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

Abstract

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.

Keywords: AMPK activation; BECN1-xCT complex; astragalus polysaccharides; ferroptosis; urothelial carcinoma.

Figure 1

APS inhibits the proliferation and migration of RT4 and T24 cells. CCK-8 assays revealed that APS significantly inhibited the viability of RT4 (A) and T24 (B) cells. The transwell results revealed that 15 and 10 μM APS significantly inhibited the migration of RT4 (C) and T24 (D) cells (Bar represents 10 μm, 20 × magnification). Scratch assays confirmed that 15 and 10 μM APS significantly reduced the migration of RT4 (E) and T24 (F) cells (Bar represents 50 μm, 4 × magnification). ^*p < 0.05, ^**p < 0.01 vs. Con.

Figure 2

APS induces ferroptosis in RT4 and T24 cells. RT4 and T24 cells were treated with 1 μM ferrostatin (Fer-1, a ferroptosis inhibitor), 20 μM Z-VAD-FMK (a pancaspase inhibitor), 20 μM necrostatin-1 (Nec-1, a necrosis inhibitor) and 10 μM 3-methyladenine (3-MA, an autophagy inhibitor). Then, RT4 and T24 cells were treated with 15 and 10 μM APS for 24 h. The CCK-8 results showed that Fer-1 significantly reversed APS-induced death in RT4 (A) and T24 (B) cells. (C) C11 BODIPY 581/591 staining revealed that Fer-1 reversed APS-induced lipid peroxidation in RT4 and T24 cells (Bar represents 20 μm, 20 × magnification). ^**p < 0.01, ^***p < 0.001 vs. Con; ^##p < 0.01, ^###p < 0.001 vs. APS.

Figure 3

Fer-1 reversed APS-induced ferroptosis. (A) FerroOrange staining showed that Fer-1 could reduce the APS-induced increase in Fe²⁺ levels (Bar represents 20 μm, 20 × magnification). (B) Statistical analysis showed that Fer-1 reversed APS-induced Fe²⁺ (C) and MDA (D) accumulation in RT4 and T24 cells. Fer-1 reversed the APS-induced decrease in GSH levels (E) in RT4 and T24 cells. (F) CCK-8 analysis showed that the APS-induced decrease in the viability of RT4 and T24 cells could also be ameliorated by Fer-1. Flow cytometry revealed that Fer-1 treatment reduced the APS-induced increase in the mortality of RT4 (G, H) and (I, J) T24 cells. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 vs. Con; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. APS.

Figure 4

APS inhibited the activity of xCT and reduced the expression of GPX4. (A) APS significantly reduced the activity of glutathione peroxidase in RT4 and T24 cells. (B) The Western blot results revealed that APS did not change the expression of xCT in RT4 and T24 cells but reduced the protein level of GPX4. (C) The IF results showed that APS did not change the fluorescence level of xCT but significantly inhibited the fluorescence expression of GPX4 (Bar represents 10 μm, 20 × magnification). (D) APS reduced the activity of xCT in RT4 and T24 cells. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 vs. Con.

Figure 5

APS activates AMPK/BECN1 signaling in RT4 and T24 cells. (A) The Western blot results showed that APS significantly increased the phosphorylation levels of AMPK and BECN1 in RT4 and T24 cells. IF staining showed that APS induced the formation of the BECN1-xCT complex in RT4 (B) and T24 (C) cells (Bar represents 10 μm, 40 × magnification). ^**p < 0.01, ^***p < 0.001 vs. Con.

Figure 6

Knockdown of AMPK reverses APS-induced ferroptosis. (A) The Western blot results showed that the transfection of si-AMPK significantly inhibited AMPK expression even in APS-treated RT4 and T24 cells. (B) the JC-1 results showed that silencing AMPK significantly ameliorated APS-induced upregulation of MMP (Bar represents 20 μm, 20 × magnification). In RT4 and T24 cells, the APS-induced increase in Fe²⁺ (C) and MDA (D) levels could be alleviated by si-AMPK to some extent. (E) In RT4 and T24 cells, the reduction of GSH induced by APS could be reversed by knockdown of AMPK. ^*p < 0.05, ^***p < 0.001 vs. Con; ^##p < 0.01, ^###p < 0.001 vs. APS.

Figure 7

APS inhibited the growth of tumors in nude mice. (A) Representative images of tumor tissues from nude mice. APS significantly inhibited tumor volume (B) and weight (C) in nude mice. APS increased the levels of Fe²⁺ (D) and MDA (E) in tumor tissues. (F) APS decreased GSH levels in tumor tissues. The RT-PCR results showed that APS increased the mRNA levels of ptgs2 (G) and Chac1 (H) in tumor tissues. (I) After APS treatment, the phosphorylation levels of AMPK and BECN1 were increased in the tumor tissues of nude mice. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 vs. Con.

Figure 8

Diagram of the molecular mechanism by which APS inhibits UC.