In vitro antitumor capacity of extracts obtained from the plants Plukenetia volubilis (Sacha inchi) and Moringa oleifera in gastric cancer

Plant material, extracts, tumor line

Dehydrated leaves (1.5 kg) of both plants and Plukenetia seeds were obtained from hydroponic cultures (Sasha Colombia SAS, Piedecuesta, Colombia) and processed in the Chromatography and Mass Spectrometry Laboratory, CROM-MASS- UIS (Bucaramanga, Colombia) for subsequent extraction of freeze-dried extracts by roto evaporation using petroleum ether, cyclohexane, and ethanol as solvents. The resulting products were stored at 4°C for later use. Dimethyl sulfoxide (DMSO) 0.2% (Scharlau Química, SU01590250) was used as a solubilization vehicle. Vegetable oil from P. volubilis was obtained by cold pressing the seeds. P. volubilis seed oil was solubilized in 1.25% ethanol and RMPI (Sciencell, 09521)

Cell line and culture

The non-metastatic gastric cancer tumor line (ATCC CRL-1739) was maintained in RPMI (Sciencell, 09521) and supplemented with 10% SFB (BIOWEST, S181B-500) and a cocktail of antibiotics and antifungal (10. 000 units/mL penicillin, 10,000 μg/mL streptomycin and 25 μg/mL amphotericin B) (Sciencell, 0533)) and incubated at 37°C in 5% CO₂ atmosphere until the sufficient confluence of cells was achieved.

Cell cytotoxicity

The effect of the extracts of M. oleifera and P. volubilis, as well as the P. volubilis oil were evaluated according to the viability of AGS cells after treatment by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Selvakumaran, Pisarcik, Bao, Yeung, & Hamilton, 2003), which is a colorimetric assay that assesses the cellular metabolic activity of NADPH-dependent mitochondrial oxidoreductase enzymes, which can reduce tetrazolium salts (yellow) to formazan (purple). Briefly, cells were seeded in 96-well plates in quadruplicate at 20,000 cells/well density, reaching the optimal population after 48 hours. The cells were treated with six different concentrations of each plant extract and P. volubilis oil for 72 hours. The concentrations tested for the extracts were 6.25, 12.5, 25, 50, 100, and 200 μg/mL, while the oil was tested at concentrations of 1.6, 6.3, 25, and 100% v/v. Once the treatment was completed, the MTT test was performed according to the instructions of the commercial company (MTT Assay Kit Cell Proliferation, ABCAM, ab211091). Briefly, 50 μL of serum-free media and 50 μL of MTT reagent were added to each well and the cells were incubated at 37°C for 3 hours. After incubation the MTT reagent-supplemented media was removed and 150 μL of MTT solvent were added to each well. The absorbance was determined at 590 nm in a multimodal plate reader (Varioskan Flash, Thermo Fisher Scientific, USA). Doxorubicin (Merck, 1225703), cisplatin (Seven Pharma M000629), and miltefosine (Abcam, ab143837) were positive controls for cytotoxicity. The assays were performed in three independent experiments with two replicates per assay. Values were normalized according to the untreated control. Results are presented as the Cytotoxic Concentration 50 (CC50) of the treatments for AGS cells (CC₅₀), which was determined by sigmoidal regression using Msxlfit software (GO Business Solution, Guildford, UK). Two experiments were performed, each treatment in triplicate.

The effect of the extracts on AGS cell viability/death is expressed as the percentage of viability using the following formula:

Cell proliferation

The proliferation of AGS cells cultured with the extracts of P. volubilis and M. oleifera leaves, and P. volubilis oil was determined using the CellTiter-Blue^® kit (Promega, G8080). An inoculum of 5,000 cells/mL per well grew for 24 hours to allow cell adhesion. Cells were then exposed to 100, 50, 25, and 10 μg/mL concentrations of extracts and oil, and serial readings were taken every 48 hours until 96 hours post-treatment. Test compounds and controls were added to get a final volume of 100μl in each well. 20μl/well of CellTiter-Blue^® Reagent were added after the desired test exposure. After 4 hours of incubation the fluorescence was quantified in a Varioskan™ LUX microplate reader (Thermo Scientific™) at excitation/emission wavelength 460/590 nm, and assays were performed in triplicate.

Cell cycle evaluation

AGS cells were seeded in 24-well plates at 1×10⁵ cells/well density for cell cycle analysis and cultured overnight. Each extract/oil/control was added at the concentration equivalent to the respective CC₅₀. After eight hours of incubation at 37 °C, 5% CO₂, cells were mechanically detached with a syringe plunger, and cells were collected and fixed with 95% ethanol and stored at -20°C overnight. The cells were washed with cold PBS and incubated in 400 μL of a solution containing PI (propidium iodide) (ThermoFisher, BMS500PI) at 50μg/mL, RNase A (ThermoFisher R1253) (100 μg/mL), EDTA (Ethylenediaminetetraacetic acid) solution (Sigma-Aldrich E8008) (0.5 mM), and Triton X-100 (0.2%) (Sigma-Aldrich, T9284) for 30 min at 37 °C. PI fluorescence of the cell suspension was analyzed for cellular DNA fragmentation on an LSR Fortessa™ cytometer (Becton Dickinson BD Biosciences, USA). Data were obtained using FlowJo 7.6.2 data analysis software (FlowJo, USA). Hypodiploid (sub-G1 phase) cells were used as a marker for DNA fragmentation (apoptotic cells). The sub-G1 phase population was subtracted from the total number of events, and cell cycle analysis was performed by Dean Jett Fox analysis (RMS<10).

Assessment of oxidative stress

ROS production in AGS cells treated or not with P. volubilis seed oil was measured using H2DCFDA (2′,7′dihydrofluorescein) (ThermoFisher, D399) in AGS cells. The experiment was performed in 24-well plates using 2 × 10⁵ cells per well; treatments were administered at CC₅₀, and PHA (phytohemagglutinin) (Sigma-Aldrich, L8902) at 20 μg/mL was used as a control for ROS production. Kinetics was performed at 12, 24, and 48 h post-treatment; after the incubation time, the medium was removed, and 400 μL of H2DCFDA solution at 5 μM was added, incubating for one hour at 37 °C; cells were then washed and resuspended in 250 μL of PBS. After this, the cells were mechanically detached and transferred to a 96-well plate and finally read by Cytomics FC 85 500MPL flow cytometry, Brea, CA, at 488 nm excitation and 525 nm emission using argon laser and counting 10.000 events.

On the other hand, ON production in AGS cells treated or not with P. volubilis seed oil was performed in 24-well plates with a cell density of 2 × 10⁵ cells per well; the treatments were subsequently administered in 10% SFB supplemented RPMI-1640 medium at CC₅₀; PMA (Phorbol 12-myristate 13-acetate) (Merck, 16561-29-8) at 1 μg/ml was used as a control for ON production, and readings were taken at 24, 48 and 72 h post-treatment. After the end of the incubation period, the medium was removed, and 400 μL of DAF-FM diacetate (4-amino-5-methylamino-2 ′,7′-difluorofluorescein diacetate) probe (ThermoFisher, D23844) was added at 5μM in RPMI-1640 without phenol red, this was incubated for one hour at 37°C. After the time was up, the cells were washed and resuspended in 250 μL of PBS and detached with a syringe plunger. Finally, the contents were transferred to 96-well plates and read by Cytomics FC 500MPL flow cytometry, Brea, CA, at 488 nm excitation and 525 nm emission using an argon laser and counting 10,000 events. The number of positive cells was determined.

Caspase activity

The activity of caspases as apoptotic markers was determined using the commercial Caspase 3, Caspase 8, and Caspase 9 Multiplex Activity Assay Kit (Abcam, ab219915). Briefly, 20,000 cells were seeded in 96-well plates for 24 hours until adhesion was achieved. Cells were treated with the CC₅₀ extracts and oil and incubated at 37 °C, 5% CO₂, and 95% of humidity. The cells were treated for 24, 48 and 72 hours and at the end of the treatment time, 100 ul of previously prepared Caspase assay loading solution was added to each well directly to the cell plate without removing culture media/treatment. Subsequently, the plates were incubated for one hour at room temperature protected from light and after this time the caspases activity was monitored. Fluorescence was measured at excitation/emission wavelengths of 535/620 nm (Caspase 3), 490/525 nm (Caspase 8), and 370/450 nm (Caspase 9).

Annexin V activity

Death mechanisms were analyzed through the performance of the commercial Real-Time Apoptosis and Necrosis Assay kit (Promega, JA1011). AGS cells (10,000 cells/mL) were seeded in 96-well plates for 24 hours until adherence. Cells were treated for 24, 48 and 72 hours with CC₅₀ of both extracts and oil and at the end of the treatment time, 100 ul of previously prepared 2X Detection Reagent were added to each well. Subsequent incubation was carried out in a Varioskan™ LUX microplate reader (Thermo Scientific, USA) at 37 °C, 5% CO₂, and 95% humidity for three days, with readings taken every 24 hours. Phosphatidylserine translocation was measured by detecting a luminescence signal (Beads/s, integration time 1000 ms) produced by annexin V-dependent assembly of two luciferase fragments. Membrane integrity was measured as fluorescence at excitation/emission wavelengths of 485/525 nm. DMSO 25% was used as a control for apoptotic induction.

Wound recovery assay

To examine whether treatment affects cell proliferation and migration, 250,000 cells/well were seeded in 24-well plates in 500 μl of growing medium. Once 80% confluency was reached, the monolayer was deliberately wounded vertically with a micropipette tip, the culture medium was changed, and the cells were seeded with the respective extract and oil at the respective CC₅₀. Microphotographs were taken at 0h, 24h, and 48h to assess wound closure. A straight line was drawn with an ultra-fine tip marker on the back of the wells to keep the field during image acquisition. The microscope used was the DMi1 (Leica Microsystems, Germany). The negative control corresponded to cells seeded in 10% FBS RPMI medium without any treatment.

Statistical analysis

Data are presented as mean value ± standard deviation. Cytotoxicity values are expressed as mean Cytotoxic Concentration (CC₅₀) calculated by linear regression analysis with GraphPad Prisma 8.0. The normality test was performed with the Shapiro-Wilk test. Differences in cell cycle were performed with the non-parametric Mann-Whitney U test.) Statistically significant differences were established with a p-value <0.05. The effect of treatment on proliferation, cell death, and caspase expression in AGS cells was assessed by comparing the relative fluorescence units by one-factor analysis of variance (ANOVA) after checking the assumptions of normality and homogeneity of variances. Where normality was not accepted, the non-parametric Kruskal-Wallis test was used. Analyses were performed in IBM SPSS Statistics software, version 25.0, using a 95% confidence level in all cases. Where significant differences were identified, post hoc tests were performed using the Tukey or Games-Howell test, as appropriate.

Cytotoxic effect of Plukenetia volubilis and Moringa oleifera derivative extracts in AGS tumor cells

Cells were exposed to various concentrations of the extracts and oil for 72 hours. The effect on viability was evidenced by the MTT assay. The results revealed that the extracts of both plants affect the survival of the AGS gastric cancer cell line, with CC₅₀ values ranging from 94.5 mg/mL to 158.3 μg/mL ( Figure 1). The extracts showed a CC₅₀ above 100 μg/mL for AGS cells, except for the M. oleifera cyclohexane extract, which showed a CC₅₀ of 94.3 μg/mL. Given the CC₅₀ values obtained for the extracts on AGS cells, it can be deduced that, except for the cyclohexane extract of M. oleifera, which showed high cytotoxicity, the extracts present moderate toxicity to these cells ( Figure 1). Similarly, the oil obtained from P. volubilis seed also showed cytotoxic potential on AGS cells with a reduction in cell viability of 47% ( Figure 1).

Figure 1. Effect of derivatives obtained from each of the plants under study on cell viability.

Data were normalized with respect to the untreated control (100% survival) and are shown as the mean and standard deviation of at least three independent experiments performed in quadruplicate for each dose. The cytotoxic concentration 50 (CC₅₀) of each derivative is shown.

The CH extract from M. oleifera was 1.6 times more effective than its counterpart from P. volubilis, while the extract obtained with PE was the most cytotoxic. However, at the highest dose tested, the latter induced more cell death (88%) than CH extract (72%) despite having the lowest CC₅₀. It was also observed that the cytotoxic effect of the extracts and the oil is dose-dependent, as detailed in the viability curves ( Figure 1).

Antiproliferative effect of Plukenetia volubilis and Moringa oleifera derivative extracts in AGS tumor cells

The effect on cell proliferation was determined by cell viability assay with the CellTiter-Blue^® kit (Promega). All derivatives, extracts, and oil affected the proliferation of tumor cells at concentrations below the CC₅₀, and this effect is maintained over time.

Moringa extracts had better inhibitory dynamics for the doses required and the time employed compared with P.volubilis extracts. For example, the EtOH and PE extracts of M. oleifera exhibited a significant inhibitory effect from the second day of treatment, compared to the same extracts from P. volubilis ( Figure 2a,b,d,e). Moreover, among all extracts tested, the EtOH extract of M. oleifera showed almost complete inhibition at the evaluated concentrations ( Figure 2a). In contrast, the antiproliferative effect of the cyclohexane of M. oleifera is not as evident since it was found that the lowest dose of this extract showed an innocuous effect on the AGS line ( Figure 2c). This behavior was also observed in the EtOH extract from P.volubilis where the 10 ug/ml dose also had no effect on inhibition of cell proliferation ( Figure 2d). Despite this, it is this extract that exhibits the most significant antiproliferative potential among the P. volubilis group, first, because it achieves an evident antiproliferative effect up to a dose of 50 ug/mL, and second, the inhibition was observed from the second day of treatment, events that do not occur with the other two extracts ( Figure 2d,e,f).

Figure 2. Effect of M. oleifera and P. volubilis extracts on cell proliferation.

Data were normalized with respect to the untreated control (100% survival) and are shown as the mean and standard deviation of at least two independent experiments performed in triplicate for each dose.

On the other hand, the P. volubilis oil generated a varied inhibitory effect on proliferation at all concentrations tested, especially at the highest concentration ( Figure 3); moreover, the effect was maintained until the end of the treatment.

Figure 3. Effect of P. volubilis oil on cell proliferation of the AGS line.

Data were normalized with respect to the untreated control (100% survival) and are shown as the mean and standard deviation of at least two independent experiments performed in triplicate for each dose.

Death inducing effect of Plukenetia volubilis oil in AGS tumor cells

Fluorescence microscopy revealed that cells treated with the different derivatives and N-ethyl nitrosourea showed a generally lower proportion of dead cells (red fluorescence) relative to live cells (green fluorescence), as shown in Figure 4. The viability of AGS cells treated with the extracts and oil at the respective CC₅₀ ranged from 43% to 69%, with the CH and EtOH extracts of M. oleifera affecting cell viability the most, with viability percentages close to 40%. All extracts produced a low mortality of AGS cells, with the percentages obtained being less than 10% ( Table 1). On the other hand, P. volubilis oil at a concentration of 25% showed viability percentages of 77% and mortality of 23%, being the highest mortality of all the plant derivatives evaluated ( Table 1, Figure 4). N-ethyl nitrosurea, used to induce stomach cancer in C57Bl6 mice, produced 89% viability and 9% mortality. With doxorubicin, viability was observed in 38% of cells, and mortality was 66%. These results suggest that P. volubilis oil is cytotoxic to AGS cells at concentrations of 25% v/v or higher. As expected, most cells of the untreated group show fluoresce green (live), and only a small proportion of cells show red fluoresce ( Figure 4j).

Figure 4. Effect of extracts and oil on the viability/cytotoxicity of AGS cells.

The figure shows a representative photograph of AGS cells treated with cyclohexane (a), ethanol (b), and petroleum ether (c) extracts of P. volubilis. Photographs (d), (e) and (f) correspond to AGS cells treated with cyclohexane, ethyl and petroleum ether extract of M. oleifera, respectively. Cells treated with P. volubilis oil 25% v/v (g), N-ethyl Nitrosurea 25% v/v (h), Doxorubicin 5μg/ml (I) and untreated (J): fluorescence microscopy, magnification 10×.

Effect of P. volubilis oil in the AGS cell cycle

The effect on the content of the DNA in AGS cells treated with the different extracts of M. oleifera and P. volubilis and the oil of P. volubilis was analyzed by flow cytometry with PI staining. The assays revealed statistically significant changes only in cells treated with P. volubilis oil compared to untreated cells. In cells treated with P. volubilis oil, the percentage of cells in the sub-G1 phase increased from 5.1 ± 2.2% in control cells to 28.2% ± 12.3 within 8 hours of treatment (p = 0.0027), and the percentage of cells in G1 phase decreased from 80% to 53% ± 12.8% (p = 0.0025). With leaf extracts, no statistically significant differences in arrest were observed in the sub-G1 phase cell population compared to untreated cells (p > 0.005) ( Figure 5).

Figure 5. Effect of extacts and oil on the cell cycle of AGS cells.

Data correspond to the X ± SD of the percentages of AGS cells treated for 8 hours with P. volubilis and M. oleifera leaf extracts and P. volubilis oil at the corresponding CC₅₀. Statistical test ANOVA two-way comparison multiple treatments, Dunnet statistical test p<0.0001 data analyzed in Graph Pad Prisma 80. A value of p>0.05 (NS), p<0.05 (*), p<0.001 (**), and p<0.001 (***) was considered statistically significant (ANOVA) to compare treatments after confirming the normal distribution of the data. Sub G1 cells: hypo diploid cell population (apoptosis); G1 cells: diploid cell population or in G0 - G1 phase; G2/M cells: tetraploid cell population; and S cells: cell population in synthesis phase.

Effect of M. oleifera petroleum ether and P. volubilis ethanolic extracts in the AGS cell membrane.

AGS cell death after treatment with M. oleifera and P. volubilis derivatives extracts was characterized by measuring phosphatidylserine translocation and cell membrane permeability, which occur sequentially during apoptosis. The PE extract of M. oleifera and the EtOH extract from P. volubilis induced phosphatidylserine externalization over time, with membrane permeability increasing between 48 and 72 hours of treatment ( Figure 6b, e). In contrast, the remaining extracts showed a reversal in the maintained curves over time, increasing progressively. The P. volubilis oil induced a membrane permeability greater than the phosphatidylserine translocation ( Figure 6d).

Figure 6. Assessment of apoptosis/necrosis induced by plants derivatives on AGS cells.

The figure shows the mean and standard deviation of luminescence and fluorescence of at least two independent experiments performed in sextuplicate for the corresponding CC₅₀ for each extract or oil compared to untreated cells as negative control (NC). DMSO was used as a positive control of the assay (h). In panels b and e, there is a significant delay between the appearance of PS: Annexin V binding and the loss of membrane integrity, suggesting an apoptotic phenotype leading to secondary necrosis.

Effect of M. oleifera and P. volubilis derivatives in the activation of the caspases in AGS cells

To complement the study of the effector mechanisms of cell death, the enzymatic activity of some apoptotic markers, such as caspases 3, 8, and 9, was evaluated. Overall, all extracts increased the enzymatic activity of the caspases studied in treated AGS cells compared to the untreated control. This activity increased gradually in all treated groups and remained consistent for 72 hours. Both extracts and oil induced higher caspase eight activity compared to caspases 3 and 9 ( Figure 7a). The CH extract from M. oleifera and P. volubilis showed the most significant effect on caspase 8 induction, but it was the oil the derivative that induced the highest caspase 8 activity in AGS cells at 72 hours ( Figure 7b). On the contrary, caspase 9 showed the lowest activity in the different treatments, exhibiting almost identical fluorescence in all derivatives, except for the M. oleifera CH extract ( Figure 7a), which induced a discrete increase in the expression of caspase 9 compared to the other treatments.

Figure 7. Enzymatic activity of caspases 3, 8, and 9 in tumor cells treated withplants derivatives.

The figure shows the mean and standard deviation of the fluorescence of at least two independent experiments performed in sextuplicate for the corresponding CC₅₀ of each plant derivative, a) extract or b) oil. Panel c shows the caspase activity on positive (DMSO) and negative (NC, Vehicle) controls. A higher activity is observed in the group treated with the apoptosis inducer regarding the three caspases in a gradual manner over time compared to the negative control and the vehicle. MOEtOH: M.oleifera ethanol extract; PVEtOH; P. volubilis etanol extract; MOPE: M.oleifera petroleum ether extract; PVPE; P. volubilis petroleum ether extract; MOCH: M.oleifera ciclohexane extract; PVCH; P. volubilis ciclohexane extract; NC: negative control (untreated cells).

Effect of treatment of Plukenetia volubilis oil in the production of ROS and NO by AGS tumor cells

The P. volubilis oil increased the production of ROS and NO in AGS cells compared to untreated cells ( Table 2). The differences between untreated and extract-treated cells were statistically significant (p = 0.037). No statistically significant differences were observed between the stimulation of cells with PHA (used as a positive control for ROS production) or PMA (used as a positive control for ON production) and treatment with P. volubilis seed oil.

Inhibition of cell migration

The potential of the biological derivatives to inhibit the migration capacity of AGS cells was studied by analysing the closure of the wound caused in the cell monolayer over time and qualitatively. Of all the derivatives studied, the ethanolic extract at a concentration of 100 μg/ml revealed through microphotographs a difference from its untreated counterpart with respect to wound closure at the end of treatment after 48 hours ( Figure 8).

Figure 8. Inhibition of migration of AGS cells.

The figure shows a photomicrograph of the scraping at 0 (a,b), 24 (c,d), and 48 (e,f ) hours: a,b: Wound in the monolayer of AGS cells at 0 hours c: Wound area in control (untreated) cells shows no significant wound closure at 24 hours. e: At 48 hours, the wound has almost healed in control cells. d,f: Wounds in treated cells (CC₅₀ dose) healed at a slower rate, with an almost negligible decrease in the area studied; wounds in treated cells remained with few cells within the area studied at 24 and 48 hours.