The Palmitic Acid Induced Ferroptosis Via CD36 Activating ER Stress In Colon Cancer Cells

Part?PA induced ferroptosis of colon cancer cells through ER stress–calcium release–iron accumulationObjective Observe the effect of PA treatment on the proliferation and death of colon cancer cell line HT29,detect the relevant indicators of ER stress and ferroptosis,clarify the specific way of PA-induced cell death and explore the specific molecular mechanism of ferroptosis.Methods First,in the colon cancer cell line HT29,the appropriate concentration was screened by PA treatment with different concentration,and the apoptosis and necrosis condition under the selected concentration were detected.Subsequently,we use Lipid ROS,MDA(malonaldehyde),transmission electron microscopy and other indicators to detect the ferroptosis,and the ferroptosis inhibitor Fer-1(ferrostatin-1)was used for verification;Iron Assay Kit and Calcein AM probe were used to detect iron ions levels to determine the molecular pathway of ferroptosis,and add DFP (deferiprone,iron-chelating drug)or FAC(ferric ammonium citrate)to verify.Then we explored the endoplasmic reticulum stress induced by PA treatment in HT29 cells through QPCR experiments and calcium ion detection.Finally,we used the confocal microscope to observe the relationship between the endocytosis-recycling transport of transferrin and PA and calcium ion levels.Results The results of CCK-8 and LDH release experiments found that 120?M PA inhibited the proliferation of HT29 cells and induced cell death;the results of flow cytometry,WB,and the addition of apoptosis inhibitor Z-VAD-FMK or necrosis inhibitor Necrostatin-1 experiments proved that apoptosis and necrosis were not the main.After PA treatment,the Lipid ROS content and MDA level of HT29 cells increased.Transmission electron microscopy showed that the volume of mitochondria decreased and the number of mitochondrial cristae decreased.The addition of Fer-1can significantly inhibit cell death;and after PA treatment,the total iron content of HT29 cells increased,and the level of Fe²⁺ions increased.DFP and FAC could reduce and aggravate cell death,respectively,confirming that the ferroptosis was caused by the increase of iron ions.PA treatment increased the expression of endoplasmic reticulum stress markers PERK,EIF2?,and ATF4,the release of endoplasmic reticulum calcium increased,and the level of cytosolic calcium increased;the results of confocal microscopy showed that after PA treatment,the transportation efficiency of transferrin was improved.The addition of BAPTA-AM(Ca²⁺chelator)could inhibit the transportation efficiency,and the addition of Thapsigargin(endoplasmic reticulum stress inducer)could promote the transportation efficiency.Conclusions PA treatment induced the death of colon cancer cell line HT29 in a non-apoptotic and necrotic manner,but induced cell ferroptosis,and the occurrence of ferroptosis was caused by excessive iron ion accumulation.PA could mediate the release of endoplasmic reticulum calcium by inducing endoplasmic reticulum stress,and the accumulation of cytoplasmic iron ions may be mediated by transferrin transportation regulated by cytosolic calcium ion levels,which provided a new idea for the study of ferroptosis.Part?CD36 mediated PA-induced ER stress's ferroptosisObjective Observe the sensitivity of different colon cancer cell lines to PA-induced ferroptosis and explore the main difference.On this basis,construct the cell model based on the interference and overexpression of differential gene to explore the role of this gene in ferroptosis.Methods First,the colon cancer cell lines HCT116,SW480,SW620 were treated with PA,the proliferation and death rate of different cell lines were observed.Subsequently,the lipid reactive oxygen species,iron ions,calcium ions,and fatty acid uptake capacity were detected to determine the differences between different cell lines;and the difference in CD36 expression between HT29 and SW620 cell lines were verified by QPCR and WB.Then constructed si RNA-CD36 model in HT29 cells to observe the changes of ferroptosis phenotype;the lentiviral-overexpression-CD36model was constructed in SW620 cells to observe the changes in sensitivity to PA-induced ferroptosis.Finally,the database was used to analyze the expression of CD36in colon adenocarcinoma and its influence on the prognosis.Results HT29 and HCT116 cell lines were sensitive to PA-induced ferroptosis,SW480 and SW620 cell lines were resistant to PA-induced ferroptosis.After PA treatment,SW620 cells did not show changes in Lipid ROS,MDA,Fe²⁺,and Ca²⁺levels,but the fatty acid uptake capacity of SW620 cells was significantly lower than that of HT29 cells.QPCR and WB results showed that after PA treatment,the expression of CD36 in HT29 cells increased,while the expression of CD36 in SW620cells did not change significantly,and the basal expression of CD36 in HT29 cells was significantly higher than that in SW620 cells.After si-1(si RNA sequence-1)and si-2(si RNA sequence-2)interfered with the expression of CD36 in HT29 cells,and then treated with PA,the ferroptosis phenomenon of HT29 cells was partially alleviated.After overexpression of CD36 in SW620 cells by lentivirus and treatment with PA,SW620-Homo-CD36(SW620-CD36-overexpression cell line)cells showed cell ferroptosis.CD36 was significantly higher in colon adenocarcinoma than in normal tissues,and the prognosis of colon adenocarcinoma patients with high CD36expression was worse.Conclusions Different colon cancer cell lines had different sensitivities to PA-induced ferroptosis,and their differential performance mainly came from the level of CD36 expression.Colon cancer cells with high expression of CD36 were more sensitive to ferroptosis under PA treatment,which provides a new strategy for tumor patients with precise treatment.Part?The anti-tumor effect of PA in nude-mice xenograft models with different CD36 expressionObjective To verify the anti-colon cancer activity of PA and its mechanism through the nude-mice xenograft model,and to evaluate the safety of PA in vivo.Methods Colon cancer cell line HT29 were planted on the left and right axillary sides of nude-mice to establish HT29-nude-mice xenograft models;the stable transfected cell lines SW620-Homo-CD36 and SW620-NC cells were respectively planted on the left and right axillary sides of nude-mice to establish SW620-nude-mice xenograft models.Set up the PA treatment group and the control group,each with 5 mouses;each of the PA treatment group was intraperitoneally injected once a day(10mg/kg),the control group was intraperitoneally injected with the same amount of BSA solution,and the tumor volume change and survival status of tumor-bearing nude-mice were monitored every three days for a total of 15 days.The total iron content of tumors was compared by Iron Assay Kit,and the MDA content of tumors was compared by MDA detection kit.Results In the HT29-nude-mice xenograft model,the volume and weight of the tumor were significantly lower than the control group under the treatment of PA intraperitoneal injection.The total iron content and MDA content of the tumor in the PA treatment group were higher.SW620-NC,SW620-Homo-CD36-nude-mice xenograft model under the treatment of PA intraperitoneal injection,the volume and weight of SW620-NC-tumor are significantly higher than SW620-Homo-CD36-tumor,and the total iron content and MDA content of SW620-Homo-CD36-tumor were significantly higher than that of SW620-NC-tumor.Conclusions PA had anti-colon cancer activity in nude-mice xenograft models with high CD36 expression,and its mechanism was related ferroptosis induced by iron excess.PA has no obvious toxic reaction in nude-mice xenograft models of colon cancer,which provides an experimental basis for the clinical application of PA.