Mechanism of baicalin anti-A549 cells by inhibiting PD-L1 based on autophagy pathway

Tumor immunotherapy represented by blocking programmed cell death protein 1/programmed cell death ligand 1 (PD-1/PD-L1) has gained better therapeutic effect in lung cancer treatment, and the development of small molecule drugs to block PD-1/PD-L1 provides a new strategy for lung cancer immunotherapy. In this study, we applied an in vitro cell model to investigate the regulation of PD-L1 expression and the mechanism of action of baicalin on A549 lung cancer cells. The effects of baicalin on the viability of A549 cells were detected by CCK8 assay; the expression of PD-L1 protein in A549 cells was detected by Western blot; the mechanism of baicalin cytotoxicity and its correlation with PD-L1 protein expression were investigated by using small-molecule inhibitors of apoptosis and autophagy; the formation of autophagic vesicles in A549 cells treated with baicalin was observed under transmission electron microscope. And the alterations of acid autophagic vesicles and autophagic lysosomes were observed under fluorescence microscope. The results of CCK8 experiments showed that baicalin (12.5-400 μmol·L^-1) inhibited the proliferation of A549 cells in a dose-dependent manner; at the same time, the elevation of PD-L1 protein expression after interferon-γ (IFN-γ) interference could be reduced with the increase of baicalin concentration; on the other hand, treatment with autophagy inhibitors, wortmannin, and chloroquine, could call back the baicalin-induced cytotoxicity, transmission electron microscopy and fluorescence microscopy showed that the number of autophagic vesicles increased after baicalin treatment of A549 cells, and Western blot results showed that baicalin promoted the expression of autophagy-related proteins LC3-Ⅱ/Ⅰ and beclin-1; the number of baicalin-induced acidic autophagic vesicles in A549 cells was attenuated after the intervention of wortmannin, and at the same time the LC3-Ⅱ/Ⅰ expression was inhibited and the inhibitory effect on PD-L1 was regulated. The above results suggest that baicalin may exert antitumor effects by activating the autophagy protein LC3 in A549 cells to reduce the expression of PD-L1. This study lays the foundation for baicalin to have the ability to be developed as a small molecule blocker of the PD-1/PD-L1 signaling pathway.