Preparation of Annona muricata Crude Extract (AMCE)
Samples of Annona muricata leaves were obtained from the Annona muricata cultivars in Johor, Melaka, Negeri Sembilan, Selangor, Perak, and Perlis in the months of September to November 2014. The plant was identified and deposited with a voucher number by Science Officer Lim Chung Lu from the Forestry Division, Forest Research Institute Malaysia. Details of the sampling sites and voucher number of each sample are shown in Additional file 1: Table S1. All of the 19 samples of old mature Annona muricata leaves were air-dried for a week before being ground to a powder using a grind mill. Later, about 10 g of each powdered samples were transferred into a Schott bottle containing 200 mL of cold sterile distilled water. The samples were incubated for 3 days with frequent agitation using an orbital shaker at room temperature. The mixture was then, filtered to discard any solid material/marc. Finally, the filtrate extract was dried using the freeze dryer/ lyophilizer machine to give the end product (AMCE).
The cell lines, MCF-7, MDA-MB-231, 4 T1 and MCF-10A were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The MCF-7 and 4 T1 cells were maintained in RPMI 1640 medium while MDA-MB-231 cell was maintained in DMEM medium. Both media were supplemented with 10 % Fetal Bovine Serum (FBS) and 1 % Penicillin/Streptomycin. MCF-10A on the other hand, was maintained in DMEM-F12 medium supplemented with hydrocortisone (0.5 μg/mL), insulin (10 μg/mL), hEGF (20 ng/mL) and 10 % FBS. The cells were grown in a humidified incubator at 37 °C in the presence of 5 % CO2. The cell was passaged upon reaching 70 % confluency.
The proliferation of the cells or cell viability was assessed by the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) dye reduction as described by Zhi-Dong et al . The cytotoxic potential of the crude extract samples could be determined from this assay based on the IC50 generated. A hundred microliter of cells at a concentration of 0.8 × 105 cells/well were placed into a 96-well plate and maintained in the respective medium (RPMI/DMEM) for 24 h. The following day, Annona muricata crude extract (AMCE) was added to the wells and then, incubated for 72 h. MTT solutions (5 mg/ml) was added at a volume of 20 μL into each wells and incubated for 3 h. Later, the solutions were removed from wells and 100 μL of DMSO were added to solubilize the formazan crystals. Finally, the plate was read using an ELISA plate reader at a wavelength of 570 nm (Bio-tek Instruments, USA).
Annexin V/FITC assay
The Annexin V/FITC assay was performed using Annexin V Kit (BD Pharmigen, USA) in order to analyse the potential of B1 AMCE in causing apoptosis. The cells were seeded in a 6-well plate at a concentration of 2.4 × 105 cells/mL and incubated overnight. On the next day, the seeded cells were treated with the IC50 value of Annona muricata crude extract (AMCE) and incubated for 48 and 72 h. The cells were harvested according to the incubation time point and the resulting pellets were resuspended in the binding buffer provided. Five microliter of FITC Annexin V and 5 uL of PI were added to stain the cells suspension and allowed to stand in a dark place at room temperature for 15 min. Afterwards, the stained cells were analysed by flow cytometry machine (Becton Dickinson, USA).
Acridine Orange/ Propidium Iodide assay (AO/PI)
Cell viability/apoptosis of the 4 T1 cells was analysed based on the AO/PI dual staining of live/dead nucleated cells. The AO/PI assay was carried out according to the protocol described by Salim et al.  with a slight modification. Cells were seeded in a 6 well-plate at a concentration of 2.4 × 105 cells/mL and incubated overnight before treating with the IC50 value of Annona muricata crude extract (AMCE) the following day. The cells were incubated for another 72 h. Afterwards, the cells were harvested and the resulted pellets were resuspended in 200 μL PBS. Six microliter of the suspended cells was then stained with 4 μL AO/PI and the mixture were loaded onto a glass slide. The images were captured with a fluorescence microscope equipped with Nikon camera.
Cell cycle assay
To further examine the effects of B1 AMCE on the induction of apoptosis, the effects on the cell cycle was tested. The cell cycle assay was carried out using CycleTEST PLUS DNA Reagent Kit (BD Pharmigen, USA). The cells were seeded at a concentration of 2.4 × 105 cells/mL in a 6 well-plate and incubated overnight. The next day, the seeded cells were treated with IC50 of Annona muricata crude extract (AMCE) and incubated for 72 h. After trypsinization, cells were collected and a volume of 250 μL of solution A (trypsin buffer) was added. After 10 min of incubation at room temperature, 200 μL of solution B (trypsin inhibitor and RNase buffer) were added and the cell suspension was mixed gently. A further 10 min of incubation time at room temperature were required before a 200 μL of cold solution C (propidium iodide stain solution) were added to stain the cells. The mixture solutions were incubated for another 10 min in the dark on ice before analysed by flow cytometer machine (Becton Dickinson, USA).
This assay was attempted based on the predicament of the 4 T1 cells are able to migrate/invade with the presence of stimulants. It was conducted based on the protocol outlined by Chen . Prior to the experiment, a 70 % confluent 4 T1 cells were serum starved for 24 h before being seeded at a density of 2 × 105 cells/mL in the insert chamber coated with solidified Matrigel (BD Biosciences) for the invasion assay whereas for the migration assay, the chamber was not coated by the Matrigel basement membrane. In the lower compartment of the chamber, 2 mL of RPMI medium supplemented with 10 % FBS and the desired concentration of Annona muricata crude extract was added. The inserts were incubated in a 37 °C CO2 incubator for 24 h. The inserts were removed afterwards and the inner side of the inserts were swabbed to remove the non-/invaded cells. The outer side of the inserts bearing the migrated/invaded cells were then fixed in methanol for 30 min before being stained with 0.5 % of crystal violet. The images appeared on the membranes were later captured with an inverted microscope equipped with a camera (Nikon, Japan).
Wound healing assay
This assay was done using the method outlined by Liang et al. . A concentration of 3.5 × 105 4 T1 cells were seeded in a 6-well plate and incubated overnight. The next day, a straight wound line was drawn across the 100 % confluent attached cell layer with pipette tips. The floating cells were removed with PBS and replaced with new fresh RPMI medium. Annona muricata crude extract (AMCE) was added to the wells and images of the closure of the wound were recorded at time point 0, 3, 6, 9, 12, and 24 h using the inverted microscope equipped with a camera (Nikon, Japan).
Animal and diet
Six to eight-week-old female BALB/c mice were used for in vivo experiments and were obtained from UPM Animal Resource Unit. Mice were divided into groups and acclimatized for 7 days, fed with normal diet and water. All methods involving the experimental use of animals have been reviewed and approved by the Institutional Animal Care and Utilize of Committee of the Faculty Veterinary and Medicine, Universiti Putra Malaysia (Reference Number: UPM/IACUC/AUP/RO55/2015). All animals were fully conducted in humane and ethical care and under the regulation of the governing body concerning the animals.
Mice were separated into 3 open-cages defining their respective groups; normal, untreated, and treated where each group bearing 7 mice per cage. Mice in the untreated and treated group were induced with 1 × 105 cells/mL of 4 T1 breast cancer cells via subcutaneous (s.c) injection using a 27 gauge needle (Teruma, USA). Mice were observed on a daily basis for about 5 days until the tumor masses develop. Treatment with Annona muricata crude extract (AMCE) of 20 mg/20 g mice was given to the treated group while the other two groups were fed with distilled water. This treatment was conducted once daily for 28 days. After 28 days of treatment, the mice were euthanized and then sacrificed by cervical dislocation. Tissue samples like tumors and vital organs which include lung and spleen were harvested and directly used in downward analysis. One-half of the tumors were placed in tubes containing 10 % formalin for fixation and histological analysis while the other half were stored in tubes containing ‘RNAlater’ solution.
Hematoxylin and eosin histology staining of the tumors
The harvested tumors were fixed in 10 % formalin and were embedded in paraffin before being sliced into thin sections. Then the paraffin sections were stained with hematoxylin and eosin (H&E) and were viewed under a bright-field microscope (Nikon, Japan). The mitotic cells present were counted and compared between the groups.
Lung clonogenic assay
The metastasis of 4 T1 cells to other parts from primary tumor site was investigated by clonogenic assay. The clonogenic assay was carried out based on DuPre et al’s protocol . Lung organ was harvested from the untreated and treated group of mice under sterile condition and was chopped into smaller pieces as to avoid the clogging of pipette. Afterwards, it was placed in the tubes containing 5 mL PBS and 100 uL (2 mg/mL) of collagenase type IV for 30 min at 37 °C. The solutions were passed through a 70 mm cell strainer and recollected in a new tube before centrifuging to obtain the pellet. The cells pellets were washed with PBS twice before being resuspended in 10 ml RPMI medium supplemented with 10 % fetal bovine serum and 60 μM 6-thioguanine (Fisher, USA). The cell suspension was plated in a 6-well plate and a 1/10 serial dilution was performed to fill the other 5 wells of the same plate. The plates were incubated for 10 days in a 37 ̊C incubator equipped with 5 % CO2. Unattached cells were rinsed with PBS twice before the attached cells/colonies were fixed in methanol for 1 h and later stained with 0.5 % crystal violet for another 1 h. The wells were washed by PBS and viewed under microscope.
The effect of B1 AMCE on the level of immune cells population from spleen was investigated by this assay. The spleens from the mice of all groups were harvested in a sterile condition. They were placed into a petri dish containing PBS solution and were mashed through 70 μL cell strainer. The single cell suspension were washed twice with ice-cold PBS and followed by the centrifugation step. The splenocytes were resuspended in a 2 mL NH4Cl lysis buffer and incubated for 10 min at 4 °C. Later, the cells were washed with PBS and centrifuged until a clean yellow pellet obtained. The pellets were dissolved in PBS and CD3, CD4, CD8, AND NK1.1 dye (Abcam, USA) were added into tubes accordingly in dark condition and were shook at 150 rpm for 2 h. Afterwards, 1 mL of PBS was added and the tubes were centrifuged. The resulted pellets were dissolved with 600 uL of 1 % paraformaldehyde and stored in the dark place at 4 °C before being analysed by flow cytometer machine (BD, USA).
MDA antioxidant assay
The effect of B1 AMCE as antioxidant against lipid peroxidation in 4 T1 tumor sample was investigated on the basis of the level of malondialdehyde (MDA). Two hundred microliter of tumor sample supernatant was mixed with 800 μL of PBS, 25 μL of butylated hydroxytoluene (BHT), and 500 μL TCA. The mixture was vortexed and incubated on ice for 2 h. After centrifuging at 2000 × g for 15 min, 1 mL of supernatant was taken out and transferred into tube containing 75 μL of 0.1 M EDTA and 250 μL of 0.05 M TBA. The tube was boiled in water bath for 15 min and then, left to cool at room temperature before read by spectrophotometer at 532 nm and 600 nm wavelengths. The result obtained was compared to MDA standard curve.
Nitric oxide/Griess reagent assay
The effect of B1 AMCE on the level of nitric oxide in 4 T1 tumor was investigated using the Griess reagent assay. It was carried out using the Griess Reagent Kit for Nitrite Determination (Life Technologies, USA). Twenty microliter of Griess reagent containing equal volume of sulfanilic acid and N-1-napthylethylenediamine dihydrochloride was mixed with 150 μL of the nitrite-containing sample and 130 μL of deionized water in a microplate and incubated for 30 min at room temperature. Standard curve was also prepared by diluting the nitrite standard solution with deionized water to give a series of concentration between 1–100 μM. In place of the nitrite- containing sample, the standards were mixed with the Griess reagent and incubated in a similar manner. The absorbance of the sample and standards were read by spectrophotometer (Beckman Coultor, USA) at 548 nm wavelength before the nitrite concentrations corresponding to the standard plot could be evaluated.
The effect of B1 AMCE on the protein level affecting the angiogenesis process in 4 T1 tumor was investigated using the Raybio Mouse Angiogenesis Kit (RayBiotech, Inc.). A volume of 100 μL of 1x Blocking Buffer is added into each well of the glass chip and incubated at room temperature for 30 min. The Blocking Buffer were decanted and aspirated before 100 μL of samples were added into the wells and incubated for 2 h at RT. Later, the samples were removed and the wells were washed with Wash Buffer I for 3 times at each 2 min interval. The glass chip assembly was submerged into a container containing Wash Buffer I and shook gently for 10 min and this step was repeated with the Wash Buffer II. The wash buffer was decanted before 70 μL of 1X Biotin-conjugated Anti-cytokines were added into each wells and incubated with gentle rocking for 2 h at RT. After washing with Wash Buffer I and followed by Wash Buffer II, a 70 μL of 1X Streptavidin-Fluor were added to each well and incubated in a dark room for another 2 h in a similar manner. The washing steps were followed after removing the streptavidin-fluor from the glass chip. Later, the glass chip was removed from its tube assembly and rinsed with deionized water. A dry glass chip was sent immediately to scanning with laser scanner (Innopsys‘InnoScan) at excitation frequency of 532 nm.
All data were expressed as the means ± standard error of mean (S.E.M.). The analysis was performed with one-way analysis of variance (ANOVA) and the group means were compared by Duncan test. Values of p < 0.05 were considered as statistically significant.