Cell lines: All cell lines used in this study were obtained from The American Type Culture Collection (ATCC; Rockville, MD, USA). The Human prostate cancer cell line DU145, and lung cancer A549 cells were cultured in RPMI-1640 (Invitrogen, Grand Island, NY, USA) with 4 mM L-glutamine and no Phenol Red, adjusted to contain 10% fetal bovine serum and 100 U/mL penicillin-streptomycin. The cells were incubated at 37˚C in a 95% air, 5% CO₂ atmosphere until they approached 80% confluence. All the chemicals used in this study were of HPLC grade and were used without further purification. Assay kits used in all the analyses in this study were purchased through Fisher Scientific Inc.

The Dioscorea alata cv. (White Yam) tubers used were grown in the Parish of St Catherine in Jamaica, harvested in December 2018 then transported to UWI Biotechnology Centre, washed with tap water and sun-dried for 2 hours. The tubers were peeled, cut in pieces and dried at 50˚C to constant weight, milled to a fine powder, and stored in sample bottles at room temperature. The powdered sample (18.802 g) was sequentially extracted in a 120 cm × 500 cm Soxhlet apparatus using 500 mL of hexane, diethyl ether, acetone, methanol them water. The extraction mixture was refluxed for 24 hours at temperatures corresponding to the boiling point of the respective solvents (hexane, 70C; diethyl ether, 35C; acetone, 56C; methanol, 65C and water, 100C). Following extraction, particulate matter was removed by filtering the samples through a 0.45 μm fritted glass filter. The extracts were then purified using an SPE-C18 column (C18-E, 55 um, 70A, 8B-S001-LEG). The SPE column was washed with 10% MeOH and eluted with 25%, 75% and 100% MeOH. The samples were then evaporated to dryness using a combination of simple distillation and rotary evaporation.

Cell viability was assayed using CellTiter 96 Aqueous One Solution (Promega, Madison, Wisconsin) according to the manufacture instructions. Cells were seeded in 96 well plates at 5 × 10³ cells/well and allowed to grow for 24 hours. They were then treated with different extracts at 5 successive 2-fold dilutions, ranging from 6.5 µg/mL to 100 µg/mL. The final DMSO content within each extract concentration did not exceed 0.1% (v/v) in the growth medium. Cells were exposed at 37C for 24 and 72 hours after which cell viability and cytotoxicity were assayed using 10% (v/v) CellTiter 96^® Aqueous One Solution Reagent. After exposure to the extracts, the effect on cell viability was determined using the CellTiter 96‚ Aqueous One non-radioactive cell proliferation assay (Promega, Madison, WI) according to manufacturer’s instructions. After incubation treated cell with Methyl Thiazolyl Tetrazolium (MTT) reagent for 1hr at 37C, the absorbance at 490 nm was measured using a ELX155 800UV universal microplate reader (Bio-Tech, Inc.). The results were then analyzed using GraphPad Prism 6.

To determine the effects of apoptosis on cells after the treatment of samples under investigation, the morphology of cells was observed via acridine orange (AO)/ethidium bromide (EB) dual staining [14]. Cells (5.0 × 10⁵) were loaded in a 24 well flask and treated with the compounds of interest. After 24 hours, cells were harvested with Trypsin and resuspended in 500 uL of PBS. Cells (25 µl of cell suspension 5.0 × 10⁵ to 2.0 × 10⁶ cells/ml) were incubated with 1 µl of AO/EB solution. Each sample was mixed prior to microscopy and quantification and was evaluated immediately. The cell suspension (10 µl) was placed on a microscope slide and covered with a glass coverslip. Four fields of view were examined under a fluorescence microscope using a fluorescein filter at 40× and 60× (higher or lower magnification may be desired depending on cell type, nuclear morphology should be discernible). The number of apoptosis cells were counted, and the results were then statistically analyzed.

Caspase protocol was conducted according to manufacturer’s instructions [15] [16]. Cells were cultured in a 96-well black plate and treated with increasing concentrations of DaJa-3 sample. Each well contained 100 µL of sample and the control contained 100 µL of growth media only. To each well 100 µL of Apo-ONE® Homogeneous Caspase-3/7 Reagent was added. Plates were gently mixed using a plate shaker at 300 rmp for 30 seconds and then incubated at 37C for 4 hours. The fluorescence of each well was detected using a fluorescence microplate reader at an excitation wavelength range of 485 ± 20 nm and an emission wavelength range of 530 ± 25 nm (Promega, 2018). Results were then analyzed using GraphPad Prism 6.

Cells were seeded in T75 flasks at 1 × 10⁶ cells/flask and treated with DaJaE3 extract with four, 10-fold dilutions. A549 cells were treated with a concentration close to its IC₅₀ value. Cells were then trypsinized and harvested after 24 hours, washed with PBS and fixed with 1 mL 80% cold MeOH. Cells were centrifuged for 5 mins at 1500 rpm, supernatant was removed, and the cell pellet was suspended in 500 µL of propidium iodide (10 μg/ml) containing 200 µg/mL of RNase (Sigma, MO, USA). The cells were then incubated on ice for 1 hour. Cell cycle distribution was determined from 20,000 cells using a Becton Dickinson FACSCalibur flow cytometer [16]. The acquisition data was analyzed using FlowJo V10 software.

Immunoblotting was carried out according to [17]. Cells were grown to 80% - 85% confluency then treated with varying concentrations of DaJaE3 extract for 24 hours. After treatment, the cells were harvested using trypsin and centrifuged for 5 min at 1500 rpm. The cell pellet was resuspended in RIPA lysis buffer containing 1% protease inhibitor and the protein concentration was determined by BCA assay according to the manufacturer’s instructions. An equal amount of protein (20 µg) was separated on 10% SDS PAGE and was transferred to a PVDF membrane. The membrane was blocked with 1% nonfat milk in TBS overnight at 4˚C then incubated with the following antibodies: anti-BAX, anti CDK4, anti-GDPH and anti-cyclin B (Santa Cruz Biotechnology) for 18 hours. The membrane was probed with Horseradish peroxidase-conjugated secondary antibody. The immunogen was detected using a chemiluminescence reagent according to the manufacturer’s instructions. The immunoblot signal was detected using an AlphaInnotech Fluorochem HD 9900 (Alpha Innotech, San Leandro, CA) equipped with a Charged -coupled Device camera.

Dioscorea spp. has been studied by many groups for its nutritional and medicinal value [1] [11] [18]. However, the compounds present in D. alata that are responsible for its biological activity are not well characterized. Since D. alata contains a myriad of active and non-active compounds located in different parts of the tuber, we used solvents of different polarities to solvate the compounds and generate a fingerprint of bioactive analytes present in the yam. To evaluate the bioactive anti-cancer compounds, D. alata was extracted with Hexane (DaJa-1), Diethyl ether (DaJa-2), Acetone (DaJa-3), Ethanol (DaJa-4) then Water (DaJa-5) for 24 hours each. Each extract was cleaned-up dissolved in DMSO and analyzed for anti-cancer bioactivity using MTT In vitro toxicity assays.

Preliminary screening of D. alata extracts demonstrated that the acetone extract (DaJa-3) possesses growth inhibitory properties DU-145 cancer cells (Figure 1(a)). The order of growth inhibition of the extracts in DU-145 cell is DaJa-3 (IC50, 31.45 µg/mL) > DaJa-4 (IC50 60 µg/mL) > DaJa-1 (IC50 > 100 µg/mL) ≥ DaJa-2 (IC50 > 100 µg/mL) ≥ DaJa-5 (IC50 > 100 µg/mL). The potency of DaJa-3 extract as an anti-cancer agent, was determined in two cancer cell lines (DU145 and A549) found in the NCI-60 Human Tumor Cell Lines Screen (Figure 1(b), Figure 1(c)). The anti-proliferative activity of DaJa-3 in these cells was performed using the Five-Dose assay method at a concentration of 0, 6.25, 12.5, 25, 50 and 100 µg/mL of DaJa-3. A plot of cell viability as a function of concentration is shown in Figure 1(b), Figure 1(c). The IC₅₀ of DaJa-3 was determined by plotting the normalized viability data to a downhill dose–response curve with a variable slope. Maximal inhibition of DU145 and A549 cell growth by DaJa-3 was achieved at 90 - 100 µg/mL. A549 was more sensitive toward DaJa-3 extract with an IC₅₀ value of 22.28 µg/mL (CI 19.89 to 24.95 µg/mL), compared to that of DU145 cells with an IC₅₀ value of 31.45 µg/mL (CI 27.58 to 35.86 µg/mL) (Figure 1(b), Figure 1(c)). A time dependent effect of DaJa-3 in the lung cells (A549) was noted when compared to the prostate cancer cells (DU145). After a 72-hour exposure to a single dose of DaJa-3, A549 cells appeared to be less sensitive to the extract with an increased IC₅₀ value from 22.22 µg/mL to 32.27 µg/mL, suggesting the extract is being metabolized. In contrast, DU145 displayed no changes in sensitivity over the same 72-hour period using a single dose of DaJa-3 (Figure 1(b), Figure 1(c), compare 24 and 72 hours).

Investigations into whether the observed cell cytotoxicity induced by DaJa-3 was a result of chemically induced cellular cytotoxicity or cell-induced apoptosis was carried out. Cells fluorescently stained with acridine orange (AO) and ethidium bromide (EB) were examined following exposure to DaJa-3 to reveal signs of nuclear condensation, nuclei fragmentation, and membrane budding, which are all hallmark features of apoptosis. Examination of both DU145 and A549 cells treated with 25 µg/mL of DaJa-3 for 24 hours revealed the presence of four distinct morphologies associated with the cells in different stages of apoptosis (Figure 2(a)). There was a decrease in the number of viable cells (VC) with an intact cell membrane and organized cell structures that fluoresced bright green (Figure 2(a), red**). Cell death was also observed in both early (single arrow), and late (double arrow) apoptotic stages (Figure 2(a)) in both cell lines following exposures to DaJa-3.

The apoptotic index was determined for both A549 and DU145 by quantifying the relationship between dose of DaJa-3 and number of apoptotic cells (Figure 2(b), Figure 2(c)). The apoptotic index was calculated by averaging the number of apoptotic cells per field (EA + LA) (70 - 60 cells) then dividing by the total number of cells per field (120 - 140 cells). DU145 and A549 cells treated with DaJa-3 and harvested after a 24-hour treatment showed a dramatic increase in apoptotic cells and a decrease in total viable cell number. Figure 2(b) shows that A549 cells had a significant increase in apoptotic cells (10% in control versus 52.3% in 10 µg/ml DaJa-3, p < 0.0001) after treatment with 10 µg/mL of Daja-3 extract. As seen with the MTT cytotoxicity assay (Figure 1), there was a significant decrease in viable cells (85% at 0 µg/ml DaJa-3 versus 35% at 10 µg/mL DaJa-3) and a significant difference in necrotic cells (Figure 2 insert). The same degree of apoptosis was observed in both A549 and DU145 cells in the presence of DaJa-3 after a 24-hour exposure (Compare Figure 2(b), Figure 2(c)). Treatment of DU145 with 25 µg/mL DaJa-3 resulted in a significant increase in apoptotic cells (17.2% in control versus 57.2% in 25 µg/ml DaJa-3, p < 0.01), and a significant decrease in viable cells (85% at 0 µg/ml DaJa-3 versus 25% at 10 µg/mL). DU145 cells also showed a significant increase in necrotic cells (7.5% in control versus 35% in 25 µg/ml DaJa-3, p < 0.01). However, there was no significant increase in necrotic cells observed in A549 cells at similar concentrations.

The mechanism by which DaJa-3 induces anti-proliferative activity was next examined using flow cytometry analysis (Figure 3(a)). DaJa-3induced proliferation blockage was determined on unsynchronized DU145 and A549 cells which were treated with 25 µg/mL of extract for 12 or 24 hours. Analysis of untreated DU145 cells, showed that the G₁ phase contained 64.4% of cells, S phase 13.8% and G₂ phase 20.0%. Upon treatment of DU145 cells with DaJa-3, there was a notable decrease (22%) in G₁ cells, which was accompanied by an increase (41%) of cells in the G₂/M phase (Figure 3). When these experiments were performed on A549 cells, no concentration dependent changes were observed in the different phases of the cell cycle (Figure 3(b)). However, a high concentration of DaJa-3 (25 µg/mL extract for 24 hours) produced a sharp (26%) decrease in cells in the G₂/M phase (Figure 3(b)). A time-dependent change in cell accumulation for A549 cells in different phases of the cell cycle was also observed. Treatment of A549 for 12 hours resulted in a 21% accumulation of cells in the G1 phase with a corresponding 61% decrease of cells in the S-phase and 52% decrease in the G₂/M phase of the cell cycle. The data suggests that DaJa-3 induces G₂ phase cell cycle arrest in DU145 cells and G₁ arrest in A549 cells. These results therefore suggest that DaJa-3 affects multiple pathways in inducing its antiproliferative effects, depending on the tumor cell type.

The morphological analysis and flow cytometry assessment of DaJa-3-induced apoptosis was validated by ascertaining if the activity of the downstream apoptotic protein Caspase-3/7 had been modulated in the presence of DaJa-3. The level of active Caspase-3/7 was measured by proteolytic cleavage of rhodamine 110, from the bis-(N-CBZ-L-aspartyl-L-glutamyl-L-valyl-L-aspartic acid amide) (Z-DEVD R110) substrate (Figure 4). There was a dose-dependent increase in DaJa-3-induced caspase activity in DU145 cells and a bi-phase response in A549 cells. There was a significant difference with the Caspase 3/7 activity in DU145 cells at 50 µg/mL of DaJa-3E3, when compared to the control. In A549 cells, the greatest activity observed was at 25 µg/mL of DaJa-3, followed by a decrease in the Caspase-3/7 activity to below 25% at 50 µg/mL (Figure 4).

The expression levels of select apoptosis and cell cycle proteins in the absence and presence of DaJa-3 was also examined. Figure 5 demonstrated inhibition of CDK4 protein levels in DU145 cells following exposure to DaJa-3 for 24 hours. However, DaJa-3 showed no modulation of CDK4 expression in A549 lung cancer cells. The levels of Bax, a pro-apoptotic regulator protein, showed a significant increase by DaJa-3 in the A549 lung cancer cells but was undetected in the DU145 prostate cancer cells. Analysis of cyclin B, a G₂/M cell cycle check protein indicated that DaJa-3 decreased its expression in DU145 prostate cancer cells and induced a slight increase in A549 lung cancer cells (Figure 5).