1.   
Introduction:

Cancer being a catastrophe of the frame of higher multicellular
organisms consequential from the abnormal growth of the cells attains
modifications in the statement of copious amendments, prompting dysregulation
of the conventional cell programming concerned with the cell division and cell
separation. This upshot in an unevenness of cell replication and cell passing
that stimulates the development of a tumor cell population. The characters that
portray a lopsided growth of a malignant tumor are the competence to strike
locally, to sweep to neighboring lymph nodes, and to metastasize far off organs
in the body. Clinically, growth seems, by all accounts, to be an extensive
assortment of diseases with various phenotypic attributes. As a cancerous
growth ensues, genetic drift in the cell population forms cell heterogeneity in
such features as cell antigenicity, invasiveness, metastatic potential, rate of
cell proliferation, differentiation state, and response to chemotherapeutic
agents. At the molecular level, all cancers have plentiful things in mutual,
which implies that the ultimate biochemical lesions steering to malignant
transformation and progression can be fabricated by a common but not analogous
pattern of alterations of gene readout. In general, malignant cancers cause
noteworthy morbidity and will be lethal to the host if not treated. Omissions
to them give the marks to be concealed, laidback cancers that may remain
clinically undetectable (or in situ), permits the host to have a standard life
expectancy. There are over 100 diverse categories of cancer, and each
is classified by the type of cell that is initially affected, and these include
breast cancer, cervical cancer, skin cancer, leukemia, lung cancer, prostate
cancer, and so on. Chronic degenerative diseases like
cancer have touched rampant proportions and are regarded as a serious medical
condition and therefore, the treatments of these diseases are of clinical
importance (WHO, 2005). In consequence, the medicinal plants play a pivotal
role and are considered as the basis for health preservation and care
worldwide. There have
been researches done on the anti-cancerous properties of plants and recognized
for centuries.

 The National Cancer Institute (NCI) has screened
approximately 35,000 plant species for conceivable anticancer activities. Among
them, about 3,000 plant species have established reproducible anticancer
activity. Various studies
have concentrated on the anticarcinogenic properties of plants, to name a few, Abrus precatorius, Albizza lebbeck Alstonia
scholaries Anacardium occidentale hepatoma, Asparagus racemosa, Boswellia
serrata, Ethyrina suberosa, Euphorbia hirta, Gynandropis pentaphylla, Nigella
sativa, Peaderia foetida, Picrorrhiza kurroa, Withania somnifera, Annona
muricata. Due to the therapeutic ability of the species Annona muricata of
the family Annonaceae, wide range of studies has been made over the last
decade. The bioactivity as well as toxicity of this species has diverted the
attention towards itself.

 

2.   
Annona muricata: Description
and ethnomedicinal uses:

Annona muricata, commonly called Soursop or Graviola, is
a tropical plant species renowned for its edible fruit which has selected
restorative benefits, yet in addition particular toxicological impacts. This plant
is a species of the genus Annona, of the Annonaceae family, order Magnoliales
and Division Magnoliophyta. The genus Annona comprises over 70 species among which A. muricata is the most
extensively grown. The Soursop tree grows up to 5– 10 m tall and 15– 83 cm in
breadth with low branches. It has a tendency to blossom and fruit a large
segment of the year, however there are more characterized seasons relying upon
the altitude. It is scattered in the tropical areas. The flowers of the plant are showy and are
yellow-green colored. The fruit here is an ovoid berry, dull green
in shading. Each fruit may comprise 55–170 black seeds when fresh and they
transform to light brown when dry. The flesh is white and creamy with
distinctive aroma and flavor.

Figure 1: A)
Annona muricata, fruit tree    B) Leaves     C) Fruit, an ovoid berry    D) Flower

 

 

Traditional
medicinal benefits of
leaves, bark, fruit and seed of A.
muricata have been the emphasis of myriad medicinal
routines. The most significantly used preparation in traditional medicine is
the decoction of bark, root, seed or leaf and applications are wide-ranging.
The ingestion of leaves decoction is used as analgesic and also it is used to
cope with discomfort accompanying colds, flu and asthma; and to treat cutaneous
(external) and internal parasites. The use of leaves to treat malaria is very
substantial. The fruit is not only cherished as food, but the juice is used as
galactogogue to treat diarrhea, heart and liver diseases, and against
intestinal parasites. Customary therapeutic qualities of A. muricata have been recognized in
tropical locales to ponder different afflictions, for example, fever, torment,
respiratory and skin disease, bacterial infections, hypertension, aggravation,
diabetes and cancer. There have been a number of reports on the
ethno-

medical uses of A.
muricata leaves
including treatments for hypertension, diabetes and cancer. Most
parts of the A. muricata tree, similar to that of the other Annona species,
namely A. squamosa and A. reticulata are
widely used as local and habitual medicines contrary to a large number of human
inflamations, infections and disorders. The pulp of the fruit is used as natural
medicine for joint pains, dysentery, neuralgia, diarrhea, rheumatism, fever,
malaria, parasites, skin rushes and worms, and 
also is eaten to increase mother’s milk after parturition. The leaves
are used to control cystitis, , body aches and diabetes. Additionally, the
administration of the leaf’s decoction is thought to display anti-rheumatic and
neuralgic effects. In addition, the cooked leaves are topically used to treat
abscesses and rheumatism that was reported by de Sousa OV and
colleagues.(9)

 The crushed
seeds are thought to have anthelmintic activities antaagonistic to external and
internal worms. In tropical Africa, the plant is employed as an astringent
pesticide, and insecticide; along this it is used to treat coughs, pain and
skin diseases. In India, the fruit and flower are used as remedies against
catarrh, while the root-bark and leaves are well known to have an anti
phlogistic and anthelmintic activities which was worked upon
by Adewole SO, Ojewole J.(2)

In
Malaysia, the crude extract of A. muricata and A.
squamosa is used as a syrup on the head to buffer from fainting. In South
America and tropical Africa, including Nigeria, leaves of A.muricata are
implemented as an ethnomedicine opposed to tumors and cancer. Moreover, the
smooth muscle relaxant, hypotensive hypoglycemic, anti-inflammatory, sedative,
and antispasmodic effects accredit to the leaves, barks and roots of A.
muricata. Moreover along with the ethnomedicinal uses, the fruits are
widely employed for the preparation of beverages, ice creams,and syrups (3, 21, 23, 26) .

 

3.   
Phytoconstituents:

There
are more than 200 chemical compounds that have been documented and isolated
from this plant; the foremost being the alkaloids, phenols, flavonoids and
acetogenins. Based on the in vitro studies, extracts and phytochemicals of A. muricata have been sorted out as
anti-microbial, anti-inflammatory, anti-protozoan, antioxidant, insecticide,
larvicide, and cytotoxic to
tumor cells. Studies on the extracts and isolated compounds of A. muricata showed contraceptive,
antitumor, anti-ulceric, wound healing, hepato-protective, anxiolytic,
anti-stress, anti-inflammatory, anti-icteric and hypoglycemic activities.
Furthermore, there have been clinical studies carried out in order to boost the
hypoglycemic activity of the ethanolic extracts of A. muricata leaves. Mechanisms of action of a few pharmacological
activities have been explicated, such as cytotoxic, antioxidant, antimicrobial,
anti-nociception and hypotensive activities. Nonetheless, some phytochemical
compounds isolated from A. muricata
have shown a neurotoxic effect in vitro and in vivo. Thus, these crude extracts
and isolated compounds requires further studies to define the magnitude of the
effects, optimal dosage, long-term safety, and potential side effects. (32)

Constant examinations on diverse parts
of the A. muricata have shown the occurrence of varieties of phyto
constituents and compounds, including flavonol triglycosides (FTGs) alkaloids
(ALKs), phenolics (PLs), megastigmanes (MGs), cyclopeptides (CPs) and essential
oils. The existence of various minerals such as Ca, Na, Fe, K, Cu and Mg imply
that regular intake of the A. muricata fruit can help furnish essential nutrients to the
human body. However, Annona species, including A. muricata, have been
shown to be a vital source of annonaceous acetogenin compounds (AGEs). Almost
all the parts inclusive of the fruits, leaves, stems and
roots of this plant are known to be rich in flavonoids, isoquinoline alkaloids
and annonaceous acetogenins. (23,25,26,28,35,42)

Acetogenins are a unique category of C-35/C37 secondary
metabolites obtained from long chain (C-32/C34) fatty acids in the polyketide
pathway. They are basically illustrated by combining fatty acids with  2-propanol unit at C-2 that gives a methyl-substituted
?, ?-unsaturated ?-lactone. Starting with the discovery of uvaricin from Uvaria
accuminata in 1982, there have been several acetogenins that are
identified. About 500 have been reported from numerous parts of the plants in
the Annonaceae family. Because of the exceptional structures and broad spectrum
of biological activities, AGEs have drawn significant scientific interest of
late. The active annonaceous acetogenins have shown to be successful in
inducing death in cancer cells that are resistant to even chemotherapeutic
drugs. Besides their remarkable anti proliferative efficacy, these annonaceous
acetogenins have been endorsed to debilitating side effects such as
neurotoxicity suggesting that these components can easily traverse the
blood–brain barrier and are known to cause atypical Parkinson’s disease, thus
restricting their development as new drug entities. Various biological
activities have been reported for AGEs, including antimalarial, anti-parasitic
and pesticidal activities. However, the physiological activities of AGEs are
initially characterized by the toxicity against cancer cells and inhibitions of
the mitochondrial complex I. (11)

4.    Anticancer activity:

There are plentiful reports that signify anti-proliferative effects of
various extracts of the plant and isolated AGEs against various cancer cell
lines. As mentioned earlier cancer is categorized based on the primary tissue
it occurs in. The antitumor effects of A.
muricata against various cancer cell lines are described here.

Breast Adenocarcinoma:

An
oncogene, the epidermal growth factor receptor (EGFR) that is quite often overexpressed
in breast cancer (BC) and is linked with poor prognosis and drug resistance. Studies
have revealed that there is a selective inhibition of breast cancer cells via
EGFR down regulation by the extract of Graviola. Thus, EGFR is a rational
target for BC therapy development. In addition, xenografts mouse model studies experiments
have showed that the fruit extract reduces the growth of BC cells. The Fruit extract
specifically suppressed the growth of EGFR-overexpressing human BC (MDA-MB-468)
cells but did not in non-tumorigenic human breast epithelial cells (MCF-10A). These reports bolster the
evidence that Graviola has selective anti-growth effects between cancer and
non-cancer cells (8). In a report by Yu-Min Koa
and colleagues it was shown that Graviola favors apoptosis in ER-related
pathways. In addition, it was also noted that Graviola had subsided MCF-7 tumor
growth while hindering ER-cyclin D1 and Bcl-2 protein expressions in nude mice
(44). A promising
antitumor effect was reported in an in vivo study
on 7,12-dimethylbenzene anthracene (DMBA)-induced cell proliferation in the
breast tissues of mice. Oral administration of the Graviola leaves supposedly have
protective effects towards the development of breast carcinogenesis was shown
by DMBA as it induced a protective effect against DNA damage.

Lung Cancer:

The A549 cells (adenocarcinomic
human alveolar basal epithelial cells) when treated with aqueous
extracts of A. muricata showed elevated levels of ROS, reduction of MMP (matrix
metalloproteinases) through the upheaval of expression of Bax and decreased
expression of Bcl-2. These events led to the release of cytochrome c release to
the cytosol. The released cyt-c activated
caspase-9 and caspase-3, thus causing apoptosis. Concomitantly, there was a
cell cycle arrest at G0G1 phase. The incubation of A549 cells with superoxide
dismutase and catalase predominantly suppressed the cytotoxicity that was induced
by the extract. This highlighted that the intracellular ROS plays an important
role in cell death. Anonna muricata also has had impacts on Lewis lung carcinoma (LLC)
tumor cell lines that were examined both in
vivo as well as in vitro was
shown in a study by Zhao GX1 et,. al. This
validated that Graviola had antitumor activity by limiting the natural growth
of the lung tumors (45). NADH oxidase inhibition in cancer cell lines, down
regulation of the P-glycoprotein pump via ATP depletion and Cell cycle arrest
at S-phase progression has shown to be affected by Graviola through its
anti-cancerous and cytotoxic mechanisms, which has been shown by additional
research.  

Liver Carcinoma:

The aqueous extracts of Annona muricata was tested for the
activation of caspases in Huh-7 human liver cancer cells. Huh-7 cells when
treated with the extracts, both caspase-9 and caspase-3 activities in the cells
were uplifted. This suggested that the extract had induced apoptosis by activating
the mitochondrial mediated intrinsic pathway. The protein analysis (western
blot) of caspases demonstrated that the expressions of the cleaved caspases was
dose dependent. Considering these results, it was concluded that the extract of
A.muricata leaves have a good
potential to play a vital role as cancer chemotherapeutic agents. Furthermore, the
reports by ____________
indicate the reduction in the viability of hepatocellular carcinoma cell lines
possibly through G0/G1 or
S phase arrest or via induction of sub-G0/G1 DNA fragmentation. This mechanism
remains yet to be confirmed.

Pancreatic Cancer (PC):

Pancreatic tumor aggressiveness is allied
with a heightened metabolic activity and glucose concentration of malignant
tumors. Also a hypoxic environment has been proven to
be necessary for the oncogenic and metabolic transformation and the survival of
the pancreatic cancer cells. In particular, it is believed that resistance
to drugs is induced by hypoxia by the activation of PI3K, Akt, NF-?B and MAPK
pathways 51.  The cells after being treated with the Graviola
extract, showed low viability as the phosphorylation of the molecules was
hindered by the extract. Accordingly, the major transcription factor that is
activated under hypoxic conditions, HIF-1a expression was investigated. The reports
stated that a product of the extract had blocked glucose transporters (GLUT1
and GLUT4), NF-?B, HIF-1a and other
glycolytic enzymes like LDHA, which reduced the uptake of glucose molecules and
the production of ATP by the PC cells. This overall reduction caused the cell
death. In accord with the ATP reduction, the extract also increased the ROS
levels within the cells that eventually led to necrosis. 52–54. Necrotic agents
by itself being less significant in the treatment of cancer as they induce a
local inflammation. But the inflammation thus caused leads to the activation of
the innate immune system to initiate anti-tumor activities. 52. This effect along
with the progression of PC cells was evaluated in the KrasG12DPdx1- Cre mice (55,56). The mice with a
developing pancreatic intraepithelial neoplastic (PanIN) lesions was fed with
the plant extract. The cells were analyzed and it was concluded that the
cytotoxic effects were particular to the tumor cells. Thus the administration
of Graviola caused glycolytic inhibition was seen in the PC cells that led to
the depletion of the tumor.

 

Prostate Cancer

The anti-proliferative effects of the water
extract of leaves of A. muricata was examined in vivo against the benign
prostatic hyperplasia (BPH-1) cell line and the rat prostates were observed. Normal
histology of all the other testes was observed. There was a significant
reduction in the size of the seminal vesicles of the test groups and displayed
a remarkable atrophy with a raised cellularity and zero secretion in the
acinii. The apoptotic characteristics of the glandular epithelium was typically
seen (pycknotic nuclei and nuclear material towards the periphery). Also, the Bax
protein was up regulated, while Bcl-2 was suppressed. Thus it was concluded
that Annona muricata
has anti-proliferative effects on BPH-1 cells and deduces the prostate size,
possibly through apoptosis (5). The effect of Graviola extract against
the prostate cancer cell lines has also been expounded in vitro. Experiments
have been performed to show that Graviola initiates necrosis in PC-3 cells
through the inhibition of cellular metabolism and tumor mobility. Further
evaluation depicted the downregulation of the expression of the hypoxia-related
factors and glycolytic factors following treatment in PC cells with Graviola
(41). The inhibition of
prostate cancer proliferation, viability and clonogenic colonies have been proved
as an outcome of the Graviola leaf extract (GLE) pharmacokinetics and
absorption kinetics (43). by Yang C et, .al

 

Colon Cancer:

In a study performed by Jaramillo MC et al., the mechanism of action of ethyl
acetate extract of A. muricata leaves against colon cancer
cells (HT-29 and HCT-116) and lung cancer cells (A549) has been illustrated. The
leaf extract was proficient to induce apoptosis in colon and lung cancer cells
through the mitochondrial-mediated pathway. This anti-proliferative effect was
alongside with cell cycle arrest in the G1 phase (20). However,
the migration and invasion of colon cancer cells were profoundly inhibited by
the leaf extract. The in vivo chemo preventive potential of the ethyl acetate
extract of the A. muricata leaves against azoxymethane-induced
colonic aberrant crypt foci (ACF) in rats was validated by Moghadamtousi and
colleagues. Oral dosage that was administered for 60days caused a significant
reduction of ACF formation in rats when tested by methylene blue staining of
the colorectal specimens. PCNA and Bcl2 proteins were down regulated whereas
Bax protein was up regulated after the administration of the extract. This was depicted
in the immunohistochemistry analysis where they
were compared with cancer control group. In addition the levels of enzymatic
antioxidants showed an increase and a suppression was seen in malondialdehyde
level of the colon tissue homogenates. This suggested the restraint of lipid
peroxidation. An AGE annomuricin E was found to inhibit the growth of HT-29 cells.
The cytotoxic effect of annomuricin E was enhanced by the G1 cell cycle arrest.
Annomuricin activated the mitochondrial events comprising the dissipation of
the mitochondrial membrane potential and caused the leakage of cytochrome c
from the mitochondria. Followed by this, annomuricin E activated caspase 3/7
and caspase 9 responsible for the apoptosis. Furthermore, Moghadamtousi and colleagues examined that
ethyl acetate extract of Annona muricata leaves (EEAM) exerted a
striking cytotoxic effects on HCT-116 cells as determined by MTT and LDH
assays. Flow cytometric analysis illucidated the cell cycle arrest at G1 phase
and also the externalization of phosphatidylserine acting as an indicator of
the induction of apoptosis. EEAM treatment activated 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.
These processes subsequently steer to
attenuation of mitochondrial membrane potential (MMP) and cytochrome c release.
Release of cytochrome c activates apotosome and the intrinsic caspase cascade
that triggers execution of apoptosis through DNA fragmentation. Immunofluorescence analysis portrayed 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. (29,30,31).  Thus, these findings verify the usage of A. muricata leaves in
ethnomedicine against cancer and emphasize annomuricin E as one of the contributing
compounds in the anticancer activity of A.
muricata leaves.

Ovarian Cancer:

Apart from its various medicinal properties, acetogenins
in Annona muricata showed potent anti-ovarian cancer activity which selectively
attack cancerous cells without harming healthy cells and preventing metastasis.
Apoptosis of granulosa cells of the ovary has been initiated by this acetogenin
in many organs of cancerous cells of humans. The plant had proved to be an
effective anti-tumor and anti-cancer medicinal plant, and thus represents a
source for new antiovarian cancer drug discovery. In vitro cytotoxicity tests
were performed using MTT assay as described by Xu-jie and Chu 11. In vivo
Pilot Experiment An in vivo pilot experiment was performed with thirty Swiss
albino mice consisting of all females (average weights 18-25g). In order to
mimic advanced ovarian cancer, the mice were injected intraperitoneally (i.p.)
with 1 x 104 OV7-96020764-CDNA-(20uL) cell line into the abdominal cavity to
form ascites. Five groups of mice were examined: four control mice (no
treatment), twelve mice treated with crude and pure compound from A. muricata
leaf and four mice treated with doxorubicin (10 mg) after ascites had formed.
Cells of ascites of two mice were frozen and stored for future experiments. To
study the reduction of swollen abdomen, 5 mg/kg doxorubicin (Rubex) and the
isolated compound at a final concentration of 20 mg/kg were administered i.p.

 

Leukemia:

Investigations
have also been done to check the ability of ethanolic extracts of Annona muricata leaves for its cytotoxicity potential and capacity of
inducing apoptosis in K562 cancer cells, a chronic myelogenous cell line. The activity of Caspase-3
was remarkably enhanced during the apoptosis stimulated by the extract at a low
quantity. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end
labelling (TUNEL) assay results verified the process of apoptosis. The Caspase-3
activity and TUNEL assay reports endorsed that the ethanolic extract of A. muricata leaves induced apoptosis in
K562 cell lines. Ezirim, A
U and colleagues thus concluded in their paper that Annona muricata could be considered as a natural source for the
preparation of pro apoptotic drugs. Also in the reports by, Constant Anatole Pieme and others, A. muricata had exhibited anti proliferative effects on HL-60 cells
(Human promyelocytic leukemia cell line) by promoting the loss
of cell viability, morphological differences, loss in membrane mitochondrial
potential and G0/G1 phase cell arrest. Their conclusions confirmed the efficacy
of A. muricata as an agent of
chemotherapeutic and cytostatic activity in HL-60 cells (7).

Cervical Cancer:

In a study by A N Artanti et,al Our result demonstrated
that polyketide derivatives from Annona
muricata have growth inhibitory and cytotoxic effect on cervical cancer
cell line. The acetogenins from Annona
muricata L. leaves performed potent cytotoxic effect on HeLa cells. Decreasing
cell viability may be because of either cell death or cell cycle arrest. The
mechanism of cell cycle distribution is also associated with some of celuller protein
especially p53 protein. p53 is a tumor suppresor protein. In this study, they
observed that AGEs from Annona muricata
treatment increased p53 level in nucleus. Therefore, AGE isolation may be amandable
as viral inhibitor agent and as competitor of vaccine to prevent the
development of cervical cancer. In conclusion, extracts of Annona muricata L.
leaves indicate has potential to be developed as a co-chemotherapeutic agent on
HeLa cell lines, it can exhibit potential abbility with p53 stabilization.
Further molecular target detection to investigate its cellular pathway needs to
be conducted.