Inhibition Of Rapamycin On Tumor Growth And Metastasis In Rat Experimental Model Of Hepatocellular Carcinoma

Among the most serious complications of general immunosuppressive therapy in organ transplantation is the high risk of the recurrence of neoplastic tumors and the development of de novo cancer. Cancer has therefore become a major cause of death in patients otherwise successfully treated by organ transplantation. Rapamycin, as a new immunosuppressive drug, not only has the characteristics of low nephrotoxicity and liver toxicity for clinical application, but also can inhibit growth and metastasis of many kinds of tumors including hepatocellular carcinoma (HCC). The anti-tumor mechanisms of rapamycin may be related to the inhibition of endothelial cell proliferation, sensitization of endothelial cell to apoptosis, blockage of cell cycle progression and anti-angiogenesis. It's well known that tumor growth and metastasis depend on angiogenesis and HCC is generally considered to be a hypervascular tumor. Therefore, inhibition of angiogenesis may be considered as an effective measure for controling HCC growth and metastasis. Moreover, hypoxia inducible factor-1α(HIF-1α) and vascular endothelial growth factor (VEGF) play important roles in angiogenesis. Recent studies have demonstrated that rapamycin can exert its anti-tumor effects by inhibiting HIF-1αand VEGF in rhabdomyosarcoma and pancreatic carcinoma. However, whether rapamycin can inhibit HCC growth and metastasis through regulating HIF-1αand VEGF has been few reported.PartΙDynamic changes of tumor angiogenic characteristics in induced rat hepatocellular carcinoma model Objective: To investigate the expression and correlation of hypoxia inducible factor-1α(HIF-1α), vascular endothelial growth factor (VEGF) and microvessel density (MVD) in rat hepatocarcinogenesis.Methods: Sixty male Spreque-Dawley (SD) rats were selected to establish an experimental rat hepatocellular carcinoma (HCC) model by using diethylnitrosamine (DEN) and N-nitrosomorpholine (NMOR). During rat hepatocarcinogenesis, 7 rats were sacrificed at 0, 4, 8, 12 and 16 weeks respectively. All rats were killed at the 20th week. The liver sections were prepared as routine and antiangiogenetic effects were assessed by CD34 immunostaining. The levels of HIF-1αand VEGF proteins and mRNAs were examined by immunohistochemistry, western blot and semi-quantitative RT-PCR respectively at different stages of rat HCC model.Results: After 8, 12 and 20 weeks of induction, typical fatty degeneration and inflammatory cell infiltration, liver cirrhosis and cancer pathological changes were observed. The expression of HIF-1αand VEGF proteins and mRNAs increased significantly in the experimental rats. MVD increased considerably at the stage of liver cancer, with a significant positive correlation between MVD and HIF-1αas well as VEGF (r =0.946, 0.951 respectively, all P <0.01), and between HIF-1αand VEGF (r =0.955, P <0.01).Conclusions: HIF-1αand VEGF play important roles in tumor occurrence and development during rat hepatocarcinogenesis possibly through promoting tumor angiogenesis. PartΙΙPrevention of hepatic tumor growth and metastasis in rats with rapamycinObjective: To test the effect of rapamycin (RAPA) on hepatic tumor growth and metastasis in Spreque-Dawley (SD) rat model and explore the possible mechanism.Methods: SD rat hepatocellular carcinoma (HCC) model with metastatic potential was induced by diethylnitrosamine (DEN) and N-nitrosomorpholine (NMOR). 120 SD rats were randomized into four groups 16 weeks after DEN and NMOR treatment, recieved 4-week intraperitoneal injection of RAPA (1.5 or 4.5 mg/kg/d), CsA (25 mg/kg/d) or equal volume of 0.9% saline, respectively. Tumor growth and metastasis were checked after the 4-week treatment. Serum vascular endothelial growth factor (VEGF) was determined by enzyme-linked immunosorbent assay (ELISA). Antiangiogenetic effects were assessed by CD34 immunostaining. The levels of hypoxia-inducible factor 1 alpha (HIF-1α) and VEGF proteins and mRNAs were detected by immunohistochemistry, western blot and reverse transcriptase-polymerase chain reaction (RT-PCR).Results: The mean liver weight (5.58%±0.42% and 5.69%±0.74%), the mean liver nodules (5.12±0.68 and 5.67±1.12) , the mean lung nodules (0.43±0.11 and 0.45±0.83), and the lung metastsis rate (17.2% and 14.8%) were lower in rats treated with RAPA 1.5 mg/kg/d or 4.5 mg/kg/d than those in rats treated with saline, which were 10.42%±1.86%, 12.36±3.45, 1.81±0.3 and 50.0% respectively (P <0.01 or P <0.05). The intratumoral microvessel density (MVD), serum VEGF, and the levels of HIF-1αand VEGF were lower in RAPA-treated rats than those in control rats. However, CsA-treated rats showed an opposite trend compared with the RAPA-treated rats.Conclusions: RAPA can repress the expression of angiogenesis-promoting factors HIF-1αand VEGF, and significantly inhibits the growth and metastasis of HCC.