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, 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.
Institute of Biomedical Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan; Department of Pathology, Kaohsiung Armed Forces General Hospital, Kaohsiung, Taiwan.
Ren-Jie
RJ
Wei
Department of Pathology, Kaohsiung Armed Forces General Hospital, Kaohsiung, Taiwan.
Su-Shuan
SS
Lin
Institute of Biomedical Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan.
Wen-Ren
WR
Wu
Department of Biotechnology, Southern Taiwan University of Science and Technology, Tainan, Taiwan; Division of Physiology, Livestock Research Institute, Council of Agriculture, Taiwan.
Lih-Ren
LR
Chen
Department of Biotechnology, Southern Taiwan University of Science and Technology, Tainan, Taiwan; Department of Pathology, Chi-Mei Medical Center, Tainan, Taiwan; National Institute of Cancer Research, National Health Research Institute, Tainan, Taiwan; Institute of Clinical Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
Chien-Feng
CF
Li
Institute of Biomedical Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan.
Han-De
HD
Chen
Institute of Biomedical Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan.
Chien-Ting
CT
Chou
Institute of Biomedical Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan.
Ya-Chun
YC
Chen
Institute of Biomedical Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan; Department of Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan.
Shih-Shin
SS
Liang
Department of Pathology, Kaohsiung Armed Forces General Hospital, Kaohsiung, Taiwan.
Shang-Tao
ST
Chien
Institute of Biomedical Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan; Department of Biological Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan; Doctoral Degree Program in Marine Biotechnology, National Sun Yat-sen University, Kaohsiung, Taiwan. Electronic address: ylshiue@mail.nsysu.edu.tw.
Yow-Ling
YL
Shiue
D017209:Apoptosis / Q000187:drug effects*; D001343:Autophagy / Q000187:drug effects*; D020169:Caspases / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D045744:Cell Line, Tumor; D016195:G2 Phase / Q000187:drug effects; D006801:Humans; D008870:Microtubules / Q000187:drug effects*; D008928:Mitochondria / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology*; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
C490492:ABT751; D017382:Reactive Oxygen Species; D013449:Sulfonamides; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; C546842:MTOR protein, human; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases; D020169:Caspases
ABT-751; Apoptosis; Autophagy; Huh-7; Microtubule