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AMDE-1 is a dual function chemical for autophagy activation and inhibition.

Li M, Yang Z, Vollmer LL, Gao Y, Fu Y, Liu C, Chen X, Liu P, Vogt A, Yin XM - PLoS ONE (2015)

Bottom Line: AMDE-1did not affect MAP kinase, JNK or oxidative stress signaling for autophagy induction.This inhibition was correlated with a reduction in lysosomal degradation capacity but not with autophagosome-lysosome fusion.The dual effects of AMDE-1 on autophagy induction and lysosomal degradation suggested that its net effect would likely lead to autophagic stress and lysosome dysfunction, and therefore cell death.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, Guangdong, China; Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.

ABSTRACT
Autophagy is the process by which cytosolic components and organelles are delivered to the lysosome for degradation. Autophagy plays important roles in cellular homeostasis and disease pathogenesis. Small chemical molecules that can modulate autophagy activity may have pharmacological value for treating diseases. Using a GFP-LC3-based high content screening assay we identified a novel chemical that is able to modulate autophagy at both initiation and degradation levels. This molecule, termed as Autophagy Modulator with Dual Effect-1 (AMDE-1), triggered autophagy in an Atg5-dependent manner, recruiting Atg16 to the pre-autophagosomal site and causing LC3 lipidation. AMDE-1 induced autophagy through the activation of AMPK, which inactivated mTORC1 and activated ULK1. AMDE-1did not affect MAP kinase, JNK or oxidative stress signaling for autophagy induction. Surprisingly, treatment with AMDE-1 resulted in impairment in autophagic flux and inhibition of long-lived protein degradation. This inhibition was correlated with a reduction in lysosomal degradation capacity but not with autophagosome-lysosome fusion. Further analysis indicated that AMDE-1 caused a reduction in lysosome acidity and lysosomal proteolytic activity, suggesting that it suppressed general lysosome function. AMDE-1 thus also impaired endocytosis-mediated EGF receptor degradation. The dual effects of AMDE-1 on autophagy induction and lysosomal degradation suggested that its net effect would likely lead to autophagic stress and lysosome dysfunction, and therefore cell death. Indeed, AMDE-1 triggered necroptosis and was preferentially cytotoxic to cancer cells. In conclusion, this study identified a new class of autophagy modulators with dual effects, which can be explored for potential uses in cancer therapy.

No MeSH data available.


Related in: MedlinePlus

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.
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pone.0122083.g004: 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.

Mentions: To elucidate how AMDE-1 might inhibit autophagy degradation we first examined the colocalization of AMDE-1-induced GFP-LC3 puncta with the lysosomes as detected by the anti-LAMP2 antibody (Fig 4A). We observed a significant level of colocalization between the two signals, implying that AMDE-1 did not block the fusion between the autophagosome and the lysosome. We then assessed whether AMDE-1 could affect the lysosome acidity. We used two sensitive pH probes that can accumulate in the lysosome. AO is a lysosomotropic metachromatic fluorochrome, and presents green fluorescence in the cytosol but red fluorescence when it is accumulated in the acidic compartment due to its being highly protonated [31]. LysoTracker Red (LTR) manifests red fluorescence in a pH-dependent manner in the lysosome. As seen in Fig 4B, while ammonium chloride (AC) neutralized the pH of the lysosome, resulting in the disappearance of the red fluorescence of both AO and LTR, AMDE-1 had no effect on the fluorescence intensity of the two probes. Thus AMDE-1 did not seem to affect the acidity of the lysosome.


AMDE-1 is a dual function chemical for autophagy activation and inhibition.

Li M, Yang Z, Vollmer LL, Gao Y, Fu Y, Liu C, Chen X, Liu P, Vogt A, Yin XM - PLoS ONE (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4403922&req=5

pone.0122083.g004: 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.
Mentions: To elucidate how AMDE-1 might inhibit autophagy degradation we first examined the colocalization of AMDE-1-induced GFP-LC3 puncta with the lysosomes as detected by the anti-LAMP2 antibody (Fig 4A). We observed a significant level of colocalization between the two signals, implying that AMDE-1 did not block the fusion between the autophagosome and the lysosome. We then assessed whether AMDE-1 could affect the lysosome acidity. We used two sensitive pH probes that can accumulate in the lysosome. AO is a lysosomotropic metachromatic fluorochrome, and presents green fluorescence in the cytosol but red fluorescence when it is accumulated in the acidic compartment due to its being highly protonated [31]. LysoTracker Red (LTR) manifests red fluorescence in a pH-dependent manner in the lysosome. As seen in Fig 4B, while ammonium chloride (AC) neutralized the pH of the lysosome, resulting in the disappearance of the red fluorescence of both AO and LTR, AMDE-1 had no effect on the fluorescence intensity of the two probes. Thus AMDE-1 did not seem to affect the acidity of the lysosome.

Bottom Line: AMDE-1did not affect MAP kinase, JNK or oxidative stress signaling for autophagy induction.This inhibition was correlated with a reduction in lysosomal degradation capacity but not with autophagosome-lysosome fusion.The dual effects of AMDE-1 on autophagy induction and lysosomal degradation suggested that its net effect would likely lead to autophagic stress and lysosome dysfunction, and therefore cell death.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, Guangdong, China; Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.

ABSTRACT
Autophagy is the process by which cytosolic components and organelles are delivered to the lysosome for degradation. Autophagy plays important roles in cellular homeostasis and disease pathogenesis. Small chemical molecules that can modulate autophagy activity may have pharmacological value for treating diseases. Using a GFP-LC3-based high content screening assay we identified a novel chemical that is able to modulate autophagy at both initiation and degradation levels. This molecule, termed as Autophagy Modulator with Dual Effect-1 (AMDE-1), triggered autophagy in an Atg5-dependent manner, recruiting Atg16 to the pre-autophagosomal site and causing LC3 lipidation. AMDE-1 induced autophagy through the activation of AMPK, which inactivated mTORC1 and activated ULK1. AMDE-1did not affect MAP kinase, JNK or oxidative stress signaling for autophagy induction. Surprisingly, treatment with AMDE-1 resulted in impairment in autophagic flux and inhibition of long-lived protein degradation. This inhibition was correlated with a reduction in lysosomal degradation capacity but not with autophagosome-lysosome fusion. Further analysis indicated that AMDE-1 caused a reduction in lysosome acidity and lysosomal proteolytic activity, suggesting that it suppressed general lysosome function. AMDE-1 thus also impaired endocytosis-mediated EGF receptor degradation. The dual effects of AMDE-1 on autophagy induction and lysosomal degradation suggested that its net effect would likely lead to autophagic stress and lysosome dysfunction, and therefore cell death. Indeed, AMDE-1 triggered necroptosis and was preferentially cytotoxic to cancer cells. In conclusion, this study identified a new class of autophagy modulators with dual effects, which can be explored for potential uses in cancer therapy.

No MeSH data available.


Related in: MedlinePlus