(±)-Baclofen br Cancer Cell Line Encyclopedia CCLE Data
Cancer Cell Line Encyclopedia (CCLE) Data Analysis
Pharmacological profiling data for irinotecan was downloaded for 12 colorectal adenocarcinoma cell lines along with the DNA methylation data from the CCLE (http://portal.broadinstitute.org/ ccle; accessed on Jun. 13, 2018)
Establishment of CHFR-Overexpressing Cells Using Plasmid DNA Vector
The recombinant plasmid DNA human CHFR (target sequence: 5′-GCGATCGCACGCGT-3′) (RC228526) was purchased from OriGene (Rockville, MD). The recombinant plasmid was then transformed into competent Escherichia coli cells. The bacteria were cultured, and the recombinant plasmids were extracted and purified using PureLink HiPure Plasmid DNA Purification kits (Invitrogen, Carlsbad, CA). HCT-116 and SNU-C5 (±)-Baclofen were plated in six-well plates at a density of 3 × 105 cells per well and incubated overnight.
Cells were then transfected with the human CHFR vector or a blank control using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Transfection was verified by Western blot analysis.
Small Interfering RNA (siRNA)–Mediated Knockdown of CHFR
siRNA against CHFR and the control sequence were purchased from Qiagen (Chatsworth, CA). The sequence of the CHFR-specific siRNA was 5′-AACCAGAGGTTTGACATGGAA-3′, and AllStars Negative Control siRNA (catalog no. 1027281) was used as the control (nonspecific). siRNA transfection was performed using HiPerFect Transfection Reagent (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Briefly, 12 μl of 20 nM siRNA solution and 20 nM HiPerFect Transfection Reagent was incubated in 100 ml of serum-free RPMI 1640 medium for 10 minutes to facilitate complex formation. The resulting mixture (final concentration 5 nM) was added to SNU-81 and CaCo-2 cells (1 × 106) and incubated in a 60-mm tissue culture dish with 4 ml of RPMI 1640. The cells were then washed at 0, 24, 48, and 72 hours after transfection.
Briefly, cell homogenates containing equivalent amounts of protein were centrifuged at 4000×g, and the supernatant fractions were subjected to SDS-PAGE. Following electrophoresis, the proteins were transferred to polyvinylidene fluoride membranes (Millipore, Biller-ica, MA) and blocked by incubation for 2 hours at 4°C in 1% Tween 20-TBS buffer containing 1.5% nonfat dry milk (Bio-Rad, Hercules, CA) and 1 mM MgCl2. Membranes were then incubated for 2 hours at room temperature with primary antibodies against CHFR (Santa Cruz bio Technology, Santa Cruz, CA) or β-actin (Cell Signaling Technology, Beverly, MA). Next, the membranes were washed thrice for 15 minutes with blocking solution and incubated with diluted HRP-conjugated secondary antibody (SouthernBiotech, Birming-ham, AL) for 1 hour at room temperature. This was followed by washing with blocking solution (thrice for 15 minutes), incubation with WEST-ZOL plus chemiluminescence reagent (iNtRON Biotechnology, Seoul, Korea) for 1 minute, and exposure to film (Kodak Blue XB-1).
The primary aim of the present study was to determine the association of methylation in CHFR, WRN, and SULF2, as well as CIMP status, with time to progression (TTP) of irinotecan-based systemic chemotherapy in patients with metastatic CRC. In addition to TTP, OS and response were analyzed as indicators of treatment outcomes with irinotecan treatment. TTP was defined as the time interval from the date of treatment initiation to the date of disease progression. OS was calculated from the date of treatment initiation to the date of death from any cause. Categorical variables were compared using Pearson’s χ 2 test or Fisher’s exact test, and continuous variables were compared using Mann-Whitney test. The Kaplan-Meier method was used for estimating TTP and OS, and comparisons were made using log-rank tests. To adjust for baseline characteristics, we performed multivariate analyses with a Cox proportional hazard model using a forward conditional variable selection method. Age (continuous variable), sex, differentiation (well-differentiated to moderately differentiated vs. poorly differen-tiated), tumor location (proximal vs. distal), number of metastatic
Neoplasia Vol. 21, No. 1, 2019 CHFR Promoter Methylation in Metastatic Colorectal Cancer Cha et al. 149
organs (1-2 vs. ≥3), serum carcinoembryonic antigen (CEA) levels (≤5 vs. N5 ng/ml), BRAF mutation status, hMLH1/hMSH2 (proficient vs. deficient), and number of prior systemic chemotherapy lines in the metastatic setting were included as covariates based on previous studies on metastatic CRC . Two-sided P values b .05 were considered statistically significant. Statistical analyses for the clinical study were conducted using R-3.3.4 software, and analyses for in vitro study were performed with GraphPad Prism version 7.0.0 for Windows (GraphPad Software, La Jolla, CA). This study was analyzed and reported according to the Reporting Recommendations for Tumor Marker Prognostic Studies .