Experimental Study On Hepatocellular Carcinoma (HCC): Radiosensitization Of Sorafenib In Vitro And In Vivo And Hepatic Regeneration After Liver Irradiation In Cirrhotic Rats

Part I:Study on radiosensitization of sorafenib in treatment of hepatocellular carcinoma (HCC) in vitro and in vivoObjective:To investigate the radiosensitization of sorafenib in treatment of HCC cell line SK-Hep-1 and its transplanted tumor in nude mice.Material & Methods:The present study was designed into two experiments in vitro and in vivo. For experiment in vitro, cell proliferation toxicity was measured by using CCK-8 method to obtain 50% inhibition concentration (IC50) of sorafenib 48 hours after treating SK-Hep-1 cell line. Clone formation assay was applied to obtain survival fraction (SF) of each treatment, i.e., IR alone as control, IR synchronizing sorafenib 48 hours afer incubing cell line and IR followed by sorafenib. Based on multi-target single-hit model, cell survival curve was gained and theoretical IR doses of D0, SF10% and SF1% could be calculated. Sensitizing enhancement ratios (SERs) of sorafenib combing IR were obtained by compared with those of group control. For experiment in vivo, nude mice transplanted tumor model was established firstly. The mice were feeded vehicle,30mg/kg and 100mg/kg sorafenib respectively for 7 days, and the tumors were irradiated with doses of OGy,2Gy,4Gy,6Gy,8Gy and 10Gy on day 8 repectively. Tumor growth delay (TGD) was assessed. Dose response curve of transplanted tumor was obtained according to Gompertz model. Given growing to double of initial tumor volume before treatment, TGD of combination group was compared with that of IR alone group, then dose modify fraction (DMF) was calculated to evaluate synergetic anti-tumor effect of sorafenib and IR.Results:Cell proliferation toxicity assay presented IC50 of sorafenib 48 hours after treating SK-Hep-1 cell line was 9.5625μM and the concentration of 1μM, less than IC10, was used in clone formation experiment. According to multi-target single-hit model, cell survival curve of treatment with sorafenib combined with IR concurrently and theoretical IR doses of D0, SF10% and SF1% were gained. SER (DO),SER(SF10%)and SER(SF1%) were 1.1138,1.2749,1.2017 repectively, which were more than 1 and radiosensitizing effect of sorafenib combined with IR concurrently was confirmed. In vivo, after irradiated with 2Gy,4Gy,6Gy,8Gy concurrently combined with 30mg/kg sorafenib, TGD were 5.3 days,15.1 days,19 days and 20.9 days, respectively. As to IR combined with 100mg/kg sorafenib, TGD were 10.9 days,16.6 days,20.3 days and 22.6 days, respectively. DMFs of 30mg/kg and 100mg/kg sorafenib combined IR were between 1.3 and 1.8. The stronger radiosensitization was shown in 100mg/kg sorafenib combined with IR.Conclusion:Sorafenib combined with IR concurrently presented radiosensitation in vitro and in vivo.Part II:The mechanism of radiosensitization of sorafinib in treatment of hepatocellular carcinoma (HCC)Objective:The previous data had shown the radiosensitization of sorafenib in treatment of HCC cell line. This study is to investigate the underlying mechanism.Material & Methods:The methods of Western blotting, enzyme linked immunosorbent assay (ELISA) and RT-PCR were used to investigate molecular mechanism of radiosensitizing effect. SK-Hep-1 cell line was treated with 1μM sorafenib for 48 hours concurrently combined with IR of OGy, 1Gy,2Gy and 6Gy respectively. Then the cells were collected, from which the total protein was extracted. The expressions of VEGFR-2, ERK, NF-κB and Ku 70 were assessed with or without phosphorylation. ELISA was applied to measure concentration of VEGF in medium 0 hour,24 hours,48 hours and 72 hours after SK-Hep-1 being irradiated with single dose of OGy, 1Gy,2Gy,4Gy,6Gy and 8Gy respectively. Otherwise, expression of VEGF mRNA of these cells 24 hours and 48 hours after treated with IR of OGy, 1Gy, 2Gy,6Gy,8Gy was assayed also.Results:(1) The data of Western blotting showed the up-regulation of phosphorylation in DNA repair protein, Ku 70 with IR alone. Phosphorylations in VEGFR-2 and Ku 70 were inhibited obviously by treatment with 1μM sorafenib alone. The treatment of sorafenib combined with IR inhibited the expressions of phosphorylated VEGFR-2, ERK and Ku 70. (2) The results of ELISA assay presented that medium concentration of VEGF increased after IR higher than that of control group. The significant increase presented 24 hours and 48 hours after IR of 2 Gy. The concentrations of VEGF at 72 hours after IR of 1Gy,2Gy,4Gy and 6Gy were significantly higher than those of control group (P<0.05), the highest value after 1 Gy IR, while the difference was not notable after 8Gy IR (P>0.05). As a whole, concentration of VEGF rose with the increase of IR dose, but decreased with high IR dose of 8Gy, of which the reason was the decline of survival cells after 8Gy IR probably. (3) The data of RT-PCR showed expression of VEGF mRNA of Sk-hep-1 was enhanced 24 hours, especially 48 hours after IR. The increase of VEGF mRNA expression presented 48 hours after IR of 1Gy,2Gy,6Gy also. The expressions of VEGF mRNA in control group showed no significant difference between 24 hours and 48 hours after sham IR. VEGF relative transcription ratio was calculated with image analysis system compared with expression ofβ-Actin mRNA, indicating that expression of VEGF mRNA upregulated accelerated parallel to the increase of dose of IR and time after IR, most evident with 8Gy IR. The data implied that the concentration of medium VEGF after IR increased owing to IR-induced enhancement of VEGF mRNA expression.Conclusion:IR could stimulate VEGF secretion of SK-Hep-1 cell line and induce upregulation of DNA repair protein to accelerate repair of DNA damage. Sorafenib combined with IR was capable of inhibiting phosphorylations of VEGFR-2, ERK and Ku 70. These mechanisms resulted in radiosensitization of sorafenib.PartⅢ:A natural process of cirrhosis resolution and deceleration of liver regeneration after thioacetamide withdrawal in a rat modelObjective:To establish a useful rat model with cirrhosis and evaluate liver injury and liver regeneration during cirrhosis resolution.Material & Methods:In a radiobiological study of partial liver irradiation on thioacetamide (TAA) induced cirrhotic liver in rat, the observation time was 120 days after TAA withdrawal. The natural process was recorded, focusing on cirrhosis and regeneration as the baseline to understand and interpret outcome. Cirrhosis in rats was induced by orally drinking 0.03% TAA water for 29 weeks. After establishment of cirrhosis model, the rats were observed for 120 days upon TAA withdrawal to investigate the dynamic changes of liver cirrhosis and regeneration. The endpoints were:(1). Histological change. (2). Liver functions. (3). Cirrhosis:Trichrome stain serum and in situ transforming growth factor beta-1 (TGF-β1), hydroxyproline content of liver. (4). Liver regeneration:liver index; hepatocyte mitotic index (MI); hepatocyte proliferation index (PI) by flow cytometry; PCNA labeling index (LI) by IHC and expression of PCNA mRNA. (5). Growth factors:serum HGF, VEGF, TGF-a and IL-6.Results:The cirrhosis model was established after 29 weeks. And upon TAA withdrawal, the cirrhosis presented resolution. Gradual improvement in liver functions was noted with decreasing ALT, AST and ALP, and increasing PA during cirrhosis resolution. The resolution of cirrhosis was evident by histological improvement with attenuation of collagen fiber and hydroxyproline and decrease of TGF-β1 IHC index, but cirrhosis was still existed on 120 days after TAA withdrawal. Significant deceleration of liver regeneration was demonstrated with TAA withdrawal, evidenced by decrease of hepatocyte MI and PI, reduced expression of PCNA mRNA and PCNA LI, and decreases of serum HGF and VEGF.Conclusion:Cirrhosis was induced by drinking 0.03% TAA water for 29 weeks in rats. Upon TAA withdrawal it was revealed that hepatic cirrhosis continuously resolved, but still existed up to 120 days, and liver regeneration significantly decelerated.PartⅣ:Hepatic Regeneration after Partial Liver Irradiation in Cirrhotic RatsObjective:To investigate if the right half liver were capable of regeneration after sublethal irradiation (IR) and if the IR would trigger regeneration of the left half liver in cirrhotic rat.Material & Methods:This study was designed into two experiments in turn. For experiment one, the cirrhotic rats were divided into control group (without IR), and 5 Gy, 10Gy and 15Gy groups. IR dose of 5 Gy, 10Gy and 15Gy were given as single dose to the right half liver of each group respectively with observations of the endpoints scheduled on 0-day (before IR),30 days,60 days,90 days and 120 days after IR. The results showed a dose-dependent liver proliferation could be stimulated by sublethal partial liver IR of 5Gy to 15Gy, in both irradiated and unirradiated part livers, most notable by 15 Gy. Consequently, the IR of 15 Gy was chosen as a initial stimulus delivered to the right half liver in the experiment two. For experiment two, the cirrhotic rats were divided into sham (OGy) group,2.5 Gy,5 Gy and 7.5 Gy groups according to the IR dose delivered to the left half liver with the right half liver irradiated by 15 Gy. The endpoints same as those in experiment one were observed every 30 days till 150 days after IR. The cirrhotic rats without radiation were chosen to serve as control. The following endpoints were evaluated:(1). Liver injury:Body weight and Serum ALT, AST, ALP and PA. (2). Liver regeneration:liver index; hepatocyte mitotic index (MI); hepatocyte proliferation index (PI); PCNA labeling index (LI); expression of PCNA mRNA; serum HGF, VEGF, TGF-α, IL-6; (3) Serum and in situ TGF-β1.Results:IR of 5 Gy, 10Gy and 15Gy to the right half liver as well as IR of 2.5 Gy, 5 Gy and 7.5 Gy to the left half liver with the right half liver irradiated by 15 Gy could induce hepatic regeneration in both right and left half livers with indicators of liver regeneration including positive staining of PCNA and PCNA mRNA expression, PI and MI, serum HGF, VEGF and IL-6 increasing after IR, and deteriorated liver injury with decrease of ALT, AST and ALP and increase of PA, expression of TGF-β1 in Serum and in situ. The IR-induced hepatic injury and liver regeneration increased accompanying with higher IR dose and whole liver irradiated.Conclusion:Dose-dependent and volume-dependent liver injury and liver proliferation could be stimulated by sublethal partial liver IR of 15Gy in both irradiated and unirradiated portion.