Proteomic analysis of effects by x-rays and heavy ion in HeLa cells

Abstract

Background: Carbon ion therapy may be better against cancer than the effects of a photon beam. To investigate a biological advantage of carbon ion beam over X-rays, the radioresistant cell line HeLa cells were used. Radiation-induced changes in the biological processes were investigated post-irradiation at 1 h by a clinically relevant radiation dose (2 Gy X-ray and 2 Gy carbon beam). The differential expression proteins were collected for analysing biological effects.

Materials and methods: The radioresistant cell line Hela cells were used. In our study, the stable isotope labelling with amino acids (SILAC) method coupled with 2D-LC-LTQ Orbitrap mass spectrometry was applied to identity and quantify the differentially expressed proteins after irradiation. The Western blotting experiment was used to validate the data.

Results: A total of 123 and 155 significantly changed proteins were evaluated with treatment of 2 Gy carbon and X-rays after radiation 1 h, respectively. These deregulated proteins were found to be mainly involved in several kinds of metabolism processes through Gene Ontology (GO) enrichment analysis. The two groups perform different response to different types of irradiation.

Conclusions: The radioresistance of the cancer cells treated with 2 Gy X-rays irradiation may be largely due to glycolysis enhancement, while the greater killing effect of 2 Gy carbon may be due to unchanged glycolysis and decreased amino acid metabolism.

Keywords: 2D-LC-MS/MS; Carbon ion; Gene Ontology enrichment; SILAC; X-rays.

FIGURE 1.

Brief SILAC experiment workflow. For the SILAC experiment, the HeLa cells were culture in DMEM containing “heavy” ¹³C₆¹⁵N₄ L-arginine, ¹³C₆¹⁵N₂ L-lysine and “light” ¹²C₆¹⁴N₄ L-arginine and ¹²C₆¹⁴N₂ L-lysine. After 100% label incorporation, heavy cells were irradiated and harvested after 1 h. Then, the cells were digested and mixed (treated vs. control) at 1:1 for mass spectrometric analysis.

FIGURE 2.

Distribution of log₂ transformed protein expression ratios of two irradiation types. The proteins represented by data points lying close to the y-axis (y-axis=1) did not show any expression changes. Outliers were considered as proteins with significantly differential expression only if they had a p-value≤0.05 and were identified with a minimum of 2 unique peptides.

FIGURE 3.

The biological process analysis for 2 Gy carbon and 2 Gy X-ray deregulated proteins. (A) the biological process distribution of up-regulated proteins in 2 Gy carbon irradiation(NNN, Nucleoside, nucleotide and nucleic acid). (B) The biological process distribution of up-regulated proteins in 2 Gy X-ray. C The biological process distribution of down-regulated proteins in 2 Gy carbon irradiation (AA, Amino acids; Pro, Protein;). (D) The biological process distribution of down-regulated proteins in 2 Gy X-ray (Chro, Chromatin; Pro, Protein).

FIGURE 4.

The highest score network post-irradiation by 2 Gy carbon and X-ray using IPA analysis. (A) The network “Cellular assembly and organization, cellular function and maintenance, post-translational modification, protein folding and cell death and survival” had a highest score of 29 post-irradiation by 2 Gy carbon. (B) The network “nucleic acid me tabolism, small molecule biochemistry, lipid metabolism, cellular assembly and organization, and DNA replication, recombination, and repair” had a highest score of 49 post-irradiation by 2 Gy X-ray. The shade of red represented significant up-regulated proteins and shade of green represented down-regulated proteins.

FIGURE 5.

The highest score network of overlap deregulated proteins underlying 2 Gy carbon and X-ray using IPA analysis. (A) The overlap proteins with significant changes underlying 2 Gy carbon. The network “cellular assembly and organization, cellular function and maintenance, amino acid metabolism” had a highest score of 26. (B) The overlap proteins with significant changes underlying 2 Gy carbon. The network “lipid metabolism, small molecule biochemistry, nucleic acid metabolism” had a highest score of 32. (red: up-regulated; green: down-regulated).

FIGURE 6.

Biological and function assays for 2 Gy X-ray and carbon beam. (A) Survival fraction of HeLa cells after 2 Gy X-ray (red) or carbon beam (green) exposure. The plating efficiency of HeLa cells were 0.279±0.020 and 0.095±0.003 respectively in 2 Gy X-ray and 2 Gy carbon. (B) Changes in ATP levels between cells exposed to different irradiation and sham control (*, p < 0.05, t-test was one-tailed). (C) Differences in lactic acid levels between sham control and irradiated cells (*, p < 0.05, t-test was one-tailed).

FIGURE 7.

The western blotting for analysis of LDHA, SCO1 and Akt. (A) The images of western blot. (B) Fold changes between treated samples and control were analyzed by gray-values.