Antitumor Activity Screening And Preliminary Mechanism Study Of Compounds Based On Naphthoquinone And Indole Structures From Chinese Herbal Medicines

Natural small molecule compounds in Chinese herbal medicine have become one of the research hotspots of anticancer drugs due to their excellent anti-tumor effects and less adverse reactions.It has become a popular method to design novel anticancer compounds based on the natural compounds in Chinese herbal medicine.In this paper,based on the research of compound library,and the human lung cancer cells A549,human prostate cancer cells PC-3,human liver cancer cell Hep G2,human chronic myeloid leukemia K562 cells and rat renal cell NRK-52E as test objects,a series of in vitro activities of naphthoquinone and indole structures were evaluated,includeing MTT assay,cell migration test,plate clone formation test,apoptosis related test,ROS level test and cycle test.Then the binding effect of the compounds with related proteins was simulated by molecular docking,and the tumor inhibition mechanism of the compounds was preliminarily explored by western blot.The research idea and methods of this project were as follows:the antiproliferation ability of naphthoquinone and indole series compounds to five cells(four cancer cells and one normal cell)lines was determined by MTT method.The results showed that most of these compounds had certain anticancer activity,and their toxicity to normal cell NRK-52E were relatively lower,indicating most of them had good selectivity.Naphthoquinone series compound 5n and indole series compound 6had obvious inhibitory effects on A549 and PC-3 cells,respectively.The scratch test was carried out on lung cancer cell A549 with strong migration ability.The results showed that compounds 5n and 6 had better anti-migration effect on A549 cells.The effects of compound 5n and 6 on the proliferation,colony formation ability of A549and PC-3 cells were tested by plate cloning experiment.The results showed that compound 5n and 6 significantly inhibited the proliferation and colony formation ability of A549 and PC-3 cells,respectively,and the inhibition ability increased with the increase of concentration.In order to analyze the effects of compounds on apoptosis of cancer cells,Hoechst 33258 staining showed that compounds 5n and 6significantly destroyed the nucleation of A549 and PC-3 cells,respectively.JC-1staining was used to analyze the changes of mitochondrial membrane potential of A549 and PC-3 cells after 24 h treatment with compounds.It was found that compounds 5n and 6 reduced the mitochondrial membrane potential of A549 and PC-3 cells in a concentration-dependent manner,respectively,and the reduction of membrane potential was the precondition of cell apoptosis.Annexin V-FITC/PI double staining assay showed that compounds 5n and 6 induced apoptosis in a dose-dependent manner in A549 and PC-3 cells,respectively.DCFH-DA was used to detect the fluorescence intensity of cells after treatment with copounds for 24 h.The dual results of fluorescence microscopy and flow cytometry showed that compounds5n and 6 increased ROS levels in A549 and PC-3 cells,respectively.Given that it had been reported in the literature that increasing the level of cellular ROS was one of the key reasons for the anti-tumor activity of 1,4-naphthoquinone compounds,two groups with antioxidant NAC(reducing ROS)and without NAC were compared to explore whether there was a certain correlation between ROS level and the cytotoxicity of the naphthoquinone series compound 5n.The results showed that ROS played an important role in compound 5n's induction of apoptosis in A549 cells.Further analysis of the effects of compounds on the cell cycle of cancer cells showed that compounds 5n and 6 inhibited A549 and PC-3 cells in the G2/M phase,respectively,and ROS had no significant effect on A549 cell cycle arrest induced by compound5n.Then molecular docking was used to simulate the binding of compounds 5n and 6to STAT3(PDB:1BG1)and EGFR^wt(PDB:2ity),respectively.The results showed that both compounds had moderate binding strength with their corresponding proteins and the small molecules were well encapsulated by proteins.Finally,Western blotting was used to investigate the effects of compound 5n on the expression of Akt,STAT3and their phosphorylated proteins in MAPK/Akt/STAT3 pathway.The results showed that compound 5n had inhibitory effects on Akt,STAT3 and their phosphorylated proteins.The effect of indole compound 6 on the expression of EGFR and its phosphorylated protein,and the expression of Akt and its phosphorylated protein in the downstream EGFR pathway PI3K/Akt was investigated.The results showed that compound 6 could reduce the expression of Akt,p-Akt and p-EGFR,and compound 6also could reduce the expression of EGFR at low concentration.Through this topic research,compound 5n,6 respectively had better inhibition effect on the growth proliferation,cell migration,colony growth of A549,PC-3 cell lines,the former may induce cell apoptosis by inhibiting the MAPK/Akt/STAT3pathway,while the latter may affect the signal transduction of the downstream signal pathway PI3K/Akt by affecting the EGFR protein and autophosphorylation,thus promoting cell apoptosis and inhibiting cell growth.