Matrine induces V-ATPase-dependent cytoplasmic vacuolation and inhibits the function of the lysosome in leukemia cells

Matrine is the main component extracted from legumes and has extensive anti-cancer effects; however, its molecular mechanism is unclear. In our study, we found that matrine induced vacuolation in leukemia cells is closely related to cell proliferation inhibition. Vacuolization was reversed after matrine removal. The neutral red staining assay indicated that the matrine-induced vacuoles were acidic, and the vacuoles originated mostly from the lysosome or endosome, as observed by transmission electron microscope (TEM) and fluorescence microscopy localization of LAMP-GFP. Furthermore, single-cell RNA sequencing (RNA-seq) demonstrated that the expression of vacuolation- and lysosomal-related genes were up-regulated after matrine treatment, and western blot (WB) and flow cytometry (FCM) analysis confirmed that matrine inhibits intracellular proteolytic enzyme expression and activity, suggesting that matrine may inhibit lysosomal function. In addition, we identified that matrine significantly up-regulated the expression levels of vacuolar ATPase (V-ATPase) subunits in cells, and the V-ATPase inhibitor effectively reversed the occurrence of cell vacuoles, suggesting that V-ATPase plays an important role in matrine-induced vacuoles. The molecular structure of matrine was further analyzed, and the protonation of matrine in lysosomes to activate V-ATPase may be a direct cause of vacuole formation. Our results revealed a new molecular mechanism by which matrine inhibit leukemia cell proliferation.