Study Of The Increased Chemotherapeutic Sensitivity Of 5-FU By HSPB1-knockdown-Induced Ferroptosis In Gastric Cancer Cells

Background and objective:5-Fluorouracil（5-FU）is the most important chemotherapeutic drug for gastric cancer（GC）,and chemotherapy resistance is still an inevitable hardship in the clinical treatment of GC.5-FU is an analogue of uracil,which affects pyrimidine synthesis by inhibiting thymidylate synthetase and thus intracellular d TTP pools are depleted,inducing apoptosis and preventing tumor growth.However,it is uncertain whether 5-FU induces ferroptosis in GC.In our previous study,we found that mutant-type P53 mediated the high expression of CXCR1 in GC cells,thereby participating in drug resistance of GC.We also screened 27 different proteins after silencing CXCR1 in GC cells,presenting a notably low expression of HSPB1 of them.After that,Our team made an in-depth study on HSPB1 and ferroptosis in order to explore the connection of them.Based on the above,this study was to explore whether the chemotherapeutic sensitivity of 5-FU was increased by HSPB1-knockdown-induced ferroptosis in GC cells,which can provide a partial experimental foundation to drug resistance.Methods:（1）To explore the influence of liproxstatin-1 on HSPB1-Knockdown cell lines in GC:（1）Screening HSPB1-knockdown（HSPB1-KD）cell lines:constructed SGC7901-HSPB1-KD and BGC823-HSPB1-KD GC cell lines were screened out by puromycin and verified by Western blot and RT-q PCR.（2）SGC7901 and BGC823 GC cell lines were severally split up into groups to treat（including HSPB1-Control group,HSPB1-KD group and HSPB1-KD+Lip-1 group）.HSPB1-Control group and HSPB1-KD group were treated with DMEM medium（10%FBS）.HSPB1-KD+Lip-1 group were treated with Liproxstatin-1（10μM）for 48 hours.（3）We measured cell viability,Lipid ROS,mitochondrial structural features,expression of protein indexes of all groups by CCK8,flow cytometry and fluorescence microscope,electron microscope,Western blot.（2）To explore whether 5-FU could affect the occurrence of ferroptosis in GC cells:（1）SGC7901 and BGC823 GC cell lines were severally split up into groups to treat（including Control group,5-FU group and 5-FU+Lip-1 group）.Control group were treated with DMEM medium（10%FBS）.5-FU group were treated with 5-FU（10 ug/ml）for24 hours.5-FU+Lip-1 group were treated with Liproxstatin-1（10μM）for24 hours in advance,and then replaced Liproxstatin-1（10μM）with 5-FU（10 ug/ml）+Liproxstatin-1（10μM）for 24 hours.（2）We measured Lipid ROS,mitochondrial structural features,expression of protein indexes of all groups by flow cytometry and fluorescence microscope,electron microscope,Western blot.（3）To explore the influence of HSPB1-knockdown-induced ferroptosis on chemotherapeutic sensitivity of 5-FU in GC cells:（1）SGC7901 and BGC823 GC cell lines were severally split up into groups to treat（including HSPB1-Control group,HSPB1-Control+5-FU group and HSPB1-KD+5-FU group）.Control group were treated with DMEM medium（10%FBS）.HSPB1-Control+5-FU group and HSPB1-KD+5-FU group were treated with 5-FU（10 ug/ml）for 24 hours.（2）We measured three cell function indexes of all groups by CCK8,wound healing assay,flow cytometry and fluorescence microscope.Results:（1）Liproxstatin-1 could inhibit the ferroptosis by HSPB1-knockdown induced in GC cells:（1）Single out the stable HSPB1-KD GC cell lines which were established in early work.（2）CCK8 showed that cell viability of HSPB1-KD group were lower than HSPB1-Control group,but the HSPB1-KD+Lip-1 group were increased by treatment with liproxstatin-1 in the 48^thhour.（3）Flow cytometry and fluorescence microscope showed that the HSPB1-KD group had the highest level of lipid ROS among the three groups.（4）Electron microscope showed that the ferroptotic mitochondrial structural features of HSPB1-KD group has increased obviously compared to the other two groups.（5）Western blot showed that HSPB1-KD group had over-expression of KEAP1 and low expression of GPX4 and SLC7A11 compared to the other two groups,which means the increasement of ferroptosis.（2）5-FU could induce the ferroptosis in GC cells and it could be inhibited by Liproxstatin-1:（1）Flow cytometry and fluorescence microscope showed that the 5-FU group had the highest level of lipid ROS among the three groups.（2）Electron microscope showed that the ferroptotic mitochondrial structural features of 5-FU group has increased obviously compared to the other two groups..（3）Western blot showed that 5-FU group had over-expression of KEAP1 and low expression of GPX4 and SLC7A11 compared to the other two groups,which means the increasement of ferroptosis.（3）HSPB1-KD could increase chemotherapeutic sensitivity of 5-FU through the influence of ferroptosis:（1）CCK8 showed that the cell inhibition of 5-FU was increased by HSPB1-KD.（2）Flow cytometry and fluorescence microscope showed that the accumulation of lipid ROS were the highest level in HSPB1-KD+5-FU group and the lowest level in Control group.（3）Wound healing assay showed inhibited migration ability of HSPB1-KD+5-FU group.Conclusion:HSPB1-Knockdown could regulate ferroptosis in GC cells by up-regulating KEAP1 or down-regulating GPX4 and SLC7A11,and then increase chemotherapeutic sensitivity of 5-FU.