title: Annona muricata leaves induce G₁ cell cycle arrest and apoptosis through mitochondria-mediated pathway in human HCT-116 and HT-29 colon cancer cells.
abstract: BACKGROUND
  Annona muricata known as "the cancer killer" has been widely used in the traditional medicine for the treatment of cancer and tumors. The purpose of this study is to investigate the anticancer properties of ethyl acetate extract of Annona muricata leaves (EEAM) on HT-29 and HCT-116 colon cancer cells and the underlying mechanisms.
  
  
  METHODS
  The effect of EEAM on the cell proliferation of HT-29 and HCT-116 cells was analyzed by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) assay. High content screening system (HCS) was applied to investigate the cell membrane permeability, mitochondrial membrane potential (MMP), nuclear condensation 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. Flow cytometric analysis was used to determine the cell cycle distribution and phosphatidylserine externalization. The protein expression of Bax and Bcl-2 was determined using immunofluorescence analysis. In addition, the potential of EEAM to suppress the migration and invasion of colon cancer cells was also examined.
  
  
  RESULTS
  EEAM exerted significant cytotoxic effects on HCT-116 and HT-29 cells as determined by MTT and LDH assays. After 24 h treatment, EEAM exhibited the IC₅₀ value of 11.43 ± 1.87 µg/ml and 8.98 ± 1.24 µg/ml against HT-29 and HCT-116 cells, respectively. Flow cytometric analysis demonstrated the cell cycle arrest at G1 phase and phosphatidylserine externalization confirming the induction of apoptosis. EEAM treatment caused excessive accumulation of ROS followed by disruption of MMP, cytochrome c leakage and activation of the initiator and executioner caspases in both colon cancer cells. Immunofluorescence analysis depicted the up-regulation of Bax and down-regulation of Bcl-2 proteins while treated with EEAM. Furthermore, EEAM conspicuously blocked the migration and invasion of HT-29 and HCT-116 cells.
  
  
  CONCLUSIONS
  These findings provide a scientific basis for the use of A. muricata leaves in the treatment of cancer, although further in vivo studies are still required.
authors.affiliation: Biomolecular Research Group, Biochemistry Program, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia.
authors.forename: Soheil
authors.initials: S
authors.lastname: Zorofchian Moghadamtousi
authors.affiliation: Department of Pharmacy, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.
authors.forename: Hamed
authors.initials: H
authors.lastname: Karimian
authors.affiliation: Department of Pharmacology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.
authors.forename: Elham
authors.initials: E
authors.lastname: Rouhollahi
authors.affiliation: Department of Pharmacology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.
authors.forename: Mohammadjavad
authors.initials: M
authors.lastname: Paydar
authors.affiliation: Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur 50603, Malaysia.
authors.forename: Mehran
authors.initials: M
authors.lastname: Fadaeinasab
authors.affiliation: Biomolecular Research Group, Biochemistry Program, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia. Electronic address: habsah@um.edu.my.
authors.forename: Habsah
authors.initials: H
authors.lastname: Abdul Kadir
mesh_terms: D031041:Annona / Q000737:chemistry*; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D003110:Colonic Neoplasms / Q000188:drug therapy / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D059585:G1 Phase Cell Cycle Checkpoints / Q000187:drug effects*; D045325:HCT116 Cells; D019073:HT29 Cells; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D010936:Plant Extracts / Q000737:chemistry / Q000494:pharmacology*; D018515:Plant Leaves / Q000737:chemistry*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects; D051028:bcl-2-Associated X Protein / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D010936:Plant Extracts; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D051028:bcl-2-Associated X Protein; D045304:Cytochromes c; D007770:L-Lactate Dehydrogenase; D020169:Caspases
keywords: Annona muricata; Apoptosis; Colon cancer; Invasion; Migration; Mitochondria