br Irinotecan HCl trihydrate was purchased from Selleckchem
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 Dexmedetomidine (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.
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 cells 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).