Fig 1.
Oleaster leaf infusion decreases tumor growth in athymic nude mice xenografted with HCT116 cells.
Cells (1×106) were injected subcutaneously into the left flank of nude mice (n = 10). Half of these animals received oleaster leaf infusion (1%) in the feeders, while tap water was given to the other half (control). Palpable tumors were measured using a Vernier caliper for 30 days. ** and *** represent p < 0.01 and p < 0.001, respectively, as compared to group of mice receiving tap water. p values were obtained by one-way ANOVA, followed by Fisher’s LSD test.
Fig 2.
Effects of PEOL on the viability and morphology of HCT116 (A), HCT8 (B), and CCD 841 CoN cells (C). The cells (6 ×103 cells/well) were incubated with different concentrations of PEOL for 24 h as described in Materials and Methods. Cell viability was measured by MTT test and morphology was assessed by light microscopy (magnification ×200). *,**,*** represent p < 0.05, p < 0.01, p < 0.001 respectively as compared to untreated cells. p values were obtained by one-way ANOVA, followed by Fisher’s LSD test.
Fig 3.
PEOL induced apoptosis in colon cancer cells.
Annexin V and 7-AAD labeling, caspase-9 activation, and PARP cleavage were monitored after 6 h, 12 h, and 24 h of HCT116 exposure to 30 μg/ml PEOL (A). HCT116 and HCT8 cells were treated with different concentrations of PEOL for 24 h and Caspase-3 and -7 activities were assessed by flow cytometry (B). HCT116 and HCT8 cells were incubated (or not) with z-VAD-fmk for 1 h, and then treated with PEOL (20 μg/ml). After 24 h, Annexin V and 7-AAD labeling was assessed by flow cytometry (C). HCT116 cells were treated with increasing concentrations of oleuropein for 24 h. then PARP cleavage and Annexin V and 7-AAD labeling were monitored were as described in Materials and methods (D). *,**,*** represent p < 0.05, p < 0.01, p < 0.001 respectively as compared to HCT116 untreated cells. ‡, ‡‡, ‡‡‡ represent p < 0.05, p < 0.01, p < 0.001 respectively as compared to HCT8 untreated cells. p values were obtained by one-way ANOVA, followed by Fisher’s LSD test.
Fig 4.
PEOL-mediated apoptosis was independent of p53 status.
HCT116 p53+/+ and HCT116 p53-/- cells were treated with increasing concentrations of PEOL for 24 h. The effect of PEOL on p53 expression in HCT116 p53+/+ was assessed by western blotting (A). Insert shows p53 expression in HCT116 p53+/+ and HCT116 p53-/- (A insert). Cell death was assessed with Annexin V and 7-AAD staining (B). Cleaved PARP were determined by western blotting in cells treated with PEOL as described in Materials and methods (C). Data represent means ± SEM (n = 3). ** and *** represent p < 0.01 and p < 0.001 respectively as compared to untreated cells. p values were obtained by one-way ANOVA, followed by Fisher’s LSD test.
Fig 5.
Increases in mitochondrial ROS associated with mitochondrial membrane potential (ΔΨm) decreases in PEOL-treated cells.
HCT116 and HCT8 cells were treated with PEOL for 24 h, and then incubated whether with MitoSOX Red or TMRM as described in Materials and Methods. Representative histograms are shown in parallel with bar graphs showing the relative TMRM mean relative fluorescence intensity (A) and MitoSOX fluorescence (C) as measured by flow cytometry. Cytoplasmic cytochrome c was determined by western blotting as described in Materials and methods (B). Data represent means ± SEM (n = 3). *,**,*** represent p < 0.05, p < 0.01, p < 0.001 respectively as compared to HCT116 untreated cells. ‡, ‡‡, ‡‡‡ represent p < 0.05, p < 0.01, p < 0.001 respectively as compared to HCT8 untreated cells. p values were obtained by one-way ANOVA, followed by Fisher’s LSD test.
Fig 6.
PEOL induced ROS-dependent ER stress.
HCT116 and HCT8 cells were treated with PEOL for 24 h then eIF2α phosphorylation and CHOP mRNA expression were determined by western blotting (A,B) and Real-time PCR (C,D), respectively. HCT116 were treated incubated with NAC (0.5 mM) before PEOL treatment for 24 h, then CHOP mRNA expression (E) or Annexin V and 7-AAD labeling were performed (F). Data represent means ± SEM (n = 6). *,**,*** represent p < 0.05, p < 0.01, p < 0.001 respectively as compared to untreated cells.‡‡‡ represent p < 0.001 for Annexin V+ comparison between NAC-treated and untreated cells. p values were obtained by one-way ANOVA, followed by Fisher’s LSD test.
Fig 7.
Ca2+ signaling modulation by PEOL in HCT8 and HCT116 cells.
Cells were loaded with the fluorescent probe, Fura-2/AM. The changes in F340/F380 were monitored under a Nikon microscope (TiU) by using S Fluor 40× oil immersion objectives as described in Materials and Methods. The colored time-lapse changes in the increases in [Ca2+]i were recorded in PEOL-treated HCT116 and HCT8 cells in 100% Ca2+-buffer (A and B). The arrow head indicates the time when PEOL was added into the wells without interruptions in recordings. Inserts show the single traces of observations of cell treatment with PEOL and oleuropein in 100% or 0% Ca2+-buffer which were reproduced independently. (C) and (D) show the effect of cells preincubation for 1 h with BAPTA-AM (5 μM), or ruthenium red (5 μM) on PEOL induced apoptosis (Annexin V and 7-AAD staining) in HCT116 an HCT8 cell lines. (E) shows the effect of cells preincubation with BAPTA-AM (5 μM) of MitoSOX fluorescence measured after 24 h incubation with PEOL in HCT116. (F) shows the effect of cells preincubation with BAPTA-AM (5 μM) on CHOP mRNA expression in response to increasing PEOL concentration. (G) represents a schematic conclusion summarizing the molecular mechanism of PEOL-induced apoptosis. Data represent means ± SEM (n = 6). ‡ and * represents p < 0.01 as compared to PEOL treated cells. p values were obtained by one-way ANOVA, followed by Fisher’s LSD test.