Hughes et al. reviewed the existing literature of 640 potential relevant papers to summarize CIMPs in CRC. Although there are many lines of evidence that have been proposed as po tential biomarkers for CRC in humans, many researchers continue to research new CRC specific methylation mark ers. Recently, methylation chip array techniques have been widely used to identify new DNA methylation biomarkers in CRC. However, array data are needed to confirm other methods such as quantitative methylation polymerase chain reaction, methylation sensitive high resolution melting, and pyrosequencing. QMSP is a sensitive tool and offers quantitative analysis of DNA methylation status. Vincristine is a vinca alkaloid from the plant Cathar anthus roseus, and mainly arrests mitosis in metaphase by binding to tubulin dimers.
It is used as a chemo therapy drug for various types of cancers, including non Hodgkins lymphoma, acute lymphoblastic leu kemia, lung cancer, breast cancer, and CRC. Re cently, cyclophosphamide, vincristine, and prednisone chemotherapy was used to significantly kinase inhibitor FH535 improve overall survival and progression free survival in primary colonic lymphoma patients. There was one report that low concentration of vincristine reduced the meth ylated cytosine in human lung adenocarcinoma cells. However, the DNA methylating based effects of vincristine are still unknown for methylation marker genes in CRC. In this study, to identify new hypermethylated candi date genes in CRC patients, we analyzed methylation profiles using bead chip array based technology and QMSP.
In addition, to identify methylation based thera peutic target inhibitor genes, the demethylating effect of vincris tine was examined using 21 hypermethylated candidate genes and 18 CIMP markers. Correlations between meth ylation status and mRNA expression were analyzed by reverse transcription PCR. Methods Tissues Thirty one pairs of colorectal cancer tissues and adjacent normal tissues and 10 normal colon tissues were obtained from the Department of Colorectal Sur gery, Korea University Medical Center. The characteris tics of each subject are summarized in Table 1. This study was approved by the institutional review board of Korea University and informed consent was obtained. The diagnosis of CRC tis sues was acquired from pathology reports, the institu tional review board, and histological evaluations.
Fresh tissue samples were frozen in liquid nitrogen after resec tion and stored at 80 C. Cell lines One normal colon cell line and three CRC cell lines were obtained from the American Type Culture Collection. CCD18Co cells were cultured in Eagles minimum essential medium and the three CRC cells were cultured in RPMI 1640 medium, all supplemented with 10% fetal bovine serum and 1% peni cillin streptomycin, and maintained at 37 C and 5% CO2 atmosphere.