Enhancement of mTOR signaling contributes to acquired X-ray and C-ion resistance in mouse squamous carcinoma cell line

Abstract

Our aim was to evaluate whether repetition of C-ion (carbon ion beam) irradiation induces radioresistance as well as repeated X-ray irradiation in cancer cell lines, and to find the key molecular pathway for radioresistance by comparing radioresistant cancer cells with their parental cells. A mouse squamous cell carcinoma cell line, NR-S1, and radioresistant cancer cells, NR-S1-C30 (C30) and NR-S1-X60 (X60), established by repetition of C-ion and X-ray irradiation, respectively, were used. X-ray and C-ion sensitivity, changes in lysosome, mitochondria, intracellular ATP and reactive oxygen species (ROS) level, and mechanistic target of rapamycin (mTOR) signaling were evaluated. Moreover, the effect of rapamycin on radioresistance was also assessed. X-ray and C-ion resistance of C30 cells was moderate, and the resistance of X60 cells was the highest in this study. In X60 cells, the amount of lysosome, mitochondria, intracellular ATP and ROS level were significantly increased, and mTOR and p70S6K (ribosomal protein S6 kinase p70) phosphorylation were enhanced compared with C30 and NR-S1 cells. The inhibition of mTOR signaling was effective for X-ray and C-ion radiosensitization in both cell lines, especially in X60 cells in which X-ray and C-ion resistance was decreased to the same level as that in NR-S1 cells. Our results indicated that the contribution to generate X-ray and C-ion resistance was less for repeated C-ion irradiations compared with repeated X-ray irradiation. Moreover, we found that activated mTOR signaling contributes to X-ray and C-ion resistance in the X60 cancer cells.

Keywords: Acquired radioresistance; energy metabolism; mTOR signaling; rapamycin; repeated X-ray and C-ion irradiations.

Figure 1

Difference in X‐ray (a) and C‐ion (b) sensitivity between NR‐S1, X60 and C30 cells. The blue, red and green lines show survival curves of NR‐S1, X60 and C30 cells, respectively. The triangles indicate statistical difference with respect to the survival curve of NR‐S1 cells (anova, P < 0.05).

Figure 2

Morphological difference between NR‐S1 (a, d), X60 (b, e) and C30 (c, f). Upper panels (a–c) show images under bright light in normal culture conditions. Lower panels (d–f) show fluorescence image of lysosome, mitochondria and nucleus, which are stained in red, green and blue, respectively. The scale bars indicate 25 μm.

Figure 3

Amount of lysosome (a), mitochondria (b), intracellular ATP concentration (c) and reactive oxygen species (ROS) level (d). Blue, red and green boxes represent the values of NR‐S1, X60 and C30 cells. The boxes and error bars show mean value and standard deviation, respectively, of at least three independent experiments. The asterisk shows statistical difference (P < 0.05, Dunnett's test).

Figure 4

Protein expression and phosphorylation of mTOR (a) and p70S6K (b) in each cell, and the phosphorylation ratio of each protein (c). Blue, red and green boxes in Figure 4(c) represent the values of NR‐S1, X60 and C30 cells. Boxes and error bars show mean value and standard deviation of at least three independent experiments. The asterisk shows statistical difference (P < 0.05, Dunnett's test).

Figure 5

Effect of rapamycin on plating efficiency (a), X‐ray (b–e) and C‐ion (f–i) sensitivity. Cells were treated with 100 nM of rapamycin or 0.1% methanol for 24 h, and then irradiated with the indicated doses of X‐ray or C‐ion. Blue, red and green boxes in (a) represent the plating efficiency of NR‐S1, X60 and C30 cells. The meshed and filled boxes, respectively, show 0.1% methanol and rapamycin treated cells. The values and error bars show mean value and standard deviation of at least three independent experiments. The asterisk shows statistical difference (P < 0.05, Dunnett's test and t‐test). The blue circle, red square and green triangle in (b–i) show survival fractions for NR‐S1, X60 and C30 cells, respectively, and the close symbols with the solid curve and the open symbols with dashed curve show the survival fractions with and without rapamycin, respectively. Asterisks adjacent to the curves indicate that the curves were statistically difference (P < 0.05, anova). N. S. means no significant difference between the curves.