Chloroform Fraction of Methanolic Extract of Seeds of Annona muricata Induce S Phase Arrest and ROS Dependent Caspase Activated Mitochondria-Mediated Apoptosis in Triple-Negative Breast Cancer
Authors: Kariyil Bibu J. , Ayyappan Usha P.T. , Gopalakrishnan Anu , George Ajith J. 
Published: September 2020

Background:
Triple Negative Breast Cancers (TNBCs) have high morbidity and shorter survival rate
in the population. These types of cancers have 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 anti-cancer effect and mechanism of action in TNBCs have not been elucidated.

Objective:
The present study was undertaken to evaluate the in vitro, in vivo, and in silico anti-cancer 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 the 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), intracellular reactive oxygen species assay, DNA fragmentation assay,
agarose gel electrophoresis, terminal deoxynucleotidyl Transferase dUTP Nick End Labeling (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 tumor model was also conducted. Phytoconstituents
in CMAM were analyzed 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±0.16μg/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+0.04μg/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 a reduction in tumor volume and serum biomarkers. Histopathologically, there was a
marked reduction in cellularity, nuclear chromatin condensation, and a few normal cells in the 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 mitochondriamediated
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 anticancerous effect of
CMAM. Thus, muricatacin could be a potential candidate for the targeted therapy of TNBCs.