title: Annona muricata leaves induced apoptosis in A549 cells through mitochondrial-mediated pathway and involvement of NF-κB.
abstract: BACKGROUND
  Annona muricata leaves have been reported to have antiproliferative effects against various cancer cell lines. However, the detailed mechanism has yet to be defined. The current study was designed to evaluate the molecular mechanisms of A. muricata leaves ethyl acetate extract (AMEAE) against lung cancer A549 cells.
  
  
  METHODS
  The effect of AMEAE on cell proliferation of different cell lines was analyzed by MTT assay. High content screening (HCS) was applied to investigate the suppression of NF-κB translocation, cell membrane permeability, mitochondrial membrane potential (MMP) and cytochrome c translocation from mitochondria to cytosol. Reactive oxygen species (ROS) formation, lactate dehydrogenase (LDH) release and activation of caspase-3/7, -8 and -9 were measured while treatment. The western blot analysis also carried out to determine the protein expression of cleaved caspase-3 and -9. Flow cytometry analysis was used to determine the cell cycle distribution and phosphatidylserine externalization. Quantitative PCR analysis was performed to measure the gene expression of Bax and Bcl-2 proteins.
  
  
  RESULTS
  Cell viability analysis revealed the selective cytotoxic effect of AMEAE towards lung cancer cells, A549, with an IC50 value of 5.09 ± 0.41 μg/mL after 72 h of treatment. Significant LDH leakage and phosphatidylserine externalization were observed in AMEAE treated cells by fluorescence analysis. Treatment of A549 cells with AMEAE significantly elevated ROS formation, followed by attenuation of MMP via upregulation of Bax and downregulation of Bcl-2, accompanied by cytochrome c release to the cytosol. The incubation of A549 cells with superoxide dismutase and catalase significantly attenuated the cytotoxicity caused by AMEAE, indicating that intracellular ROS plays a pivotal role in cell death. The released cytochrome c triggered the activation of caspase-9 followed by caspase-3. In addition, AMEAE-induced apoptosis was accompanied by cell cycle arrest at G0/G1 phase. Moreover, AMEAE suppressed the induced translocation of NF-κB from cytoplasm to nucleus.
  
  
  CONCLUSIONS
  Our data showed for the first time that the ethyl acetate extract of Annona muricata inhibited the proliferation of A549 cells, leading to cell cycle arrest and programmed cell death through activation of the mitochondrial-mediated signaling pathway with the involvement of the NF-kB signalling pathway.
authors.forename: Soheil Zorofchian
authors.initials: SZ
authors.lastname: Moghadamtousi
authors.affiliation: Biomolecular Research Group, Biochemistry Program, Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia. habsah@um.edu.my.
authors.forename: Habsah Abdul
authors.initials: HA
authors.lastname: Kadir
authors.forename: Mohammadjavad
authors.initials: M
authors.lastname: Paydar
authors.forename: Elham
authors.initials: E
authors.lastname: Rouhollahi
authors.forename: Hamed
authors.initials: H
authors.lastname: Karimian
mesh_terms: D031041:Annona / Q000737:chemistry*; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D059447:Cell Cycle Checkpoints; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D045304:Cytochromes c / Q000378:metabolism; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D010936:Plant Extracts / Q000494:pharmacology*; D018515:Plant Leaves / Q000737:chemistry; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D051028:bcl-2-Associated X Protein / Q000378:metabolism
chemical_list: D016328:NF-kappa B; D010936:Plant Extracts; D017382:Reactive Oxygen Species; D051028:bcl-2-Associated X Protein; D045304:Cytochromes c; D053148:Caspase 3; D053453:Caspase 9