Combination Effect of Sorafenib with Ionizing Radiation on Human Oral Carcinoma In Vitro and In Vivo

• Main Authors: Betty Chang, 張明宇
• Other Authors: Jeng-Jong Hwang
• Format: Others
• Language: en_US
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/74545404841045378262

碩士 === 國立陽明大學 === 生物醫學影像暨放射科學系 === 102 === Background: Oral cancer is ranked the fourth cancer incidence in Taiwan. The major modalities for the treatment of oral cancer are radiotherapy (RT), chemotherapy and surgery, in which RT is the standard, however, all with poor prognosis. Radiation damages cellular DNA, leading to an increase in NF-κB/DNA binding activity if the double strand breaks are not repaired. NF-κB has been reported as a key molecule involved in radioresistance and tumorigenesis. Sorafenib, a multikinase inhibitor, has been shown to have antitumor effects and to be able to inhibit radiation-induced NF-κB activation in several types of cancers. It is noteworthy that OSCC often causes bone invasion and results in a poor quality of life for patients. Here, the therapeutic efficacy of sorafenib combined with ionizing radiation in tumor progression and bone erosion was investigated in a human OSCC cell line in vitro and in vivo. Materials and Methods: Human oral carcinoma SAS cell line was used in this study. SAS cells were treated with sorafenib (15 uM) alone and combined with radiation (10 Gy, pre-treat, concurrent and post-treat), respectively, and determined by MTT assay for cytotoxicity. Apoptosis was determined by DNA fragmentation. NF-κB/DNA binding activity and cell migration were assayed by electrophoretic mobility shift assay (EMSA) and transwell assay, respectively. Protein expressions of NF-κB regulated effector proteins were evaluated by Western blot. Six-week-old male Balb/C mice were injected with 1.5 x 106 SAS/luc2 cells into their right masseter. Two weeks later, mice were randomly separated into six groups: (a) normal; (b) control; (c) sorafenib alone (10 mg/kg/d by gavage); (d) radiation alone (single dose of 6 Gy on day 1); (e) pretreatment (10 mg/kg/d of sorafenib three days prior to a single dose of 6 Gy), (f) combination (10 mg/kg/d sorafenib and 6 Gy irradiation on day 1). Five weeks after tumor cell inoculation, mice were sacrificed, and tumors were removed. Tumor growth was monitored by bioluminescent imaging (BLI). Results: Cytotoxicity of SAS cells induced by sorafenib was increased in a dose-dependent manner with IC50 (inhibition concentration at 50% survival) at 15 uM. Sorafenib inhibited NF-κB/DNA binding activity and the expressions of its regulated downstream proteins while up-regulating pro-apoptotic proteins expressions. DNA laddering assay showed that sorafenib combined with radiation induced DNA fragments. BLI in vivo showed that tumor growth was suppressed most significantly in the pretreatment group. The body weight change of mice of all groups throughout the experimental period were within ± 20%. Ex vivo EMSA and Western blot showed that DNA/NF-κB activity and the expressions of NF-κB associated proteins were down-regulated. The expression level of p65 in tumor tissues of mice was suppressed most significantly in the group pre-treated with sorafenib and radiation. The mandible and zygoma of mice treated with sorafenib alone and combination showed little and no damage, respectively, assayed by micro-computed tomography (micro-CT). Conclusions: Synergistic effect was found when sorafenib was combined with radiation for the treatment of human SAS oral carcinoma in vitro and in vivo. Our results suggest that sorafenib combined with radiation not only suppresses radiation-induced NF-κB activity but can also diminish the level of bone destruction, indicating that this could be a potential treatment strategy for OSCC.