Secondary metabolites of mulberry leaves exert anti-lung cancer activity through regulating the PD-L1/PD-1 signaling pathway

Lung cancer ranks the top of malignancies that cause cancer-related deaths worldwide. The leaves of Morus alba L are traditional Chinese medicine widely applied in respiratory diseases. Our previous work has demonstrated the anti-lung cancer effect of secondary metabolites of mulberry leaf. But their mechanism of action has still not fully elucidated. We synthesized Moracin N (MAN) probe conjugated with alkyne to label lung cancer cells and identified protein targets by chemical proteomic analysis. MAN and its probe exerted similar growth-inhibitory effect on human lung cancer cells. Chemical proteomic results showed that MAN targeted the programmed death ligand 1 (PD-L1) checkpoint pathway and T cell receptor (TCR) signaling pathway, indicating its immune-regulatory function. Cell-free surface plasmon resonance (SPR) results showed the direct interaction of MAN with PD-L1 protein. Molecular docking analysis demonstrated that MAN bound to E158 residue of PD-L1 protein. MAN downregulated the expression levels of PD-L1 in a time- and dose-dependent manner and disrupted the PD-L1/programmed death 1 (PD-1) binding, including other secondary metabolites of mulberry leaves Guangsangon E (GSE) and Chalcomoracin (CMR). Human peripheral blood mononuclear cells (PBMCs) co-cultured with MAN-treated A549 cells, resulting in the increase of cluster of differentiation (CD)8⁺ Granzyme B (GZMB)⁺ T cells and the decrease of CD8⁺PD-1⁺ T cells. It suggested that MAN exerts anti-cancer effect through blocking the PD-L1/PD-1 signaling. In vivo, MAN combined with anti-PD-1 antibody significantly inhibited lung cancer development and metastasis, indicating their synergistic effect. Taken together, secondary metabolites of mulberry leaves target the PD-L1/PD-1 signaling, enhance T cell-mediated immunity and inhibit the tumorigenesis of lung cancer. Their modulatory effect on tumor microenvironment makes them able to enhance the therapeutic efficacy of immune checkpoint inhibitors in lung cancer.