Role Of Heat Shock Protein 70 In Iron-dependent Oxidative Stress

Iron-dependent oxidative stress means that iron ions in cells catalyze the Fenton reaction,causing lipid peroxide accumulation,protein denaturation,and misfolding.Heat shock protein70(HSP70)is a stress protein,which can help the misfolded protein to refold or degrade and protect the cell under the oxidative stress state of the cell.However,the relationship between the HSP70 family and iron-dependent oxidative stress is unclear.Polyunsaturated fatty acids(PUFAs)are essential fatty acids for humans.Under cellular pathological conditions,dietary intake of PUFAs can also cause lipid peroxidation in the presence of iron ions.This study mainly explored the HSP70 family in the iron-dependent oxidative stress model of PC3 cells induced by docosahexaenoic acid(DHA)superimposed with iron death inducer erastin and RSL3.The role and possible mechanisms of HSPA1 A,HSPA8,and HSPA9 provide a theoretical basis for applying iron-dependent oxidative stress mechanisms to cancer treatment and the prevention of neurodegenerative diseases.The main conclusions are as follows:(1)Exploring HSP70 in PC3 cells by constructing plasmids to overexpress HSPA1 A,HSPA8 and HSPA9 intracellularly and construct small interfering RNA to knock down HSPA8 and HSPA9,using MTT method to measure cell survival rate and LDH method to determine cell death rate.The results showed that in DHA+erastin and DHA+RSL3 induced iron-dependent oxidative stress models,overexpression of HSPA8 and HSPA9 could inhibit cell death,knockdown of HSPA8,HSPA9 could aggravate cell death,but HSPA1 A had no effect.(2)Exploring the role of HSPA8 in iron-dependent oxidative stress through proteasome inhibitors and lysosomal inhibitors.Exploring the location of HSPA8 through cellular immunofluorescence technology.Then we used small molecule peptide P140 that can be combined with its nuclear import sequence.It was found that HSPA8 enters the nucleus during iron-dependent oxidative stress,but inhibiting HSPA8 entry into the nucleus by P140 treatment does not exacerbate cell death.The protection mechanism of HSPA8 in iron-dependent oxidative stress was not affected by the lysosomal pathway and the proteasome pathway,and its nuclear entry was not the core mechanism of protection.(3)Exploring the function of HSPA9 in iron-dependent oxidative stress by measuring mitochondrial function-related indicators such as ATP,mitochondrial membrane potential,and ROS in PC3 cells.To investigate whether knockdown of HSPA9 can reduce the expression of iron-sulfur cluster synthetase(NFS1)in mitochondria through Western Blot.As a result,it was found that overexpression of HSPA9 can stabilize the generation of ATP.Knockdown of HSPA9 could decrease the mitochondrial membrane potential,increase ROS and increase intracellular iron ions.Knockdown of HSPA9 could down-regulate the expression of NFS1.Knockdown of NFS1 could increase the free iron in cells and increase cell death.The results of animal transplanted tumor experiments also proved that the effect of HSPA9 in vivo is consistent with the results of in vitro experiments.This experiment mainly explored the protective effects of HSPA8 and HSPA9 on iron-dependent oxidative stress and explored their related mechanisms.HSPA8 and HSPA9 are involved in protecting against iron-dependent oxidative stress.HSPA9 not only protects mitochondrial function when iron-dependent oxidative stress occurs,but also regulates intracellular free iron content by stabilizing the key enzyme NFS1 that synthesizes iron-sulfur clusters.HSPA8 enters the nucleus during iron-dependent oxidative stress and its protection is independent of the lysosomal and proteasome pathways.In addition,this experiment found that HSPA1 A can also be used as a tumor proliferation target.The above conclusions provide experimental basis and theoretical reference for the application of iron-dependent oxidative stress in the treatment of cancer and neurodegenerative diseases,and provide a theoretical basis for the future development of anti-tumor drugs.