Cell culture and acrolein treatment.
Mouse fibroblast cell lien (NIH/3T3) and human normal colorectal cell CCD 841 CoN (ATCC® CRL-1790™) were purchased from ATCC and maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% BCS and 15% FBS, respectively. Acrolein stock solution (Sigma-Aldrich) was prepared freshly before use. Cells at 70% confluency were treated with different concentrations of acrolein (0–10 µM) in complete culture medium for 1–3 months at 37 °C in the dark and acrolein-containing medium was changed every two days.
Cell proliferation assay.
Cell proliferation was determined using a modified 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT; Sigma, St. Louis, MO) assay 57. Briefly, cells (1000/ well) were seeded in 96-well plates overnight, and measured every day for 7 days. The resulting formazan dissolved with DMSO were measured at 570 nm and results were presented as the percentage of the control values. All of these experiments were performed in triplicate and were repeated independently at least three times.
Flow cytometry analysis of cell cycle phases.
Cells were washed twice in ice-cold PBS and fixed in ice-cold 70% ethanol for 30 min or overnight at 4 °C. Cells were then washed in PBS and digested with DNase-free RNase A (50 U/ ml) at 37 °C for 30 min. Before flow cytometry analysis, cells were re-suspended in 500 µl propidium iodide (PI, 10 µg/ ml; Sigma) for DNA staining. PI staining was used to measure for cell cycle status using a Becton-Dickinson FACScan instrument and Cell Quest software.
Soft agar colony formation assay.
Soft agar colony formation assay was performed as described previously 58. Briefly, a 3-ml aliquot of 1.2% agar in a culture medium was plated in 60-mm dishes. Then 1,000 cells of transformed malignant or untransformed cells were mixed with 3 ml of 0.35% agar in a medium and plated on the solidified bottom agar. When the top agar solidified, the dishes were transferred to an incubator and cultured for 30 days. Two or three drops of the medium were added to each dish three times a week. After culturing for 30 days, the visible cell colonies were photographed and counted.
Tumor sphere culture assay.
Acrolein-transformed NIH/3T3 clones were trypsinized, and re-suspended at 1000 cells/ Ultra-Low Attachment 96-well Plate (Corninig) in culture medium containing 2 mM L-glutamine, N2 supplement, B27 supplement, 20 ng/mL hrEGF (Sigma), 20 ng/mL hrbFGF (Sigma) for two weeks. Fresh growth factors were added to the cells twice a week. Cumulative total numbers of cells from the spheroid cultures were calculated.
Cell migration assay.
The cell migration assay was performed in vitro utilizing modified Boyden chambers with a Transwell apparatus (polycarbonate membranes with 8-mm pores, Corning)59. Parental NIH/3T3 or NIH/3T3 Acr-clones (5x104 in 500 µl of growth medium/well, 6-well plates) were added into the upper chamber and the lower chamber contained 750 µl growth medium supplemented with 10% FCS. Cells on the upper membrane surface were wiped with a cotton swab after 24 h incubation at 37 °C in a 5% CO2 incubator. Membranes were then fixed, stained with crystal violet and cells that migrated to the lower membrane surface were counted in nine random fields using a microscope at 200x magnification. These experiments were performed in triplicates and were repeated at least three times.
Cells were washed twice with ice-cold PBS and lysed on ice for 20 minutes in radioimmunoprecipitation assay (RIPA) lysis buffer (20 mM Tris HCl, 150 mM NaCl, 1% (v/v) NP-40, 1% (w/v) sodium deoxycholate, 1 mM Ethylenediaminetetraacetates (EDTA), 0.1% (w/v) sodium dodecyl sulfate polyacrylamide (SDS) plus protease and phosphatase inhibitors). Lysates were then centrifuged at 13,200 rpm for 10 min, and the protein concentrations of supernatant were determined by BCA™ Protein Assay Kit. Protein samples (30 µg) were run on 8-100% SDS-polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene difluoride (Bio-Rad, U.S.A.) at 90 V for 120 min. Proteins were transferred onto nitrocellulose membranes (Bio-Rad). Blots were probed with primary antibodies overnight at 4 °C. Primary antibodies included: P-EGFR (Tyr1148, 1:1000, Cell signaling #4404); EGFR (1:1000, Cell signaling#2232); RAS (1:1000, Cell signaling #3965); p-AKT (1:1000, Cell signaling#4058); AKT (1:1000, Cell signaling#4685); P-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (1:1000, Cell signaling#9101); p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody #9102; Cyclin D1 (1:1000, Cell signaling#2978); c-myc (1:500, Santa cruz, sc-42). After primary antibody incubation, the membrane were washed and incubated with a horseradish peroxidase-conjugated secondary IgG (1:3,000; Millipore) for 1 h at room temperature. Immunoreative bands were detected using Amersham Enhanced Chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ, U.S.A.). The bound primary and secondary antibodies were stripped by incubating the membrane in stripping buffer (100 mM 2-mercaptoethanol, 2% SDS) for 30 min at room temperature. The membrane was then re-probed with GAPDH (1:1000, Cell signaling, #5174).
Xenograft mouse model.
All animal experiments were approved by the Institutional Animal Care and Use Committee of National Yang-Ming University and the study was carried out in compliance with the ARRIVE guidelines (IACUC#1070208rr). All methods in animal experiments were carried out according to the Guidelines for Animal Research of National Yang-Ming University. Fifteen 6-week-old male Balb/c nude mice, weighing 25–30 g, were used. Tumors were induced by injecting acolein-transformed NIH/3T3 cells (5x106 in 100 µl PBS per animal) subcutaneously into the right axillary fossa of mice as described previously with slight modification59. In order to generate the tumor growth curve, measurement of tumor was performed twice a week with a digital caliper and volumes were calculated by (length x width2)/2. Body weight was also evaluated twice weekly. Tumor samples were collected after sacrifice. Each sample was cut in halves; one half was saved in 10% formaldehyde and one half was stored at − 80 °C until further use.
RNA isolation and cDNA Microarray analysis.
The total RNA was isolated from cells TRIzol® Reagent (Thermo Fisher Scientific), according to the manufacturer’s instructions. RNA samples were quantified using an ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, U.S.A.) and the quality was using an Agilent 2100 Bioanalyzer with a Nanochip (Agilent, Santa Clara, CA) following the manufacture's instruction. The microarray hybridizations were performed using total RNA prepared from the NIH/3T3 and NIH/3T3 Acr-clone#4 as described previously48. GeneChip Mouse Genome 430 2.0 Affymetrix oligonucleotide Gene Chips (Affymetrix) were analyzed at the Microarray & Gene Expression Analysis Core Facility (VYM Genome Research Center, National Yang-Ming University) according to the Affymetrix protocols. Microarray datasets were analyzed using Ingenuity Pathway Analysis (IPA version 57662101) (QIAGEN).
Collection of formalin-fixed paraffin-embedded (FFPE) tissues and tissue microarray collection of CRC patients.
254 CRC FFPE tissues with corresponded tissue microarray collected at Taipei Veterans General Hospital were recruited for participation in the study and informed written consent was given by each participant or his (or her) relative. For 18 cases, tumor tissue and adjacent non-tumor tissue samples were surgically dissected and sent to the Department of Pathology of examination. Patients with a diagnosis of CRC were included in the present study. Our study protocol was approved by the Institutional Review Board of Taipei Veterans General Hospital (IRB#2020-01-010BC) and the study was carried out in accordance with the Declaration of Helsinki principles.
Immunohistochemistry (IHC) analysis for Acr-dG adduct and c-myc.
For the tissue microarray (TMA), hematoxylin and eosin-stained sections from each paraffin-embedded, formalin-fixed block were used to define diagnostic areas, and a representative 0.6 mm core was obtained from each case and inserted in a grid pattern into a recipient paraffin block49,50. IHC analysis was carried out as previously described with slight modification60. Briefly, sections (4 µm) were then deparaffinized in xylene and rehydrated in a descending ethanol series. In order to enhance immunoreactivity, sections were incubated in Tris-EDTA, pH 6.0, and boiled for 12 min. Endogenous peroxidase activity was eliminated by incubation in hydrogen peroxide. Incubation with primary antibodies for Acr-dG antibody (generated in house), c-myc (Santa Cruz, sc-40) was performed overnight at 4 °C in 1% BSA in phosphate buffer saline (PBS). Bound antibodies were visualized with DAB (diaminobenzidine) used as a chromogen and omission of the primary antibody was served as a negative control. Positive controls (normal liver) were stained in parallel with each set of TMA studied. Assessment of Acr-dG and c-myc immunoexpression was performed by light microscopy at x400 magnification by a pathologist.
Descriptive statistics were presented as the mean ± standard deviation or as the number (percentage). Student’s t-tests were used to determine statistical significance, and two-tailed P-values are shown. A minimum of three independent replicate experiments was performed to justify the use of statistical tests. Survival was analyzed using Kaplan Meier survival analysis, and the log rank test was used for comparison between the two groups. Multivariate analysis was performed using Chi-Square analysis. Statistical significance was defined as a p < 0.05. All analyses were performed with the IBM SPSS Statistics software package, version 23.0.