Experimental Study On The Effect Of Sorafenib Combined With Radiation On Hepatocellular Carcinoma

Part Ⅰ Study on the in vitro effect of sorafenib combined with radiation on Hepatocellular carcinomaPurpose:The multikinase-inhibition action of sorafenib provides strong rationales for its combination use with radiotherapy.ln the present study, we used two Hepatocellular carcinoma (HCC) cell lines of different radiosensitivity,SMMC-7721and SK-HEP-1, to investigate whether a combination of sorafenib and radiation will enhance the treatment response in vitro.Methods:Cell viability assay was carried out using CCK-8to calculate sorafenib concentrations producing percentages of cell growth inhibition (inhibition concentration, IC).To minimize the potential influence of drug toxicity, an appropriate less toxic dose of sorafenib, that is10%of IC50, was chosen to be used in colony formation assays. Then, radio-modulating effect of sorafenib was assessed by performing colony formation assays, and sensitizing enhancement ratio (SER) was determined.Results:Sorafenib treatment of48h inhibited cell proliferation with an IC50of4.82μM in SMMC-7721and9.55μM in SK-HEP-1cells. According to that, colony formation assays were performed using selected sorafenib doses of low toxicity (10%of IC50), i.e.,0.5μM for SMMC-7721and1μM for SK-HEP-1.Post-irradiation sorafenib produced radiosensitization obviously for SMMC-7721(SER=1.21) but did not alter the shape of the radiation dose-response curve for SK-HEP-1(SER=1.00). Pre-irradiation sorafenib only increased the β component of the survival curve at higher radiation doses for both cells, resulting SER of1.10and1.18for SMMC-7721and SK-HEP-1, respectively.Conclusion:Sorafenib enhanced radiosensitivity of two HCC cells in a schedule-dependent manner. Part Ⅱ Mechanism of radiosensitization by sorafenib in Hepatocellular carcinomaPurpose:To investigate the underlying mechanism based on which sorafenib sensitizes HCC cells to irradiation.Methods:The expression of several proteins associated with cell proliferation (p-ERK1/2), DNA damage repair (p-Ku70, p-BRCA1), apoptosis (p-NF-KB) and angiogenesis (p-VEGFR2) were tested by Western blotting, comparing combination treatment with radiation alone. Micronuclei (MN) assay was carried out to determine whether sorafenib increases radiation-induced DNA damage. The effect of sorafenib on radiation-induced apoptosis was examined using both Annexin V-FITC/PI staining flowcytometry and assay of caspase-3activation.Results:Sorafenib inhibited radiation-induced phosphorylation of VEGFR2and activation of downstream ERK pathway in HCC cells. MN frequency increased markedly with combination treatment of radiation and sorafenib comparing to separate treatment alone. Combination treatment downregulated the expression of p-BRCA1and p-Ku70, suggestive of both HR and NHEJ repair pathway blockage by sorafenib in irradiated HCC cells. Additionally, sorafenib blocked radiation-activated NF-KB and significantly increased radiation-induced early apoptotic level as well as caspase-3activation. However, these observations were not consistent, and the exception occurred in SK-HEP-1when sorafenib given following radiation, which explained the failure of radiosensitization in this group.Conclusion:Sorafenib inhibited post-irradiation cell proliferation, increased radiation-induced DNA damage and suppressed DNA repair capacity, and also promoted radiation-induced apoptosis, all of which contributed to the potential effect of irradiation and sorafenib in HCC cells. Part III Study on the in vivo effect of sorafenib combined with radiation on Hepatocellular carcinoma xenograftsPurpose:To examine the combination antitumor effect of radiation and sorafenib on HCC xenografts.Methods:SMMC-7721and SK-HEP-1cells were implanted subcutaneously into nude mice to establish HCC xenograft models. Tumor-bearing mice were treated with various schedules of sorafenib and radiation, including vehicle control, radiation alone, radiation followed by7days of daily sorafenib at30mg/kg/d or100mg/kg/d p.o.(R→30S or R→100S, sequential schedule) and7days of daily sorafenib at two above dose levels followed immediately by radiation (30S+R or100S+R, concurrent schedule). Tumor growth curves were fitted with Gompertz or Cubic model. Tumor growth delay (TGD) was defined as the time required for the average relative tumor volume to double from the first day of treatment (V0). Then, TGD of each radiation dose with or without sorafenib was normalized for the drug toxicity by subtracting that for sorafenib alone of corresponding dose level or vehicle control. Finally, normalized TGDs were plotted against radiation doses to set up a dose-response curve, and dose modifying factor (DMF) was calculated.Resluts:For SMMC-7721tumors, compared with radiation alone, sequential treatment with radiation followed by7days of daily sorafenib delayed tumor growth in a dose-dependent manner, with DMFs increasing with sorafenib dose (maximum,1.57). However, concurrent treatment with7days of sorafenib followed immediately by radiation exhibited almost the same effectiveness in delaying the growth of SMMC-7721tumors at both dose levels of sorafenib with DMFs averaging about1.4. In SK-HEP-1tumors, concurrent treatment produced dose-dependent growth inhibition, with DMF of up to1.82for higher sorafenib dose. Sequential schedule also increased the effectiveness of radiation, but higher sorafenib dosage seemed to fail to further enhance this radiosensitization (DMFs averaging about1.5).Conclusion:Sorafenib potentiated radiation antitumor effect in vivo significantly no matter what schedule was taken. The effectiveness of sorafenib against tumor vasculature and angiogenesis would play additional role in delay of tumor growth in vivo, which merits further confirmation in the future.