PMC

F4 fraction induces morphological changes in tumor cells. A. A375 cells treated with ethanol 0.2% (left panel), vincristine 0.1 μg/ml (middle panel) or F4 fraction 31.2 μg/ml (right panel). Morphological changes were analyzed under invert microscope. Results represent three independent performed experiments. B. A375 (10⁴) treated with ethanol 0.2% (left panel) or F4 fraction 31.2 μg/ml (middle and right panels) for 24 h. Cells were stained with Oregon Green-phalloidin were analyzed under fluorescent microscope. Results show photos representing four independent experiments.

Petiveria alliacea F4 fraction characterization. A. Upper panel shows compound UV spectra and retention time. Lower panel shows peak area and retention time. B. F4 fraction was subjected to MALDI-TOF-MS analysis. Numbers above the peaks correspond to m/z ratios. The horizontal axis represents the mean relative intensity and the abscissa m/z ratios. HCCA peaks correspond to matrix (4-cyano-4 hydroxy-cinnamic acid) signals. C. m/z ratio of compounds reported for Petiveria and compounds found in F4 fraction matching within (± 7) units of the m/z ratio.

F4 fraction abrogates K562 erythroleukemic cells colony forming ability. K562 cells plated at 2.5 × 10⁵ cells/well were treated with ethanol (0.2%), or F4 fraction (from 7.8 to 31.2 μg/ml), or etoposide (100 μg/ml), or vincristine (0.1 μg/ml) for 24 h. Afterwards, cells were stained with crystal violet (0.4% in ethanol). Data represents number of colonies ± SEM representing two independent experiments. ***p < 0.001 versus control (ethanol 0.2%) Unpaired Student's t-test).

Effect of Petiveria alliacea F4 fraction on cell cycle. A. A375 cells treated with ethanol (0.2%), or F4 fraction (31.2 μg/ml) or vincristine (0.1 μg/ml) for 24 h, were permeabilized, stained with propidium iodide (PI) 50 μg/ml and analyzed through flow cytometry (FACScalibur CellQuest software program) (Becton Dickinson). Bars represent relative percentage of cell-cycle distribution ± SEM and represent three independent performed experiments. ***p < 0.001 versus control (ethanol 0.2%; Unpaired Student's t-test). B. A375 cells treated with ethanol (0.2%) or F4 fraction (31.2 μg/ml) or vincristine 0.1 μg/ml for 12, 18, 24 and 48 h, were permeabilized, stained with PI 50 μg/ml and analyzed through flow cytometry (FACScalibur CellQuest software program) (Becton Dickinson). Histograms represent relative cell DNA content representing two independent experiments. C. A375 cells treated with ethanol (0.2%) or F4 fraction(62.5, 31.2, 15.6 μg/ml) or vincristine (0.1 μg/ml) for 48 h, were permeabilized, stained with PI 50 μg/ml and analyzed through flow cytometry (FACScalibur CellQuest software program) (Becton Dickinson). Histograms represent relative cell DNA content representing two independent experiments.

Petiveria alliacea F4 fraction is cytotoxic to tumor cell lines without affecting human normal cells. A. A375 B. Mel Rel C. K562 D. Human fibroblasts E. PBMC stimulated with PHA or F. PBMC-PBS treated with F4 fraction concentrations (white) at 125 (1), 62.5 (2), 31.2 (3), 15.6 (4), 7.8 (5), 3.9 (6), and 1.8 μg/ml (7); or vincristine (black) 0.1 (1), 0.05 (2), 0.025 (3), 0.0125 (4) 0.00625 (5), 0.0031 (6) and 0.0015 μg/ml (7), for 24 h. Cell viability was determined by MTT assay as described in the methods section. Data represent cell viability percentage (%), where the vehicle-treated cells are regarded as 100%. The values are mean ± SEM from three independent experiments.

F4 fraction has no activity on mitochondrial membrane depolarization. A. K562 human cells were treated with F4 fraction (31.2 μg/ml), or positive control S2 fraction (15.6 μg/ml) or ethanol (0.2%) for 4, 8 and 12 h. All cells were stained with JC-1 (Sigma) dye and analyzed by flow cytometry (FACScalibur CellQuest software program) (Becton Dickinson). Bars represent cell percentage (%) ± SEM of depolarized (filled bars) or non-depolarized (hatched bars) cells, representing two independent experiments. ***p < 0.001 versus control (ethanol 0.2%; Unpaired Student's t-test). B. A375 cells treated with ethanol (0.2%) (left panel) or F4 fraction (31.2 μg/ml) (right panel) for 24 h were permeabilized, stained with DAPI and analyzed under fluorescence microscope (Olympus). Results show photos representing four independent experiments.