Fig 1.
(A) The molecular structure of AMDE-1. (B-C) A549, wild type MEFs and Atg5 KO MEFs expressing GFP-LC3 were treated with or without AMDE-1 (10 μM) for 6 h, and then analyzed by GFP-LC3 puncta formation (B) or by immunoblotting (C). (D-E) A549, wild type MEFs and Atg5 KO MEFs were treated with 1 μM rapamycin (Rap) or 10 μM AMDE-1 for 6 h, followed by immunostaining with an anti-Atg16L1 antibody (D). Atg16L1 puncta were quantified (E). Values represent means ± SD from three independent experiments. ***: p<0.001. CM: control medium.
Fig 2.
AMDE-1 can induce autophagy through AMPK-mTOR-ULK1 pathway.
(A) Wild-type MEFs and HeLa cells were treated with or without 10 μM of AMDE-1 for 6 h. Phosphorylation of S6 was detected by immunoblotting assay. (B-C) MEFs were treated with 10 μM AMDE-1 (B), or chloroquine (CQ, 40 μM) or E/P (E64D, 25 μM plus pepstatin A, 50 μM) (C) for different times as indicated and analyzed by Western blot. The zero time point represents no treatment. (D-E) MEFs expressing GFP-LC3 were treated with AMDE-1 alone or together with N-acetyl cysteine (NAC, 20 μM), U0126 (30 μM), or SP600125 (20 μM) for 6 h. GFP-LC3 puncta formation (D) and endogenous LC3 level were analyzed (E).
Fig 3.
AMDE-1 can inhibit autophagic degradation.
(A-B) MEFs were treated with or without AMDE-1 (10 μM) and CQ (40 μM) for 20 h. Endogenous LC3 was analyzed by immunoblot assay (A) and densitometry (B). (C-D) MEFs were treated with AMDE-1 (10 μM) for 0–20 h as indicated. The level of SQSTM1/p62 was analyzed (C) and quantified (D). (E-F) HEK-293A cells expressing GFP-RFP-LC3 were treated with CQ (40 μM), Rap (1 μM) and AMDE-1 (10 μM) for 6 h (E). Puncta showing both green and red fluorescence (indicated as yellow), or showing only the red fluorescence (indicated as red) were quantified (F). (G-H) MEFs were incubated in complete medium (CM), or in EBSS with or without AMDE-1 (10 μM), 3-MA (10 mM), CQ (10 μM), Rap (1 μM) or CBZ (50 μM) for 6 h (G) or 16 h (H). The long-lived protein degradation was measured. For panel B, D, F, G and H, values represent means ± SD from three independent experiments. ***: p<0.001; **: p<0.01.
Fig 4.
AMDE-1 can block lysosome degradation.
(A) GFP-LC3-expressing MEFs were treated with AMDE-1 (10 μM) for 6 h, followed by immunostaining with anti-LAMP2. Arrows indicated colocalized GFP-LC3 (green) and LAMP2 (red) puncta. (B) HeLa cells were treated with AMDE-1 (10 μM) for 6 or 20 h or with ammonium chloride (AC, 20 mM) for 2 hr. as indicated, followed by staining with acridine orange (AO, 1 μg/ml) or Lyso Tracker Red (LTR, 50 ng/ml) for 30 min. (C) HeLa cells were pre-incubated with self-quenched bodipy-conjugated BSA (DQ-BSA, 10 μg/ml) for 1 h and then treated with AMDE-1 (10 μM), E64D (25 μM) plus pepstatin A (50 μM) (E+P) or CQ (40 μM) for 6 h. (D) HeLa cells were starved in DMEM for 1.5 h and incubated with or without EGF (200 ng/ml) together with Baf (0.5 μM) or AMDE-1 (10 μM) for 0–4.5 h. EGFR level was detected by immunoblot. (E) Hela cells were treated with AMDE-1 (10 μM) for 0–20 h. The lysosome-enriched fraction was analyzed for the expression of cathepsin D (CTSD) and cathepsin B (CTSB). (F) Cells were treated with or without AMDE-1 for 20 hrs, and the activity of cathepsin B and cathepsin D at 20 h was analyzed using the lysosome-enriched fraction. Cathepsin activities were standardized to that of the untreated sample, which was set to 100%. Values represent means ± SD from three independent experiments. ***: p<0.001.
Fig 5.
AMDE-1 is a potent cytotoxic agent.
(A-B) HeLa cells were treated with AMDE (A) or CQ (B) at different doses for 48 hours. Cell death was measured. The dose responses were fitted with a logarithmic regression (A) or a second order polynomial regression (B) algorithm, respectively. (C) Cells were treated with CQ (50 μM) or AMDE (2.5 μM) for 48 hours. Cell death was then measured. (D) HCT116 and CCD-18Co cells were treated with AMDE at different concentrations for 48 hours. Cell death was measured. The dose response was fitted with a logarithmic regression algorithm. (E) HCT16 was treated with or without AMDE-1 (2.5 μM), zVAD (20 μM), and/or necrostatin-1 (necro, 40 μM) for 48 hours. Cell death was measured. Values represent means ± SD from three independent experiments. *: p<0.05, ***: p = 0.001.
Fig 6.
A proposed mode for the action of AMDE-1.
AMDE-1 can trigger autophagy by the AMPK-mTOR-ULK1 pathway. However, AMDE-1 can also suppress lysosomal function and thus autophagic flux. Consequently, the lysosome dysfunction and/or the accumulation of non-degraded cellular material can promote cell death. The dashed arrow suggests that AMDE-1 may also affect autophagy by other unknown mechanisms.