Riccardin D-N induces lysosomal membrane permeabilization by inhibiting acid sphingomyelinase and interfering with sphingomyelin metabolism in vivo.

Lysosomes are important targets for anticancer drug discovery. Our previous study showed that Riccardin D-N (RD-N), a natural macrocylic bisbibenzyl derivative produced by Mannich reaction, induced cell death by accumulating in lysosomes. Experiments were performed on human lung squamous cell carcinoma tissue from left inferior lobar bronchus of patient xenografts and H460 cells. RD-N was administrated for 25days. The specimens of xenografts in Balb/c athymic (nu+/nu+) male mice were removed for immunohistochemistry, subcellular fractionation, enzyme activities and Western blotting analysis. mRFP-GFP-LC3 reporter was used to examine autophagy in H460 cells. Sphingomyelin assay was evaluated by thin-layer chromatography and assay kit. Lysosomal membrane permeabilization (LMP) caused by acid sphingomyelinase (ASM) inhibition and subsequent changes of sphingomyelin (SM) metabolism selectively destabilized the cancer cell lysosomes in RD-N-treated H460 cells in vitro and tumor xenograft model in vivo. The destabilized lysosomes induced the release of cathepsins from the lysosomes into the cytosol and further triggered cell death. These results explain the underlying mechanism of RD-N induced LMP. It can be concluded that a more lysosomotropic derivative was synthesized by introduction of an amine group, which could have more potential applications in cancer therapy.