| Objective: Ferroptosis is a new kind of programmed cell death,which is different from apoptosis and necrosis.The research on new tumor treatment model based on this is endless.But during ferrotosis,stress-induced heat shock proteins play a role in protecting tumor cells from death.In this paper,we investigated the synergistic sensitization effects of HSP70 family protein inhibitor Pifithrin-?(PFT)and ferroptosis inducer Erastin on ferroptosis in glioma cells,and designed a nano probe based PDA to explore the applications in cancer diagnosis and therapy.Methods: The effects of ferroptosis inducer Erastin on glioma cell viability were detected by CCK-8 assay and fluorescence staining;The effects of PFT combined Erastin on cell ferroptosis were investigated by CCK-8 assay,MDA assay,GSH assay and combined index;The expression levels of the related heat shock protein and glutathione peroxidase 4(GXP4)were detected by Western blot;The role of HSPA5 in the combined drug effect was investigated by knockdown and overexpression of HSPA5;A nude mouse model was constructed to evaluate the efficacy of combined drug treatment;Melanin nanoparticles were prepared by the method of dopamine self-polymerization into pellets,and loaded by PFT and Erastin to formate PDA/PE;PDA/PE size,Zata potential and in vitro stability were measured by dynamic light scattering(DLS);The loading and in vitro release rates of PFT and Erastin in PDA/PE were determined by high performance liquid chromatography(HPLC);The uptake rate of PDA/PE from tumor cells was determined by laser confocal microscopy(CLSM);The biocompatibility of PDA/PEG and the cytotoxicity of PDA/PE were determined by CCK-8 assay;The effects of PDA/PE on ferroptosis related proteins were detected by Western blot;By constructing the nude mouse model and injecting PDA/PE into the tail vein,the tumor mri was located and the therapeutic effect was evaluated.Reaults: The results of CCK-8 assay and fluorescence staining showed that Erastininduced ferroptosis in glioma cells SW1783,U87 MG and LN229,and different cells had different tolerance to Erastin;Compared with Erastin treated alone,PFT combined with Erastin significantly reduced cell viability,increased MDA content,reduced GSH content,reduced HSPA5 and GXP4 protein expression levels,and effectively reversed cell death after ferroptosis specific inhibitor was added;Compared with the control group,knockdown of HSPA5 further decreased the cell viability and further decreased the GPX4 expression level after Erastin treatment;Compared with control group,overexpression of HSPA5 reverse the cell viability and GPX4 expression level after Erastin treatment,and the cell death of sensitized cells was also reversed after PFT and Erastin treatment;Animal studies have shown that combination therapy is more effective in inhibiting tumor growth than drug treatment alone.The nanomaterial PDA/PE were successfully prepared,the average size of the material was 81.7 nm,the Zeta potential was-29.2±1.1 mv,and the stability was good in vitro;The drug loading rates of PFT and Erastin by HPLC were 16% and 9%respectively,and the 72 h release rates were 63% and 79% respectively;CLSM detected a good uptake of PDA/PE;PDA/PE at low concentrations caused cell death and leaded to decreased GPX4 expression level;In vivo experiments showed that PDA/PE could achieve tumor localization imaging under MRI and effectively inhibit tumor regrowth.Conclusion: This study demonstrated that Erastin could induce ferroptosis in glioma cells,and HSPA5 played a negative role in regulating iron death.Thus,a small molecule compound PFT was screened out,and the experiments proved that PFT could enhance Erastin induced ferroptosis through HSPA5-GPX4 axis.Moreover,a novel nanomaterial was successfully prepared,which was loaded with PFT and Erastin simultaneously by using polydopamine as the carrier,and was able to achieve tumor localization imaging and induce ferroptosis to treat tumors,providing a new strategy for cancer treatment. |
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