Inhibition of NF-κB is required for oleanolic acid to downregulate PD-L1 by promoting DNA demethylation in gastric cancer cells.

ABSTRACT

Gastric cancer is one of the most common causes of cancer-related death worldwide. Immunotherapy via programmed cell death protein 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) blockade has shown benefits for gastric cancer. Epigenetic DNA methylation critically regulates cancer immune checkpoints. We investigated how the natural compound oleanolic acid (OA) affected PD-L1 expression in gastric cancer cells. Interleukin-1ß (IL-1ß) at 20 ng/mL was used to stimulate human gastric cancer MKN-45 cells. IL-1ß significantly increased PD-L1 expression, which was abolished by OA. Next, OA-treated MKN-45 cells were co-cultured with activated and PD-1-overexpressing Jurkat T cells. OA restored IL-2 levels in the co-culture system and increased T cell killing toward MKN-45 cells. Overexpression of PD-L1 eliminated OA-enhanced T cell killing capacity; however, PD-1 blocking antibody abrogated the cytotoxicity of T cells. Moreover, OA abolished IL-1ß-increased DNA demethylase activity in MKN-45 cells. DNA methyltransferase inhibitor 5-azacytidine rescued OA-reduced PD-L1 expression; whereas DNA demethylation inhibitor gemcitabine inhibited PD-L1 expression, and, in combination with OA, provided more potent inhibitory effects. Furthermore, OA selectively reduced the expression of DNA demethylase TET3 in IL-1ß-treated MKN-45 cells, and overexpression of TET3 restored OA-reduced PD-L1 expression. Finally, OA disrupted nuclear factor κB (NF-κB) signaling IL-1ß-treated MKN-45 cells, and overexpression of NF-κB restored OA downregulation of TET3 and PD-L1. The cytotoxicity of T cells toward MKN-45 cells was also weakened by NF-κB overexpression. Altogether, OA blocked the IL-1ß/NF-κB/TET3 axis in gastric cancer cells, leading to DNA hypomethylation and downregulation of PD-L1. Our discoveries suggested OA as an epigenetic modulator for immunotherapy or an adjuvant therapy against gastric cancer.