Role Of Mismatch Repair Protein In Estradiol-induced Colon Cancer Cells Apoptosis And Performance Of Methylated SEPT9 Test In Detecting Colorectal Neoplasm

Background and Aim:The incidence and mortality of colorectal cancer(CRC) has increased rapidly in China and has been a major public health threat in the past three decades. As a national strategy, the role of prevention and early detection in reducing the incidence and mortality of CRC has been focused on.Chemoprevention should be at the first line of defence against CRC. Epidemiological studies suggest estrogen to be protective against colon carcinogenesis, and this effect may be dependent on mismatch repair(MMR) status. The underlying mechanism remains unclear. The first part of our study investigated the roles of MMR proteins in apoptosis induced by estrogen in colon cancer cells.There is sufficient evidence to support the notion that screening and early detection reduce mortality due to CRC. While fecal occult blood tests(FOBT) are the most widely used CRC screening methods, the sensitivity of FOBT is low. Methylated Septin 9(SEPT9) is a novel blood-based biomarker for CRC, however, it has never been used in China. We evaluated the performance of the second generation SEPT9 assay for the detection of colorectal neoplasm, and compared it with fecal immunochemical test(FIT) in the second part of this study. Methods:The h MLH1-deficient HCT116 cells, h MSH2-deficient Lo Vo cells and paired MMR-proficient cells established by transient transfection with wild type c DNAs of h MLH1/h MSH2 were used. After treatment by estradiol, the cells viability and apoptosis were assessed. The expression of Bax, caspase-9 and caspase-3 were detected by western blot. The effects of overexpression of estrogen receptor β(ERβ) on apoptosis were observed too.A total of 135 patients with CRC, 169 with adenomatous polyps(84 advanced adenomas and 85 non-advanced adenomas), 81 with hyperplastic polyps and 91 healthy controls were included. The clinical status of all subjects was verified by colonoscopy. In all patients, peripheral blood samples were taken for SEPT9 testing using Epi pro Colon 2.0 test. For 177 patients, both SEPT9 and FIT were performed. Sensitivity and specificity for each test was calculated with 95% confidence intervals(CI) based on the exact binomial distribution. Mc Nemar’s test was used to compare the detection rates of SEPT9 and FIT for CRC and advanced adenoma. Results:After reexpression of h MLH1, estradiol could induce more notable apoptosis and loss of cell viability in h MLH1-proficient cells than in these h MLH1-deficient HCT116 cells. The increase of cleaved caspase-9 and caspase-3 induced by estradiol was more marked after transfection with h MLH1. Overexpression of h MSH2 and ERβ could induce apoptosis in HCT116 cells; however, the effects were independent of estradiol. Overexpression of h MLH1 upgraded estradiol-induced apoptosis related proteins even in h MSH2-deficient Lo Vo cells.The sensitivity and specificity of SEPT9 for CRC were 74.8%(95% CI: 67.0%-81.6%) and 87.4%(vs non-CRC, 95% CI: 83.5%-90.6%), respectively. The false positive rate of SEPT9(positive in healthy control and hyperplastic polyps) was 4.7%(95%CI: 2.2%-8.6%). SEPT9 was positive in 66.7% of stage I, 82.6% of stage II, 84.1% of stage III, and 100% of stage IV CRCs. The sensitivity of SEPT9 for advanced adenomas was 27.4%(95% CI: 18.7%-37.6%). The sensitivity and specificity of FIT for CRC was 58.0%(95% CI: 46.1%-69.2%) and 82.4%(95% CI: 74.4%-88.7%), respectively. SEPT9 detected 25 cancers that were missed by FIT, whereas FIT detected 12 cancers missed by SEPT9. SEPT9 showed better performance in CRC detection than FIT, but similar among advanced adenomas. Conclusions:The proteins of h MLH1 might enhance the estradiol-induced apoptosis in colonic cancer cells via caspases; however, the mismatch repair capacity of MMR proteins may not be required in h MLH1-dependent estrodial-induced cell death. MMR status should be assessed before hormone replacement therapy or future application of estrogen-based chemoprevention to maximize the benefit/risk ratio.With improved performance characteristics in detecting CRC than FIT, the second generation SEPT9 assay could play an important role in CRC screening and early detection. However, the sensitivity of SEPT9 for advanced adenomas was not better than FIT. The combination of SETP9 assay and FIT may represent a promising direction of future CRC screening.