Studies On Anti-hepatoma Activity And Molecular Mechanisms Of Arenobufagin

Aim:To evaluate in vivo anti-hepatoma effect of arenobufagin and explore probable molecular mechanisms of arenobufagin-induced apoptosis in doxorubicin-sensitive and -resistant human hepatoma cells (HepG2 and HepG2/ADM), and further study inhibitory effect of arenobufagin on the invasion and migration of HepG2 cells. Methods:S180 sarcoma and H22 hepatoma animal model in KM mice, together with orthotopic transplantation tumor model of HepG2 and HepG2/ADM in nude mice were established for the evaluation of in vivo anti-hepatoma effect of arenobufagin. Changes in body weights, spleen index, number of spleen cells and white cells in blood were measured. Increase of ROS production and intracelluer calcium level in HepG2 cells was observed by a fluorescence microscope and quantified by a fluorescence microplate reader. Expression levels of mitogen-activated protein kinase (MAPK) and nuclear factor-KB (NF-κB) signaling pathways related proteins were determined by western blot. Cell adhesion assay, cell scratch assay and Transwell chamber assay were used to determine changes in adhesion, invasion and migration of HepG2 and Western blot was used to detect the expression of matrix metalloproteinase 2 and 9 (MMP 2 and MMP 9). Results:In vivo studies suggested that treatment with arenobufagin could significantly inhibit the growth of H22 or S180 tumor-bearing KM mice as well as HepG2/ADM tumor-bearing nude mice, but its inhibitory effect was not remarkable on HepG2 tumor xenografts. The body weights of nude mice were not shown to be markedly changes in arenobufagin treatment group compared with control group, whereas the spleen index, the total number of spleen cells and white cells in blood all obviously decreased, indicating that arenobufagin had toxicity to immune system. Studies on molecular mechanisms showed that arenobufagin induced ROS generation, increase of intracellular calcium level, downregulation of p-Erk, upregulation of p-p38 and p-JNK. While arenobufagin could also inhibit IκB expression and phosphorylation, downregulate nuclear factor-KB (NF-κB) p50 and p65 subunites expressions, and trigger translocation of p65 subunit from cytosol to nuclear. Furthermore, it was found that PD98059 (Erk inhibitor) could augment arenobufain-induced cell death, but SP600125 (JNK inhibitor) had no effect. In addition, arenobufagin could also obviously suppress the adhesive, invasive and migrative ability of HepG2 cells in a dose-dependent manner, which was associated with decrease of the expressions of MMP 2 and MMP 9. Conclusion:Taken together, our results indicate that arenobufagin has anti-hepatoma activity in vivo, and ROS generation, intracellular calcium increase, activations of mitogen-activated protein kinase (MAPK) and NF-κB pathways may be related with the arenobufagin-induced apoptosis. Arenobufagin also can inhibit the adhesion, invasion and migration of hepatoma cells.