Discovery Of Natural Product EGCG As A Human Flap Endonuclease 1 Inhibitor And Its Role In The Synthetic Lethal Antitumor

Human flap endonuclease 1(hFEN1)is a typical member of the 5’-nuclease superfamily,which plays an important role in the process of DNA replication and repair,and is very important for maintaining the stability and integrity of the genome.In fact,hFEN1 is overexpressed in a variety of tumor tissues,which is related to the malignancy of the tumor and a poor prognosis.In addition,a large amount of evidence indicates that it is also involved in the acquired resistance of tumor cells,so it is considered to be a tumor treatment important target.In this paper,we take hFEN1 as a research target and devote ourselves to the exploration of its inhibitors,and the antitumor activity of inhibitors was evaluated from the perspectives of synthetic lethality.First,according to the TCGA database,hFEN1 was found to be more highly expressed in gastric cancer tissues than normal tissues,and its expression level was positively correlated with the malignancy of the tumor.Based on the substrate specificity of hFEN1,by constructing a fluorescent screening model,it is found that in polyphenols natural products(including EGCG,ECG,EGC,EC)in green tea,epigallocatechin gallate(EGCG)is hFEN1 Potential inhibitor.Subsequently,through enzymatic kinetics,differential scanning fluorimetry and molecular docking studies,it was confirmed that in the presence of Mg2+,EGCG inhibited the activity of hFEN1 in a non-competitive inhibitory mode;and EGCG possibly shared the same pocket with the terminal nucleotide of the DNA product in the hFEN1 protein,so the inhibition of hFEN1 may be achieved by blocking the base unpairing of DNA substrate.Then,we found that the expression level of hFEN1 is related to the proliferation and migration ability of tumor cells.Overexpression of hFEN1 significantly enhances the proliferation and migration ability of MGC-803 cells.MRE11A is highly mutated in gastric cancer.Based on the synthetic lethal partnership between hFEN1 and MRE11A,we created an MRE11A-deficient environment in gastric cancer cells by siRNA.It was found that EGCG had obvious synthetic lethal effect on MRE11A-deficient MGC-803 gastric cancer cells through MTT,wound healing,cellular immunofluorescence and flow cytometry.Finally,we used the combination of EGCG and cisplatin or 5-fluorouracil,which wre DNA damaging chemotherapy drugs,to sensitizing gastric cancer cells,and found that there was a synergistic lethality between EGCG and cisplatin or 5-fluorouracil,which was achieved by inhibiting hFEN1.In summary,EGCG can achieve anti-tumor effects by targeting hFEN1 with a synthetic lethal mechanism.Our original findings not only expanded the means of new targeted anti-tumor therapies,but also provided scientific basis for the development of natural products of polyphenols in adjuvant anti-cancer treatment.