Effects of dietary Se, green tea and the combination of Se and green tea on <i>SFRP5</i> and <i>Cyclin D1</i> mRNA expression (tumor study).

<p>Quantitative RT-PCR analysis of expression of <i>SFRP5</i> (A) and <i>Cyclin D1</i> (C) mRNA in AOM-untreated normal colons and AOM-induced tumors (n = 6); expression of <i>SFRP5</i> (B) and <i>Cyclin D1</i> (D) mRNA from the control, green tea, Se and the combination diet (n = 6) in AOM-treated normal-appearing crypts. Significant repression of <i>SFRP5</i> and marked increase <i>Cyclin D1</i> mRNA in AOM-induced colon tumors were noted compared with normal colons. <i>SFRP5</i> was significantly increased by the combination diet, Se alone and green tea alone. <i>Cyclin D1</i> was also significantly inhibited by the combination diet and Se alone, but not green tea. Statistical significance of dietary intervention between the groups was analysed by ANOVA, values with different superscripts in each column were statistically different (<i>P</i><0.05), Bars: mean ± SEM.</p

Effect of Se and green tea alone, and their combination on tumor incidence, multiplicity and size in AOM-treated rats.

<p>AOM, azoxymethane; Se, selenium; SEM, standard errors of mean.</p><p>Tumor study was undertaken by feeding animals with 4 different diets (25 rats per group), containing either control diet, green tea diet, Se diet and the combination diet. Animals received two weekly AOM (15 mg/kg) injections to induce tumor formation and killed 30 wk later.</p>#<p>No statistical analysis undertaken for adenocarcinomas due to the small numbers.</p><p>Values with different superscripts (a, b, c) in each column were statistically different (<i>P</i><0.05).</p

Effects of dietary Se, green tea and the combination of Se and green tea on β-catenin, DNMT1 and Ac-H3 expression (tumor study).

<p>A representative section of immunohistochemical staining β-catenin, DNMT1 and Ac-H3 in AOM-untreated normal crypts (A), AOM-treated normal-appearing crypts from the control, green tea, Se and the combination diet (B) and tumors; labelling index for β-catenin (membrane and nuclear positive cells) (n = 12) (C) and labelling index for DNMT1 and Ac-H3 (n = 12) (D). Western blot analysis of β-catenin, DNMT1 and Ac-H3 for colon samples (n  = 6) (E), expression of β-catenin and DNMT1 was normalized to that of β-actin and expression of Ac-H3 was normalized to that of H3, data was presented as precent of control (F). Increased strong β-catenin nuclear staining and DNMT1 overexpression were displayed in AOM-induced tumors, whereas weaker Ac-H3 expression was noted in colon tumors. β-catenin nuclear staining was significantly decreased by the combination diet and Se alone, but not green tea; DNMT1 was significantly decreased by the combination diet and green tea alone, but not Se; Ac-H3 nuclear staining was significantly increased by the combination diet and Se alone, but not green tea. Statistical significance of dietary intervention between the groups was analysed by ANOVA, values with different superscripts in each column were statistically different (<i>P</i><0.05), Bars: mean ± SEM.</p

Experimental design for evaluation of Se, green tea and the combination diet for chemopreventive effects against colonic carcinogenesis in an AOM-induced rat CRC model.

<p>ACF study (A): groups of rats (n = 15) were fed control, Se, green tea or diet containing Se and green at age of 5 wk. 2 wk later, rats were given 15 (mg/Kg/body weight) AOM, once a week for 2 wk. 8 wk after last AOM treatment, rats were euthanized and colons were evaluated for ACF; normal-appearing crypts were also examined for β-catenin, COX-2 and Ki-67 expression. Tumor study (B) : groups of rats (n = 25) were fed control, Se, green tea or diet containing Se and green at age of 5 wk. Rats were given two weekly AOM treatments similar to ACF study. 30 wk after AOM treatment, rats were euthanized and colons were historically evaluated for tumor outcomes; normal-appearing crypts were also examined for β-catenin, DNMT1, Ac-H3 as well as <i>SFRP5</i> and <i>Cyclin D1</i> expression.</p

Table_1_The Use of Circulating Tumor DNA for Prognosis of Gastrointestinal Cancers.docx

<p>Gastrointestinal cancers, including oesophageal, gastric and colorectal cancers (CRC) have high rates of disease recurrence despite curative resection. There are a number of recent studies that have investigated the use of circulating tumor DNA (ctDNA) for prognostic value in these cancers. We reviewed studies that had been published prior to March 2018 that assessed the prognostic values of ctDNA in patients with oesophageal and gastric cancers, gastrointestinal stromal tumors (GIST) and CRC. We identified 63 eligible clinical studies that focussed on recurrence and survival. Studies assessed investigated various ctDNA biomarkers in patients with different stages of cancer undergoing surgical resection, chemotherapy and no treatment. For oesophageal squamous cell carcinoma and oesophageal adenocarcinoma, methylation of certain genes such as APC and DAPK have been highlighted as promising biomarkers for prognostication, but these studies are limited and more comprehensive research is needed. Studies focusing on gastric cancer patients showed that methylation of ctDNA in SOX17 and APC were independently associated with poor survival. Two studies demonstrated an association between ctDNA and recurrence and survival in GIST patients, but more studies are needed for this type of gastrointestinal cancer. A large proportion of the literature was on CRC which identified both somatic mutations and DNA methylation biomarkers to determine prognosis. ctDNA biomarkers that identified somatic mutations were more effective if they were personalized based on mutations found in the primary tumor tissue, but ctDNA methylation studies identified various biomarkers that predicted increased risk of recurrence, poor disease free survival and overall survival. While the use of non-invasive ctDNA biomarkers for prognosis is promising, larger studies are needed to validate the clinical utility for optimizing treatment and surveillance strategies to reduce mortality from gastrointestinal cancers.</p

Composition of experimental diets (g/100 g diet).

<p>The experimental diets consisted of a modified AIN-76A diet achieved by adding 19% sunflower oil and 20% protein.</p>#<p>Milk protein was used as source of protein for control diet and green tea diet</p>†<p>Se-enriched milk protein was used as source of protein and Se for Se diet and the combination diet.</p>‡<p>No Se was included in mineral mix in the diets.</p

Effects of dietary Se, green tea and the combination of Se and green tea on β-catenin, COX-2 and Ki-67 expression (ACF study).

<p>A representative section of immunohistochemical staining of β-catenin, Ki-67and COX-2 in AOM-untreated normal crypts (A), AOM-treated normal-appearing crypts from control, green tea, Se and the combination diet (B); labelling index for β-catenin (membrane and nuclear positive cells, calculated as the number of positive cells per crypt column divided by the total number of cells and multiplied by 100 (n = 12) (C); labelling index for COX-2 and Ki-67 (n = 12) (D). β-catenin and Ki-67 nuclear staining was significantly decreased by the combination diet and Se alone, but not green tea. COX-2 cytoplasmic staining was significantly decreased by green tea, Se and the combination diet. Statistical significance of dietary intervention between the groups was analysed by ANOVA, values with different superscripts in each column were statistically different (<i>P</i><0.05), Bars: mean ± SEM.</p

Effect of Se alone, green tea alone, and their combination on the formation of AOM-induced ACF in rats.

<p>AOM, azoxymethane; Se, selenium; ACF, aberrant crypt foci; SEM, standard errors of mean.</p><p>ACF study was undertaken by feeding rats with 4 experimental diets (15 rats per group), containing either control diet, green tea diet, Se diet and the combination diet. Animals received two weekly AOM (15 mg/kg) injections to induce ACF and were killed 8 wk later.</p>#<p>Total number of ACF was calculated as the sum of the small and large ACF.</p>†<p>Small ACF (Crypt multiplicity) was classified by the number of crypts per focus (1–3).</p>‡<p>Large ACF (Crypt multiplicity) was classified by the number of crypts per focus (≥4).</p><p>Values with different superscripts (a, b, c) in each column were statistically different (<i>P</i><0.05).</p

A Two-Gene Blood Test for Methylated DNA Sensitive for Colorectal Cancer

<div><p>Background</p><p>Specific genes are methylated with high frequency in colorectal neoplasia, and may leak into blood. Detection of multiple methylated DNA biomarkers in blood may improve assay sensitivity for colorectal cancer (CRC) relative to a single marker. We undertook a case-control study evaluating the presence of two methylation DNA markers, <i>BCAT1</i> and <i>IKZF1</i>, in circulation to determine if they were complementary for detection of CRC.</p><p>Methods</p><p>Methylation-specific PCR assays were developed to measure the level of methylated <i>BCAT1</i> and <i>IKZF1</i> in DNA extracted from plasma obtained from colonoscopy-confirmed 144 healthy controls and 74 CRC cases.</p><p>Results</p><p>DNA yields ranged from 2 to 730 ng/mL plasma (mean 18.6ng/mL; 95% CI 11-26 ng/mL) and did not correlate with gender, age or CRC status. Methylated <i>BCAT1</i> and <i>IKZF1</i> DNA were detected in respectively 48 (65%) and 50 (68%) of the 74 cancers. In contrast, only 5 (4%) and 7 (5%) controls were positive for <i>BCAT1</i> and <i>IKZF1</i> DNA methylation, respectively. A two-gene classifier model (“either or” rule) improved segregation of CRC from controls, with 57 of 74 cancers (77%) compared to only 11 of 144 (7.6%) controls being positive for <i>BCAT1</i> and/or <i>IKZF1</i> DNA methylation. Increasing levels of methylated DNA were observed as CRC stage progressed.</p><p>Conclusions</p><p>Detection of methylated <i>BCAT1</i> and/or <i>IKZF1</i> DNA in plasma may have clinical application as a novel blood test for CRC. Combining the results from the two methylation-specific PCR assays improved CRC detection with minimal change in specificity. Further validation of this two-gene blood test with a view to application in screening is now indicated.</p></div

Detection of methylated <i>BCAT1</i> and <i>IKZF1</i> DNA in plasma specimens.

<p><i>BCAT1</i> (top panel, black triangles) and <i>IKZF1</i> (bottom panel, white triangles) methylation specific assays were used to assess the appearance of circulating methylated <i>BCAT1</i> and <i>IKZF1</i> DNA in 218 plasma specimens including 144 healthy controls and 74 cancers. Mann-Whitney t-tests were performed on calculated positivity rates for each phenotypic class to derive the P-values. The calculated 95% confidence intervals (95% CI) are indicated.</p