Fig. 4. Metformin inhibits cancer-enhanced PD-1 expression via AMPK-dependent deregulation of PD-1 protein stability. (A) NK-92 and Jurkat cells were treated or not with CM (50%) or metformin (10 mM) for 12 hrs. Immunoblotting analyses were performed using the indicated antibodies. (B) NK-92 and Jurkat cells were pretreated with DMSO or MG132 (10 μM) for 1 hr and then treated or not with CM (50%) or metformin (10 mM) for 12 hrs. Immunoblotting analyses were performed using the indicated antibodies. (C) NK-92 and Jurkat cells were pretreated or not with CM (50%) or metformin (10 mM) for 12 hrs and then treated with CHX (100 μg/mL) for the indicated periods. Immunoblotting analyses were performed using the indicated antibodies (left panel). Quantification of PD-1 levels relative to tubulin levels is shown (right panel). Band intensity was quantified using the ImageJ software. Data are presented as mean±SD of three independent experiments. *P<0.05 and **P<0.01, Student’s t-test. (D) NK-92 and Jurkat cells were treated or not with CM (50%) and metformin (10 mM) for 12 hrs. Immunoprecipitation was performed using an anti-PD-1 antibody, followed by immunoblotting analyses using the indicated antibodies. (E) NK-92 and Jurkat cells were treated or not with CM (50%) or metformin (10 mM) for 12 hrs. The cells were incubated with MG132 (10 μM) for 6 hrs before they were harvested using a guanidine-HCl-containing buffer. Immunoprecipitation was performed using an anti-PD-1 antibody, followed by immunoblotting analyses using the indicated antibodies. (F) 293T cells stably expressing V5-PD-1 and Flag-vector or Flag-CA-AMPK were treated or not with CM (50%) for 12 hrs. The cells were incubated with MG132 (10 μM) for 6 hrs before they were harvested using a guanidine-HCl-containing buffer. Immunoprecipitation was performed using an anti-V5 antibody, followed by immunoblotting analyses using the indicated antibodies. (G) AMPK WT and AMPK DKO MEFs were transfected or not with V5-PD-1. These cells were incubated with MG132 (10 μM) for 6 hrs before they were harvested using a guanidine-HCl-containing buffer. Immunoprecipitation was performed using an anti-V5 antibody, followed by immunoblotting analyses using the indicated antibodies. (H) NK-92 and Jurkat cells were pretreated with DMSO or compound C (5 μM) for 1 hr and then treated or not with CM (50%) and metformin (10 mM) for 12 hrs. Immunoblotting analyses were performed using the indicated antibodies. (I) 293T cells stably expressing V5-PD-1 were pretreated with DMSO or compound C (5 μM) for 1 hr and then treated or not with CM (50%) or metformin (10 mM) for 12 hrs. The cells were incubated with MG132 (10 μM) for 6 hrs before they were harvested using a guanidine-HCl-containing buffer. Immunoprecipitation was performed using an anti-V5 antibody, followed by immunoblotting analyses using the indicated antibodies.
Abbreviations: PD-1, programmed death 1; AMPK, adenosine monophosphate-activated protein kinase; CM, conditioned medium; DMSO, dimethyl sulfoxide; CHX, cycloheximide; CA, constitutively active; WT, wild-type; DKO, double knockout.

Ann Lab Med 2024;44:426~436 https://doi.org/10.3343/alm.2023.0443