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

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.

SUBMITTER: Li M

PROVIDER: S-EPMC4403922 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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

Li Min M Yang Zuolong Z Vollmer Laura L LL Gao Ying Y Fu Yuanyuan Y Liu Cui C Chen Xiaoyun X Liu Peiqing P Vogt Andreas A Yin Xiao-Ming XM

PloS one 20150420 3

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 ...[more]

PMID: 25894744

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Project description:Proteostasis mechanisms, such as proteotoxic-stress response and autophagy, are increasingly recognized for their roles in influencing various cancer hallmarks such as tumorigenesis, drug resistance, and recurrence. However, the precise mechanisms underlying their coordination remain not fully elucidated. The aim of this study is to investigate the molecular interplay between Hsp70 and autophagy in lung adenocarcinoma cells and elucidate its impact on the outcomes of anticancer therapies in vitro. For this purpose, we utilized the human lung adenocarcinoma A549 cell line and genetically modified it by knockdown of Hsp70 or HSF1, and the H1299 cell line with knockdown or overexpression of Hsp70. In addition, several treatments were employed, including treatment with Hsp70 inhibitors (VER-155008 and JG-98), HSF1 activator ML-346, or autophagy modulators (SAR405 and Rapamycin). Using immunoblotting, we found that Hsp70 negatively regulates autophagy by directly influencing AMPK activation, uncovering a novel regulatory mechanism of autophagy by Hsp70. Genetic or chemical Hsp70 overexpression was associated with the suppression of AMPK and autophagy. Conversely, the inhibition of Hsp70, genetically or chemically, resulted in the upregulation of AMPK-mediated autophagy. We further investigated whether Hsp70 suppression-mediated autophagy exhibits pro-survival- or pro-death-inducing effects via MTT test, colony formation, CellTiter-Glo 3D-Spheroid viability assay, and Annexin/PI apoptosis assay. Our results show that combined inhibition of Hsp70 and autophagy, along with cisplatin treatment, synergistically reduces tumor cell metabolic activity, growth, and viability in 2D and 3D tumor cell models. These cytotoxic effects were exerted by substantially potentiating apoptosis, while activating autophagy via rapamycin slightly rescued tumor cells from apoptosis. Therefore, our findings demonstrate that the combined inhibition of Hsp70 and autophagy represents a novel and promising therapeutic approach that may disrupt the capacity of refractory tumor cells to withstand conventional therapies in NSCLC.

Project description:TRAF6 (TNF receptor associated factor 6) plays a pivotal role in NFKB activation and macroautphagy/autophagy activation induced by TLR4 (toll like receptor 4) signaling. The objective of this study was to determine the functional role of PRDX1 (peroxiredoxin 1) in NFKB activation and autophagy activation. PRDX1 interacted with the ring finger domain of TRAF6 and inhibited its ubiquitin-ligase activity. The inhibition on TRAF6 ubiquitin-ligase activity by PRDX1 induced the suppression of ubiquitination of an evolutionarily conserved signaling intermediate in Toll pathways (ECSIT) essential for NFKB activation and BECN1 (beclin 1) required for autophagy activation. An inhibitory effect of PRDX1 on TRAF6 was clearly evidenced in PRDX1-knockdown (PRDX1KD) THP-1, PRDX1KD MDA-MB-231, and PRDX1KD SK-HEP-1 cells. PRDX1KD THP-1 cells showed increases of NFKB activation, pro-inflammatory cytokine production, NFKB-dependent gene expression induced by TLR4 stimulation, and resistance against Salmonella typhimurium infection. Additionally, migration and invasion abilities of PRDX1KD MDA-MB-231 and PRDX1KD SK-HEP-1 cancer cells were significantly enhanced compared to those of control cancer cells. Taken together, these results suggest that PRDX1 negatively regulates TLR4 signaling for NFKB activation and autophagy functions such as bactericidal activity, cancer cell migration, and cancer cell invasion by inhibiting TRAF6 ubiquitin-ligase activity.Abbreviations3-MA: 3-methyladenine; BECN1: beclin 1; CHUK/IKKA: conserved helix-loop-helix ubiquitous kinase; ECSIT: ECSIT signalling integrator; ELISA: enzyme-linked immunosorbent assay; NFKB: nuclear factor kappa-light-chain-enhancer of activated B cells; IB: immunoblotting; IKBKB/IKKB: inhibitor of nuclear factor kappa B kinase subunit beta; IL1B: interleukin 1 beta; IL6: interleukin 6; IP: immunoprecipitation; LPS: lipopolysaccharide; MAP1LC3/LC3: microtuble associated protein 1 light chain 3; MAP3K7/TAK1: mitogen-activated protein kinase kinase kinase 7; MAPK14/p38: mitogen-activated protein kinase 14; mROS: mitochondrial reactive oxygen species; PRDX1: peroxiredoxin 1; PRDX6: peroxiredoxin 6; RELA/p65: RELA proto-oncogene, NF-kB subunit; TRAF6 TNF: receptor associated factor 6.

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

Publications

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

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