The mechanism of triptolide-induced cuproptosis in lung cancer cells
Objective To explore the molecular mechanism of cuproptosis in lung cancer cells induced by triptolide.Methods Human lung cancer cell lines A549 and H460 were cultured,and cell viability and proliferation was detected with MTT assay and EdU experiment,respectively.Cell migration ability was detected by transwell migration assay.Copper ion fluorescence probe was used to determine the intracellular copper concentration.JC-1 fluorescent probe was applied to test the changes of mitochondrial membrane potential.Western blot was used to examine the expression levels of FDX1,POLD1,SDHB,LIAS,DLAT monomer and oligomer proteins,and the expression levels of copper homeostasis regulatory proteins CTR1,ATP7A and ATP7B.Results The results from MTT assay showed that triptolide inhibited the viability of lung cancer cells in a concentration-dependent manner,and the IC50 of triptolide on A549 and H460 cells were 74.67 nmol/L and 34.12 nmol/L,respectively.Triptolide could significantly inhibit the proliferation and migration of lung cancer cells(P<0.05)confirmed by EdU experiment and transwell migration assay.After treatment with triptolide,the copper concentration in A549 and H460 cells was increased and the mitochondrial membrane potential was decreased under fluorescence microscope.Furthermore,the data from Western blot demonstrated that the expression of FDX1,POLD1,SDHB,LIAS and DLAT monomer in A549 and H460 cells was down-regulated,while the expression of DLAT oligomer was increased(P<0.05).There was no obvious change in the expression of copper homeostasis regulatory protein CTR1,but the expressions of ATP7A and ATP7B were down-regulated(P<0.05).Conclusion Triptolide can increase the intracellular copper concentration by down-regulating the expression of ATP7A and ATP7B,and then down-regulate the expression level of iron-sulfur cluster protein,increase the expression level of DLAT oligomer,induce copper death of lung cancer cells,and inhibit the proliferation and migration of lung cancer cells.
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