Abstract:

Objective To investigate the biological characteristics of proliferation, apoptosis, migration and invasion of radiation-induced polyploid cervical cancer HeLa cells, and to analyze the potential facilitation of polyploid HeLa cells in cervical cancer recurrence after radiotherapy. Methods HeLa cells were irradiated by 6 MV-X ray with 7 Gy and 14 Gy, the cells were cultured until the third day, and then they were recorded as 7 Gy group and 14 Gy group respectively. The unirradiated HeLa cells were recognized as the control group. The cell morphology was checked under optical microscope. Flow cytometry was used to determine cell ploidy. MTT assay was applied to detect cell proliferation. Flow cytometry by AnnexinⅤ-FITC/PI double labeling was used to detect apoptosis. The ability of migration and invasion was detected by Transwell assay. The expression levels of STAT3 signal pathway-related proteins were analyzed by Western blotting. Results Compared with the control group, the volume of HeLa cells in 7 Gy group and 14 Gy group increased significantly. The percentages of polyploid HeLa cell subsets in the control group, 7 Gy group and 14 Gy group were （6.33±1.26）%, （21.13±0.50）% and （46.07±1.68）% respectively, with a statistically significant difference （F=780.47, P<0.001）. The absorbance values in the control group, 7 Gy group and 14 Gy group of polyploidy HeLa cells were 0.21±0.01, 0.23±0.02, 0.16±0.01 at 24 h, 0.37±0.03, 0.38±0.06, 0.21±0.00 at 48 h, 0.66±0.02, 0.55±0.01, 0.28±0.01 at 72 h, and there were statistically significant differences （F=31.62, P=0.001; F=20.10, P=0.002; F=708.52, P<0.001）. Further pairwise comparison showed that the proliferation abilities of polyploidy HeLa cells of the 14 Gy group at 24, 48 and 72 h were significantly lower than those of the control group and the 7 Gy group （all P<0.05）. The proportions of apoptotic cell subset in the control group, 7 Gy group and 14 Gy group were （3.67±1.16）%, （3.07±0.81）%, （3.83±0.91）%, the proportions of early apoptotic subset were （2.33±0.35）%, （2.13±0.61）%, （2.23±0.32）%, and the proportions of late apoptotic subset were （1.33±0.81）%, （0.93±0.31）%, （1.60±0.60）% respectively. There were no statistically significant differences （F=0.52, P=0.620; F=0.15, P=0.864; F=0.92, P=0.450）. The migrated numbers of cells in the control group, 7 Gy group and 14 Gy group were 297.40±26.53, 121.33±15.16, 18.40±4.79, and the invaded numbers were 195.67±20.26, 63.60±6.91, 9.47±3.23 respectively, with statistically significant differences （F=647.28, P<0.001; F=213.94, P<0.001）. Compared with the control group, the migration and invasion abilities of polyploid HeLa cells in the 7 Gy and the 14 Gy groups were significantly decreased, and the migration and invasion abilities of polyploid HeLa cells in the 14 Gy group were significantly lower than those in the 7 Gy group （all P<0.001）. The expression levels of P-STAT3 （Tyr 705） and Bcl-2 in radiation-induced polyploidy HeLa cells were higher than those in the control group, and the expression levels were further increased with the increase of radiation dose. Compared with the control group, the expression levels of Survivin and Mcl-1 in polyploid HeLa cells in the 14 Gy group were up-regulated （both P<0.05）. There was no significant difference in Bcl-xL expression among the three groups （F=0.52, P=0.618）. Conclusion The proliferation, migration and invasion abilities of polyploid HeLa cells are reduced by radiation, and the proportion of apoptotic subset is not significantly changed, but the activation of STAT3 signaling pathway is accompanied by up-regulation of downstream anti-apoptotic related proteins, which is favorable for the polyploid tumor cells to be the potential risk factor of recurrent cervical cancer after radiotherapy.

Key words: Cervical neoplasms, Polyploid tumor cells, Radiation, STAT3