A microtubule inhibitor, ABT-751, induces autophagy and delays apoptosis in Huh-7 cells

The objective was to investigate the upstream mechanisms of apoptosis which were triggered by a novel anti-microtubule drug, ABT-751, in hepatocellular carcinoma-derived Huh-7 cells. Effects of ABT-751 were evaluated by immunocytochemistry, flow cytometric, alkaline comet, soft agar, immunoblotting,...

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Published in:Toxicology and applied pharmacology 2016-11, Vol.311, p.88-98
Main Authors: Wei, Ren-Jie, Lin, Su-Shuan, Wu, Wen-Ren, Chen, Lih-Ren, Li, Chien-Feng, Chen, Han-De, Chou, Chien-Ting, Chen, Ya-Chun, Liang, Shih-Shin, Chien, Shang-Tao, Shiue, Yow-Ling
Format: Article
Language:English
Subjects:
60 APPLIED LIFE SCIENCES
ABT-751
AGAR
APOPTOSIS
Apoptosis - drug effects
Autophagy
Autophagy - drug effects
Caspases - metabolism
CELL CYCLE
Cell Division - drug effects
Cell Line, Tumor
DNA
DNA DAMAGES
DRUGS
FLUORESCENCE
G2 Phase - drug effects
HEPATOMAS
Huh-7
Humans
INHIBITION
LYMPHOMAS
Microtubule
MICROTUBULES
Microtubules - drug effects
MITOCHONDRIA
Mitochondria - metabolism
MODULATION
PHOSPHOTRANSFERASES
POLYMERASE CHAIN REACTION
POLYMERASES
Proto-Oncogene Proteins c-akt - metabolism
Reactive Oxygen Species - metabolism
RNA
SERINE
Sulfonamides - pharmacology
THREONINE
TOR Serine-Threonine Kinases - metabolism
Tumor Suppressor Protein p53 - metabolism
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Summary:The objective was to investigate the upstream mechanisms of apoptosis which were triggered by a novel anti-microtubule drug, ABT-751, in hepatocellular carcinoma-derived Huh-7 cells. Effects of ABT-751 were evaluated by immunocytochemistry, flow cytometric, alkaline comet, soft agar, immunoblotting, CytoID, green fluorescent protein-microtubule associated protein 1 light chain 3 beta detection, plasmid transfection, nuclear/cytosol fractionation, coimmunoprecipitation, quantitative reverse transcription-polymerase chain reaction, small-hairpin RNA interference and mitochondria/cytosol fractionation assays. Results showed that ABT-751 caused dysregulation of microtubule, collapse of mitochondrial membrane potential, generation of reactive oxygen species (ROS), DNA damage, G2/M cell cycle arrest, inhibition of anchorage-independent cell growth and apoptosis in Huh-7 cells. ABT-751 also induced early autophagy via upregulation of nuclear TP53 and downregulation of the AKT serine/threonine kinase (AKT)/mechanistic target of rapamycin (MTOR) pathway. Through modulation of the expression levels of DNA damage checkpoint proteins and G2/M cell cycle regulators, ABT-751 induced G2/M cell cycle arrest. Subsequently, ABT-751 triggered apoptosis with marked downregulation of B-cell CLL/lymphoma 2, upregulation of mitochondrial BCL2 antagonist/killer 1 and BCL2 like 11 protein levels, and cleavages of caspase 8 (CASP8), CASP9, CASP3 and DNA fragmentation factor subunit alpha proteins. Suppression of ROS significantly decreased ABT-751-induced autophagic and apoptotic cells. Pharmacological inhibition of autophagy significantly increased the percentages of ABT-751-induced apoptotic cells. The autophagy induced by ABT-751 plays a protective role to postpone apoptosis by exerting adaptive responses following microtubule damage, ROS and/or impaired mitochondria. •An anti-microtubule agent, ABT-751, induces autophagy and apoptosis in Huh-7 cells.•ABT-751 induces early autophagy and delays apoptosis.•ABT-751 induces autophagy via modulations of nuclear TP53 and AKT/MTOR pathways.•ABT-751-induced apoptosis is ROS-, mitochondria- and caspase-dependent.•Inhibition of autophagy enhances ABT-751-induced apoptosis.
ISSN:0041-008X
1096-0333
DOI:10.1016/j.taap.2016.09.021