The concentration-related inhibitory effects of MGN (a) and DCT (b) on the viability of human GC cells incubated with selected concentrations of these compounds for 72 h. Results are expressed as the mean ± the standard error of the mean (SEM) of 24 samples (n = 24) from at least three independent experiments. Statistical differences were analyzed with Student’s t-test (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 vs. control).

The concentration-dependent inhibitory effects of MGN on the proliferation of human GC cells incubated with selected concentrations of this compound (2–75 μg/mL) for 72 h. Results are expressed as the mean ± SEM of 24 samples from at least three independent experiments. Statistical differences were analyzed using Student’s t-test (** p < 0.01; *** p < 0.001; **** p < 0.0001 vs. control).

Representative flow cytometry dot plot graphs of ACC-201 (a), AGS (b), MKN-74 (c) and NCI-N87 (d) GC cell lines after the treatment with a medium (ctr) and MGN for 72 h. Regions R2 and R3 included apoptotic cells with active caspase-3. All results are expressed as mean ± SEM of three independent experiments. Statistical differences were analyzed using Student’s t-test (* p < 0.05; *** p < 0.001; **** p < 0.0001 vs. control).

Representative flow cytometry dot plot graphs of ACC-201 (a), AGS (b), MKN-74 (c) and NCI-N87 (d) GC cell lines after the treatment with a medium (ctr) and MGN for 72 h. Regions R2 and R3 included apoptotic cells with active caspase-3. All results are expressed as mean ± SEM of three independent experiments. Statistical differences were analyzed using Student’s t-test (* p < 0.05; *** p < 0.001; **** p < 0.0001 vs. control).

The number of cleaved caspase-3-positive ACC-201, AGS, MKN-74 and NCI-N87 GC cell lines after the treatment with a medium (ctr) and MGN for 72 h. All results are expressed as mean ± SEM of three independent experiments. Statistical differences were analyzed using Student’s t-test (* p < 0.05; *** p < 0.001; **** p < 0.0001 vs. control).

Representative cell cycle progression of the human GC cell lines following MGN treatments for 72 h using a flow cytometer. Representative histograms of PI-stained nuclear DNA content of each cell cycle phase vs. cell counts in untreated control and treated ACC-201 (a), AGS (b), MKN-74 (c) and NCI-N87 (d) cells, respectively. M1, M2, M3 and M4 in each histogram denote the number of cells in sub-G1, G1, S and G2/M phase, respectively. All results were expressed as means ± SEM of three independent experiments. Statistical differences were analyzed using Student’s t-test (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 vs. control).

The anti-proliferative effect for the mixture of DCT and MGN administered in combination in ACC-201 (a), AGS (b), MKN-74 (c) and NCI-N87 (d) cell lines. Cell viability for the mixture of DCT and MGN administered for 72 h at the fixed ratio of 1:1 was measured by the MTT assay with various increasing concentrations of both active agents. All GC cell lines were exposed to DCT and MGN mixture treatment using different ratios of their IC₅₀ values. Data are expressed as mean ± SEM. All results were analyzed using a one-way ANOVA test followed by Dunnett’s multiple comparisons test. Statistical differences were considered relevant at **** p < 0.0001 vs. the control group.

Concentration–effect lines for MGN and DCT administered alone and in combination for 72 h at the fixed-ratio of 1:1, illustrating the anti-proliferative effects of the drugs and their mixture in ACC-201 (a), AGS (b), MKN-74 (c) and NCI-N87 (d) cell lines, measured in vitro through the use of the MTT assay. The dashed line on each graph represents in approx. the IC₅₀ values of the studied drugs administered either alone or in combination.

Isobolograms illustrating additive interactions between MGN and DCT with respect to their anti-proliferative effects on ACC-201 (a), AGS (b), MKN-74 (c) and NCI-N87 (d) cell lines measured in vitro using the MTT assay for 72 h. The IC₅₀ ± SEM for MGN and DCT are plotted on the Y- and X-axes, respectively. The points A′ and A″ depict the theoretically calculated IC_50add values (±SEM) for both lower and upper isoboles of additivity. The point M on each graph represents the experimentally derived IC_50mix value (±SEM) for the mixture, which produced a 50% anti-proliferative effect (50% isobole) in the tested cell lines.

(a) Amount of MGN (concentrations: 100, 175, 250, 500 or 750 µg/mL) absorbed by zebrafish larvae after 95 h of exposure (n = 3 samples per group, n = 50 larvae per sample). Effect of MGN (concentrations: 100, 175, 250 or 500 µg/mL) on: (b) swim bladder inflation, (c) yolk sac necrosis, (d) body axis and (e) touch response at 96 hpf (n = 27–31 per group) (* p < 0.05, *** p < 0.001 vs. control group; Fisher’s exact test). (f) Effect of MGN (concentrations: 100, 175 or 250 µg/mL) on locomotor activity of larvae after 95 h long incubation (one-way ANOVA). Ctrl: control; MGN: magnoflorine.

Representative photographs of larvae incubated in MGN (250 or 500 µg/mL). MGN:magnoflorine. Scale bar: 1 mm.