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

Sang Bum Kim
Sang Bum Kim
, 
Lu Zhang
Lu Zhang
 and 
Jerry W. Shay
Jerry W. Shay
   | Jan 18, 2022
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Article Category: Research Article
Published Online: Jan 18, 2022
Page range: 32 - 41
DOI: https://doi.org/10.2478/gsr-2014-0003
© 2014 Sang Bum Kim et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Figure 1.

HZE particles increase growth advantage. HCEC CT7 cells were seeded in 6-well plate in triplicate and counted at 2 day intervals over a 7 day period. Irradiation promotes cellular proliferation rate, whereas CDDO-Me pretreatment attenuates this effect. Data represent mean ± s.d. of repeated experiments. ***p<0.005 in Student’s t-test.
HZE particles increase growth advantage. HCEC CT7 cells were seeded in 6-well plate in triplicate and counted at 2 day intervals over a 7 day period. Irradiation promotes cellular proliferation rate, whereas CDDO-Me pretreatment attenuates this effect. Data represent mean ± s.d. of repeated experiments. ***p<0.005 in Student’s t-test.

Figure 2.

HZE particles increase neoplastic transformation. (A) HZE particles increase number of colonies formed in soft agar. (B) Sizes of soft agar colonies are bigger in HZE particle-irradiated cells than in unirradiated controls, as shown in the representative photographs and quantification. Colony size was measured from 15 to 25 colonies in each group. (C) HZE particles enhance cell migration in wound healing assay. (D) HZE particles promote invasion through Matrigel®. Data represent mean ± s.d. of repeated experiments. ***p<0.005, ****p<0.001 in Student’s t-test.
HZE particles increase neoplastic transformation. (A) HZE particles increase number of colonies formed in soft agar. (B) Sizes of soft agar colonies are bigger in HZE particle-irradiated cells than in unirradiated controls, as shown in the representative photographs and quantification. Colony size was measured from 15 to 25 colonies in each group. (C) HZE particles enhance cell migration in wound healing assay. (D) HZE particles promote invasion through Matrigel®. Data represent mean ± s.d. of repeated experiments. ***p<0.005, ****p<0.001 in Student’s t-test.

Figure 3.

HZE particles activate β-catenin signaling and affect DNA repair genes expression. (A) HZE particles decrease level of phospho-Stat3 (Y705), whereas increase level of active-β-catenin and cyclin D1 expression. CDDO-Me treatment reverses these effects. GAPDH is used as loading control. (B) HZE particles decrease series of DNA repair genes expression using DNA repair gene array. *p<0.05 in Student’s t-test in repeated experiments.
HZE particles activate β-catenin signaling and affect DNA repair genes expression. (A) HZE particles decrease level of phospho-Stat3 (Y705), whereas increase level of active-β-catenin and cyclin D1 expression. CDDO-Me treatment reverses these effects. GAPDH is used as loading control. (B) HZE particles decrease series of DNA repair genes expression using DNA repair gene array. *p<0.05 in Student’s t-test in repeated experiments.

Figure 4.

CDDO-Me protects cells against HZE particles. Treatment of CDDO-Me before and during irradiation reverses transformative properties of irradiated HCEC CT7 cells in terms of number of soft agar colonies (A), sizes of soft agar colonies (B), cell migration (C), and invasion through Matrigel® (D). CDDO-Me treatment alone does not have any significant effects. Data represent mean ± s.d. of repeated experiments. *p<0.05, **p<0.01, ***p<0.005, ****p<0.001 in Student’s t-test.
CDDO-Me protects cells against HZE particles. Treatment of CDDO-Me before and during irradiation reverses transformative properties of irradiated HCEC CT7 cells in terms of number of soft agar colonies (A), sizes of soft agar colonies (B), cell migration (C), and invasion through Matrigel® (D). CDDO-Me treatment alone does not have any significant effects. Data represent mean ± s.d. of repeated experiments. *p<0.05, **p<0.01, ***p<0.005, ****p<0.001 in Student’s t-test.

Supplementary Figure S1.

Schematic flow-chart of experimental timeline. 4×104 cells were seeded into T25 flask 6 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 18 h 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 UT Southwestern. Irradiated cells were split, a half of the cells were frozen and stored for further assays, and the other half of cells were passage cultured for 4 weeks and then subjected to proliferation and soft-agar assays.
Schematic flow-chart of experimental timeline. 4×104 cells were seeded into T25 flask 6 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 18 h 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 UT Southwestern. Irradiated cells were split, a half of the cells were frozen and stored for further assays, and the other half of cells were passage cultured for 4 weeks and then subjected to proliferation and soft-agar assays.

Supplementary Figure S2.

The first proliferation and soft-agar assays were performed 4 weeks after irradiation. A. HCEC CT7 cells which were passage-cultured for 4 weeks after irradiation were seeded in triplicate 6-well plates and counted at 2 day interval over a 7 day period. B. Cells which were passage-cultured for 4 weeks after irradiation were seeded in 0.375% soft-agar in triplicate 24-well plates. 4 weeks later, colonies larger than 0.1-mm diameter were counted and normalized to unirradiated control.
The first proliferation and soft-agar assays were performed 4 weeks after irradiation. A. HCEC CT7 cells which were passage-cultured for 4 weeks after irradiation were seeded in triplicate 6-well plates and counted at 2 day interval over a 7 day period. B. Cells which were passage-cultured for 4 weeks after irradiation were seeded in 0.375% soft-agar in triplicate 24-well plates. 4 weeks later, colonies larger than 0.1-mm diameter were counted and normalized to unirradiated control.
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