Journal: Carcinogenesis

Article Title: Extra-virgin olive oil contains a metabolo-epigenetic inhibitor of cancer stem cells

doi: 10.1093/carcin/bgy023

Figure Lengend Snippet: A phenolic-enriched crude olive oil extract inhibits the mammosphere-initiating capacity of BC CSC-like states. ( A ) Three-dimensional map of phenolic compound separation obtained by HPLC-ESI-TOF in a crude EVOO-PE obtained via isolation protocols described in , available at Carcinogenesis Online. ( B ) Representative ALDEFLUOR ® assay to identify SUM-159 cells with high ALDH activity (ALDH + ) in the absence or presence of 10 μg/mL of the crude EVOO-PE for 3 days. The ALDH inhibitor diethylaminobenzaldehyde (DEAB) was used as negative control. Monolayer cultures were fed with the crude EVOO-PE every other day. Results are representative of two technical replicates per n ; n = 3 biological replicates. MSFE is expressed as percentages means (columns) ± SD (bars); three technical replicates per n ; n = 3 biological replicates. MTT uptake-based measurement of cell viability is expressed as percentages uptake (OD 570 ) relative to untreated control cells (=100% cell viability). The results are expressed as percentages means (columns) ± SD (bars); three technical replicates per n ; i = 3 biological replicates. ( C ) Figure shows representative light microscope representations (×20 magnification) of mammospheres formed by HMLER ShControl and HMLER ShEcad cells growing in sphere medium for 7 days in the absence or presence of graded concentrations of EVOO-PE. MSFE and MTT calculations were performed as described for SUM-159 cells in the left panels; three technical replicates per n ; n = 3 biological replicates. (* P < 0.01 and ** P < 0.001, statistically significant differences from the untreated (control) group).

Article Snippet: Approximately 2 × 10 6 SUM-159 cells were subcutaneously injected into the dorsal flanks of female athymic nude mice (4- to 5-week-old mice, 23–25 g; Harlan Laboratories).

Techniques: Isolation, Activity Assay, Negative Control, Light Microscopy

Journal: Carcinogenesis

Article Title: Extra-virgin olive oil contains a metabolo-epigenetic inhibitor of cancer stem cells

doi: 10.1093/carcin/bgy023

Figure Lengend Snippet: DOA specifically and potently decreases the mammosphere-initiating capacity of BC CSC-like states. ( A ) Representative ALDEFLUOR ® assay to identify SUM-159 cells with high ALDH activity (ALDH + ) in the absence or presence of 20 μmol/L DOA for 3 days. The ALDH inhibitor DEAB was used as negative control. Monolayer cultures were fed with the DOA every other day. Results are representative of two technical replicates per n ; n = 3 biological replicates. ( B ) Figure shows representative light microscope representations (×20 magnifications) of mammospheres formed by HMLER ShEcad cells growing in sphere medium for 7 days in the absence or presence of graded concentrations of DOA. MSFE and MTT calculations were performed as described in ; three technical replicates per n ; n = 3 biological replicates. (* P < 0.01 and ** P < 0.001, statistically significant differences from the untreated (control) group; n.s. not statistically significant). ( C ) Anoikis-resistant cells obtained as described in , available at Carcinogenesis Online were cultured in DOA-free mammosphere medium for 7 days, and MSFE were calculated following the same procedure as that described in and 2; three technical replicates per n ; n = 2 biological replicates.

Article Snippet: Approximately 2 × 10 6 SUM-159 cells were subcutaneously injected into the dorsal flanks of female athymic nude mice (4- to 5-week-old mice, 23–25 g; Harlan Laboratories).

Techniques: Activity Assay, Negative Control, Light Microscopy, Cell Culture

Journal: Carcinogenesis

Article Title: Extra-virgin olive oil contains a metabolo-epigenetic inhibitor of cancer stem cells

doi: 10.1093/carcin/bgy023

Figure Lengend Snippet: DOA blocks tumor-initiating capacity of CSC-like cells in vivo . ( A ) Tumor growth rate was calculated by measuring volumes along several weeks after injection of DOA-pretreated of untreated control cells. Shown are the mean volumes (±SD) for at least 9 weeks. Tumor-free survival in mice bearing SUM-159 xenografts with volumes ≥10 and ≥50 mm 3 is shown as a function of time. [* P < 0.01 and ** P < 0.001, statistically significant differences from the untreated (control) group; n.s. not statistically significant]. ( B ) Mammosphere-initiating cells were isolated and exposed to graded concentrations of DOA for 2 h. Viable single cell suspensions (1 × 10 4 cells) were orthotopically injected into the mammary fat pads of SCID/Beige mice, and tumor growth was monitored for at least 4 months. Tumor-free survival in mice bearing orthotopically implanted SUM-159 CSC-like cells is shown as a function of time.

Article Snippet: Approximately 2 × 10 6 SUM-159 cells were subcutaneously injected into the dorsal flanks of female athymic nude mice (4- to 5-week-old mice, 23–25 g; Harlan Laboratories).

Techniques: In Vivo, Injection, Isolation

Journal: Carcinogenesis

Article Title: Extra-virgin olive oil contains a metabolo-epigenetic inhibitor of cancer stem cells

doi: 10.1093/carcin/bgy023

Figure Lengend Snippet: ( A ) DOA synergistically interacts with mTOR and DNMT inhibitors. Shown are representative microphotographs of PM arrays (microplates PM-M11–PM-M14) from two biological replicates. The different interactions were defined as described in , available at Carcinogenesis Online. ( B ) DOA decreases mTOR and DNMT activities in cultured cells. Left. Representative western blotting analyses of phospho-p70 S6 Kinase (Thr389)/phospho-p85 S6 Kinase (Thr412) in SUM-159 cells cultured in the absence of presence of DOA or rapamycin for up to 6 h, as specified. Right. Nuclear extracts of SUM-159 were exposed to 20 μmol/L DOA for 2 h before assessing DNMT activity using the DNMT activity/Inhibition Assay Kit of Active Motif as per manufacturer’s instructions. The results are expressed as percentages of the means (columns) ± SD (bars); three technical replicates per n ; n = 2 biological replicates. (** P < 0.001 versus DNMT activity in untreated nuclear extracts). ( C ) DOA binds the ATP-binding pocket in mTOR kinase domain and inhibits mTOR kinase activity. Overall structures and views of the interaction between DOA (red) and well-known TORKinhibs with the ATP-dependent catalytic pocket of mTOR (PDB code 4JT6). Figure shows in sticks all the interaction residues involved in the binding of DOA/TORKinhibs using PLIP. Hydrogen bond interactions are represented by orange dashed lines; salt bridges are represented by yellow dashed lines and charge centers by yellow spheres; Cation-π interactions are represented by blue dashed lines and white spheres for the center of the aromatic ring. ( D ) DOA directly inhibits the ATP-dependent catalytic activity of mTOR. A dose-response curve of ATP-dependent mTOR kinase activity was created by plotting FRET signal of the Z′-LYTE Kinase assay as the function of DOA concentration.

Article Snippet: Approximately 2 × 10 6 SUM-159 cells were subcutaneously injected into the dorsal flanks of female athymic nude mice (4- to 5-week-old mice, 23–25 g; Harlan Laboratories).

Techniques: Cell Culture, Western Blot, Activity Assay, Inhibition, Binding Assay, Kinase Assay, Concentration Assay