The Effect Of ATRA On RSL3-induced Ferroptosis In Gastric Cancer Cells And Its Mechanisms

Ferroptosis,a newly discovered of cell death method,is regulatory necrosis mediated by iron catalysis and over-oxidation of polyunsaturated fatty acids.It has unique morphological,biochemical and genetic characteristics.Although the research on ferroptosis is still in the early stage and the relevant mechanism is still unclear,many studies have shown that ferroptosis is related to cancer,neurodegenerative diseases,ischemia-reperfusion injury and other diseases.Compared with normal cells,tumor cells have a higher demand for iron,so tumor cells are more prone to iron-catalyzed ferroptosis.Many drugs such as sorafenib,apatinib,artemisinin and its derivatives have been found to exert anti-tumor effects by inducing ferroptosis.Therefore,the induction of ferroptosis of tumor cells by drugs has become one of the new strategies for treating tumors.All-trans retinoic acid(ATRA)is a metabolic intermediate product of vitamin A in animals and has a wide range of pharmacological and physiological activities.At present,ATRA has been successfully applied to the treatment of acute promyelocytic leukemia.It can also be used in combination with conventional chemotherapy drugs in gastric cancer to prolong the overall survival of patients.The latest research found that ATRA can affect the expression of iron homeostasis-related proteins,such as transferrin receptor(TFRC),microtransferrin(MTF)and ferritin.In addition,ATRA can also enhance the ferroptosis of liver cancer cells induced by sorafenib.Based on the above research background,this thesis explored the anti-tumor effect of ferroptosis inducers in gastric cancer,and analyzed the correlation between ATRA and ferroptosis and ATRA influence on the anti-tumor effect of the ferroptosis inducer RSL3.The main findings are as follows:(1)RSL3 can induce ferroptosis of gastric cancer cellsFlow cytometry and SRB method were used to detect the effects of ferroptosis inducers RSL3 and Erastin on gastric cancer cells.The results showed that compared to Erastin,RSL3 is more likely to induce the increase of intracellular lipid ROS and total ROS levels and cell death.The ferroptosis inhibitor Fer-1 can inhibit this death,but not by the autotropic inhibitor Bafilomycin A1,apoptosis inhibitor Z-VAD-FMK and necrosis inhibitor Necrostatin-1.Therefore,RSL3 inhibits tumor growth by inducing ferroptosis in gastric cancer cells.(2)ATRA does not cause ferroptosis of gastric cancer cellsRNA-seq results showed that ATRA could affect the expression of ferroptosis-related genes in HGC-27 cells,such as up-regulating the transcription levels of genes such as ACSL4,GPX4 and TP53,and down-regulating the transcription levels of genes such as TFRC,SLC7A11,ACSL3 and FTH1.In HGC-27,MKN-1,BGC-823 and MGC-803 cells,Western blot further confirmed that ATRA could up-regulate the protein expression of ACSL4 and down-regulate the protein expression of TFRC to a certain extent,which is consistent with the results of RNA sequencing,but the effect of ATRA on the expression of GPX4 protein has cellular differences.Detection by flow cytometry and MDA detection kit showed that ATRA had almost no effect on cell viability,lipid ROS level,total ROS level and MDA content,and would not induce ferroptosis of gastric cancer cells.(3)ATRA can antagonize RSL3-induced ferroptosis and there are cellular differencesThe SRB method data showed that RSL3 can inhibit the proliferation of seven gastric cancer cell lines in a concentration-dependent manner,including AGS,BGC-823,HGC-27,MGC-803,MKN-1,MKN-45 and NCI-N87.When ATRA and RSL3 are used together,ATRA can antagonize the death of HGC-27 and MKN-1 cells induced by RSL3,but cannot antagonize the effects of RSL3 on other cells.The cell viability,lipid ROS level,total ROS level and MDA content were detected by flow cytometry and MDA kit.Results suggested that ATRA can antagonize the ferroptosis of HGC-27 and MKN-1 cells induced by RSL3,but cannot antagonize the ferroptosis of BGC-823 and MGC-803 cells induced by RSL3.Western blot detection revealed that RSL3 could significantly down-regulate the protein expression of GPX4.ATRA can antagonize the down-regulation of GPX4 induced by RSL3 in HGC-27 and MKN-1 cells,but not in BGC-823 and MGC-803 cells.Therefore,ATRA may antagonize the ferroptosis induced by RSL3 by up-regulating the expression of GPX4 protein.Besides,ATRA can also partially antagonize the effects of RSL3 on cell clone formation and cell cycle arrest.There are also cellular differences,which is consistent with its antagonism of RSL3 to induce ferroptosis.(4)In vivo experiments further confirmed that ATRA could antagonize the anti-tumor effect of RSL3The HGC-27 nude mouse xenograft model was constructed,and the anti-tumor effect of the combination of ATRA and RSL3 was further analyzed in vivo.The experimental results showed that compared with the blank control group,the ATRA single-agent group had almost no inhibitory effect on tumor growth,and the RSL3single-agent group significantly inhibited tumor growth.Compared with the RSL3single-agent group,the ATRA and RSL3 combination group’s tumor volume not only did not decrease,but also increased to a certain extent.This result indicates that ATRA antagonizes the anti-tumor growth effect of RSL3.The detection of tumor tissue in mice further confirmed that RSL3 down-regulated the expression of GPX4 and increased the level of MDA,while ATRA can antagonize the increase in MDA level caused by RSL3 and restore the expression of GPX4 protein.There was no significant difference in the body weight of the mice and they were in good growth state,indicating that the drugs had no apparent toxic and side effects.In summary,RSL3 exerts an anti-tumor effect by inducing ferroptosis in gastric cancer cells.Although ATRA cannot induce ferroptosis,it can antagonize the ferroptosis induced by RSL3 in gastric cancer.This regulation was controlled by up-regulating GPX4 protein expression and inhibiting the increase of intracellular lipid peroxidation.