Endostatin, an angiogensis inhibitor, was discovered by Dr. Judah Folkman¡¦s group in 1997. From their series studies, they demonstrated that the angiogenesis inhibition approach, which abolished the formation of new blood vessels and led to starvation of cancer cells, is a safe, effective anticancer method without side effect and drug resistance. Phase clinical trial on endostatin was carried out in 1999 and completed in 2001, heralding the approaching of a new arsenal of cancer therapy drugs. Endostatin is also a proteolytic fragment (~20 kDa) from an extracellular protein, collagen XVIII. It potently inhibits endothelial cell proliferation and angiogenesis, but has no cytotoxic effects on other cells. Above all, cycled therapy of experimental cancer in rodents with endostatin led to tumor dormancy without drug resistance. However, the exact mechanism on how endostatin inhibited endothelial cells proliferation remains largely unknown. We have cloned mouse endostatin cDNA from mice liver by RT-PCR. After verification by DNA sequencing, endostatin cDNA was subcloned in to E. coli expression vector to express and generate large quantities of recombinant GST-fused endostatin. Unlike His-tagged endostatin, GST-endostatin is soluble and capable of inhibiting endothelial cell lines EA.hy926 with a half-maximal inhibition concentration (IC50) of 20 nM. In present study, we investigated whether GST-endostatin caused alterations in cytoskeleton in endothelial cells. By using a fluorescence dye to visualize the actin filament under confocal microscope, it was found that endostatin induced the corruption of actin network in endothelial cells. Western blot analysis revealed that GST-endostatin treatment caused downregulation of cytoskeleton proteins such as tubulin, vimentin and ECM-related signaling molecules such as focal adhesion kinase (FAK), mitogen activated protein kinse (MAPK), Erk in a dose-dependent manner. Moreover, GST-endostatin decreased the levels of cell survival factor such as AKT and NF-£eB. Since GST-endostatin induced sustained calcium rise, the effect of endostatin on protein kinase Cs (PKCs) were studied and revealed that endostatin reduced the levels of PKCK1¡BPKC eta¡BPKC iota and PKC lamda. Other than endothelial cell, the cytotoxicity of GST-endostatin in hepatoma cells were investigated since liver the primary expression site of collagen XVIII, precursor of endostatin. Unexpectedly, endostatin also inhibited the proliferation of hepatoma cells. Flow cytometry and nucleus staining indicated that GST-endostatin also induced apoptosis in hepatoma cells. Moreover, GST-endostatin exhibited differential cytotoxic effect against well-differentiated (such as HepG2, Hep3B) and poor differentiated (such as Mahlavu, Sk-hep-1) hepatoma cells that the IC50 for well differentiated hepatoma cells were 8-10 folds lower than for poor-differentiated cells. Above all, GST-endostatin inhibited the migration of SK-hep-1 and modulated the secretion of matrix-metalloproteinases (MMPs) by Mahlavu and SK-hep-1 cells. In summary, present study explored the role of alterations in cytoskeleon network in the cytotoxic mechanism of GST-endostatin. Moreover, the inhibitory effects of GST-endostatin on proliferation of hepatoma cells were reported for the first time.