Oxygen and Silicon Ion Particles Induce Neoplastic Transformation in Human Colonic Epithelial Cells

Telomerase immortalized, but non-cancerous HCEC CT7s were established and maintained under low oxygen (2% oxygen and 5% carbon dioxide) on Primaria® plates (BD Bioscience, San Jose, CA) as described previously (Ly et al., 2011; Roig et al., 2010). For HZE irradiation, 4×10⁴ cells were seeded into T25 flask six days prior to irradiation. Cells were shipped to the NASA Space Radiation Laboratory (NSRL) (Upton, NY) and maintained in a low oxygen incubator. HZE particle irradiations were performed at the NSRL with a mixed-field radiation scheme in which cells were exposed to 1-Gy of 250 MeV/nucleon ¹⁶O particles and then exposed to 1-Gy of 300 MeV/nucleon ²⁸Si particles on the following day.

To address radioprotection activity of CDDO-Me, the cells were treated with 50 nM CDDO-Me (Kim et al., 2012) 18 hours before first irradiation. The medium was changed after first irradiation with medium containing 50 nM CDDO-Me. After the last irradiation, cells were changed with fresh medium and shipped back to University of Texas (UT) Southwestern. An equivalent amount of DMSO was used as a vehicle control.

At UT Southwestern, half of the cells were frozen and stored for further assays and the other half of cells were passage cultured for four weeks in the absence of CDDO-Me. Unirradiated control cells were also sent along with other cells to the NSRL, maintained in the same facility, and shipped back to UT Southwestern; they were placed on the radiation platform but were not irradiated. The first proliferation and soft agar assays were performed four weeks after irradiation. Several months later, the second proliferation and soft agar assays were validated using frozen/stored cells four weeks after passage culture. Other assays were also performed using frozen/stored cells four weeks after passage culture (Supplementary Figure S1).

Cells were seeded at a density of 10⁴

cells/well in triplicate (technical replicate) 6-well plates and incubated. Cells were detached using 0.05% Gibco® Trypsin-EDTA (Life Technology, Grand Island, NY) and suspended cells were counted at two day intervals over a seven day period using a Z1 Coulter Counter® (Beckman, Indianapolis, IN). Each experiment was repeated using cells from different cell suspensions (biological replicate).

1CT7 cells were seeded at a density of 5 x 10⁵ cells/well in triplicate 6-well plates. A single wound was created in the center of the confluent cell monolayer by removal of the attached cells with a sterile plastic pipette tip. The debris was removed by washing with phosphate buffered saline (PBS) and cells photographed using an EVOS® microscope (Westover Scientific, Life Technology) to measure the distance of the wounded area. After 24 hours of incubation with growth medium containing 1 mM thymidine (proliferation inhibitor, Sigma), the same areas were photographed to measure a ratio of wound closure. The wound closure was quantified by measuring the length between the wound borders by ImageJ software (NIH, Bethesda, MD) per 4x fields of view measuring three fields per plate. The ratio of wound closure was calculated by [(length of closed are) / (length of wounded area) × 100 (%)]. Each experiment was repeated independently using cells from different cell suspensions.

Cells were suspended in 0.375% Noble agar (Difco, Detroit, MI) in supplemented basal medium at a density of 1,000 cells and overlaid on 0.75% Noble agar in 24-well plates. Each sample was seeded in triplicate and incubated at 37° C for four weeks. Colonies larger than 0.1 mm were counted. The experiment was repeated independently using cells from different cell suspensions. Colonies were photographed using a stereo microscope (SteREO Discovery, Carl Zeiss, Thornwood, NY) and colony size was determined using a measuring tool in ImageJ software.

10⁵ cells were serum-starved overnight, suspended in 300-μl of basal medium, and plated onto 8.0-μm pore BD Matrigel^TM Invasion Chamber (#354480, BD Biosciences, Bedford, MA). 500-μl of medium containing 2% serum and growth supplements was added to the bottom well. After incubation for 24 hours, cells were fixed and stained using a mixture of 6.0% (vol/vol) glutaraldehyde and 0.5% crystal violet for 10 min. Non-migratory cells on the upper surface on the filter were scraped off and migratory cells on the lower compartment were photographed by Axioskop 2 (Carl Zeiss, Thornwood, NY) and counted using ImageJ. Experiments were performed twice using different cell suspension for biological replicates in triplicate invasion chambers. Invading cells were quantified by averaging the number of stained cells per 4x field of view counting five fields per chamber.

Passage cultured cells four weeks after irradiation were harvested and total cell lysates were prepared in Laemmli SDS reducing buffer (50 mM Tris-HCl (pH 6.8), 2% SDS, and 10% glycerol), boiled, and resolved on an 4-15% Mini-PROTEAN TGX^TM Precast Gel, and transferred to a polyvinylidinefluoride membrane using the Trans-Blot^® Turbo^TM Transfer System (Bio-Rad, Hercules, CA). The following antibodies were used: anti-Active-β-catenin (1:1000, Millipore, 05-665), anti-cyclin D1 (1:1000, Cell Signaling, 2978S), and anti-phospho-Stat3 (Tyr705, 1:1000, Cell Signaling, 9145S). HRP-conjugated goat, anti-mouse, or anti-rabbit (Jackson ImmunoResearch) were used as secondary antibodies at 1:5000 and detected with the SuperSignalWest Pico Chemiluminescent Substrate Kit (Thermo Scientific) and the gel documentation system, G:BOX (Syngene, Frederick, MD). Experiments were performed twice using different cell suspension.

Total RNA was isolated from cells passage-cultured for four weeks after irradiation using RNeasy Plus Mini kit (Qiagen, Chatsworth, CA), according to the manufacturer’s protocol. Then 1 μg total RNA was converted to cDNA in 20 μl reaction using RT² First Strand Kit (Qiagen, Chatsworth, CA) and 91 μl RNase-free water was added to dilute. The Human DNA Repair RT²Profiler^TM PCR Array (Qiagen, Chatsworth, CA) was used for screening, per the manufacturer’s protocol. Real-time quantitative PCR reactions were set up with RT² qPCR Master Mixes (Qiagen, Chatsworth, CA) and run on the LightCycler 480 II (Roche) with 45 cycles of denaturation (95°C, 15 sec) and annealing (60°C, 1 min) cycle. Experiments were performed twice using different cell suspension.

We previously reported that combined radiation exposure to 2-Gy ¹H followed by 0.5-Gy ⁵⁶Fe ion particles increased proliferation and anchorage-independent growth in HCEC CT7 cells (Eskiocak et al., 2010). In this study, we set out to investigate whether radiation exposure to other ions, such as ¹⁶O and ²⁸Si particles, had similar effects on HCEC CT7 cells. This is important since in space, astronauts will be exposed to mixed fields of varying ion types, and while there is mounting data on single ion exposures, mixed-field experiments are mostly lacking. We employed a mixed-field radiation scheme in which cells were exposed to 1-Gy ¹⁶O radiation at an energy of 250 MeV/nucleon, followed 24 hours later by 1-Gy ²⁸Si radiation at an energy of 300 MeV/nucleon. Irradiated cells were split; half of the cells were frozen and stored for further assays and the other half of cells were passage cultured for four weeks and then subjected to a series of functional cell transformation assays.

The first proliferation and soft agar assays, which were done four weeks after irradiation, show the irradiated cells exhibit a proliferative and anchorage-independent growth advantage compared to unirradiated control cells (Figure S2). The proliferation and soft agar data were validated using frozen/stored cells four weeks after passage culture. The irradiated and differently passage-cultured cells also exhibit a proliferative advantage in a short-term growth assay compared to unirradiated cells. These cells also show enhanced anchorage-independent growth, which is a characteristic of malignant cells. The number of colonies (1.8-fold, p<0.001), as well as the sizes of the colonies (1.4-fold, p<0.001), were significantly increased in the irradiated cells (Figure 2A and Figure B). Additionally, irradiation promotes increased migratory capability (2.3-fold, p<0.0001) of these cells, as is demonstrated by the scratch migration assay (Figure 2C). Finally, the invasion ability (3.0-fold, p<0.001) through extracellular matrices (Figure 2D) was significantly increased, another indicator of the cells’ metastatic ability.

Signal transducer and activator of transcription 3 (Stat3) mediates the expression of a variety of genes in response to cell stimuli, and thus plays a key role in many cellular processes, such as cell growth and the regulation of apoptosis. Recently, it has been reported that loss of Stat3 activity enhances cell proliferation and induces cancer progression by regulating expression of important regulators of colon cancer, such as β-catenin and cyclin D1 (Musteanu et al., 2010). Abnormally activated β-catenin and cyclin D1pathway are well-known biomarkers for most colorectal cancers and for a proportion of other neoplasias (Clevers and Nusse, 2012). In the current study we observed decreased level of Tyr-705-phosphorylated Stat3, resulting in increased level of active-β-catenin and cyclin D1 in the transformed cells (Figure 3A), indicating increased tumorigenesis.

Defects in expression of DNA repair genes result in chromosome instability and may be an additional high risk factor for progression from benign to malignant colorectal cancer (Jin et al., 2005). Using a qPCR-based DNA repair gene array, we found that several DNA repair genes (XRCC3, MSH5, PARP2, TOP3B, and MMS19) were significantly (p<0.05) under-expressed in the irradiation transformed cells, but remained at control levels in CDDO-Me-mediated protected cells (Figure 3B).

Since CDDO-Me has been shown to protect cells and animals against irradiation (Eskiocak et al., 2010; Kim et al., 2012), we next examined if it has the same protective effects for cells exposed to ¹⁶O and ²⁸Si particles. Compared to unprotected irradiated cells, treatment with CDDO-Me before or during irradiation reduced the cell proliferation (Figure 1). CDDO-Me also reduced anchorage-independent growth, cell migration, and invasion ability compared to the irradiated cells without the radioprotector (Figure 4A-D). CDDO-Me treatment alone has undetectable effects in unirradiated cells. These data document that nontoxic pretreatment with CDDO-Me can potentially prevent HZE particle-induced cell transformation.

Figure 1.