550198 ICTXXX10.1177/1534735414550198Integrative Cancer TherapiesAsare et al research-article2014 Article Integrative Cancer Therapies Antiproliferative Activity of Aqueous 2015, Vol. 14(1) 65– 74© The Author(s) 2014 Reprints and permissions: Leaf Extract of Annona muricata L. on the sagepub.com/journalsPermissions.nav DOI: 10.1177/1534735414550198 Prostate, BPH-1 Cells, and Some Target ict.sagepub.com Genes George Awuku Asare, PhD1, Dan Afriyie, MSc, MPhil1, Robert A. Ngala, PhD2, Harry Abutiate, MPhil3, Derek Doku, MSc1,2, Seidu A. Mahmood, PhD1, and Habibur Rahman, PhD1,4 Abstract Background. Annona muricata L. has been reported to possess antitumor and antiproliferative properties. Not much work has been done on its effect on BPH-1 cell lines, and no in vivo studies targeting the prostate organ exist. The study determined the effect of A muricata on human BPH-1 cells and prostate organ. Methods. The MTT assay was performed on BPH-1 cells using the aqueous leaf extract of A muricata. Cells (1 × 105 per well) were challenged with 0.5, 1.0, and 1.5 mg/mL extract for 24, 48, and 72 hours. Cell proliferation and morphology were examined microscopically. BPH-1 cells (1 × 104 per well) were seeded into 6-well plates and incubated for 48 hours with 0.5, 1.0, and 1.5 mg/mL A muricata extract. Reverse transcriptase polymerase chain reaction was performed using mRNA extracted from the cells. Possible target genes, Bax and Bcl-2, were examined. Twenty F344 male rats (≈200 g) were gavaged 30 mg/mL (10 rats) and 300 mg/mL (10 rats) and fed ad libitum alongside 10 control rats. Rats were sacrificed after 60 days. The prostate, seminal vesicles, and testes were harvested for histological examination. Results. Annona muricata demonstrated antiproliferative effects with an IC of 1.36 mg/mL. Best results were obtained after 48 50 hours, with near cell extinction at 72 hours. Bax gene was upregulated, while Bcl-2 was downregulated. Normal histological architecture was observed for all testes. Seminal vesicle was significantly reduced in test groups (P < .05) and demonstrated marked atrophy with increased cellularity and the acinii, empty of secretion. Prostate of test groups were reduced with epithelial lining showing pyknotic nucleus, condensation, and marginalization of the nuclear material, characteristic of apoptosis of the glandular epithelium. Furthermore, scanty prostatic secretion with flattening of acinar epithelial lining occurred. Conclusion. Annona muricata has antiproliferative effects on BPH-1 cells and reduces prostate size, possibly through apoptosis. Keywords Annona muricata, BPH-1, prostate, proliferation, apoptosis, rats Introduction States in 2000, there were 4.5 million visits to physicians with issues relating to BPH. Globally and nationally, more Prostate cancer is the most frequent cancer in men and the and more people are turning to complementary and alterna- second highest cause of mortality by cancer for the male tive medicine for various ailments of which the use of population. Approximately 29% and 9% of leading new medicinal plants is foremost. cancer cases and deaths, respectively, in the United States Annona muricata L., commonly called soursop, is a were attributed to the prostate in 2012.1 A 1.33-fold increas- small erect evergreen tropical fruit tree plant belonging to ing trend of incidence rate between 1999 and 2002 was reported in Korean men.2 Furthermore, prostate cancer may become problematic if a less than 15-year survival is pre- 1University of Ghana, Accra, Ghana 2 dicted.3 In Ghana, 17.35% of male cancer death is attributed Kwame Nkrumah University of Science and Technology, Kumasi, Ghana3West Africa Postgraduate College of Pharmacists, Lagos, Nigeria Ghana to the prostate.4 Treatment, on the other hand, has adverse 4 5 Bangladesh Agricultural University, Mymesingh, Bangladesheffects, and in some cases unneeded, as some men do not die from their cancer and may harbor tumors that are indo- Corresponding Author: George Awuku Asare, Chemical Pathology Unit, Department of Medical lent even in the absence of therapy.3,6 Benign prostatic Laboratory Sciences, School of Allied Health Sciences, College of Health hyperplasia (BPH) affects more than 50% of men in their Sciences, University of Ghana, PO Box KB 143, Korle Bu, Accra, Ghana. 60s and as much as 90% in their 70s and 80s. In the United Email: gasare@chs.edu.gh 66 Integrative Cancer Therapies 14(1) the family Annonaceae, growing 5 to 6 meters in height. Leaves were milled and soaked by the proportion of 1 kg of The leaves of A muricata have been reported to contain sev- the milled substance soaked in 4000 mL of water for 24 eral groups of substances collectively called annonaceous hours. The mixture was then boiled for 1 hour and filtered acetogenins. Monotetrahydrofuran annonaceous aceto- through fine linen gauze. The marc was then soaked with genins, cis-corossolone (4) annocatalin (5), annonacin, another 3000 mL of water for another 24 hours and filtered. annonacinone, solamin, and corossolone have been isolated The 2 filtered solutions were then pooled and freeze-dried. from the leaves of A muricata. The first 2 isolates have sig- The yield from 1 kg of ground substance was 25.2 g. nificant cytotoxic activity in vitro against 2 human hepa- toma cell lines, Hep G(2) and 2,2,15. Compound 5 showed High-Performance Liquid Chromatography a high selectivity toward the Hep 2,2,15 cell line.7 Additionally, acetogenins 1 (annoreticuin-9-one) and 2 (cis- (HPLC) Analysis annoreticuin) isolated from A reticulata and A montana, Different batches of the extract were monitored by chro- respectively, have been reported to have cytotoxicity against matographic fingerprint. Samples were analyzed on a certain cancer cell lines. Acetogenin 1 targets the human Shimadzu HPLC system (Kyoto, Japan), Ultimate XB-C pancreatic tumor cell line (PACA-2), human prostate ade- 18column (150 × 4.6 mm, 5 µm), and the absorbance was nocarcinoma (PC-3), and human lung carcinoma (A-549), measured at 208 nm. The mobile phase solvent A was water while acetogenin 2, targets human hepatoma carcinoma cell and solvent B acetonitrile (ACE) at a flow rate of 1 mL/min line (Hep G2). The dichloromethane extract of the seeds of and an injection volume of 1 µL. The gradient run ACE– A muricata yielded annoreticuin-9-one (1), while the flesh H O was as follows: from 10%:90% to 10%:90% (0-10 of the fruit yielded cis-annoreticuin (2).8 The presence of 2minutes); from 10%:90% to 85%:15% (10-30 minutes); Annonaceous acetogenins, muricoreacin (1) and murihexo- from 85%:15% to 85%:15% (30-40 minutes). An optimum cin C (2) (mono-tetrahydrofuran acetogenins) in the leaves easily controlled and reproducible procedure of extraction of A muricata (Annonaceae) with significant cytotoxic described previously was established from the fingerprint activities targeting human prostate adenocarcinoma (PC-3) results. and pancreatic carcinoma (PACA-2) cell lines has been demonstrated.9 Leaves of A muricata in ethyl acetate showed a higher death rate to HeLa cells than the ethanol Effect of A muricata on BPH-1 Cell Viability distilled water extract. Similarly, chloroform extract appli- Cell viability assays were performed on BPH-1 cells. In cation to HeLa cells showed a higher death rate than ethyl brief, cells were seeded into 96-well plates at a density of acetate extract. The chloroform extracts appear to be a bet- 1 × 105 cells/well in 0.1 mL RPMI 1640, 10% fetal bovine ter option for cancer causing viruses.10 The aqueous extract serum (FBS) medium. Cells were treated with 0.5, 1.0, is said to contain general glycosides, saponins, and flavo- and 1.5 mg/mL extract (in phosphate-buffered saline noids.11 In an acute toxicity study (LD < 5000 mg/kg body 50 [PBS]) and incubated at 37°C for 24, 48, and 72 hours. At wt), the aqueous extract did not show any toxicity on sys- the end of treatment time for various plates, the medium temic organs.11 However, the use of an aqueous infusion of was replaced by 100 µL MTT (Sigma, St Louis, MO) per about 140 µg/cup is said to have caused neurotoxicity well and incubated for an additional 4 hours at 37°C. The related to atypical parkinsonism in Guadeloupe.12 The etha- reaction was stopped by adding 100 µL DMSO, AR grade nolic extract of the leaves of A muricata is said to have (Sigma) to each well to dissolve the purple-blue MTT hypoglycemic and antidiabetic effects.13 Furthermore, its formazan precipitate. The absorbance was read at 570 nm protective effect on lipid profile has been documented.14 on an ELISA microplate reader (BioTek, Elx800, VT). The aim of the study therefore was to investigate the The inhibition of growth was assessed as percent viability effect of A muricata on human benign prostate cells (BPH- where vehicle treated cells were considered as 100% 1) and whole prostate organ in male F344 rats. viable. Materials and Methods RNA Extraction and Reverse Transcriptase Plant Material Extraction Polymerase Chain Reaction (RT-PCR) Analysis The leaves of A muricata were collected from the outskirts BPH-1 cells were seeded into 6-well plates at a density of 1 × of the capital city Accra from July to August 2013 and 104 per well in 2 mL medium (in 10% FBS) and treated with authenticated by the national herbarium. Specimens were 0.5, 1.0, and 1.5 mg/mL plant extract (in PBS) for 48 hours. deposited with voucher number UG 00178.AM.215/13. Total RNA was isolated using TriZol reagent (Invitrogen, Leaves were hand-washed by rubbing the surface gently Carlsbad, CA). Oligo(dT)-primed RNA (1 µg) was reverse- under running water. They were later sun-dried for 3 days. transcribed using the SuperScript II transcriptase kit (RR047A, Asare et al 67 Takara, Shiga, Japan) according to the manufacturer’s instruc- Table 1. HPLC quantitative results showing area under the tions. cDNA obtained was amplified by PCR with TaqDNA curve (AUC) and peak ratios of the 8 peaks isolated. polymerase (Fermentas, Burlington, Canada). The presence Area Under Peak of possible target genes, Bax and Bcl-2, was determined using Name the Curve Ratio the obtained cDNA and glyceraldehyde-3-phosphate dehy- drogenase (GAPDH) as the internal control. The sequence of 1 1.1492 7.8 primers used for amplification were as follows: Bcl-2—for- 2 1.6811 11.4 ward 5′-GG TGGTGGAGG AACTCT TCA-3′ and reverse 3 3.9991 27.1 5′-GAGCAGCGTCT TCAGAGACA-3′; Bax—forward 4 2.7795 18.8 5 1.8501 12.5 5′-CCAAGAAGCTG AGCGAG TGT-3′ and reverse 5′-TC 6 2.298 15.6 ACGGAG GAAGTCCAG TGT-3′; GAPDH—forward 5′ 7 0.5016 3.4 TGCTGAGTATGTCGTGGAG-3′ and reverse 8 0.5138 3.5 5′-GTGTTCTGAGTGGCAGTGAT-3′ (bcl2—268 bp; bax— 248 bp; GAPDH—240 bp). The PCR reaction was performed under the following conditions: Bcl-2, denaturation at 94°C Statistical Analysis and Data Evaluation for 30 seconds, annealing at 58°C for 60 seconds, and exten- sion at 72°C for 60 seconds. For Bax, denaturation at 94°C for Statistical analysis of the data was done using Graph Pad 30 seconds, annealing at 55°C for 30 seconds, and extension Software, Version 5.0, for Windows (Graph Pad software, at 72°C for 45 seconds; for GAPDH, denaturation at 94°C for San Diego, CA). Results were expressed as mean ± SEM, n = 30 seconds, annealing at 58°C for 60 seconds, and extension 10. Significance of difference between controls and dose at 72°C for 60 seconds. The samples were analyzed by run- groups were evaluated by performing a 1-way ANOVA. Post ning 1.5% agarose gel electrophoresis and DNA bands exam- hoc analysis was performed with Bonferroni multiple com- ined using a Bio-Rad 2000 gel documentation system. parison test where ANOVA showed significant differences. P values ≤.05 were considered statistically significant. Animal Study Results The protocol adopted followed the OECD15 document on the use of laboratory animals and was approved by the ethics In Vitro Assays committee of the Noguchi Memorial Institute for Medical Research. Thirty male F344 rats were divided into 3 groups From Figure 1 and Table 1, it can be seen that the areas of of 10 rats each and housed in stainless steel cages. Group I peaks 3, 4, 6, 5, 2, 1, 8, 7 were in the ratio of 27.1:18.8:15.6 (normal control) was fed the standard diet and water. Rats in :12.5:11.4:7.8:3.5:3.4. Results from the study are therefore group II (low dose [LD]) were orally administered A muri- reproducible if the fingerprint and its ratios are the same. cata extract at a dose of 30 mg/kg body wt of A muricata, Cell morphology showed diminishing cells as the concen- while rats in groups III (high dose [HD]) were orally admin- tration of the plant extract increased from 0 mg/mL to 1.5 mg/ istered extract at 300 mg/kg body wt. Plant extract adminis- dL (Figure 2). BPH-1 cell viability using MTT assay demon- tration was repeated for 60 days. After 60 days of extract strated significant dose-dependent decrease. Cell viability administration, all animals were sacrificed and the prostate, reduced from 100% to 80.1%, 65%, and 47% as the dose seminal vesicles, and testes were harvested. increased from 0, 0.5, 1.0 to 1.5 mg/mL, respectively (Figure 3). Statistical differences of each dose (0.5, 1, and 1.5 mg/mL) compared to the control was significant (P < .05, P < .05, P < Histopathological Analysis .001, respectively). IC recorded was 1.36 mg/mL. 50 Fat- and connective tissue-free prostate, seminal vesicles, PCR results demonstrated a decrease in band intensity and testes were harvested, blotted with clean tissue, exam- for Bcl-2 gene downregulation as the concentration of A ined, and weighed to obtain organ to body weight ratios. muricata increased from 0 mg/mL to 1.5 mg/mL. Thereafter, prostate and seminal vesicle were immediately Conversely, there was an upregulation of Bax in a dose- fixed in 10% buffered formaldehyde solution. Testes were dependent manner. The internal control GAPDH gave fixed in Bouin’s solution. Three-micrometer sectioned strong positive bands (see Figure 4). slides of prostate were hematoxylin and eosin (H&E) stained and evaluated microscopically for histological Effect of A muricata on Prostate Organ and changes using Olympus BX 51TF (Olympus Corporation, Seminal Vesicle Tokyo, Japan) light microscope connected to a digital cam- era. Images of selected sections were captured at 100×, The extract reduced the size of the prostate in the test groups. 200×, and 400× magnifications. Prostatic index (PI) for the Control, LD, and HD groups were 68 Integrative Cancer Therapies 14(1) mAU(x100) 245nm,4nm (1.00) 3 1.00 1 2 4 6 7 8 0.75 5 0.50 0.25 0.00 -0.25 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0min Figure 1. A Shimadzu HPLC system with DAD detector and an Ultimate XB-C column was used. 18 Chromatographic profile of the aqueous extract of A muricata leaf is seen in the figure. To control the quality of A muricata extract, the peak areas of 1 to 8 were in the optimum ratio of 7.8, 11.4, 27.1, 18.8, 12.5, 15.6, 3.4, 3.5, respectively. The mobile phase consisted of solvent A (water) and solvent B (acetonitrile [ACE]). The gradient ACE–H O procedure used was as follows: 10%:90% to 10%:90% (0-10 minutes); from 10%:90% to 85%:15% (10-30 2 minutes); from 85%:15% to 85%:15% (30-40 minutes). The flow rate was 1.0 mL/min and the column temperature was set at 30°C. Figure 2. BPH-1 cell proliferation at various concentrations. Figure 3. BPH-1 cell viability test after 48-hour treatment with A reduction in cell density as concentration increases from 0 to 1.5 A muricata. mg/mL is seen (A to D). The morphology of the cell remains the A dose-dependent decrease in cell proliferation is seen as the dose same. Thus, the cell growth inhibition seen is a direct effect of the increases from 0 to 1.5 mg/mL. At the highest dose of 1.5 mg/mL, antiproliferative effect of A muricata. BPH-1 cell viability was almost 50%. Similar patterns were obtained for MTT viability test at 24 and 72 hours. Differences of dose 0.5 and 1.0 mg/mL compared to the control were significant (P < .05 and P < .05, 0.178 ± 0.086, 0.152 ± 0.075, and 0.157 ± 0.061, respectively respectively). Greater significance was observed between control and (Figure 5). Although there was a slight reduction in PI 1.5 mg/mL (P < .001). Asare et al 69 0.60 0.50 0.40 0.30 † * 0.20 0.10 0.00 C LD HD Various groups Figure 4. Shows a downregulation of BCl-2 and upregulation of Bax mRNA extracted from BPH-1 cells after 48 hours of Figure 6. “Seminal vesicle index” (SVI) was significantly treatment with A muricata at doses of 0 mg/mL (lane 1), 0.5 mg/ reduced from 0.44 ± 0.07 (Control group) to 0.18 ± 0.04 mL (lane 2), 1.0 mg/mL (lane 3), and 1.5 mg/mL (lane 4). GAPDH (LD) and 0.23 ± 0.05 (HD). Thus, A muricata caused about was used as a positive control. 50% relative reduction in the seminal vesicle size. Differences between control and LD as well as control and HD were statistically significant (*P = .004 and †P = .009, respectively). 0.30 0.25 0.20 0.15 0.10 0.05 0.00 C LD HD Various groups Figure 5. The figure demonstrates the reduction in prostatic index (PI) at day 60 of A muricata administration. There was a reduction in PI both with the LD (30 mg/kg body wt.) and HD (300 mg/kg body wt.). However, decreases were not statistically significant. Figure 7A. Section through the seminal vesicle of a rat fed on control rat chow for 60 days. Note normal histology, with differences they were not statistically significant. The “seminal presence of pseudostratified epithelium of low cylindrical cells vesicle index” (wet wt. of seminal vesicle/total body wt. × 100) that were identified in the base line. H&E, 100×. for the control, LD, and HD were 0.437 ± 0.069, 0.184 ± 0.041, and 0.227 ± 0.052, respectively. Statistical differences between eosinophilic substances were found in the acinii. The central control and LD, and control and HD were significant (P = .004 lumen of the gland showed occasional pyknotic nuclei as and .009, respectively; Figure 6). seen in the lining epithelium, characteristic of apoptosis. Nonetheless, rats from either low- and/or high-dose groups Microscopic Changes showed no evidence of inflammatory changes in the seminal vesicle (Figure 7D and E). Microscopically, the acinii of the seminal vesicle of the con- The prostate of rats fed on controlled chow showed nor- trol rats had the normal structure where nuclei are basal and mal histological structures (Figure 8A and B). The alveoli the cytoplasm appeared eosinophic (Figure 7A). Many of showed tall columnar epithelial cells with an apparent high the cells showed vacuolation in the cytoplasm, which is the ratio of cytoplasm to nucleus. However, representative sec- indication of maturity. However, both low- and high-dose tions of tissues of prostate obtained from rats that received levels of leaf extract resulted in atrophy and loss of secretion low dose of leaf extract showed apoptosis in the epithelium in the seminal vesicle (Figure 7B and C). The nuclei of the of the glandular acinii (Figure 8C and D). They include dis- acinar cells appeared to be smaller, and structureless crete condensation of the chromatin, to sharply delineated Prostatic index (PI) Seminal vesicle index (SVI) 70 Integrative Cancer Therapies 14(1) Figure 7D. Section through seminal vesicle of a rat fed 30 Figure 7B. Shows thickening of the basal epithelium of mg of leaf extract for 60 days (low-dose group). Note fluid connective tissue and reduction of secretion in the acinii. separates the epithelium from the submucosa (arrow). The Representative section of seminal vesicle from a rat fed 30 mg of nuclei are densely colored and packed. H&E, 400×. aqueous leaf extract of A muricata per day for 60 days (low-dose group). H&E, 100×. Figure 7C. Section through the seminal vesicle of a rat from Figure 7E. Section through the seminal vesicle of a rat fed 300 high-dose group for a period of 60 days. Marked atrophy of the mg of leaf extract (high dose) for 60 days. Note epithelial lining seminal tissues were noticed. Note increased cellularity and the showed pyknotic nucleus (arrow). H&E, 400×. acinii are empty of secretion. H&E, 100×. acinar lumens. The lumens were empty and there was flat- granular masses along the nuclear envelope, shrinking of tening of the internal lining of the acinar lumen. This led to the cells, twisting of the cellular and nuclear outlines, and an apparent decrease in the average cell number per unit fragmentation of the nucleus. The affected cell disintegrated area, and this decreased the thickness of the prostate epithe- into membrane-bound apoptotic bodies that remained as a lium. Treatment with high dose of leaf extract for 60 days ghost space along with its neighboring cells. Furthermore, caused reduction of the stroma, acini size, and shrinking of the cell membrane and the membrane encasing the apop- the epithelium, but it appeared flat with a decrease in the totic fragments retained their integrity. However, there was thickness of the fibromuscular layer (Figure 8E and F). In no associated inflammation in the apoptotic areas. Rats that comparison with the control, representative sections of tes- received low dose of the leaf extract showed marked reduc- tes examined from rats fed on both low and high levels of tion in cytoplasm and secretory activity of the acinii. leaf extracts showed active spermatogenesis (not shown in Compared to those of control prostate glands, represen- the figure). Representative sections of tissues showed nor- tative sections of prostrate obtained from the high-dose mal histological structures of the testes, expressed seminif- group showed dramatic decrease in prostatic fluid in the erous tubules containing different kinds of germ cells: Asare et al 71 Figure 8A. Normal glandular structure of the prostate from Figure 8C. Section through prostate of rat that received 300 rats fed on control rat cow. Note conspicuous microvilli. H&E, mg of aqueous leaf extract of A muricata per day for a period 200×. of 60 days (high-dose group). Note scanty prostatic secretion with flattening of the acinar epithelial lining. There is a significant reduction in the epithelial thickness. H&E, 200×. Figure 8D. Section through prostate from a rat fed on control Figure 8B. Section through prostate from a rat fed 30 mg rat chow. Note conspicuous microvilli projection with an of leaf extract/day (low-dose prostate) for 60 days. Note apparent high ratio of cytoplasm to nucleus. H&E, 400×. condensation of nuclear material (apoptosis) of the glandular epithelium. H&E, 200×. powder extracted with n-butanol yielded 5.4 g of the crude extract.17 It appears the yield is higher with polar solvents spermatogonia, spermatocytes, spermatids, spermatozoa, among other factors. and somatic Sertoli cells. The interstitial tissues found The leaf, stem, bark, and seeds of A muricata contain between seminiferous tubules showed interstitial cells and varying amounts of biologically active chemicals called Leydig cells. There was no evidence of adverse effects of Annonaceous acetogenins.18,19 So far 14 different tested any kind including degenerative, inflammatory, and/or atro- acetogenins have demonstrated ATP-blocking properties phic alterations in the testicular tissues. and were found to be more potent against multidrug resis- tant cancer cells.20 Discussion Profiling of medicinal plant extract is important and crit-ical in order to establish the authenticity and quality of the In this study, a yield of 2.52% (w/w) was obtained. A simi- extract. The process has been used in many studies in order lar yield of 2.62% of the aqueous extract of A muricata was to ensure that results produced from such extracts are a true obtained by Adewole and Ojewole.16 However, 50 g of leaf reflection of the said plant.21 HPLC fingerprinting of the 72 Integrative Cancer Therapies 14(1) breast carcinoma cell line (MCF-7), the IC was 4.34 µg/ 50 mL for the ethanol extract, whereas colon carcinoma cell line (CACO) displayed IC50 values 2.82 µg/mL, and liver carcinoma cell line (HEPG2) showed values of 3.43 µg/ mL.25 Acetogenins from A muricata have been reported to exhibit cytotoxic activities against the human pancreatic tumor cell line (PACA-2), human lung carcinoma, and human prostate adenocarcinoma (PC-3) (A-549).8 In this study, proliferation of benign prostatic hyperplasia cell line (BPH-1) treated with A muricata showed a reduction in the number of cells in a dose-dependent manner that was not due to direct cytotoxicity but growth inhibition (0.0 mg/mL > 0.5 mg/mL > 1.0 mg/mL > 1.5 mg/mL), indicating that the aqueous plant extract was capable of exerting inhibition of human benign prostate cell proliferation and, possibly, its Figure 8E. Prostate section from a rat fed 30 mg of aqueous tumor. leaf extract of A muricata per day for a period of 60 days (low- Other aqueous plant extracts such as Artemisia vulgaris dose group). Note condensation and marginalization of the inflorescence have been reported to exert an inhibitory nuclear material. H&E, 400×. effect on cell growth and colony formation of prostate can- cer PC-3 cells. A vulgaris demonstrated 8% to 65% inhibi- tion at similar doses for 24 hours. Furthermore, an increased cell growth inhibition was observed after incubation of these cells from 24 to 72 hours at various doses.26 Such dose-dependent increases with corresponding morphologi- cal changes were also observed in this study. Near cell extinction was observed at the highest dose of 1.5 mg/mL after 72 hours Some studies on human breast carcinoma cells (MDA- MB-435S) and human immortalized keratinocyte cells (HaCaT) have confirmed the presence of therapeutically active antineoplastic compounds in the n-butanolic leaf extract of A muricata. Isolation of the active metabolites from the extract is a prospect17 as they could possibly sup- press proliferation of prostate cancer cells as observed in Figure 8F. Microphotograph of prostate from a rat that BPH-cells in this study. The antitumor effects of A muricata received 300 mg aqueous leaf extract of A muricata per day for leaves may be due to the presence of acetogenins. Previous a period of 60 days (high-dose group) showing pyknotic nuclei studies revealed that most of the acetogenins act as a DNA and decreased secretion in acinii with flattening of the epithelial topoisomerase I toxin, causing cancer cell arrest at the G1 linings. H&E, 400×. phase and inducing apoptotic cell death in a Bax- and cas- pase-3-related pathway. Furthermore, the inhibition of the aqueous extract of leaf revealed 8 peaks with Rf values in enzyme NADH-ubiquinone oxidoreductase (complex I) in the range of 0.1 to 0.3 under conditions previously described. mitochondria was suggested.27-29 This was reevaluated anytime a new batch of extract was Imbalance of molecular mechanisms of proliferation and received. The practice was adopted for correct identifica- apoptosis has been found to be associated with the develop- tion of the plant, as a marker of the active phytochemicals ment of BPH and its cancer, with evidence of reduction of and also a reliable indicator of genetic variability in plant apoptosis as a major underpinning factor. Several studies populations.22 Similar practices were done for A squamosa have revealed that there is upregulation of pro-apoptotic L.23 including DNA fingerprinting of 4 Annona proteins Bax and Bak and downregulation of anti-apoptotic species.24,25 protein Bcl in cell lines undergoing apoptosis. In this study, 2 The antiproliferative potential of A muricata on BPH-1 administration of the aqueous leaf extract of A muricata cells was seen in a dose-dependent manner. Cell viability induced Bax upregulation and bcl-2 downregulation in a decreased as the dose increased from 0.5 mg/mL to 1.5 mg/ dose-dependent manner in BPH-1 cells using RT-PCR. mL. The effect of A muricata on various cell lines have The histological changes observed in the prostate of rats been reported before from hydrodistilled oils. Regarding given 30 and 300 mg of leaf extract of A muricata appear to Asare et al 73 agree with the findings of Olamide et al30 and Dhanotiya as pathologic substrates of the disease, thereby arresting the et al.31 These authors used methanolic and/or ether extracts disease, reducing the prostate volume, and improving of Citrullus lantatus and C colocynthis Schrad, respectively. symptoms.41 The significant decrease in SVI in the treat- Cell apoptosis from prostate and seminal vesicle do not ment group therefore implies a possible reduction in DHT appear to have been described for rats feeding plant extracts. and the shrinkage of the prostate as seen in the reduced PI However, the present experiment showed apoptosis in the of the treated groups. glandular epithelium in both prostate and seminal vesicles. The evidence presented here on the aqueous leaf extract It would seem that the onset of apoptotic effect in the semi- of A muricata and its possible anti-BPH properties provides nal vesicle may not necessarily correlate with the appear- the basis for further studies using BPH animal models. ance of prostatic cell apoptosis, although the nature of induction of the effect may well be changed in the latter Declaration of Conflicting Interests organ, a matter that needs careful investigation. In addition, The author(s) declared no potential conflicts of interest with respect the absence of ill effects toward spermatogenesis in the rats to the research, authorship, and/or publication of this article. so gavaged shows that A muricata would have an advantage over other plant extracts so far used to prevent prostate Funding hyperplasia.32 The changes in the seminal vesicle showed accumula- The author(s) received no financial support for the research, tion of fluid in the submucous layer. The authors regard authorship, and/or publication of this article. such changes to be due to different cause or causes. In the case of the seminal vesicle, the immediate cause of apopto- References sis observed is likely due to accumulation of fluid deep into 1. American Cancer Society. Cancer Facts & Figures. Atlanta, the submucous layer and thus isolated epithelial cells must GA: American Cancer Society;2012. have been deprived of nutrients. This effect has the resem- 2. Won YJ, Sung S, Lee JS. Nationwide cancer incidence in blance to those produced in the vas deferens of mouse by Korea, 2003-2005. Cancer Res Treat. 2009;41:122-131. the administration of oestrogen.33 Although, in the present 3. Johansson JE, Andren O, Andersson SO, et al. Natural history of study, the androgen level in the rats were not measured, it is early, localized prostate cancer. JAMA. 2004;291:2713-2719. 4. Wiredu EK, Armah HB. 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