The hypermethylation of p16 gene exon 1 and exon 2: potential biomarkers for colorectal cancer and are associated with cancer pathological staging.

BACKGROUND: Tumor suppressor gene p16 promoter hypermethylation has been widely studied in colorectal cancer (CRC), yet its clinicopathological significance remains controversial. The methylation alterations of other regions within p16 gene are still rarely researched. The present study aimed to explore the methylation changes of p16 gene body in CRC and to find whether they were associated with clinicopathological staging of CRC. METHODS: Paired colorectal cancer tissues and corresponding adjacent normal tissues from 30 CRC patients were collected. The methylation levels of two CpG islands within p16 gene body, exon 1 and exon 2, were accurately assessed simultaneously by a LC-MS/MS method. The p16 protein expressions were assessed by immunohistochemistry assay. Statistical analyses were carried out using SPSS 17.0 software. Heat-map analysis was carried out by HemI 1.0 software. RESULTS: In the present study, CRC tissues showed more highly methylated than adjacent normal tissues at both CpG islands of p16 gene. And exon 2 hypermethylation was higher and more frequent than exon 1. The ROC curve analysis showed that the simultaneous use of both indicators had excellent sensitivity and specificity for distinguishing CRC tissues and adjacent normal tissues. Following, the methylation level of p16 exon 1/2 was negatively related to p16 protein expression. Further correlation analysis revealed that p16 exon 1 hypermethylation was associated with N/Dukes staging (p = 0.033), and p16 exon 2 hypermethylaiton was associated with T staging (p = 0.035). CONCLUSIONS: The p16 gene body was remarkably hyper-methylated in CRC tissues and associated with p16 protein expression and cancer clinicopathological staging. The combination of p16 exon 1 and exon 2 could better reflect the overall methylation status of p16 gene body and provide potential biomarkers of CRC.

Distinct differences in serum eicosanoids in healthy, enteritis and colorectal cancer individuals.

BACKGROUND: Eicosanoids as inflammatory mediators take part in the regulation of disease progression. However, the application of serum eicosanoid in disease progression identification was still uncertain. METHODS: Serum from 52 healthy volunteers, 34 enteritis patients and 55 colorectal cancer (CRC) patients were collected. Ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was used to analyze the change of serum eicosanoids. RESULTS: Of 158 eicosanoids, we found that lower levels of anti-inflammatory eicosanoids 13-HOTrE, 9-HOTrE, DHA, 11-HETE and 12-HHT were observed in enteritis and CRC group compared with healthy group, meanwhile the content of 5-iPF2α-VI as oxidative stress mediator in enteritis and CRC group was greater than that in healthy groups. Moreover, 9-HODE, 13-HODE, 12,13-diHOME, 8-HETE and 15-HETE were dramatically decrease in CRC group compared with non-CRC group. Additionally, the change of 5-, 12- and 15-HETE content in serum sample was associated with progression from healthy to enteritis, finally to CRC. No significant difference between serum eicosanoids and the expression of CerbB-2 and Ki67 were observed. CONCLUSION: Serum eicosanoids might be used as a possible biomarker for identifying among health, enteritis and CRC.

Denaturation manner of sarcoplasmic proteins in Pale, Soft and Exudative meat determines their positive impacts on myofibrillar water-holding capacity.

The aim of the study was to investigate the denaturation manner of sarcoplasmic proteins (SP) under PSE condition to explain their positive impacts on water-holding compacity. We found that the SP precipitation under PSE-like condition (pH 5.5, 40 °C) and heating conditions (pH 5.5, 7.0, 8.0, 55 °C) were similar, but the myofibrillar water-holding capacity was improved only under PSE-like condition (pH 5.5, 40 °C). To understand the denaturation mechanism of SP, their physicochemical properties were examined. Results demonstrated that PSE-denaturation and heat-denaturation of SP were two different processes. At pH 7.0 and 8.0, the unfolding of SP due to temperature elevation did not alter the overall net surface negative charges but only increased hydrophobicity, whereas at pH 5.5, the net surface positive charges and hydrophobicity increased dramatically. We hypothesized that in PSE meat, denatured SP became highly positively charged and hydrophobic and easier to bind to the negatively charged MF, which is related to the improvement on water-holding capacity.

Hierarchical bioresponsive nanocarriers for codelivery of curcumin and doxorubicin.

Hierarchical responsive nanocarriers have received much attention for targeted delivery of chemotherapeutics. In this study, we designed pH and redox dual-stage responsive nanocarriers in the different delivery stages for co-delivery phosphorylated curcumin (p-Cur) with doxorubicin (Dox). The MSNs nanocarriers were functionalized via specific cleavable PEGylation and hydrogel coating crosslinked by disulfide bonds: MSNs as core load Dox; p-Cur encapsulated in hydrogel coating. In blood circulation, PEGylation endow the nanocarriers with long time during blood circulation; while in tumor tissue, PEG shells could be cleaved due to the pH-sensitive bond and expose the cationic hydrogel coating to improve cell uptake; while inside tumor cells, hydrogel coating could be cleaved due to the GSH and release the drugs. The results showed that the dual-responsive shells endowed the nanocarriers with tumor extracellular pH-triggered cell uptake and specific cancer cell target release. The synergistic effects of the p-Cur and Dox enhanced cellular apoptosis in Hela cells.

The Anticancer Role of Omega-3 Polyunsaturated Fatty Acids was Closely Associated with the Increase in Genomic DNA Hydroxymethylation.

BACKGROUND: Omega-3 polyunsaturated fatty acids (omega-3 PUFAs) have significant multiple antitumor roles. However, whether epigenetic DNA hydroxymethylation enrolls in the anticancer process of omega- 3 PUFAs is still not clear yet. OBJECTIVE: To expound the interaction between the anti-tumor role of omega-3 PUFAs and the DNA demethylation pathway and thus provide a firm foundation for deepening our understanding on anticancer mechanism of omega-3 PUFAs. METHODS: Colorectal Cancer (CRC) model rats were induced to generate tumor by N-methyl-N-nitrosourea and their counterparts treated with omega-3 PUFAs during the induction. The blood samples from different treatment groups of rats [Normal Control group (NC), colorectal cancer model group (CRC) and omega-3 PUFAs Medication Group (MG)] were used as experimental materials. Genomic 5-hydroxymethylocytosine (5hmC) content was quantified using LC-MS/MS, and the expression of ten-eleven translocation dioxygenase 1 (TET1), catalyzing the generation of 5hmC, was also evaluated by quantitative real-time PCR. RESULTS: We observed lower tumor incidence and small tumor size in MG group when compared with CRC group, supporting the effective anticancer role of omega-3 PUFAs. Due to the formation of CRC, 5hmC level was dramatically dropped in CRC group when compared with the NC group. Notably, 5hmC percentage in MG group remarkably increased close to NC group and was significantly higher than that in the CRC group. Consistent alteration pattern of TET1 expressions in mRNA was also observed in the tested groups of rats. CONCLUSION: The anticancer effect of omega-3 PUFAs was positively correlated with global 5hmC accumulation and TET1 expression, suggesting DNA hydroxymethylation pathway was factually involved in the anticancer process of omega-3 PUFAs.

Quantification of DNA methylation and hydroxymethylation in Alzheimer's disease mouse model using LC-MS/MS.

Ambiguous alteration patterns of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) involved in Alzheimer's disease (AD) obstructed the mechanism investigation of this neurological disorder from epigenetic view. Here, we applied a fully quantitative and validated LC-MS/MS method to determine genomic 5mC and 5hmC in the brain cortex of 3 month-aged (12, 15, and 18 month) AD model mouse and found significant increases of 5mC and 5hmC levels in different months of AD mouse when compared with age-matched wild-type control and exhibited rising trend from 12-month to 18-month AD mouse, thereby supporting genomic DNA methylation and hydroxymethylation were positively correlated with developing AD.

Screening of exon methylation biomarkers for colorectal cancer via LC-MS/MS strategy.

The identification of biomarkers would be of benefit for the diagnosis and treatment of colorectal cancer. DNA methylation in specific genomic regions, which had shown strongly association with disease genotypes, was an effective indicator to reveal the occurrence and development of cancers. To screen out methylation biomarkers for colorectal cancer (CRC), genomic DNA was isolated from colorectal cancerous and corresponding cancer-adjacent tissues collected from 30 CRC patients and then bisulfite-converted. The exon regions of 5 targeted genes (CNRIP1, HIC1, RUNX3, p15, and SFRP2) were amplified by using nested polymerase chain reaction with specific primers, and the amplicon was purified and hydrolyzed. The methylation levels of these specific regions were detected by liquid chromatography tandem mass spectrometry (LC-MS/MS). The results showed that 5 targeted exon regions were successfully amplified and confirmed by sequencing. The methodological validations indicated that LC-MS/MS was highly sensitive and accurate. The methylation levels of CNRIP1 and RUNX3 were remarkably high in CRC tissues with statistical difference when compared with corresponding cancer-adjacent individuals, while that of HIC1, p15, and SFRP2 had no difference between 2 subjects. These findings supported CNRIP1 and RUNX3 as potential DNA methylation biomarkers for CRC diagnosis and treatment, and our LC-MS/MS approach exhibited great advantages in the identification of regional DNA methylation biomarkers.

A quantitative method for detecting DNA methylation over targeted genomic regions using isotope dilution liquid chromatography tandem mass spectrometry.

Aberrant DNA methylation is associated with various diseases. Quantitative analysis of regional DNA methylation levels of some specific genes would aid in diseases diagnosis and risk stratification. In this study, we developed a robust method for detecting DNA methylation level over targeted genomic regions using nucleobases quantification in bisulfite amplicons by isotope dilution liquid chromatography tandem mass spectrometry coupled with a simple equation. This method had wide detection range (from 0% to 100% methylation) and high accuracy while more time-saving compared to clonal bisulfite sequencing method. The application for clinical tissue samples showed good applicability and cost effectiveness. This analytical method is suitable for quantifying average DNA methylation level over targeted genomic regions and expected to be a useful tool for detecting DNA methylation biomarkers.