Xiamen University First Affiliated Hospital Xiamen, Fujian
L. Gong1, M. CAI2, J. Wang2, Y. Lu2, X. Tan2, and Q. Lin1; 1Department of Radiation Oncology, Xiamen Cancer Center, Xiamen Key Laboratory of Radiation Oncology, The First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, China, 2Department of Oncology, The First Affiliated Hospital, Xian Jiaotong University, Xian, China
Purpose/Objective(s): Radiotherapy is one of the important methods in the treatment of liver cancer. The aim of this study is to explore the mechanism of Apoptin mimics short peptide (AP97-110) combined with ATR inhibitor (AZD6738) to promote the sensitivity of liver cancer radiotherapy. Materials/
Methods: In vitro, the intracellular distribution of AP97-110 and XPO1 was observed by confocal microscopy. The binding of AP97-110 and XPO1 to XPO1 was studied by flow fluorescence assay. The binding force between AP97-110 and XPO1 was quantitatively analyzed by fluorescence polarization, the spatial structure of AP97-110 was analyzed by AlphaFold2, and the molecular docking of AP97-110 was analyzed by Pymol and PDBePISA. The effects of AP97-110 and AP97-110 combined with AZD6738 on radiotherapy sensitivity were investigated using a cell counting kit. The apoptosis and cell cycle of hepatocellular carcinoma cells was studied by flow cytometry. The effects of AP97-110 combined with AZD6738 on the proliferation and migration of hepatocellular carcinoma cells were studied by cloning and scratching experiments. In vivo, a mouse model of liver cancer was constructed and given 4Gy IR, AP97-110+4Gy IR, AP97-110+ AZD6738+4Gy IR, respectively. The growth and tumor volume of mice were recorded to study the effect of AP97-110 combined with AZD6738. The relationship between AP97-110 combined with AZD6738 and DNA damage repair was further studied by WB and immunofluorescence experiments. Results: AP97-110 colocated with XPO1 with Kd value of 6.9±4.6nM. AP97-110 competitively inhibited XPO1 antibody from binding to XPO1. The AP97-110 contained an NES structure that allowed it to be bound by XPO1 with energy (?G) was -11.1kcal/mol and interface area (A2) was 715.6, which proveed that there was molecular docking between AP97-110 and XPO1. The results of the cell counting kit experiment showed that AP97-110 could increase the radiotherapy sensitivity by about 30%. AP97-110 combined with AZD6738 increased radiotherapy sensitivity by about 80%. Flow cytometry results showed that AP97-110 combined with AZD6738 promoted apoptosis and inhibited cell cycle of hepatocellular carcinoma after radiotherapy. AP97-110 combined with AZD6738 could inhibit the proliferation and migration of liver cancer cell. The results of in vivo experiments showed that AP97-110 combined with AZD6738 could promote the radiation sensitivity of liver cancer tissue without affecting the growth of mice. AP97-110 combined with AZD6738 up-regulated the expression levels of DNA damage protein ?-H2AX, and down-regulated the expression levels of DNA damage repair proteins Rad51 and p-chk1. Conclusion: AP97-110 combined with AZD6738 promotes radiotherapy sensitivity of liver cancer by inhibiting XPO1 and ATR to promoting DNA damage and inhibiting DNA damage repair.
