Study On Anti-tumor Activity And Potential Mechanism Of Compound RY10-4

The partial mechanism and anti-tumor activity of the novel compound RY10-4which have a specific anti-tumor effect were studied in this dissertation, and the main content was divided into two parts. In the first part, we studied the effect and related signal pathway of RY10-4-induced autophagic cell death in human breast cancer MCF-7cell line. And we also compared the difference between RY10-4and protoapigenone in the induction of autophagy in autophagy defect breast cancer MCF-7cell line, explored new insights for the potential mechanism of RY10-4induced cell death and the cause of RY10-4showing better antitumor activity than protoapigenone. In the second part, we studied the effects of RY10-4against human hepatocellular carcinoma HepG2cells in vitro and in vivo, and explored some related and potential mechanism of the antitumor activity of RY10-4against liver cancer.FART I:Study on the effect of RY10-4induced autophaic cell death in breast cancer MCF-7cell lineObjective: In consideration of the protoapigenone analog RY10-4showing better antitumor activity in vitro and lower side effects in vivo, the potential mechanism was explored here. The activity of RY10-4induced autophagy in human breast cancer MCF-7cell was studied, and compared it with the effect of protoapigenone. We also discussed the role of RY10-4induced autophagy in cell death and the related signaling pathways in the further researches.Methods:Employing immunofluorescence assay for microtubule-associated protein light-chain3(LC3B), monodansylcadaverine staining, western blot analyses for LC3B and p62as well as ultrastructural analysis by transmission electron microscopy, to observe whether RY10-4or protoapigenone induced autophagy in MCF-7cells. For exploring the role of RY10-4induced autophagy in cell death, inhibitions of autophagy by pharmacological and genetic approaches were performed. Moreover, the signaling pathways involved in RY10-4induced autophagy were studied through detecting the expressing of some related signal proteins by Western blot.Results:RY10-4could induce autophagy in MCF-7cells, but protoapigenone could not. When the autophagy activity was inhibited by autophagy inhibitor3-MA or siRNA specific for autophagic-related gene ATG7, the viability of MCF-7cells exposed to RY10-4significantly increased. In addition, the expression of phosphorylated-mTOR (p-mTOR) and p-p70S6K decreased in a concentration-dependent manner, the expression of p-Akt decreased in both concentration and time-dependent manners, while the activity of AMPK did not alter. Conclusion:RY10-4inhibited mTOR signal and induced autophagy in MCF-7cells via Akt/mTOR pathway, and the induction of autophagy enhanced RY10-4induced antitumor effect, promoted the cell death of tumor cells.PART Ⅱ:Study on the antitumor activity of RY10-4against liver cancer in vitro and in vivoObjective:To study the cell proliferation inhibition of RY10-4on hepatocellular carcinoma HepG2cells in vitro and in vivo, and to determine the biological activity and the underlying mechanisms of RY10-4on hepatocellular carcinoma cancer cells by using in vitro and in vivo experimental models.Methods:Sulforhodamine B (SRB) assay and clonogenicity assay were performed to determine the antiproliferation of RY10-4on hepatocellular carcinoma HepG2cells in vitro. Flow cytometry assay with propidium iodide (PI) staining was used to determine the effect of RY10-4on cell cycle distribution. Fluorescent staining with Hoechst33342and flow cytometry assay with Annexin V/PI staining were performed to evaluate the effect of RY10-4on the induction of apoptosis. Reactive oxygen species (ROS) assay kit and mitochondrial membrane potential (MMP) assay kit with JC-1were used to detect the ROS generation and changes in MMP induced by RY10-4, and these effects of RY10-4were confirmed by using the antioxidant NAC. Furthermore, the human hepatocellular tumor xenograft model established with HepG2cell line was used to investigate the antitumor activity of RY10-4in vivo.Results:The viability of HepG2cells in vitro significantly decreased after the treatment of RY10-4, and a concentration-dependent inhibition in colony formation was also observed. The percentage of cells in the S phase and G2/M phase as well as the ratio of apoptotic cells significantly increased in groups treated with RY10-4, and the typical morphological characteristics of apoptotic cell was observed. RY10-4could induce the ROS generation and the decrease of MMP, while these effects will be inhibited by NAC. Moreover, RY10-4treatment significantly inhibited the growth of HepG2tumor in tumor-bearing nude mice without significant hematological toxicity as well as hepatotoxicity and nephrotoxicity.Conclusion:RY10-4exhibited high antiproliferation and antitumor activity toward HepG2cell line in vitro and in vivo. The underlying mechanism for the antitumor activity of RY10-4included: blocking the cell cycle process, inducing ROS generation, decreasing the MMP, inducing apoptosis.