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  • br Heparan sulfate O endosulfatase gene SULF encodes an


    Heparan sulfate 6-O-endosulfatase gene (SULF2) encodes an oncoprotein with heparin-degrading endosulfatase activity, which activates receptor tyrosine kinases and downstream pathways including MAPK, AKT, and WNT [33]. While the role of SULF2 methylation has been mostly unknown in CRC, SULF2 methylation 
    was associated with irinotecan sensitivity in patients with gastric cancer [32]. In addition, SULF2 silencing increased sensitivity to topoisomerase 1 inhibitors via increased expression of interferon-inducible genes, including ISG15, in non–small cell lung cancer [34].
    In this study, we aimed to investigate the association of treatment outcomes for irinotecan-based systemic chemotherapy with methyl-ation in CHFR, WRN, and SULF2 as well as CIMP status in patients with metastatic CRC.
    Patients and Methods
    Patients and Irinotecan-Based Systemic Chemotherapy
    Patients who underwent surgical resection of CRC at the National Cancer Center (NCC), Korea, from 2001 to 2004 were eligible for this retrospective biomarker study if the following criteria were met: pathologically confirmed diagnosis of colorectal adenocarcinoma, age ≥19 years, synchronous or metachronous metastasis, systemic chemotherapy with one of the irinotecan-containing regimens, tumor tissues available at NCC Tumor Bank/Pathology Department, and presence of evaluable lesion(s) before initiation of irinotecan-containing chemotherapy. This study protocol was reviewed and approved by the Institutional Review Board of NCC (IRB No: NCC2014-0075). The study was conducted in accordance with the recommendations of the Declaration of Helsinki for biomedical research involving human subjects.
    of each E-64-c of FOLFIRI or 7.5 mg/kg i.v. on day 1 of each cycle of XELIRI) or cetuximab (400 mg/m2 i.v. on day 1 and 250 mg/m2 on day 8 and weekly thereafter) was combined with the cytotoxic chemotherapy. Computed tomography was performed after every four cycles for biweekly regimens and after three cycles for three-weekly regimens during the chemotherapy period or earlier if disease progression was suspected. Disease progression was defined based on the computed tomographic findings.
    Methylation Analyses
    Analysis of DNA methylation was performed as described previously [35]. Genomic DNA samples from the tumor and adjacent normal tissue were bisulfite-modified using the EZ DNA methylation kit (Zymo Research, Orange, CA) and analyzed for methylation in five CIMP-specific CpG island loci (CACNA1G, IGF2, NEUROG1, RUNX3, SOCS1), as well as CHFR, WRN, and SULF2 using a methylation-specific polymerase chain reaction (MSP) method. Briefly, 1 μg of DNA was denatured using sodium hydroxide, modified by sodium bisulfite, treated again with sodium hydroxide, precipitated with ethanol, and resuspended in water. For each locus, two primer pairs were used for the MSP analysis; the first recognizes and anneals to methylated sequences only, whereas the second set anneals to and amplifies unmethylated alleles. The detailed primer information is provided in Supplementary material 1. The
    148 CHFR Promoter Methylation in Metastatic Colorectal Cancer Cha et al. Neoplasia Vol. 21, No. 1, 2019
    PCR products were then purified using the Wizard DNA purification resin (Promega, Madison, WI). Each PCR product was directly loaded on an 8% acrylamide gel, stained with ethidium bromide, and visualized under UV illumination. CIMP status was considered positive when at least three methylated promoters were identified and as negative when zero to two methylated promoters were identified.
    The Cancer Genome Atlas (TCGA) Data Analysis
    CHFR DNA methylation (Illumina Infinium HM27 bead array; HM27) and mRNA expression microarray) data from 223 colorectal adenocarcinoma samples from TCGA project were downloaded through cBioPortal (; accessed on Jun. 12, 2018).
    Cell Lines and Cell Culture
    Irinotecan HCl trihydrate was purchased from Selleckchem (Houston, TX). The DNA methylation inhibitor 5-aza-2′-deoxycy-tidine (5-Aza-CdR) was purchased from Sigma-Aldrich (St. Louis, MO). Stock solutions were prepared in dimethyl sulfoxide and stored at −20°C.
    Growth Inhibition Assays
    A colorimetric assay using the tetrazolium salt MTS was used to assess cell proliferation after treatment with irinotecan. Equivalent numbers of cells (5 × 103 cells/well) were incubated in 0.2 ml culture medium in each well. After 1, 2, and 3 days of culture, 0.1 mg MTS solution (Promega, Madison, WI) was added to each well followed by incubation at 37°C for a further 4 hours. Plates were centrifuged at 450×g for 5 minutes at room temperature, and the medium was removed. Dimethyl sulfoxide (0.15 ml) was added to each well to solubilize the crystals, and the plates were immediately read at 540 nm using a scanning multiwell spectrometer (Bio-Tek instruments Inc., Winooski, VT). The cell proliferation rate was obtained from three biological replicates, and all experiments were performed three times.