Plant material and extraction procedures
The plant species (Annona muricata) collected from Ipoh, Malaysia, was authenticated by Dr. Yong Kien Thai, an ethnobotanist from the department of Biological Sciences, University of Malaya. The voucher specimen number for this plant is KLU47978. The air-dried leaves of A. muricata (1 kg) were cut into fine pieces using a mill grinder and soaked in n-hexane (1500 mL, three times) in conical flasks for four days at room temperature (25–27°C). The n-hexane extract was filtered and the residues were sequentially re-extracted with ethyl acetate (1500 ml, three times) and methanol (1500 ml, three times) using the same method. The resultant filtrate was concentrated to dryness by a Buchi R110 Rotavapor (Buchi Labortechnik AG, Flawil, Switzerland) at 40°C and stored at – 30°C until use. The isolated extracts were dissolved in dimethyl sulfoxide (DMSO) for further experiments.
Cell culture and MTT assay
MCF-7 (human breast cancer cells), MDA-MB-231 (human breast cancer cells), A549 (human lung cancer cells), HepG2 (human hepatoma cells) and WRL-68 (human hepatic cells) cell lines were obtained from American Type Cell Collection (ATCC, Manassas, VA, USA). Cells were cultured in RPMI-1640 medium (Sigma, St. Louis, MO, USA) supplemented with 10% FBS (PAA, Pasching, Austria), 100 U/mL penicillin (PAA) and 50 μg/mL amphotericin B (PAA) at 37°C with 5% CO2. The negative control for all the assays was represented by the untreated medium containing vehicle DMSO (0.1%).
The cytotoxicity of the extracts was determined using the MTT assay as originally described by Mossman . Briefly, cells were treated with different concentrations (1.56, 3.12, 6.25, 12.5, 25, 50 and 100 μg/mL) of three isolated extracts (hexane, ethyl acetate and methanol) and curcumin (positive control) in 96-well plates and incubated for 72 h. After the incubation time, MTT dye (20 μL, 5 mg/mL, Sigma) was added to the cells for 4 h followed by incubation with DMSO for 10 min. The colorimetric assay was measured at the absorbance of 570 nm using a microplate reader (Asys UVM340, Eugendorf, Austria). The antiproliferative potential of the extracts was expressed as IC50 values. As an ethyl acetate extract of the leaves (AMEAE) demonstrated the lowest IC50 value against lung cancer A549 cells, we used only AMEAE to continue this study against A549 cells.
LDH release assay
To confirm the cytotoxicity of AMEAE, we carried out lactate dehydrogenase (LDH) release assay using Pierce™ LDH Cytotoxicity Assay Kit (Thermo Scientific™, Pittsburgh, PA, USA). In brief, A549 cells were treated with AMEAE at different concentrations for 48 h. The supernatant of treated A549 cells was transferred into 96-well plate to assess the LDH activity. Triton X-100 (2%) served as a positive control was used to completely lyse the cells and release the maximum LDH. Next, the LDH reaction solution (100 μl) was added to the cells for 30 min. The red color intensity presenting the LDH activity was measured by the absorbance at 490 using a Tecan Infinite®200 Pro (Tecan, Männedorf, Switzerland) microplate reader. The level of released LDH from treated cells was expressed as a percentage of positive control.
Acridine orange/propidium iodide (AO/PI) double staining assay
Morphological changes induced by AMEAE in A549 cells were analyzed using Acridine orange/Propidium iodide (AO/PI) double staining assay. Briefly, A549 cells were seeded in 60 mm2 culture dishes followed by treatment with AMEAE (10 μg/mL) for 24, 48 and 72 h. After the incubation time, extract-untreated and treated A549 cells were harvested and washed with PBS. Then, the pellets were stained with 10 μg/mL of AO/PI (1 mg/mL). The stained cells were then observed under a BX51 UV-fluorescent microscope (Olympus, Tokyo, Japan) within 30 min.
Induction of the early and late apoptosis by AMEAE was further studied via Annexin-V/PI staining assay. Briefly, A549 cells (1 × 106) were plated in 60 mm2 culture dishes and treated with vehicle DMSO and AMEAE (10 μg/mL) for 24, 48 and 72 h. After harvest of adherent and suspension cells and washing them twice with PBS, they were re-suspended in Annexin-V binding buffer (BD Biosciences, San Jose, CA, USA) and stained with Annexin-V-FITC (BD) and PI (Sigma) according to the manufacturer’s instructions. The fluorescence intensity of A549 cells was then analyzed by flow cytometry (BD FACSCanto™ II, San Jose, CA, USA) through quadrant statistics for necrotic and apoptotic cell populations. PI was used for detection of the late apoptosis and necrosis while Annexin-V was consumed for the detection of the early and late apoptosis.
Cell cycle assay
A flow cytometry analysis was carried out to determine the cell cycle distribution in treated A549 cells with AMEAE. In brief, A549 cells (5 × 104 cells/mL) were treated with AMEAE (10 μg/mL) for 24, 48 and 72 h. After fixation with cold ethanol, cells were washed with PBS and stained with PI (50 μl, 10 mg/mL) for 1 h at 37°C. In addition, RNase A (10 mg/mL) was also used to limit the ability of the PI to bind only to DNA molecules. The stained cells were analyzed for DNA content using flow cytometer (BD FACSCanto™ II).
Reactive oxygen species (ROS) assay
The effect of AMEAE on the ROS formation in A549 cells was determined by ROS assay. Briefly, treated lung cancer cells with AMEAE at different concentrations in 96-well plates were incubated for 24 h. After the incubation time, the treated cells were stained with dihydroethidium (DEH) at 2.5 μg/mL and Hoechst 33342 (500 nM) dyes for 30 min. Then, cells were fixed with paraformaldehyde (3.5%) for 15 min and washed with PBS twice. The Cellomics ArrayScan HCS reader was used to measure the ROS generation in treated A549 cells.
To further determine the role of ROS generation in AMEAE-induced antiproliferative effect, A549 cells were treated with antioxidants prior to treatment with AMEAE and the cell viability was measured after 24 h. In brief, A549 cells in the exponential phase of growth were supplemented with antioxidants superoxide dismutase (SOD, 300 U/mL) and catalase (400 U/mL) for 1 h prior to AMEAE (20 μg/mL) treatment for 24 h. After incubation time, the cell viability analysis was carried out using a microplate reader (Asys UVM340, Eugendorf, Austria).
Multiple cytotoxicity assay
To simultaneously determine the crucial apoptotic events in A549 cells after treatment with AMEAE, we used Cellomics Multiparameter Cytotoxicity 3 Kit (Thermo Scientific™, Pittsburgh, PA, USA). Briefly, lung cancer A549 cells were seeded into 96-well plates for 24 h. The cells were treated with AMEAE at different concentrations prior to staining the cells with cell permeability and mitochondrial membrane potential (MMP) dyes. Then, cells were fixed and blocked with 1X blocking buffer according to the manufacture’s protocol. Next, primary cytochrome c antibody and secondary DyLight 649 conjugated goat antimouse IgG were added for 1 h. Nuclei of treated cells were also stained with Hoechst 33342 dye. Stained A549 cells in 96-well plates were analyzed using ArrayScan high content screening (HCS) system.
Bioluminescent assays for caspase-8, -9 and -3/7 activities
A dose-dependent study on the caspase-8, -9 and -3/7 activation was carried out using Caspase-Glo® 3/7, 8 and 9 kit (Promega, Madison, WI, USA). In brief, a total of 5 × 103 A549 cells were seeded per well in a white 96-well microplate and incubated with different concentrations of AMEAE for 24 h. Then, caspase-Glo reagent (100 μl) was added to the cells for 30 min. The induced activation of caspases was measured using a Tecan Infinite®200 Pro (Tecan, Männedorf, Switzerland) microplate reader.
To determine the protein expression of cleaved caspase-3 and -9, western blot analysis was carried out as previously described in detail . In brief, A549 cells treated with vehicle DMSO or AMEAE at different concentrations were washed with PBS and lysed in ice-cold Radio Immuno Precipitation Assay (RIPA) buffer. Cell extracts (80 μg protein) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), transferred to nitrocellulose membrane, probed with anti-β-actin, anti-cleaved caspase-9 and anti-cleaved caspase-3 (Cell Signaling Technology, Danvers, MA, USA). HRP-conjugated secondary antibodies were used followed by the detection of protein expression using the ECL plus chemiluminescence kit (Amersham Biosciences, Piscataway, NJ, USA).
Quantitative PCR analysis
The expression of the Bax and Bcl-2 in treated A549 cells was analyzed by quantitative PCR analysis. After treatment of A549 cells with the AMEAE extract at different concentrations for 24 h, Zymo Research Quick-RNA™ MiniPrep kit (Zymo Research, Freiburg, Germany) was used to isolate total RNAs according to the manufacture’s protocol. Then, High Capacity RNA-to-cDNA™ kit (Applied Biosystems, Foster City, CA, USA) was used to synthesize complementary DNAs. Quantitative PCR was carried out with TaqMan® Gene Expression Assays and TaqMan® Fast Advanced Master Mix using the Applied Biosystems StepOnePlus™ system. GAPDH was used to normalize all data. The IDs for TaqMan® Gene Expression Assays used in this study are GAPDH: Hs02758991_g1, Bcl-2: Hs00608023_m1 and Bax: Hs00180269_m1.
Measurement of NF-κB activity
The Cellomics ArrayScan HCS system was used to analyze the suppressive effect of AMEAE on the nuclear translocation of NF-κB induced by TNF-α. The experiment was carried out using Cellomics nucleus factor-κB (NF-κB) activation kit (Thermo Scientific) as previously described . In brief, A549 cells (1.0 × 104 cells/well) were treated with AMEAE at different concentrations in a 96-well plate for 3 h. The treated A549 cells were stimulated by TNF-α (1 ng/mL) for 30 min. Then, cells were fixed and stained according to the manufacturer’s protocol and analyzed using Array Scan HCS Reader and Cytoplasm to Nucleus Translocation Bioapplication software.
Data are presented as mean ± SEM of three individual experiments. Statistical analysis was performed with a one-way ANOVA analysis using the Prism statistical software package (GraphPad Software, USA). Differences were considered as being significant at *p < 0.05.