Fig 1.
(Panel A) Structure of the natural pentacyclic triterpene, maslinic acid [(2α, 3β)-2,3-dihydroxyolean-12-en-28-oic acid]. (Panel B) Inhibitory effect of maslinic acid on the viability of Caco-2 cells. IC50 is the concentration of MA required for 50% growth inhibition. Each point represents the mean value ± S.D. of at least three independent experiments performed in triplicate. (Panel C)Top: histograms of Caco-2 cell cycle after 72 h treatment with MA. Bottom: percentage of cells in each of the cell-cycle phases. Caco-2 cells were untreated (first column) or treated with MA at IC50 (second column) or IC80 (third column) concentrations. Cell-cycle analysis was conducted after propidium iodide staining. Values represent means ± S.D. of at least three independent experiments performed in triplicate. Key: (*) p<0.05 and (**) p<0.01, respect to the untreated control cells.
Fig 2.
Top: western blotting of the levels of pro-caspase-9 and caspase-9; centre: pro-caspase-8 and caspase-8; and bottom: pro-caspase-3 and caspase-3 proteins.
Caco-2 cells were treated with MA at IC50 and IC80 concentrations for 4 h. The levels of protein expression are expressed as arbitrary intensity units of each band compared to arbitrary intensity units of actin. The values represent means ± S.D. of at least three independent experiments performed in triplicate. Key: (*) p<0.05, (**) p<0.01 and (***) p<0.001, with respect to the untreated control cells.
Fig 3.
(Panel A) Maslinic acid induced apoptosis through activation of caspase-8 in Caco-2 cells (Top), but not in HT29 cells (Bottom). Caco-2 and HT29 cells were treated for 4 h with their corresponding IC50 or IC80 concentrations. Values represent means ± S.D. of four experiments performed in triplicate. Key (***) P<0.001, with respect to the untreated control cells. (Panel B) Diagrams of rhodamine 123/propidium iodide flow-cytometry. The right quadrants of each diagram (Q2 and Q4) represent positive cells stained with Rh123, the left quadrants (Q1 and Q3) represent negative cells stained with Rh123. Top-right, percentage of Rh123 positive cell population in control cells (CT), IC50 and IC80 concentrations, after 4 h of treatment. The values represent means ± SD. of four independent experiments performed in triplicate. Not significant differences were found.
Fig 4.
(Panel A) Western blotting to determine Bax levels. Caco-2 cells were treated with MA at IC50 and IC80 concentrations for 4 h. (Panel B) Western blotting to determine cytosolic cytochrome-c release levels in Caco-2 cells (Top) and HT29 cells (Bottom). Caco-2 and HT29 cells were treated with MA at IC50 and IC80 concentrations for 4h. The levels of protein expression are expressed as arbitrary intensity units of each band compared to arbitrary intensity units of actin. The values represent means ± SD. of at least three independent experiments performed in triplicate. Key: (*) p<0.05, respect to the untreated cells.
Fig 5.
Western blotting to determine Bid and t-Bid levels in Caco-2 cells (Top) and HT29 cells (Bottom).
Caco-2 and HT29 cells were treated with MA at IC50 and IC80 concentrations for 4 h. The levels of protein expression are expressed as arbitrary intensity units of each band compared to arbitrary intensity units of actin. MA produced clear effects on this protein in Caco-2 cells. However, this effect was not observable in HT29 cells. The values represent means ± SD. of at least three independent experiments performed in triplicate. Key: (*) p<0.05 and (**) p<0.01, with respect to the untreated cells.
Fig 6.
Schematic representation of the different mechanisms proposed for the induction of apoptosis by MA in colon-cancer Caco-2 cells (right) and HT29 cells (left).
MA is able to activate both intrinsic and extrinsic apoptotic mechanisms according to the type of cell involved. Abbreviations: Cps, caspase; cit c, cytochrome-c.