title: MG-2477, a new tubulin inhibitor, induces autophagy through inhibition of the Akt/mTOR pathway and delayed apoptosis in A549 cells.
abstract: We previously demonstrated that MG-2477 (3-cyclopropylmethyl-7-phenyl-3H-pyrrolo[3,2-f]quinolin-9(6H)-one) inhibits the growth of several cancer cell lines in vitro. Here we show that MG-2477 inhibited tubulin polymerization and caused cells to arrest in metaphase. The detailed mechanism of action of MG-2477 was investigated in a non-small cell lung carcinoma cell line (A549). Treatment of A549 cells with MG-2477 caused the cells to arrest in the G2/M phase of the cell cycle, with a concomitant accumulation of cyclin B. Moreover, the compound induced autophagy, which was followed at later times by apoptotic cell death. Autophagy was detected as early as 12h by the conversion of microtubule associated protein 1 light chain 3 (LC3-I) to LC3-II, following cleavage and lipid addition to LC3-I. After 48h of MG-2477 exposure, phosphatidylserine externalization on the cell membrane, caspase-3 activation, and PARP cleavage occurred, revealing that apoptotic cell death had begun. Pharmacological inhibition of autophagy with 3-methyladenine or bafilomycin A1 increased apoptotic cell death, suggesting that the autophagy caused by MG-2477 played a protective role and delayed apoptotic cell death. Additional studies revealed that MG-2477 inhibited survival signaling by blocking activation of Akt and its downstream targets, including mTOR, and FHKR. Treatment with MG-2477 also reduced phosphorylation of mTOR downstream targets p70 ribosomal S6 kinase and 4E-BP1. Overexpression of Akt by transfection with a Myr-Akt vector decreased MG-2477 induced autophagy, indicating that Akt is involved. Taken together, these results indicated that the autophagy induced by MG-2477 delayed apoptosis by exerting an adaptive response following microtubule damage.
authors.affiliation: Department of Pediatrics, Laboratory of Oncohematology, University of Padova, Italy. giampietro.viola.1@unipd.it
authors.forename: Giampietro
authors.initials: G
authors.lastname: Viola
authors.forename: Roberta
authors.initials: R
authors.lastname: Bortolozzi
authors.forename: Ernest
authors.initials: E
authors.lastname: Hamel
authors.forename: Stefano
authors.initials: S
authors.lastname: Moro
authors.forename: Paola
authors.initials: P
authors.lastname: Brun
authors.forename: Ignazio
authors.initials: I
authors.lastname: Castagliuolo
authors.forename: Maria Grazia
authors.initials: MG
authors.lastname: Ferlin
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Basso
mesh_terms: D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D001343:Autophagy / Q000187:drug effects* / Q000502:physiology; D045744:Cell Line, Tumor; D006801:Humans; D007211:Indoles / Q000494:pharmacology*; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015363:Quinolones / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013997:Time Factors; D050257:Tubulin Modulators / Q000494:pharmacology*
chemical_list: C569394:3-cyclopropylmethyl-7-phenyl-3H-pyrrolo(3,2-f)quinolin-9(6H)-one; D007211:Indoles; D015363:Quinolones; D050257:Tubulin Modulators; C546842:MTOR protein, human; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases