Fig 1.
Oleocanthal induces rapid necrotic cell death in a variety of cancer cells.
(A) The indicated cell lines were treated with 20 μM oleocanthal (OC) for 24 hours and viability was measured via the reduction of XTT. ****P < 0.0001 (One-way ANOVA). (B) PC3 cells were treated with 20μM oleocanthal or DMSO control for either 24 hours without media change, or 1 hour followed by a media change into full growth medium. Viability was measured 24 hours post treatment via the reduction of XTT. C and D) MDA-MB-231 cells (C) and PC3 cells (D) were treated with vehicle only (DMSO), or 20 μM oleocanthal for the indicated time points, and double-stained with Annexin-V FITC and PI. Fluorescence was measured on a flow cytometer (MoxiGo II). Treatment with 1μM Staurosporine (St) for 4 hours is presented as a positive control for apoptotic cells. Representative scatter plots from 3 independent experiments are shown, as well as bar graph quantifications: the lower right quadrant (apoptosis) is shown in green, and upper quadrant (necrosis) is shown in red. Bar graphs represent the mean ± SEM (n = 3).
Fig 2.
Oleocanthal induces LMP and cathepsin leakage.
(A) MCF-7 cells were treated with DMSO, 30μM oleocanthal for 2 hours, or 2 mM LLOMe and stained for Galectin-3. Nuclei were labeled with Hoechst 33,342. Scale bars 20 μM. Green Galectin punctea indicate compromised lysosomes. (B) PC3 cells were treated with 20 μM oleocanthal for one hour, or 2mM LLOMe for 15 minutes, then loaded with Lysotracker green. Fluorescence intensity was measured via flow cytometry. Histogram shows a representative shift in Lysotracker fluorescence associated with perturbation to the lysosomal compartment. Bar graph shows mean fluorescence intensity of three replicate experiments. (C) PC3 cells were treated with 20 μM oleocanthal, and two hours later their cytosolic fractions (Cyto), and light membrane fractions containing lysosomes (Lyso) were separated. Level of cathepsin B (CTSB) and cathepsin D (CTSD) in the various fractions or whole cell lysates is shown. LAMP2 is a lysosomal marker and GAPDH is a cytosolic marker. (D) Lysosomes isolated from overnight serum-deprived PC3 cells were incubated for 20 min with the indicated concentrations of oleocanthal or vehicle (DMSO). At the end of the incubation, lysosomes were filtered through a vacuum manifold and b-hexosaminidase activity was measured in the flow through and in the total lysosomal fraction. Broken lysosomes were calculated as the percentage of total lysosomal hexosaminidase activity detected in the flow-through and plotted in logarithmic scale. (E) PC3 cells were infected with HSP70-1 Lentiviral Activation Particles, or control (scrambled) particles, and treated with 20 μM oleocanthal. Viability was assayed using reduction of XTT. *P < 0.05, **P < 0.01 (Two-tailed unpaired t-test). Bar graphs represent the mean ± SEM (n = 3).
Fig 3.
Oleocanthal induces minimal LMP in MCF10A cells.
(A) MCF10A cells were treated with increasing concentration of oleocanthal for 24 hours and viability was measured via the reduction of XTT. (B) MCF10A cells were treated with 20 μM Oleocanthal, and two hours later their cytosolic fractions (Cyto), and light membrane fractions containing lysosomes (Lyso) were separated. Level of cathepsin B (CTSB) and cathepsin D (CTSD) in the various fractions or whole cell lysates is shown.
Fig 4.
Oleocanthal increases life span of mice with PNET tumors.
(A) Kaplan-Meier survival curve for RIP-Tag mice receiving DMSO or oleocanthal. The mice were treated with DMSO (n = 11) or oleocanthal (5 mg/kg, n = 15), 7 days a week. Mice were treated starting from 9 weeks of age. Both the Gehan-Breslow-Wilcoxon method and the Log-rank (Mantel-Cox) method were used to calculate statistical significance *P < 0.05. (B) Tumor burden from mice treated with DMSO or oleocanthal (n = 7 for each group) starting from 9 weeks of age and ending at 14 weeks of age. ns P > 0.05. (C) A cell-line derived from a murine PNET tumor, was established (N134). Cells were treated with DMSO or oleocanthal and analyzed for cytosolic cathepsin L (CTSL) via Western blot as in Fig 2C.
Fig 5.
Oleocanthal-rich olive oils are toxic to cancer cells via LMP.
(A) Relative oleocanthal concentration in various oils was measured by 1H NMR as described in Materials and Methods. (B, C, and F) PC3 cells (B) MDA-MB-231 cells (C) and MCF10A cells (F) were treated with 20 μM oleocanthal, or the specified oils for 24 hours. Viability was measured via the reduction of XTT. (D and E) Cytosolic lysates were collected as in Fig 2C and subjected to Western blot analysis of cathepsin B (CTSB) and cathepsin D (CTSD) in the cytosol. Bar graphs represent the mean ± SEM (n = 3).