Hepatocellular carcinoma (HCC) has a high incidence and mortality. Radiotherapy and sorafenib have proven effective for HCC. Here, we investigated whether sorafenib modulated the response of HCC cells to irradiation in vitro, effect of timing of sorafenib, and the underlying mechanisms.
Cell viability of the HCC cell lines, SMMC-7721 and Bel-7402, was examined by the 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2(4-sulfophenyl)-2H-terazolium (MTT) assays. Clonogenic growth assays of SMMC-7721 and Bel-7402 were determined by colony formation assays. DNA damage was assessed by monitoring γ-HAX foci in irradiated cells with immunofluorescence microscopy, and cell cycle distribution changes were examined by flow cytometry. Effects of sorafenib (15μM) added 30min prior to radiation (pre-irradiation sorafenib) of SMMC-7721 and BEL-7402 or 24h post-irradiation (post-irradiation sorafenib) on irradiated SMMC-7721 and BEL-7402 cells were compared to those of radiation alone or no treatment.
The effect of sorafenib was dependent on its time of addition in relationship to irradiation of cells. Pre-irradiation sorafenib did not significantly affect the viability of SMMC-7221 and BEL-7402 cells compared with irradiation treatment alone. In contrast, post-irradiation sorafenib increased the sensitivity of irradiated SMMC-7221 and BEL-7402 cells significantly in a time-dependent manner. Pre-irradiation sorafenib significantly increased the surviving fraction of SMMC-7221 and BEL-7402 cells in clonogenic assays whereas post-irradiation sorafenib significantly reduced the surviving fractions of SMMC-7221 and BEL-7402 cells. SMMC-7721 cells treated with sorafenib 30min before irradiation had significantly fewer cells with γ-H2AX foci (23.8±2.9%) than SMMC-7721 cells receiving radiation alone (59.9±2.4; P<0.001). Similarly, BEL-7402 cells receiving sorafenib prior to irradiation had significantly fewer cells with γ-H2AX foci (46.4±3.8%) than those receiving radiation alone (25.0±3.0%; P<0.001). In addition, irradiation (6Gy) caused a significant increase in the percentage of both SMMC-7721 and BEL-7402 cells in G2/M at 12 to 16h post irradiation, which was markedly delayed by pre-irradiation sorafenib.
Sorafenib combined with irradiation exerted a schedule-dependent effect in HCC cells in vitro, which has significant implications for the combined use of sorafenib and radiotherapy for HCC patients.
Keywords: Hepatocellular carcinoma, Radiation, Sorafenib, Apoptosis, DNA damage repair