Compound 7-5 Targets Nur77 To Induce Methuosis In Ras-mutated Tumor Cells

Cancer is still one of the most common killer endangering human life and health.The global cancer burden is still increasing year by year,and colon cancer has become the third cancer leading death.Traditional treatment programs,including chemotherapy,radiotherapy,and surgical resection,but still appear tumor chemotherapy drug resistance and postoperative tumor metastasis and recurrence,poor prognosis,which is also the difficulty to cure colon cancer.Orphan nuclear receptor Nur77 is widely involved in the occurrence and development of tumor.Although no endogenous ligand has been found,its biological activity can be regulated by small molecule compounds,which has attracted much attention as a good drug target.Compounds 7-5 involved in this study is designed and synthesized according to the structure and function regulation of Nur77 protein,aiming at targeting Nur77 and influencing signal pathway mediated by Nur77,so as to specifically induce apoptosis of malignant tumor cells.The binding constant Kd of 7-5 to Nur77 protein was 46.9 nM.In Biotin competition experiment,compound 7-5 also showed binding Nur77 in vivo,proved that compound 7-5 is an excellent new ligand for Nur77.7-5 induced cell death,and IC50 value was 1.153 μM.However,no obvious PARP cleavage was found after 7-5 induction,suggesting that 7-5-induced death is an apoptosis independent pathway,and in the cytoplasm the accumulation and fusion of time-dependent vacuoles,which led to the extremely vacuolization of the cytoplasm,and eventually led to cell detachment,rupture and death.The cell morphology was observed under electron microscope,immunofluorescence and flow cytometry were used to identify the death way as methuosis.As a new way of death,the essence of methuosis can be summarized as the dysfunction of macropinocytosis,which is different from apoptosis.This way of death has no DNA damage and is not caspase dependent,so it will not be inhibited by broad-spectrum protease inhibitors.Immunofluorescence experiments confirmed that 7-5 could induce Nur77 translocate to lysosome,resulting dysfunction of late endosome fusion with lysosome,resulting methuosis.7-5 induced methuosis in a Nur77 dependent manner.Finally,significant differences in the production of methuosis were observed in several groups of different Ras-mutated colon cell lines.7-5 specifically induced Ras-mutated colon cancer cells methuosis,but normal colorectal fibroblasts and colon cancer cells without Ras-mutated had no effect.7-5 has a strong killing effect on HCT-116(Ras-mutated)transplanted tumor,but almost no effect on HT-29(non Ras-mutated).In our study,a new Nur77 ligand compound 7-5 was found.In Ras mutant tumor cells,it can change Nur77 subcellular localization,translocating to lysosome,block late endosome fusion with lysosome,and induce the occurrence of methuosis by binding Nur77.The study expanded the non genomic function of Nur77,provided a new feasibility of non apoptosis dependent death methuosis for the clinical treatment of drug-resistant tumor cells,provided a new idea for targeting Nur77 to treat Rasmutated malignant tumors,and also proved the research value of compound 7-5.