ABSTRACT
Annona muricata (Linn.) is an
evergreen tropical tree of the Annonaceae family that posseses
phytotherapeutic bioactive compounds known as annonaceous acetogenins effective
for the treatment of cancers and other conditions. This study aimed at
investigating the effect of the aqueous leaf extract of A. muricata on
human BPH-1 cells, the prostate organ as well as certain target cellular genes
of proliferative activity. Dried A. muricata leaves were pulverized, and
the aqueous crude extract obtained. HPLC was used for monitoring various
batches of the A. muricata leaf extract (AMLE). The MTT assay was
performed on BPH-1 cells (1 x 105 per well) using AMLE concentrations of 0.5,
1.0 and 1.5 mg/mL for 24, 48 and 72 hours. Microscopic examination of
proliferation as well as morphology of the cells was carried out. RT-PCR was
used to examine possible target genes, Bax and Bcl-2, using mRNA extracted from
cells. Fifteen (15) F344 male rats (150-200 g) were placed in three groups of
five (5). The low dose (LD) and high dose (HD) groups were gavaged 30 mg/mL and
300 mg/mL respectively and fed ad libitum alongside five (5) control
(ctrl) group rats. Rats were sacrificed after 60 days. Whole blood was sampled
by cardiac puncture and processed for biochemical assay. Prostate, seminal
vesicles and testes were harvested, weighed and stored for histological
examination. HPLC chromatographic fingerprint monitoring of different batches
of AMLE yielded eight peaks. In vitro cell viability analysis showed a
dose dependent growth inhibitory effect of AMLE on BPH-1 cells. The IC50
obtained for AMLE was 1.36 mg/ml. Increasing doses of AMLE directly
up-regulated the levels of the pro- apototic protein Bax,
whiles down-regulating the levels of the anti-apoptotic protein Bcl-2. In
vivo studies showed statistically significant seminal vesicle indices
recorded for both (LD) and (HD) groups (p=0.004 and p=0.009 respectively)
compared with control (ctrl) group. There was no statistically significant
difference between PSA levels of test groups compared with control groups.
Histological examinations of the prostatic and seminal vesicle tissues however
showed dose dependent morphological changes with AMLE treatment. The acinii
were empty of secretions and there was marked atrophy with increased
cellularity observed. AMLE exerts decreased secretory activity on prostate with
flattening of acinar epithelial linings being demonstrated. The prostate
epithelial and stromal cells were flattened with scanty prostatic secretions in
the lumen. Thus, AMLE has antiproliferative activity against BPH-1 cells.
CHAPTER ONE
1.0 INTRODUCTION
1.1 BACKGROUND
Prostate cancer (PCa) is a disease
of the prostate in which the cells differentiate, become malignant and rapidly
proliferate to form a tumor mass. PCa comes second only to lung cancer with
respect to male cancer deaths. It however has the highest incidence among all
male cancers. PCa accounted for approximately 29% of newly reported cases of
cancer and 9% of all reported cancer deaths among men in the USA in 2012
(American Cancer Society, 2012). Furthermore, it was reported as the most
frequently occuring male cancer in the UK making up to a quarter of all newly
reported male cancer cases in 2012 (Cancer Statistics Registration, 2012). It
accounts for approximately 30% of cancers diagnosed each year (Australian
Institute of Health and Welfare Cancer Incidence Projections, 2012) in
Australian men. This makes it follow lung cancer closely as the second highest
cause of male cancer deaths (Smith, 2012). In Ghana, it is reported that
prostate cancer is responsible for 17.35% of all male cancer deaths making it
second only to liver cancer (Wiredu and Armah, 2006).
The treatment of this common
condition has however been the major point of controversy. Conventional
treatment regimens have produced adverse effects (Steineck et al., 2002). Apart
from being expensive, causing urinary and erectile problems, some of the
adverse effects of conventional therapy include toxicity and growth inhibition
to normal cells, (Singh et al., 2006). It is reported also that a lot of people
have prostate
cancer without they and their doctors knowing initially because it does not
present with any deleterious symptoms, thus interventional therapy is unneeded
(Johansson et al., 2004; Albertsen et al., 2005). Therefore, there is
increasing advocacy for what is termed “watchful waiting” (Johansson et al.,
2004; Andren et al., 2006), or the even newer alternative called “active
surveillance” in early cases of prostate cancer. Radical prostatectomy, radiation
therapy, cryotherapy and high intensity frequency ultrasound are all employed
in treating localized prostate cancer. Hormonal therapy and chemotherapy are
the main remedies in advanced cancer treatment regimen.
Benign prostatic hyperplasia (BPH),
also known as nodular hyperplasia of the prostate, is an abnormally increased
growth in volume of the prostate gland causing urethral compression by the
proliferating cells that are found in the periurethral area of the prostate
gland. The prevalence of BPH is very high among older men. As high as 90%
incidence is reported for men between the age range of 80 to 90 (Nickel, 2006).
As many as 4.5 million visits to the clinic in the United States in 2000 were
related to BPH (Wei et al., 2005). Among elderly Ghanaian men (aged 50 – 74
yrs), the prevalence of DRE-detected enlarged prostate was reported to be
62.3%, and that of PSA≥1.5 ng/ml was 35.3% (Chokkalingam et al., 2012).
Open surgery and procedures such as
the transurethral needle ablation (TUNA) and transurethral microwave
thermotherapy (TUMT) which are less severely invasive are among the major means
of treatment. Chemotherapeutic agents used in the treatment of BPH are mainly alpha
blockers and 5 α-reductase inhibitors. There is an ever increasing shift
towards patronizing complementary and alternative medicine (CAM) all over the
world for a wide number of ailments. And the use of medicinal plants is one of
the major access avenues of CAM for various ailments.
Annona muricata (Linn.), referred
to commonly in English as soursop or “Apre” in Akan, is a member of the family
Annonaceae. It is an evergreen tropical fruit tree that grows to about 5-6
metres in height and produces a green edible heart-shaped fruit of about 15-20
cm in diameter with a white flesh and dark seeds inside. The leaves, stem,
bark, root and seeds of A. muricata possesses several of a bioactive group of
substances known as annonaceous acetogenins. A number of these acetogenins have
been isolated and their biological activities have been well documented. The
monotetrahydrofuran annonaceous acetogenins, cis-corossolone, annocatalin,
annonacin, annonacinone, solamin, and corossolone have been isolated from A.
muricata leaves. Acetogenins 1 (annoreticuin-9-one) and 2 (cis-annoreticuin)
isolated initially from other species, A. reticulata and A. Montana
respectively, have both been reported to show significant cytotoxic activity in
vitro against two human hepatoma cell lines namely Hep G2 and 2,2,15.
Acetogenin 1 targets the human pancreatic tumor cell line (PACA-2), human
prostate adenocarcinoma (PC-3) (Kim et al., 1998; Woo et al. 1999; Ragasa et al.,
2012) and human lung carcinoma (A-549) (Zhao et al., 1993; Ragasa et al.,
2012). The dichloromethane extract of the seeds of A. muricata yielded
annoreticuin-9-one (1), while the flesh of the fruit yielded cis-annoreticuin (2)
(Ragasa et al., 2012). Kim et al. (1998) demonstrated
the presence of the Annonaceous acetogenins – muricoreacin as well as
murihexocin C (mono-tetrahydrofurans) in A. muricata, particularly in the
leaves showed significant cytotoxic activities that targets PCa cell line PC-3,
and pancreatic carcinoma cell line PACA-2. A. muricata (Linn.) leaf extracts of
ethyl acetate showed a higher death rate to HeLa cells than the ethanol and
distilled water extracts. The chloroform extract also showed a higher death
rate in HeLa cells than ethyl acetate extract. The chloroform extracts seems to
have a superior preference for cancer causing viruses (Astirin et al., 2013).
The extract was moderately cytotoxic to normal cells (WRL-68 normal human
hepatic cells), compared to cancerous cells of human breast carcinoma,
(MDA-MB-435S) as well as human immortalized keratinocyte cells (HaCaT). This
may be due to the presence of antineoplastic substances obtained in
therapeutically active amounts from n-butanolic leaf extract of A. muricata
(George et al., 2012). The aqueous leaf extract is said to contain general
glycosides, condensed tannins, saponins and flavonoids, and did not show any
toxicity on systemic organs in an acute toxicity study (LD50<5000 2011="" 2014="" a.="" al.="" and="" are="" b.wt="" contained="" differ="" different="" et="" extracts="" flavonoid="" flavonol="" ieme="" in="" kg="" levels="" mg="" muricata="" of="" parts="" phenol="" plant="" reported="" rthur="" span="" the="">
However, the use of an aqueous
infusion of about 140 μg/cup was said to have caused neurotoxicity related to
atypical parkinsonism in Guadeloupe (Champy et al., 2005). The A. muricata
ethanolic leaf extract has been shown to have hypoglycaemic and antidiabetic
effects (Gupta et
al., 2005), as well as a protective effect on the lipid profile (Adewole and
Ojewole, 2009).
Apoptosis is also called programmed
cell death and describes the normal and controlled growth phemenon of generally
seen in eukaryotic cells which involves cellular death. It is a genetically
controlled process that may produce neoplasm in the event of an alteration or
defect in the process (Berges et al., 1995). Among the several proteins
involved in the cellular apoptotic pathway, the Bcl-2 family of proteins have
emerged as vital regulators involved in the intrinsic pathway
(mitochondria-mediated apoptosis). The proteins of this group function either
as apoptosis-promoting proteins such as Bax and Bak, or as apoptosis-inhibiting
proteins such as Bcl-2 and Bcl-xL (Isaacs and Coffey, 1989; Chao and Korsmeyer,
1998).
Studies have shown an excessive proliferation
of the stromal (37-fold) and epithelial (9- fold) cells in some BPH cases
(Claus et al., 1993). The likely imbalance of molecular mechanisms of
proliferation and apoptosis underpins the development of BPH and related
cancers. Too little apoptosis in malignant cells is a major culprit for the
development of cancers (Wong, 2011). It is crucial that research is geared at
evaluating a cell-population’s response to chemotherapeutic agents and growth
factors in terms of cell viability, proliferation and apoptosis in conducting
BPH and related cancer drug development studies (Kyprianou et al., 1998; Duan et
al., 2012). Due to the widely held perception that phytotherapeutic agents are
more cost-effective, safer and have fewer side effects compared with conventional
therapy for the management
of ailments including BPH and its related cancers, there is a growing interest
in their development (Wilt et al., 1998; Thompson, 2003).
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