The chemopotential effect of Annona muricata leaves against azoxymethane-induced colonic aberrant crypt foci in rats and the apoptotic effect of Acetogenin Annomuricin E in HT-29 cells: a bioassay-guided approach

PLoS One. 2015 Apr 10;10(4):e0122288. doi: 10.1371/journal.pone.0122288. eCollection 2015.

Abstract

Annona muricata has been used in folk medicine for the treatment of cancer and tumors. This study evaluated the chemopreventive properties of an ethyl acetate extract of A. muricata leaves (EEAML) on azoxymethane-induced colonic aberrant crypt foci (ACF) in rats. Moreover, the cytotoxic compound of EEAML (Annomuricin E) was isolated, and its apoptosis-inducing effect was investigated against HT-29 colon cancer cell line using a bioassay-guided approach. This experiment was performed on five groups of rats: negative control, cancer control, EEAML (250 mg/kg), EEAML (500 mg/kg) and positive control (5-fluorouracil). Methylene blue staining of colorectal specimens showed that application of EEAML at both doses significantly reduced the colonic ACF formation compared with the cancer control group. Immunohistochemistry analysis showed the down-regulation of PCNA and Bcl-2 proteins and the up-regulation of Bax protein after administration of EEAML compared with the cancer control group. In addition, an increase in the levels of enzymatic antioxidants and a decrease in the malondialdehyde level of the colon tissue homogenates were observed, suggesting the suppression of lipid peroxidation. Annomuricin E inhibited the growth of HT-29 cells with an IC50 value of 1.62 ± 0.24 μg/ml after 48 h. The cytotoxic effect of annomuricin E was further substantiated by G1 cell cycle arrest and early apoptosis induction in HT-29 cells. Annomuricin E triggered mitochondria-initiated events, including the dissipation of the mitochondrial membrane potential and the leakage of cytochrome c from the mitochondria. Prior to these events, annomuricin E activated caspase 3/7 and caspase 9. Upstream, annomuricin E induced a time-dependent upregulation of Bax and downregulation of Bcl-2 at the mRNA and protein levels. In conclusion, these findings substantiate the usage of A. muricata leaves in ethnomedicine against cancer and highlight annomuricin E as one of the contributing compounds in the anticancer activity of A. muricata leaves.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aberrant Crypt Foci / chemically induced
  • Aberrant Crypt Foci / drug therapy*
  • Aberrant Crypt Foci / pathology
  • Animals
  • Annona / chemistry*
  • Annona / metabolism
  • Apoptosis / drug effects*
  • Azoxymethane / toxicity
  • Caspase 3 / metabolism
  • Caspase 7 / metabolism
  • Caspase 9 / metabolism
  • Cell Proliferation / drug effects
  • Colon / metabolism
  • Colon / pathology
  • Cytochromes c / metabolism
  • Down-Regulation / drug effects
  • Furans / chemistry
  • Furans / toxicity*
  • G1 Phase Cell Cycle Checkpoints / drug effects
  • HT29 Cells
  • Humans
  • Immunohistochemistry
  • Lactones / chemistry
  • Lactones / toxicity*
  • Lipid Peroxidation / drug effects
  • Male
  • Malondialdehyde / metabolism
  • Membrane Potential, Mitochondrial / drug effects
  • Mitochondria / metabolism
  • Plant Extracts / chemistry
  • Plant Extracts / pharmacology
  • Plant Extracts / therapeutic use*
  • Plant Leaves / chemistry
  • Plant Leaves / metabolism
  • Proliferating Cell Nuclear Antigen / metabolism
  • Proto-Oncogene Proteins c-bcl-2 / genetics
  • Proto-Oncogene Proteins c-bcl-2 / metabolism
  • Rats
  • Rats, Sprague-Dawley
  • Up-Regulation / drug effects
  • bcl-2-Associated X Protein / genetics
  • bcl-2-Associated X Protein / metabolism

Substances

  • Furans
  • Lactones
  • Plant Extracts
  • Proliferating Cell Nuclear Antigen
  • Proto-Oncogene Proteins c-bcl-2
  • annomuricin E
  • bcl-2-Associated X Protein
  • Malondialdehyde
  • Cytochromes c
  • Caspase 3
  • Caspase 7
  • Caspase 9
  • Azoxymethane

Grant support

This research was supported by the University of Malaya High Impact Research Chancellery (UM.C/625/1/HIR/175), University of Malaya Research Grant (RP001-2012C), and Postgraduate Research Fund (PG118-2013A). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.