Astragalus polysaccharide inhibits the development of urothelial carcinoma by activating AMPK signaling to induce BENC1-xCT complex formation

In recent years, the incidence of urothelial carcinoma (UC) has been high in men. The aim of this study was to investigate whether astragalus polysaccharide (APS) could inhibit the development of UC and the specific molecular mechanism. Our data showed that APS inhibited the proliferation of UC cells in a dose-dependent manner, and APS reduced the migratory capacity of RT4 and T24 cells. Further studies revealed that the ferroptosis inhibitor ferrostatin-1 (Fer-1) reversed APS-induced cell death, intracellular Fe2+ and malondialdehyde (MDA) accumulation, and lipid peroxidation product deposition. The Western blot and immunofluorescence results showed that APS significantly inhibited the expression of glutathione peroxidase 4 (GPX4) but did not alter the protein level of solute carrier family 7 member 11 (xCT, SLC7A11). Further analysis revealed that APS reduced the activity of xCT in RT4 and T24 cells. Moreover, APS significantly increased the phosphorylation levels of protein kinase AMP-activated catalytic subunit alpha 1 (AMPK) and BECN1 in RT4 and T24 cells, which induced the formation of the BECN1-xCT complex. However, when AMPK was silenced in RT4 and T24 cells, APS-induced ferroptosis was reversed to some extent, indicating that APS-mediated ferroptosis involves AMPK signaling. Moreover, APS has been shown to inhibit tumor growth in nude mice in vivo. In summary, our study demonstrated for the first time that APS could promote the formation of the BECN1-xCT complex in UC cells by activating AMPK/BECN1 signaling, which inhibited the activity of xCT to reduce GPX4 expression, thereby inducing ferroptosis and ultimately inhibiting UC progression.