Radiosensitizing Effect And Toxicity Of Docetaxel On Human Hepatocarcinoma Cell Line SMMC-7721

Backgrounds Primary hepatocarcinoma is a common cancer, its mortality rate ranks secondly in various tumors. Modern precise radiation therapy is a common means of treating liver cancer. However, enough dose radiation to kill the tumor may damage important tissue around. Due to a lot of hypoxic cells insensitive to radiation in many tumors, even adequate dose radiation is still unable to control the tumor effectively, therefore, it has important clinical significance to study the radiation sensitizer. Docetaxel is a new semi-synthetic derivatives of paclitaxel, vitro studies showed that docetaxel can induce apoptosis, influence distribution of cell cycle. Clinical research had shown that docetaxel could increase radiation sensitivity of tumor cells without increasing the radiation injury of normal tissue. However, there were rare reports that docetaxel could increase radiation sensitivity of hepatoma cells.Objectives This study aims to explore the radiosensitizing effect and toxicity of docetaxel on human hepatocellular carcinoma cell line SMMC-7721, which provides experimental basis for enhancing the therapeutic effect of liver cancer.Methods (1)Recovered, Passaged and Cultured SMMC-7721. Observed cell morphology by inverted microscope. Liquided and passaged regularly and experimented when cell entered the logarithmic growth phase. (2) Inhibition effect of docetaxel on the growth of SMMC-7721 was determined by MTT assay. (3) The differences of morphology changes between docetaxel of different time was observed by inverted fluorescence microscope. (4) Radiation sensitizing effect of docetaxel on SMMC-7721 cell was determined by MTT assay. (5) The effect of docetaxel combiningwith radiation on the cell cycle was detected by flow cytometry. (6) Timeliness effects of docetaxel plus radiation on the cell cycle was detected by flow cytometry.Results (1) After cultured for three days, SMMC-7721 growed well and entered the logarithmic growth phase. (2) After treated by docetaxel of different concentration for 3 h, 6 h, 9 h, 12 h, 24 h, 48 h respectively, inhibition rate increased with the increase of concentration and treating time. Results of analysis of variance showed that there were significant differences of inhibition rate among differentent treating time groups ( F=11740.167, P=0.0000), different concentration groups except 200μg/ml group, 400μg/ml group, showed a significant difference ( F=15723.685, P=0.0000), IC50 was 21.69μg/ml, 95% confidence interval was 19.12μg/ml~24.60μg/ml. There were significant differences of inhibition rate among cooperative groups of different concerntration and time(F=739.916, P=0.0000).(3) Observed by inverted fluorescence microscope (200×): normal tumor cells was blob-like arrangement, the cell number in blob is different, cell ranged closely, size was different, profile was obvious, cell were shuttle or polygon; structures within the cell such as membrane, cytoplasm and so on were clear. So were oval nucleus, nuclear membrane and nucleus. After treated by docetaxel for 3 h: Volume of tumor cell augmented, the membrane, cytoplasm, the nuclear membrane and other structures were unclear, but nucleus was still clear; 6 h : Most cells were fused and arranged like blob, structures within the cell of blob was unclear, there were uniform cavitation on the surface of cells; 9 h: Intracellular vacuole began fusing and increasing, structure within the cell revealed even more dim; 12 h: The number of cells decreased, there were black uniform particles on the surface of cells and structures within the cell could not be resolved, some cells shrank, rounded; 24 h: Cells ranged loose and intercell space increased, a vague outline of the whole cell can be seen only, the cell structure could not be resolved, the membrane was still visible. Cell surface scattered vacuoles, there was no cytoplasmic. Cell began to shrink and dissolve and there were typical nuclear pyknosis, nuclear fragmentation and cell necrosis; 48 h: Cells ranged more loose, no whole cell membrane can be seen, cells presented degeneration or dissolved necrosis. (4) Pretreated bydocetaxel for 3 h, 6 h, 12 h, 24 h, 48 h before exposure, it presented significant differences in SER among groups (F = 32.43935, P = 0.0000), it presented significant differences in SER between groups (P<0.05). SER was 1.21,1.33,1.50,1.68,1.72 and 1.83 respectively. With the increase in time of pretreatment, SER gradually increase, pretreatment of 3 h~12 h, SER increase fastest, SER subsequently growed slowly. (5)Only treatment of 4μg/ml docetaxel or radiation of 2 Gy caused significant G2/M block, it presented a synergistic effect of docetaxel and irradiation. (6) With the extension of pretreatment time, percentage of G2/M cell increased gradually, pretreatment of 12 h, 24 h, 48 h, percentage of G2/M was 32.23%,50.23% and 60.13% respectively.Conclusion Docetaxel had significant radiosensitizing effect and toxicity on SMMC-7721, IC50 was 21.6μg/ml, 95% confidence interval was 19.12μg/ml ~24.60μg/ml, mechanism of radiosensitizing effect may be related to the induction of G2/M block and redistribution of cell cycle.