| Research background:Colorectal cancer(CRC),also known as colorectal cancer,is a common malignant gastrointestinal cancer.According to the national cancer statistics published by the National Cancer Center in 2009,the incidence of colorectal cancer ranks third in the country,accounting for 9.8% of the total cancer incidence population.Compared with previous years,data continue to grow,which shows that colorectal cancer has seriously threatened people’s lives and health,looking for new drugs for colorectal cancer is imminent.Inducing the death of colorectal cancer cells will be an effective way to develop new anti-colorectal cancer drugs.At present,it has been found that the main methods of programmed cell death are autophagy,apoptosis,autophagy,necrosis,ferroptosis and so on.Among them,ferroptosis is a new type of cell death reported by Scott et al in 2012.The mode of death is completely different from apoptosis,autophagy and necrosis in biochemistry,morphology and heredity.ferroptosis is a non-apoptotic programmed death pathway in which iron-ion-dependent lipid peroxidation produces reactive oxygen species.Its morphological characteristics are mainly cell membrane integrity,no blistering,no rupture,cell aggregation,mitochondria concentration,mitochondrial membrane thickening,ridge reduction or disappearance,while mitochondrial outer membrane rupture,normal nucleus,no chromosome condensation.IMCA(2-imino-6-methoxy-2H-chromene-3-carbothioamide)was selected by the NR4A1 pharmacophore model for the Specs diversity small molecule compound library.In vivo and in vitro experiments confirmed that IMCA had significant colorectal growth.However,molecular interaction experiments showed that there was no significant interaction between IMCA and NR4A1.Using transcriptome sequencing analysis,we found that the inhibition of IMCA on the viability of colorectal cancer cells was related to its induction of ferroptosis in colorectal cancer cells.Therefore,we propose "the scientific problem of IMCA blocking colorectal cancer through ferroptosis pathway".Focusing on this scientific issue,the molecular mechanism of IMCA blocking colorectal cancer through ferroptosis pathway is discussed in this paper.Research objective:This study will apply the in vitro and in vivo models of colorectal cancer cell lines,using Western blot,qRTPCR and serological evaluation methods to explore the effect of IMCA on iron death in colorectal cancer cells in vitro and in vivo,and further explore IMCA regulation xCT/SLC7A11 The mechanism of action of TFR-mediated ferroptosis in colorectal cancer cells ultimately clarifies the molecular mechanism of IMCA blocking colorectal cancer.Through this study,it will help to provide new methods and ideas for the development of new drugs for colorectal cancer.Research methods:In this study,we first used MTT assay to detect the activity of colorectal cancer cell lines DLD-1,HCT116,and SW480 at different concentrations(0(Control),200,100,50,25,12.5 μM).BALB/C Nude nude mice were used to establish a subcutaneous tumor-bearing model of colorectal cancer DLD-1 cells,The mice were treated with IMCA for 30 days by tail vein injection of 1 mg/kg/3 days.The inhibitory effect of IMCA on colorectal cancer proliferation was studied in vitro.The influence of liver and kidney function and organ index.The effect of IMCA on apoptosis of colorectal cancer cells was detected by Annexin V-FITC/PI double staining and Western blot.Molecular interactions,computer simulations and immunofluorescence techniques were used to observe and photograph under the fluorescence microscope.The interaction between IMCA and NR4A1 was examined at the molecular and cellular levels.Transcriptome analysis explores the mechanism by which IMCA blocks colorectal cancer.In vitro detection of ferroptosis markers such as reactive oxygen species,glutathione and iron content,and the use of Western blot and qPCR experimental techniques to verify that IMCA blocked colorectal cancer by ferroptosis pathway.Using siRNA and overexpression plasmid transfection methods,we investigated the mechanism by which IMCA regulates colorectal cancer by colorectal cancer cells by regulating xCT and TFR.Research results:1.IMCA significantly inhibits the viability of colorectal cancer cells in vitro.The effect of IMCA on the proliferation of colorectal cancer cells DLD-1,HCT116 and SW480 was studied by MTT assay.The results showed that IMCA significantly inhibited the proliferation of colorectal cancer cells DLD-1,HCT116 and SW480 in vitro.2.IMCA significantly inhibits the growth of colorectal cancer in vivo.The subcutaneous model of colorectal cancer DLD-1 cells was established in BALB/C Nude nude mice.The effect of IMCA on the growth of colorectal cancer in vivo was studied by intravenous injection of 1 mg/kg/3 day IMCA for 30 days.The results showed that IMCA significantly inhibited the growth of colorectal cancer in vivo.3.IMCA has no interaction with NR4A1.The interaction between IMCA and NR4A1 was studied by immunofluorescence,computer simulation docking and microcalorimetry.The results showed that there was no interaction.4.IMCA-induced ferropotsis in colorectal cancer is associated with down-regulation of TFR and xCT/SLC7A11 expression.After the intervention of IMCA by confocal microscopy and transmission electron microscopy,the content of active oxygen increased and the mitochondria of cells disappeared.In vitro detection of reactive oxygen species,iron content,glutathione and cysteine ferroptosis markers,the results show that IMCA induces increased levels of reactive oxygen species and total ions in colorectal cancer cells,glutathione and cysteine The acid content is reduced.Western blot and qPCR experiments showed that IMCA up-regulated TFR expression in colorectal cancer and down-regulated xCT/SLC7A11 expression.Application of siRNA silencing TFR and plasmid transfection overexpressing xCT/SLC7A11 to detect ROS,iron content,glutathione and cysteine again.The results showed that after silencing TFR,IMCA induced reactive oxygen species and total oxygen in colorectal cancer cells.The content of ions decreased,the content of glutathione and cysteine decreased,and the content of glutathione and cysteine increased after overexpression of xCT/SLC7A11.Research conclusions:1.The new benzopyran derivative IMCA significantly blocked the growth of colorectal cancer in vivo and in vitro.2.IMCA blocks colorectal growth through ferroptosis.3.IMCA-induced ferroptosis in colorectal cancer is associated with the production of reactive oxygen species mediated by TFR and xCT/SLC7A11. |
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