| Objective:Angiogenin（ANG）has been demonstrated to play an essential role in tumor angiogenesis as well as in cancer cell survival,proliferation,migration and invasion.Neamine,a degradation product of neomycin,has been found to hold a dual anti-tumor effect by simultaneously inhibiting tumor angiogenesis and tumor growth through blocking the nuclear translocation of ANG in vascular endothelial cells and tumor cells,and thus to be a potential and promising new anti-cancer agents.The aim of this study was to determine the toxic target organs and the therapeutic effects on liver cancer and the mechanism of neamine through the sub-chronic toxicity test on SD rats and the anti-liver cancer research in vitro and in vivo.Method:According to the result of the acute toxicity test,SD rats were injected intraperitoneally with low,medium or high dose of neamine（6,12 or 60 mg·kg-11 for the females;8,16 or 80 mg·kg-11 for the males）in the sub-chronic toxicity experiment,and after a 90-day treatment period and a 28-day recovery period,the effects of neamine on the body weight,food consumption rate,parameters of hematology,serum biochemistry and urinalysis,organ coefficient and histopathology were evaluated to determine the toxic target organ of neamine.Tissue microarray assay was performed to examine the difference of ANG expression in hepatocellular carcinoma tissues and normal liver tissues;immunofluorescence assay was used to confirm the inhibitory effect of neamine on the nuclear translocation of ANG;MTT assay was conducted to observe the inhibition of neamine on cell proliferation of HUVECs and HepG2 cells;in vivo xenograft tumor experiment in nude mice was done to evaluate the therapeutic effect of neamine on liver cancer and the expression of ANG,CD31and Ki67 was also detected to explore the possible mechanism of anti-liver activity of neamine.Results:The sub-chronic toxicity experiment in SD rats showed that the No Observed Adverse Effect Level（NOAEL）of neamine is 12 and 16 mg·kg-1·d-11 for female and male rats,respectively,and no mortality was found throughout the experiment.The adverse effects of neamine included increased organ coefficients of spleen and kidney,increased BUN in both female and male rats at high dose,increased monocytes count（Mon%）in female rats at high dose,and increased TC,CR but decreased organ coefficients of heart and liver in male rats at high dose.All of which,except the kidney coefficient and BUN in males receiving high dose of neamine,returned to normal levels after the 28-day recovery period.Histopathological examination revealed vacuolar degeneration of glomerulus,degeneration of renal tubules and cast in the kidneys of treated rats,which were also recovered except the males in high dosing group.Tissue microarray analysis validated up-regulated ANG expression and increased nuclear expression in human hepatocellular carcinoma tissues than in normal hepatic tissues,suggesting that ANG is a possible new target for liver cancer treatment;in vitro experiments confirmed that neamine effectively blocked the nuclear translocation of ANG in HUVEC and HepG2 cells,specifically inhibited ANG-induced cell proliferation without effect on basal level cell proliferation;in vivo test verified that neamine significantly inhibited the progression of HepG2 xenografts in athymic mice accompanied by decreased expression of ANG,CD31 and Ki67 in tumors.Conclusion:（1）Kidney is the most susceptible organ for neamine toxicity;（2）Neamine holds a potential anti-liver cancer activity in vitro and in vivo;（3）Neamine exhibits dual anti-cancer effect by suppressing both tumor angiogenesis and cancer cell proliferation through blocking nuclear translocation of ANG in endothelial and cancer cells. |
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