Background:
Triple negative breast cancers (TNBCs) are having high morbidity and shorter survival rate in the population.
These types of cancers are having high aggressiveness, lymphatic invasion and absence of receptors. The treatment options for these
types of cancers are also scarce. Several studies have been conducted to investigate the effectiveness of seeds of Annona muricata for its
anti cancer activities in various cancer cell lines such as lung A549, breast MCF7, colon HT-29, oral KB and human hepatoma cell lines.
But works related to its anticancer effect and mechanism of action in TNBCs has not been elucidated.
Objective:
The present study was undertaken to evaluate the in vitro, in vivo and in silico anticancer potential of chloroform fraction of
methanolic extract of seeds of Annona muricata (CMAM) against TNBC along with elucidation of its mechanistic pathway.
Methods:
In vitro cytotoxicity- and antiproliferative- studies in three triple negative breast cancer cell lines were conducted using MTT
and SRB assays respectively. The mechanism through which CMAM exerts its pharmacological effect was elucidated in vitro employing
cell morphological assessment studies using acridine orange/ ethidium bromide (AO/EB), intra cellular reactive oxygen species
assay, DNA fragmentation assay, agarose gel electrophoresis, terminal deoxynucleotidyl transferase dUTP nick end labelling
(TUNEL) assay, cell cycle analysis, annexin binding assay and caspase activated mitochondria mediated apoptotic assays using western
blot. In vivo evaluation in 4T1 induced murine mammary tumour model was also conducted. Phytoconstituents in CMAM was analysed
using liquid chromatography mass spectroscopy. In silico binding studies with various annonaceous acetogenins against BCL-2 and
cyclin E were performed.
Results:
Cytotoxicity studies in MDA-MD-231, 4TI and BT-549 revealed the IC50 value of CMAM to be 2.5±0.14, 4.8±0.3 and 4.5±0.16
µg/mL respectively. Anti proliferative studies in 4T1, MDA-MB-231 and BT-549 revealed the GI50 values to be 0.128+0.03, 18.03+0.20,
0.95+0.04 µg/mL respectively. CMAM exhibited its cytotoxicity through the lysis of cell membrane, ROS dependent caspase activated
mitochondria mediated apoptosis, and arresting the S phase of the cell cycle. In vivo evaluation also supported the tumoricidal property of
CMAM as evidenced by reduction in tumour volume and serum biomarkers. Histopathologically there was a marked reduction in
cellularity, nuclear chromatin condensation and a few normal cells in group treated with CMAM at a dose of 31mg/Kg. Phytoconstituent
evaluation has revealed the presence of annonaceous acetogenins in CMAM. Among the various annonaceous acetogenins, muricatacin
alone showed lipophilicity and binding affinity towards BCL-2 and cyclin E1.
Conclusion:
The current study shows the effectiveness of CMAM against TNBC both in vitro and in vivo. This anticancerous effect of
CMAM could be by virtue of its ROS dependent caspase activated mitochondria mediated apoptosis and the S-phase arrest of the cell
cycle in the TNBCs. Our results indicate that the presence of annonaceous acetogenins, especially muricatacin, could be contributing to
this anticanceros effect of CMAM. Thus muricatacin could be a potential candidate for the targeted therapy of TNBCs.
Apolar Annonaceous Acetogenins from the Fruit Pulp of Annona muricata