Daphnoretin induces ferroptosis in 786-O cells by suppressing glutathione peroxidase 4 and FTH1 expression,thereby inhibiting cell proliferation and invasion independently of the GSK/FoxO3a pathway
Objective This study examines the effects of daphnoretin(DAP)on the proliferation and invasion of 786-O cells and its ability to induce ferroptosis,exploring the underlying molecular mecha-nisms.Methods The impact of DAP on 786-O cell proliferation and invasion was measured using cell counting kit-8(CCK-8)and Transwell assays at various concentrations(0,12.5,25.0,50.0,100.0 µmol/L).Additionally,cell viability was assessed with a CCK-8 assay following co-treatment with 0.2 µmol/L Erastin or 30.0 nmol/L RSL3 and 12.5 µmol/L DAP for 24 hours.Cells were also treated with 12.5 μmol/L DAP alongside 10 µmol/L deferoxamine(DFO)or 50 mg/L ferric ammonium citrate(FAC)for 24 hours,with subsequent measurement of total iron and Fe2+concentrations using assay kits.Protein expressions were analyzed via western blot.Statistical analysis involved t-tests for two-group com-parisons and one-way ANOVA for multiple groups.Results CCK-8 assays revealed that the absorbance values for the 0 µmol/L DAP group exceeded those of the 12.5,25,50,and 100 μmol/L groups after 24 and 48 h of treatment(0.10±0.15 vs.0.85±0.12,0.79±0.11,0.78±0.12,0.65±0.10,respec-tively;2.23±0.28 vs.1.93±0.16,1.43±0.13,1.29±0.20,0.92±0.34,respectively).Similarly,Transwell assays indicated higher absorbance values for the 0 μmol/L DAP group compared to the 12.5,25,50,and 100 μmol/L groups(2.93±0.36 vs.2.24±0.40,1.90±0.44,1.18±0.29,0.97±0.37,respectively;all P＜0.05).GPX4 protein expression in HK-2 cells was significantly lower than in 786-O cells(100％vs.742％,F=40.48,P＜0.01).The GPX4 protein expression in the 0 μmol/L DAP group exhibited higher relative gray values compared to the 12.5,25,50,and 100 µmol/L groups,with 1,0.84,0.81,0.73,and 0.49,respectively.The absorbance values for DAP combined with Erastin were lower than those observed with DAP or Erastin alone[(1.61±0.46)L/(g·cm)vs.(2.43±0.11)L/(g·cm)or(2.38±0.41)L/(g-cm),bothP＜0.05].The combination of DAP and RSL3 resul-ted in lower absorbance compared to DAP or RSL3 alone[(0.62±0.53)L/(g·cm)vs.(1.89±0.53)L/(g-cm)or(1.52±0.26)L/(g-cm),bothP＜0.05].Additionally,GPX4 protein expression was reduced in the DAP combined with Erastin or RSL3 groups compared to control,DAP,Erastin,or RSL3 groups(relative gray values of 0.71,0.46,1,0.92,0.84,and 1.18,respectively).No significant difference was found in FTH1 protein expression between the 786-O and HK-2 cell groups(141.33％vs.100％,F=1.49,P＞0.05),though the gray value of FTH1 protein in the 0 µmol/L DAP group was higher than in the 12.5,25,50,and 100 μmol/L groups(relative gray values of 1,0.95,0.82,0.62,and 0.47,respectively).The results of Fe2+level assay showed that the Fe2+level in the DFO group was lower than that in the control and the DAP group[(0.21±0.03)nmol/106 vs.(0.56±0.14)nmol/106 and(1.22±0.26)nmol/106,all P＜0.05].Whereas,the Fe2+level in the DAP+DFO group was high-er than that in the DFO group[(0.62±0.15)nmol/106 vs.(0.21±0.03)nmol/106,P＜0.05].In ad-dition,the relative expression of FoxO3a protein in the 0 μmol/L DAP group was lower than that in the oth-er concentration groups(1.00 vs.1.45±0.32,P＞0.05;1.00 vs.1.65±0.40,1.55±0.32,and 1.55±0.13,all P＜0.05);the relative expression of pGSK-3[3(Ser9)protein in the 0 μmol/L DAP group was higher than that in the other concentration groups(1.00 vs.1.02±0.15,P＞0.05;1.00 vs.0.82±0.04,0.72±0.03,0.67±0.11,all P＜0.05),and GSK-3β,TIGAR,KIF18A,and PSMA/FOLH1/NAALADaseI proteins were expressed among the groups differences were not statistically significant(P＞0.05).The relative expression of GSK-3β and pGSK-3β proteins in the 786-O cell group was lower than that in the HK-2 cell group[(58.00±3.00)％vs.100％,F=588,P＜0.01;(43.67±1.53)％vs.100％,F=4080.14,P＜0.01],whereas the relative expression of FoxO3a proteins was higher than that in the HK-2 cell group[(164.67±23.54)％vs.100％,F=22.63,P＜0.01].The differences in protein expression of GPX4 and FTH1 in the up-regulated pGSK-3β group were not statistically significant compared with the vector group(P＞0.05).The relative expression of GPX4 and FTH1 proteins in the down-regulated FoxO3a group was lower than that in the shNC group[GPX4:100％vs.(69.33±10.07)％,(48.00±10.82)％,(54.67±26.58)％and(29.33±6.43)％,F=10.87,all P＜0.05;FTH1:100％vs.(90.33±33.25％P＞0.05;100％vs.(64.67±16.26)％,(55.00±12.53)％and(48.00±13.23)％,F=4.49,all P＜0.05].Importantly,the pGSK-3β protein gray value in the DAP group was smaller than that in the shNC group,and the pGSK-3β protein gray value in the pcDNA3.1-GSK-3β group was larger than that in the shNC and the DAP group;however,the pGSK-3β protein gray value in the pcDNA3.1-GSK-3β+DAP group was not larger than that in the DAP group(relative gray val-ues for GSK-3β were 1.00,1.21,1.25,1.53;pGSK-3β relative gray values were 1.00,0.79,1.34,0.57).The FoxO3a protein gray values of the DAP group were larger than those of the shNC group,and the FoxO3a protein gray values of the shFoxO3a-811 and shFoxO3a-1156 groups were smaller than those of the shNC and the DAP group,but the gray value of FoxO3a protein in the combined group was not smaller than that in the DAP group(relative gray values of the groups were 1.00,1.36,0.52,1.33,0.81,1.27).Conclusion DAP induces ferroptosis in 786-O cells by reducing GPX4 and FTH1 protein levels and increasing Fe2+concentrations,which suppresses cell proliferation and invasion.However,DAP does not appear to directly affect the pGSK-3β/FoxO3a signaling pathway.
DaphnoretinRenal cell carcinomaGlutathione peroxidase 4/Ferritin heavy chain 1Phosphorylated glycogen synthase kinase-3β/Forkhead box transcription factor 3aFerroptosisProliferation and Invasion
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