 5, 65°C and 50% RH for 192 h. The melanoidins isolated from black garlic after the different thermal processing steps were divided into different melanoidin fractions, i.e., melanoidins, pure melanoidin, bound melanoidin compounds (BMC). The antioxidant activity of the melanoidins bound to low molecular weight compounds (BMC fraction) was generally higher than those of the pure melanoidins. Notably, the antioxidant activity of various soluble melanoidins differed according to the thermal processing steps. The results may be useful in predicting the behavior of various soluble melanoidins during thermal processing of garlic.

Enolization and racemization reactions of glucose and fructose on heating with amino-acid enantiomers and the formation of melanoidins as a result of the Maillard reaction.

This study investigated the enolization and racemization reactions of glucose and fructose on heating with amino acid enantiomers and the formation of melanoidins as a result of the Maillard reaction. The study measured reducing sugars and L- and D- amino acids using HPLC as an index for the amount of enolization of the sugars and isomerization of the amino acids. Additionally, the absorption of melanoidins was measured at different wavelengths (420, 450, 470, 490 nm); the UV-Vis spectra and the extinction coefficient were determined for the formation of melanoidins. Melanoidins were, rather arbitrarily, defined by a high-molecular-weight (HMW) if it was above a lower limit of 12.4 kDa, which was the nominal cut-off value in the dialysis system used. A remarkable enolization reaction of the sugars was observed in the course of the Maillard reaction. Especially, in the Fru/D-Asn model system, the degree of sugar enolization was more than in the other model systems. All of the FDAA (1-fluoro-2, 4-dinitrophenyl-5-L-alanine amide) amino acids were separated by TLC. The racemization of the amino acids was higher in the fructose-amino acids systems. Isomer formation was the highest in the Fru/D-Asn system. The L- and D- isomers showed different absorptions in the UV-Vis spectra, although these had similar shapes. The absorption of the melanoidins formed from glucose was higher than that formed from fructose. In particular, the sugar-asparagine system showed different characteristics according to the L- and D-isomers. The differences in the extinction coefficients of the melanoidins was significant (P<0.05), except for the sugar-lysine system.

Comparative Analysis of Ginsenoside Profiles: Antioxidant, Antiproliferative, and Antigenotoxic Activities of Ginseng Extracts of Fine and Main Roots.

The aim of this study was to compare ginsenosides profiles, and antioxidant, antiproliferative, and antigenotoxic activities of ginseng extract derived from fine and main roots. The result of the analysis showed a higher total content of ginsenoside in fine roots than in main roots; differences in levels between the different extracts were also confirmed. The oxygen radical absorbance capacity (ORAC) assay showed that H2O main root extract had a significantly higher activity than that from fine roots. MeOH and H2O extracts from the fine and main roots also exhibited stronger cellular antioxidant capacity 2,2'-azobis(2-amidinopropane) dihydrochloride-induced oxidative stress in HepG2 cells compared with the positive control. Through calculating the half-maximal inhibitory concentration values, the cytotoxicity of the main root extracts were ranked as follows: MeOH (6.1±1.2 µg/mL)> H2O (6.6±0.1 µg/mL)> ethanol (10.4±0.6 µg/mL); however, the cytotoxicity of all fine root extracts did not significantly differ. All the fine root extracts showed an inhibitory capacity against 4-hydroxynonenal-induced DNA damage, however only the MeOH extract of the main root showed a decrease in DNA damage. All three solvent extracts from the fine roots reduced DNA damage more in the H2O2-treated group, whereas only the MeOH and H2O extracts of the main roots produced a significant reduction. Levels of Rg3 ginsenoside were positively correlated with indices of the ORAC value, and total ginsenoside contents showed a negative correlation with DNA damage induced by H2O2. This study suggests that ginseng and the extraction solvent both affect levels of ginsenoside. Furthermore, the antioxidant potency of ginseng can be attributed to the content of some ginsenosides.

ZNF492 and GPR149 methylation patterns as prognostic markers for clear cell renal cell carcinoma: Array­based DNA methylation profiling.

The present study aimed to identify novel methylation markers of clear cell renal cell carcinoma (ccRCC) using microarray methylation analysis and evaluate their prognostic relevance in patient samples. To identify cancer­specific methylated biomarkers, microarray profiling of ccRCC samples from our institute (n=12) and The Cancer Genome Atlas (TCGA) database (n=160) were utilized, and the prognostic relevance of candidate genes were investigated in another TCGA dataset (n=153). For validation, pyrosequencing analyses with ccRCC samples from our institute (n=164) and another (n=117) were performed and the potential clinical application of selected biomarkers was examined. We identified 22 CpG island loci that were commonly hypermethylated in ccRCC. Kaplan­Meier analysis of TCGA data indicated that only 4/22 loci were significantly associated with disease progression. In the internal validation set, Kaplan­Meier analysis revealed that hypermethylation of two loci, zinc finger protein 492 (ZNF492) and G protein­coupled receptor 149 (GPR149), was significantly associated with shorter time­to­progression. Multivariate Cox regression models revealed that hypermethylation of ZNF492 [hazard ratio (HR), 5.44; P=0.001] and GPR149 (HR, 7.07; P<0.001) may be independent predictors of tumor progression. Similarly, the methylation status of these two genes was significantly associated with poor outcomes in the independent external validation cohort. Collectively, the present study proposed that the novel methylation markers ZNF492 and GPR149 could be independent prognostic indicators in patients with ccRCC.

Effect of reaction pH on enolization and racemization reactions of glucose and fructose on heating with amino acid enantiomers and formation of melanoidins as result of the Maillard reaction.

This study investigates the effect of reaction pH on enolization and racemization reactions of glucose and fructose on heating with amino acid enantiomers, can influence the formation of melanoidins as result of the Maillard reaction. Remarkable enolization reaction of sugars was observed in the course of the Maillard reaction. Especially, the degree of sugar enolization was increased as the pH levels increased, which was higher in fructose than glucose systems. Otherwise, enolization of sugars on heating with amino acid was higher in glucose than fructose systems. Formation of isomer in Glc/d-Lys, Fru/d-Asn and Fru/d-Lys were increased upon increase of pH levels. The relative amounts of isomers in Glc/l-Asn and Glc/d-Asn were decreased upon increase of pH levels. Browning development was dependent on the pH levels, being more significant for model systems apart from heated glucose solution alone. Browning development of fructose systems was higher than glucose-amino acid systems. The l- and d-isomers both showed different absorption in the UV-vis spectra and that these occur at similar shape. Every peak has a stable absorbance appeared in the range between 260 and 320nm, characteristic of melanoidins.

Antioxidant Activities of Selected Berries and Their Free, Esterified, and Insoluble-Bound Phenolic Acid Contents.

To explore the potential of berries as natural sources of bioactive compounds, the quantities of free, esterified, and insoluble-bound phenolic acids in a number of berries were determined. In addition, the antioxidant activities of the berries were determined using 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity, ferric reducing antioxidant power, and Trolox equivalent antioxidant capacity assays, in addition to determination of their metal ion chelating activities. Furthermore, several phenolic compounds were detected using high-performance liquid chromatography. Of the 6 tested berries, black chokeberry and blackberry exhibited the strongest antioxidant activities, and the various berry samples were found to contain catechin, caffeic acid, p-coumaric acid, epicatechin, vanillic acid, quercitrin, resveratrol, morin, naringenin, and apigenin. Moreover, the antioxidant activities and total phenolic contents of the fractions containing insoluble-bound phenolic acids were higher than those containing the free and esterified phenolic acids. The results imply that the insoluble-bound fractions of these berries are important natural sources of antioxidants for the preparation of functional food ingredients and preventing diseases associated with oxidative stress.

Investigation of Phenolic, Flavonoid, and Vitamin Contents in Different Parts of Korean Ginseng (Panax ginseng C.A. Meyer).

This study investigated the phenolic, flavonoid, and vitamin constituents in the main root, root hair, and leaf of ginseng. The total individual phenolic and flavonoid contents were the highest in the leaf, followed by the main root and root hair. Ferulic acid and m-coumaric acid were found to be the major phenolics in the main root and root hair, while p-coumaric acid and m-coumaric acid were the major phenolics in the leaf. Catechin was the major flavonoid component in the main root and root hair, while catechin and kaempferol were the major flavonoid components in the leaf. Pantothenic acid was detected in the highest quantity in the non-leaf parts of ginseng, followed by thiamine and cobalamin. Linolenic acid and menadione were the major components in all parts of ginseng.

Comparison of Ginsenoside Contents in Different Parts of Korean Ginseng (Panax ginseng C.A. Meyer).

The present study was conducted to investigate the ginsenoside profiles of the main root, root hair, and leaf of ginseng in order to demonstrate their possible application in medicine. The total ginsenoside content of the leaf was up to 12 times than that in the main root, and the content of protopanaxadiol groups was higher than that of protopanaxatriol groups in all the samples. The leaf was shown to contain high amounts of ginsenosides Rb3 and Rh1, whereas the main root contained large amounts of ginsenosides Rb1 and Rc. Moreover, Rb2, Rb3, and Rg1 were only detected in the root hair, leaf, and main root, respectively. The ginsenoside Re content of Panax ginseng leaf and root hair was 2.6~4 times higher than that of the main root. Therefore, the results indicate that the ginsenoside content of Panax ginseng is higher in the leaf and root hair, and lower in the main root.

RSPH9 methylation pattern as a prognostic indicator in patients with non-muscle invasive bladder cancer.

DNA methylation is a frequent and early epigenetic event with potential application as a biomarker for cancer detection and an indicator of disease evolution. The aim of the present study was to identify novel methylation markers for the prediction of patient outcomes using microarray analysis of DNA methylation in samples from long-term follow-up patients with non-muscle invasive bladder cancer (NMIBC). Candidate methylation markers were selected from our previously published genome-wide methylation profiles. The clinical relevance of candidate methylation markers was determined by quantitative pyrosequencing analysis of 136 human bladder specimens (8 normal controls and 128 NMIBCs). The reversibility of DNA methylation was examined by 5-Aza-CdR treatment in human bladder cancer cell lines. The methylation patterns of candidate markers were significantly associated with aggressive clinicopathological features. In multivariate regression analysis, hypermethylation of radial spoke head 9 homolog (RSPH9) was an independent predictor of disease recurrence (hazard ratio, 3.02; P=0.001) and progression (hazard ratio, 8.25; P=0.028). The methylation level of RSPH9 decreased with 5-Aza-CdR treatment and progressively increased in its absence in bladder cancer cell lines. RSPH9 methylation is an independent prognostic indicator in NMIBC patients, and could be of value for the assessment of disease recurrence and progression and for clinical decision-making regarding treatment.

Bone Flap Resorption Following Cranioplasty after Decompressive Craniectomy: Preliminary Report.

OBJECTIVE: Resorption of autologous bone flap grafts is a known long-term complication of cranioplasty following decompressive craniectomy (DC). We analyzed our data to identify risk factors for bone flap resorption (BFR) following cranioplasty. METHODS: A total of 162 patients who underwent cranioplasty following DC due to life-threatening elevated intracranial pressure between October 2003 and December 2012, were included in our investigation. Follow-up exceeded one year. RESULTS: BFR occurred as a long-term complication in 9 of the 162 patients (5.6%). The affected patients consisted of individuals who had undergone DC for traumatic brain injury (TBI; n=4), for subarachnoid hemorrhage (SAH; n=3), for cerebral infarction (n=1), and intracerebral hemorrhage (n=1). Logistic regression analysis identified no significant risk factors for BFR. CONCLUSION: TBI and SAH as initial diagnoses are more often associated with BFR than other diagnoses. This finding may influence future surgical decision making, especially in patients with possible risk factors for BFR. A prospective study with a large number of patients is needed to identify potential predictors of BFR such as bone flap sterilization and preservation.

Cytotoxic and anti-inflammatory effects of onion peel extract on lipopolysaccharide stimulated human colon carcinoma cells.

The present study investigated the cytotoxic activity of ethanol extract of onion peel (OPE) in HT-29 human colon carcinoma cells. High-performance liquid chromatography (HPLC) analysis was performed to determine the amounts of phenolic acids and flavonoids in OPE. In addition, the influence of OPE on antioxidant- and inflammation-associated gene expression was also determined in a model of lipopolysaccharide (LPS)-stimulated HT-29 cells. HPLC analysis showed that OPE contained well-known antioxidant compounds, including p-coumaric acid, vanillic acid, epicatechin, and morin. After incubation with OPE, HT-29 cells showed either a loss of normal nuclear architecture or detachability from each other. The cytotoxic effects of OPE on HT-29 cells were confirmed by MTT and LDH release assays. LPS-induced oxidative conditions effectively downregulated TNF-α mRNA expression in OPE pretreated HT-29 cells compared with cells only stimulated with LPS. In addition, the expression of heme oxygenase-1 (HO-1) and glutathione S-transferase (GSTs) detoxification genes (i.e., GSTM1, GSTT1, and GSTP1) was upregulated after treatment with LPS at sublethal concentrations. However, the LPS-induced mRNA expression of HO-1 and GSTs was significantly attenuated by treatment with OPE. Therefore, onion peel extract is a promising component of future nutraceuticals and value-added products.

Ras association domain family 1A: a promising prognostic marker in recurrent nonmuscle invasive bladder cancer.

UNLABELLED: The aim of this study was to investigate the value of RASSF1A methylation as a prognostic marker in bladder cancer. RASSF1A hypermethylation from 301 specimens of primary BC tissue was assessed using methylation-specific PCR. Among patients with recurrent NMIBC, RASSF1A methylation was identified as an independent predictor of cancer progression. INTRODUCTION: Aberrant methylation of promoter CpG islands is an important inactivation mechanism of tumor suppressors and tumor-related genes. Ras association domain family 1A (RASSF1A) promoter hypermethylation was shown to be associated with bladder cancer (BC), but its prognostic value remains unclear. The aim of the present study was to investigate the value of RASSF1A methylation as a prognostic marker in BC. MATERIALS AND METHODS: Primary BC tissues were obtained from 301 patients and included 186 specimens of nonmuscle invasive bladder cancer (NMIBC) and 115 specimens of muscle invasive bladder cancers (MIBC). RASSF1A hypermethylation was assessed using methylation-specific polymerase chain reaction (MS-PCR). The association between RASSF1A hypermethylation and clinicopathologic features, and the prognostic significance of RASSF1A hypermethylation were evaluated by Kaplan-Meier and multivariate Cox regression analyses. RESULTS: RASSF1A promoter hypermethylation was detected in 33.6% of BCs and occurred more frequently in MIBC (46.1%) than in NMIBC (25.8%) (P < .001). In NMIBC, RASSF1A methylation was associated with advanced tumor stage (P = .026) and high grade (P < .001). Among patients with recurrent NMIBC, RASSF1A methylation was associated with shorter time to progression by Kaplan-Meier analysis (log-rank test; P = .004) and identified as an independent predictor of cancer progression by multivariate Cox regression analysis (hazard ratio [HR], 8.559; P = .014). CONCLUSIONS: Our results suggest that methylated RASSF1A may be a potential prognostic marker in patients with recurrent NMIBC.

Tumorigenic and prognostic significance of RASSF1A expression in low-grade (WHO grade 1 and grade 2) nonmuscle-invasive bladder cancer.

OBJECTIVE: To investigate RASSF1A expression in an attempt to understand the effect of RASSF1A in low-grade, nonmuscle-invasive bladder cancer (NMIBC). METHODS: A total of 101 tumor tissues and normal donor-matched adjacent tissues from patients with primary low-grade NMIBC were selected. RASSF1A expression was measured by quantitative real-time polymerase chain reaction and is expressed as the ratio between the normal tissue level and the tumor tissue level (N/T ratio). RASSF1A promoter methylation was assessed using methylation-specific polymerase chain reaction. RESULTS: RASSF1A mRNA expression was significantly lower in patients with RASSF1A methylation than in those with no methylation (P = .046), and RASSF1A mRNA expression was lower in the tumor tissues than in the matched normal bladder mucosa (P < .001). The RASSF1A N/T ratio was greater in grade 2 and large tumors than in grade 1 and small tumors (P < .05 for each). Kaplan-Meier estimates revealed that the differences in the RASSF1A N/T ratio were associated with significant differences in the interval to tumor recurrence (P = .018) in low-grade NMIBC. Multivariate Cox regression analysis showed that the RASSF1A N/T ratio (hazards ratio 2.354, P = .015) was an independent predictor of recurrence. CONCLUSION: Decreased RASSF1A expression correlates with the transition from normal mucosa to bladder cancer and has prognostic value in low-grade NMIBC.

Runt-related transcription factor 3 methylation as a possible prognosticator in muscle-invasive bladder cancer.

This study sought to determine if runt-related transcription factor 3 (RUNX3) methylation could accurately predict the overall survival in patients with muscle invasive bladder cancer (MIBC). Forty-seven patients with a longer followup period (cohort 1; reported previously) were analyzed. Tumor samples (cohort 2; newly collected) were obtained from 139 MIBC patients. The prognostic significance of RUNX3 methylation was evaluated by Kaplan-Meier analysis and a multivariate Cox regression model. RUNX3 methylation affected the overall survival in cohort 1 (log-rank test; P=0.030). Among the patients in cohort 2, RUNX3 promoter methylation was observed in 93 of the 139 tumor samples (66.9%). Kaplan-Meier estimates showed a significant difference in overall survival according to RUNX3 methylation (P=0.020). By multivariate Cox regression analysis, positive RUNX3 methylation was an independent predictor of overall survival. These results suggest that RUNX3 promoter methylation is a significant prognostic factor for overall survival in MIBC patients.