ANTIPROLIFERATIVE ACTIVITY OF AQUEOUS LEAF EXTRACT OF ANNONA MURICATA (LINN.) ON RAT PROSTATE, BPH-1 CELLS AND SOME TARGET GENES

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).

For more Molecular Medicine Projects Click here
===================================================================
Item Type: Ghanaian Project Material  |  Attribute: 148 pages  |  Chapters: 1-5
Format: MS Word  |  Price: GH50  |  Delivery: Within 30Mins.
===================================================================

Share:

No comments:

Post a Comment

Note: Only a member of this blog may post a comment.

Search for your topic here

See full list of Project Topics under your Department Here!

Featured Post

HOW TO WRITE A RESEARCH HYPOTHESIS

A hypothesis is a description of a pattern in nature or an explanation about some real-world phenomenon that can be tested through observ...

Popular Posts