title: The novel synthesized 2-(3-(methylamino)phenyl)-6-(pyrrolidin-1-yl)quinolin-4-one (Smh-3) compound induces G2/M phase arrest and mitochondrial-dependent apoptotic cell death through inhibition of CDK1 and AKT activity in HL-60 human leukemia cells.
abstract: 2-Phenyl-4-quinolone series compounds have exhibited growth inhibitory influence on several human cancer cell lines. In this study, we investigated the effects of 2-(3-(methylamino)phenyl)-6-(pyrrolidin-1-yl)quinolin-4-one (Smh-3) on viability, cell cycle and apoptotic cell death which occurred in different leukemia cell lines (HL-60, U937 and K562) in a dose- and time-dependent manner, but which did not obviously impair the viability of normal human umbilical vein endothelial cells (HUVEC) in vitro. The approximate IC50 was 103.26 ± 4.59 nM for a 48 h treatment in HL-60 cells. Cell cycle analysis showed that 100 nM Smh-3 induced signi-ficant G2/M arrest in examined cells. Within 0, 12, 24 and 48 h of treatment, Smh-3 inhibited CDK1 activity and decreased protein levels of CDK1, cyclin A and cyclin B. Smh-3-induced chromatin condensation and DNA fragmentation were determined by DAPI and TUNEL staining. Cell apoptosis was significantly reduced after pretreatment with a pan-caspase inhibitor (Z-VAD-fmk) and results indicated that Smh-3-induced apoptosis was mainly mediated by activation of the caspase cascade in HL-60 cells. Results from colorimetric assays and Western blot analysis indicated that activities of caspase-9, -7 and -3 were promoted in Smh-3-treated HL-60 cells during cell apoptosis. Smh-3-induced apoptosis in HL-60 cells was accompanied by an apparent increase in ROS production, and protein levels of cytosolic cytochrome c, apoptotic protease activating factor-1 (Apaf-1) and apoptosis-inducing factor (AIF). Strikingly, Smh-3 induced apoptosis in HL-60 cells by simultaneously suppressing protein levels of AKT, p-AKT, p-mTOR and p-BAD and inducing BAD protein levels. Taken together, we conclude that Smh-3 acts against leukemia cells in vitro via G2/M phase arrest, down-regulation of AKT activity and induction of mitochondrial-dependent apoptotic pathways.
authors.affiliation: Graduate Institute of Pharmaceutical Chemistry, China Medical University, Taichung 404, Taiwan, ROC.
authors.forename: Shih-Ming
authors.initials: SM
authors.lastname: Huang
authors.forename: Jai-Sing
authors.initials: JS
authors.lastname: Yang
authors.forename: Shih-Chang
authors.initials: SC
authors.lastname: Tsai
authors.forename: Ming-Hua
authors.initials: MH
authors.lastname: Chen
authors.forename: Mei-Hua
authors.initials: MH
authors.lastname: Hsu
authors.forename: Hui-Yi
authors.initials: HY
authors.lastname: Lin
authors.forename: Li-Chen
authors.initials: LC
authors.lastname: Chou
authors.forename: Jo-Hua
authors.initials: JH
authors.lastname: Chinag
authors.forename: Kuo-Hsiung
authors.initials: KH
authors.lastname: Lee
authors.forename: Li-Jiau
authors.initials: LJ
authors.lastname: Huang
authors.forename: Sheng-Chu
authors.initials: SC
authors.lastname: Kuo
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D016203:CDC2 Protein Kinase / Q000037:antagonists & inhibitors*; D020169:Caspases / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects*; D016195:G2 Phase / Q000187:drug effects*; D018922:HL-60 Cells; D006801:Humans; D007938:Leukemia / Q000188:drug therapy*; D008928:Mitochondria / Q000502:physiology*; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors*; D011759:Pyrrolidines / Q000494:pharmacology*; D015363:Quinolones / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism; D051025:bcl-Associated Death Protein / Q000378:metabolism
chemical_list: C559958:2-(3-(methylamino)phenyl)-6-(pyrrolidin-1-yl)quinolin-4-one; D000970:Antineoplastic Agents; C494819:BAD protein, human; D011759:Pyrrolidines; D015363:Quinolones; D017382:Reactive Oxygen Species; D051025:bcl-Associated Death Protein; D051057:Proto-Oncogene Proteins c-akt; D016203:CDC2 Protein Kinase; D020169:Caspases