Dihydroartemisinin Promotes Ferroptosis In CcRCC Via CMA-dependent Degradation Of FTH1

Objective: Study on the mechanism of Ferroptosis induced by Dihydroartemisin(DHA)in clear cell renal cell carcinoma.Methods:(1)After treating clear cell renal cell carcinoma 786-0 and Caki-1 with DHA in a dose gradient,CCK-8 and PI staining were used to clarify the effect of DHA on the vitality of clear cell renal cell carcinoma cells and to determine the drug IC50.(2)After treating 786-0 and Caki-1 cell lines with DHA(50 μ M)for 24 h,flow cytometry was used to detect the level of Lipid reactive oxygen species(ROS)in the cells,inverted fluorescence microscopy was used to photograph changes in Labile iron pool(LIP)levels to confirm induction of Ferroptosis by DHA in cc RCC.(3)Western blotting was used to detect changes in protein levels of Ferroptosis related proteins GPX4,FTH1 and SLC7A11 after treatment with DHA(50 μ M)respectively for 786-0 and Caki-1 cells,verifying possible mechanisms of induction of Ferroptosis by DHA in clear cell renal cell carcinoma cells.(4)After treating 786-0 cells with DHA(50 μ M)for 24 Hours,transmission electron microscopy was used to photograph morphological changes to confirm induction of autophagy by DHA in cc RCC.(5)Both CQ and 3-MA were used respectively to monitor ROS and intracellular LIP levels,confirming recovery effects induced by DHA while distinguishing degree of Ferroptosis mediated by Autophagy or Chaperone-Mediated Autophagy(CMA).(6)After treating 786-0 and Caki-1 cell lines with DHA(50 μ M)according to time gradient respectively,western blotting was used to detect protein level changes of CMA related proteins HSPA8,Lamp2 a,confirming induction effect on CMA by DHA.(7)Lipo3000 transfection reagent were applied for knockdown core gene HSPA8,western blotting was applied for detection Lamp2 a &FTH1 protein level change after treated with DHA(50μm).(8)immunofluorescence was applied for determination colocalization situation between HSPA8 & FTH1 inside 786-0& Caki-1cell lines.Results:(1)The results of the CCK-8 assay and PI staining assay demonstrate that DHA has toxic effects on clear cell renal cell carcinoma cells.(2)ROS flow cytometry results showed that DHA induced accumulation of large amounts of ROS in 786-0 and Caki-1cells.(3)inverted fluorescence microscopy LIP results indicated that DHA induced an increase in LIP within the cells.(4)WB experiment results revealed that DHA mediates Ferroptosis of 786-0 and Caki-1 cells via inducing degradation of FTH1.(5)transmission electron microscopy results demonstrated that DHA induced autophagy in 786-0 cells.(6)CQ rescue experiment results showed that compared with the DHA group,CQ could significantly restore the iron-induced death of clear cell renal cell carcinoma cells induced by DHA.(7)ROS,LIP and WB results indicated that DHA induced CMA in clear cell renal cell carcinoma cells.(8)WB experiment and immunofluorescence experiment results showed that knockdown of HSPA8 could inhibit degradation of FTH1,and they had obvious co-localization in the cytoplasm.Conclusions: In summary,Dihydroartemisinin DHA can induce Ferroptosis of renal clear cell carcinoma cells via CMA-dependent FTH1 degradation.