Radiosensitization By Pentoxifylline On Human Hepatoma Cell Line And Its Mechanism

Objective: To enhance the radiosensitivity of cancer cell is one of the most important ways to improve the effects of radiotherapy. There is evidence that the duration of the G2/M delay following irradiation is correlated with cell survival. G2/M arrest provides an extended time for repair of sublethal DNA lesions and thus leads to an improved radiation response. This study is designed to investigate the effects of pentoxifylline (PTX) on radiation induced-cell cycle redistribution in human hepatocellular carcinoma cell lines which have different types of p53 gene and the radiosensitization by PTX. Its possible mechanism and clinical significance are to be discussed.Methods: HepG2 (wild-type p53) and Hep3b (p53-defective) of human hepatocellular carcinoma cell lines were studied. MTT assay was used to evaluate the cytotoxicity of PTX and clonogenic assay employed to observe its effects on the radiosensitivity of the HepG2 and Hep3b quantified by calculating the sensitive enhancement ratio (SER). Flow cytometry was performed to observe the cell-cycle of HepG2 and Hep3b in response to X-ray irradiation and the effect of PTX intervention. Optical microscope, fluorescence microscope, transmission electron microscope and flow cytometry were used to assess apoptosis of HepG2 and Hep3b. Results: Cytotoxicity: The cytotoxicity of PTX increased in a dose-dependent manner following a 48-hour treatment, with the optimal dose range of 1-5 mmol/L. A sub-toxic dose of PTX at 2 mmol/L was used in subsequent experiments. Clonogenic Assays: Clonogenic survivalassays up to 12 Gy demonstrated that p53-defective Hep3b cells (sensitizer enhancement ratio [SER] = 2.68+0.24) were sensitized by PTX (2 mmol/L) to a significantly higher degree than p53 wild-type HepG2 (SER = 1.31+0.16) cells. Flow Cytometry: Exposure of irradiation (6 Gy) cells to PTX (2 mmol/L) resulted in abrogation of the radiation-induced G2/M arrest in the p53-defective Hep3b cells, whereas the p53 wild-type-expressing HepG2 cells showed less significant impairment of G2/M checkpoint. The proportions of HepG2 cells and Hep3b cells in G2/M phase were, respectively, 53.2%, 25.2% and 86.8%, 14.8% in 6 Gy alone and 6 Gy plus 2 mmol/L PTX. Apoptosis: G2/M abrogation was accompanied by a higher increase of apoptosis in Hep3b cells than in HepG2 cells through optical microscope, fluorescence microscope, transmission electron microscope and flow cytometry. Conclusion Radiosensitization by PTX is possibly associated with the abrogation of G2/M arrest in HepG2 cells and Hep3b cells following radiation exposure, while p53-defective Hep3b cells are sensitized by PTX to a significantly higher degree than p53 wild-type HepG2 cells. These results support consideration of the clinical evaluation of PTX to enhance the efficacy of radiotherapy of hepatocellular carcinoma in the future.