Combination of the cuproptosis inducer disulfiram and anti‑PD‑L1 abolishes NSCLC resistance by ATP7B to regulate the HIF‑1 signaling pathway

Abstract

Disulfiram (DSF) is used to treat non-small cell lung cancer (NSCLC). DSF significantly increases expression of programmed death-ligand 1 (PD-L1), which may enhance immunosuppression and immune escape of tumors. Therefore, the present study aimed to investigate the role of combined treatment of DSF and anti-PD-L1 in NSCLC resistance. The viability and apoptosis of A549 cells were detected by the Cell Counting Kit-8 assay and flow cytometry, respectively. The expression levels of ATPase copper-transporting β (ATP7B) and PD-L1 in A549 cells were detected by reverse transcription-quantitative PCR and western blot analysis. The levels of reactive oxygen species (ROS), malondialdehyde (MDA) and superoxide dismutase (SOD) in A549 cells were detected by respective assay kits. The expression levels of cuproptosis-associated proteins ferredoxin-1 (FDX1), ATP7B, solute carrier family 31 member 1 (SLC31A1), succinate dehydrogenase B (SDHB), PD-L1 and hypoxia inducible factor (HIF)-1A were analyzed by western blotting in A549 cells. DSF inhibited the viability of A549 cells and promoted expression levels of ATP7B and PD-L1 at both mRNA and protein levels in A549 cells. The viability of cisplatin (DPP)-treated A549 cells was increased following DSF treatment. JQ-1 (a PD-L1 inhibitor) suppressed the viability of DPP-treated A549 cells pretreated with DSF. DSF increased expression levels of ATP7B and PD-L1. The combination treatment of DSF and JQ-1 in A549 cells increased levels of ROS and MDA, as well as expression levels of FDX1 and SLC31A1; however, combination treatment decreased levels of SOD, as well as expression levels of ATP7B, SDHB, PD-L1, and HIF-1A. PX478 (an HIF-1 inhibitor) acted with DSF to enhance the inhibitory effects on the viability and on the induction of apoptosis of A549 cells. PX478 upregulated the levels of ROS and MDA, while it downregulated levels of SOD in DSF-treated A549 cells. PX478 promoted expression levels of FDX1 and SLC31A1, while it suppressed expression levels of ATP7B, PD-L1, and HIF-1A in DSF-treated A549 cells. In conclusion, the combined treatment of A549 cells with anti-PD-L1 and DSF enhanced the effect of cuproptosis on the inhibition of NSCLC cell viability.