Invasion-inhibiting Effects of Gaseous Components in Cigarette Smoke on Mouse Rectal Carcinoma Colon-26 Cells

Materials and Methods

Materials. Winston XS Caster FR One Box cigarettes containing 1 mg of tar and 0.1 mg of nicotine per cigarette were purchased from Japan Tobacco, Inc. (Tokyo, Japan). Cambridge filters were used to remove almost all particles and nicotine from cigarette smoke, and they were obtained from Heinr. Borgwaldt GmbH (Hamburg, Germany). MVK was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Fetal bovine serum (FBS) and RPMI 1640 medium were from Invitrogen Corp. (Carlsbad, CA, USA). EDTA trypsin solution (EDTA: 2.2 mM, trypsin: 0.25%) was from Mediatech, Inc. (Manassas, VA, USA). Dulbecco's phosphate-buffered saline without calcium and magnesium [DPBS (–)] was from Nissui Pharmaceutical Co., Ltd. (Tokyo, Japan). Matrigel (Corning® Matrigel® Basement Membrane Matrix Growth Factor Reduced, #356230) and transwells (FALCON® cell culture inserts, #353097) were purchased from Corning (Corning, NY, USA). Bovine serum albumin (BSA) and fibronectin were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

Animals. Specific pathogen-free female BALB/cCrSlc mice (7 weeks old) were purchased from Japan SLC, Inc. (Shizuoka, Japan) and used in experiments after 1-week acclimation. Mice were maintained in an air-conditioned room (23±2°C and 60±10% humidity) under an artificial 12-hour light/dark cycle (7:00 a.m.–7:00 p.m.). Food and water were given ad libitum during the experimental period. All procedures followed the Guidelines for the Care and Use of Laboratory Animals at Mukogawa Women's University (approval number: P-11-2014-03-A).

Cells. A mouse cell line derived from rectal cancer (Colon-26 cells) was obtained from RIKEN BioResource Center (Ibaragi, Japan). Colon-26 cells were grown in RPMI 1640 medium containing 10% heat-inactivated FBS.

Preparation of CSE. Cigarettes were continuously combusted by reduced pressure generated using an aspiration pump (Nippon Rikagaku Kikai Co., Ltd., Tokyo, Japan), with the flow rate set at 1.0 l/min. The main stream of the cigarette smoke was passed through a Cambridge filter to remove the tar phase and nicotine, and subsequently, bubbled into 15 ml of DPBS (–). The combustion of the cigarette was repeated, unless otherwise specified, until the dry weight of the tar phase trapped on the Cambridge filter reached 150 mg (10). This solution was regarded as 100% CSE. The CSE was immediately filtered through a 0.22-μm filter and stored at −80°C, and diluted to the required concentration with DPBS (–) when necessary. The final concentrations of these solutions are expressed as a percentage of the initial content.

Assay of experimental hepatic metastasis of Colon-26 cells in mice. Sub-confluent Colon-26 cells were harvested with EDTA trypsin solution and suspended at appropriate densities in DPBS (–). The left lower back of mice was minimally incised and the spleen was exposed under anesthesia with urethane. Cells (1×10⁵/50 μl) were injected into the spleen of syngeneic BALB/cCrSlc mice.

CSE (0, 10, 30, and 100%, 16 ml/kg body weight) was daily administered intraperitoneally to the mice for 14 days after tumor cell inoculation. The date of death of mice in each group was recorded. The spleen and liver were excised and weighed.

Proliferation assay. Cells were seeded at 1×10⁵ cells/2 ml in each well of a 12-well culture plate. Cells were treated with different concentrations of CSE (0.1, 0.3 and 1%) and MVK (0.3, 1, 3, 10 and 30 μM), and incubated for 24, 48, and 72 hours in a CO₂ incubator at 37°C. Viable cells were enumerated with a Coulter counter.

Invasion assay. Transwells (6.4-mm diameter) were used with tracked-etched polyethylene terephthalate (PET) membrane filters (8-μm pore size) coated with 25 μg/filter of matrigel basement membrane matrix extracted from Engelbreth-Holm-Swarm mouse sarcoma. Sub-confluent cells were harvested with EDTA trypsin solution and resuspended at appropriate density in DMEM containing 0.1% BSA. Five-hundred-microliter samples of 5×10⁵ cells were placed in the upper-chamber compartments. The lower chambers contained 750 μl of DMEM added to 20 μg/ml of fibronectin (Sigma Chemical Co.) as a chemoattractant. CSE (0.03 and 0.1%) and MVK (0.1, 0.3, and 1 μM) were added to the upper and lower compartments. These concentrations of CSE and MVK did not have cytotoxic effects on Colon-26 cells. After 72 hours of incubation in a tissue culture incubator, non-invading cells on the upper side of the filter were completely removed by wiping with a cotton swab. Cells that had penetrated the matrix protein and adhered to the lower surface of the filter were counted microscopically after fixing and stained with a Diff-Quik kit (Sysmex, Hyogo, Japan).

Migration assay. The migration assay was performed in the same way as the invasion assay, without matrigel coating. Cells (5×10⁴ cells/500 μl) were added to the upper-chamber compartments. After 24 hours, cells which had migrated to the lower surface of the filter were stained and counted.

Statistical analyses. The data are expressed as the mean±SE (n=3-15). Statistical analyses were performed with the t-test and Dunnett's test using PRISM Version 4 (GraphPad Software, Inc., San Diego, CA, USA). Kaplan–Meier survival analysis was used to calculate survival curves, followed by the log-rank test to determine significance of differences in survival using PRISM Version 4. Differences were considered significant at p<0.05.