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tabAnti-HER2 (erbB-2) oncogene effects of phenolic compounds directly isolated from commercial Extra-Virgin Olive Oil (EVOO).

Menendez JA, Vazquez-Martin A, Garcia-Villalba R, Carrasco-Pancorbo A, Oliveras-Ferraros C, Fernandez-Gutierrez A, Segura-Carretero A - BMC Cancer (2008)

Bottom Line: Among the fractions mainly containing the single phenols hydroxytyrosol and tyrosol, the polyphenol acid elenolic acid, the lignans (+)-pinoresinol and 1-(+)-acetoxypinoresinol, and the secoiridoids deacetoxy oleuropein aglycone, ligstroside aglycone, and oleuropein aglycone, all the major EVOO polyphenols (i.e. secoiridoids and lignans) were found to induce strong tumoricidal effects within a micromolar range by selectively triggering high levels of apoptotic cell death in HER2-overexpressors.EVOO polyphenols drastically depleted HER2 protein and reduced HER2 tyrosine autophosphorylation in a dose- and time-dependent manner.The ability of EVOO-derived polyphenols to inhibit HER2 activity by promoting the proteasomal degradation of the HER2 protein itself, together with the fact that humans have safely been ingesting secoiridoids and lignans as long as they have been consuming olives and OO, support the notion that the stereochemistry of these phytochemicals might provide an excellent and safe platform for the design of new HER2-targeting agents.

View Article: PubMed Central - HTML - PubMed

Affiliation: Catalan Institute of Oncology (ICO)-Health Services Division of Catalonia, Catalonia, Spain. jmenendez@ico.scs.es

ABSTRACT

Background: The effects of the olive oil-rich Mediterranean diet on breast cancer risk might be underestimated when HER2 (ERBB2) oncogene-positive and HER2-negative breast carcinomas are considered together. We here investigated the anti-HER2 effects of phenolic fractions directly extracted from Extra Virgin Olive Oil (EVOO) in cultured human breast cancer cell lines.

Methods: Solid phase extraction followed by semi-preparative high-performance liquid chromatography (HPLC) was used to isolate phenolic fractions from commercial EVOO. Analytical capillary electrophoresis coupled to mass spectrometry was performed to check for the composition and to confirm the identity of the isolated fractions. EVOO polyphenolic fractions were tested on their tumoricidal ability against HER2-negative and HER2-positive breast cancer in vitro models using MTT, crystal violet staining, and Cell Death ELISA assays. The effects of EVOO polyphenolic fractions on the expression and activation status of HER2 oncoprotein were evaluated using HER2-specific ELISAs and immunoblotting procedures, respectively.

Results: Among the fractions mainly containing the single phenols hydroxytyrosol and tyrosol, the polyphenol acid elenolic acid, the lignans (+)-pinoresinol and 1-(+)-acetoxypinoresinol, and the secoiridoids deacetoxy oleuropein aglycone, ligstroside aglycone, and oleuropein aglycone, all the major EVOO polyphenols (i.e. secoiridoids and lignans) were found to induce strong tumoricidal effects within a micromolar range by selectively triggering high levels of apoptotic cell death in HER2-overexpressors. Small interfering RNA-induced depletion of HER2 protein and lapatinib-induced blockade of HER2 tyrosine kinase activity both significantly prevented EVOO polyphenols-induced cytotoxicity. EVOO polyphenols drastically depleted HER2 protein and reduced HER2 tyrosine autophosphorylation in a dose- and time-dependent manner. EVOO polyphenols-induced HER2 downregulation occurred regardless the molecular mechanism contributing to HER2 overexpression (i.e. naturally by gene amplification and ectopically driven by a viral promoter). Pre-treatment with the proteasome inhibitor MG132 prevented EVOO polyphenols-induced HER2 depletion.

Conclusion: The ability of EVOO-derived polyphenols to inhibit HER2 activity by promoting the proteasomal degradation of the HER2 protein itself, together with the fact that humans have safely been ingesting secoiridoids and lignans as long as they have been consuming olives and OO, support the notion that the stereochemistry of these phytochemicals might provide an excellent and safe platform for the design of new HER2-targeting agents.

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Influence of the activation (phosphorylation) status of HER2 tyrosine kinase activity on the cytotoxic activity of EVOO polyphenols. ~5 × 103 cells (96-well plates) overnight serum-starved SKBR3 (left) and MCF-7/HER2 (right) cells were treated with 0.1 μM lapatinib before exposure to 25 μM 1-[+]-acetoxypinoresinol or 25 μM DAOA. The metabolic status of treated and untreated control cells was evaluated using a MTT-based cell viability assay and Interaction Indexes were calculated by dividing expected cytotoxicities (additive models: sum of cell toxicities induced by each agent alone) by those obtained experimentally in the actual combination (Interaction Indexes < 1, = 1, > 1 < 2, and > 2 denote a nature of the interaction synergistic, additive, antagonistic and protective, respectively). Results are means (dots) and 95% confidence intervals (bars) of three independent experiments made in triplicate.
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Figure 10: Influence of the activation (phosphorylation) status of HER2 tyrosine kinase activity on the cytotoxic activity of EVOO polyphenols. ~5 × 103 cells (96-well plates) overnight serum-starved SKBR3 (left) and MCF-7/HER2 (right) cells were treated with 0.1 μM lapatinib before exposure to 25 μM 1-[+]-acetoxypinoresinol or 25 μM DAOA. The metabolic status of treated and untreated control cells was evaluated using a MTT-based cell viability assay and Interaction Indexes were calculated by dividing expected cytotoxicities (additive models: sum of cell toxicities induced by each agent alone) by those obtained experimentally in the actual combination (Interaction Indexes < 1, = 1, > 1 < 2, and > 2 denote a nature of the interaction synergistic, additive, antagonistic and protective, respectively). Results are means (dots) and 95% confidence intervals (bars) of three independent experiments made in triplicate.

Mentions: Since apigenin and luteolin, two naturally occurring flavonoids structurally related to EVOO polyphenols, have been found to inhibit tyrosine phosphorylation and deplete HER2 protein through HER2 protein degradation [41-43], we envisioned that an analogous posttranslational mechanism may also contribute to the anti-HER2 actions of EVOO polyphenols. First, to evaluate whether the biological activity of EVOO polyphenols in terms of cell viability not only related to HER2 protein overexpression but further required an enhanced tyrosine kinase activity of HER2 as a molecular "hallmark" in EVOO polyphenols-sensitive breast cancer cells, we used a pharmacological approach to determine the effect of "HER2 activity-dose" on the tumoricidal effects of EVOO polyphenols. Similarly to siRNA HER2-induced depletion of HER2 protein, specific blockade of HER2 constitutive hyperactivation (autophosphorylation at Tyr1248; Figure 2, Additional file 1) using the dual-HER1/HER2 tyrosine kinase inhibitor lapatinib (Tykerb™) was sufficient to preclude any further cytotoxic action of EVOO polyphenols. Thus, a combination of sequential lapatinib (0.1 μM) followed by treatment with sub-optimal doses of the 1-[+]-acetoxypinoresinol-rich fraction 6 and DAOA-rich fraction 4 demonstrated an antagonistic to protective nature in their cytotoxic interactions (Interaction Indexes > 2.0; Figure 10). To investigate the kinetics of inhibition/depletion of HER2 activity/expression, we treated SKBR3 and MCF-7/HER2 cells with EVOO polyphenols for different time periods and harvested them for ELISA-based analysis of HER2 protein expression. HER2 tyrosine autophosphorylation levels (i.e. activation status of HER2 Tyr1248) were measured by immunoblotting procedures. The HER2 protein levels decreased in a time-dependent manner following exposure to EVOO polyphenols, with significant HER2 depleting effects occurring as early as 6 hr after treatment with the fraction 6 containing mainly 1-[+]-acetoxypinoresinol (Figure 11). Fraction 6-decreased HER2 protein led to a coordinate decrease in HER2 autophosphorylation, which significantly declined after 6 hr and was barely detectable after 24 hr (Figure 11). Although less markedly than fraction 6, the fraction 4 containing mainly the EVOO secoiridoid DAOA also decreased HER2 protein expression and HER2 tyrosine kinase activity in a time-dependent manner, with the most significant anti-HER2 expression/activity effects occurring after 48 hr (Figure 11). These findings, altogether, strongly suggest that EVOO polyphenols could inhibit HER2 protein kinase activity by depleting the HER2 protein kinase itself.


tabAnti-HER2 (erbB-2) oncogene effects of phenolic compounds directly isolated from commercial Extra-Virgin Olive Oil (EVOO).

Menendez JA, Vazquez-Martin A, Garcia-Villalba R, Carrasco-Pancorbo A, Oliveras-Ferraros C, Fernandez-Gutierrez A, Segura-Carretero A - BMC Cancer (2008)

Influence of the activation (phosphorylation) status of HER2 tyrosine kinase activity on the cytotoxic activity of EVOO polyphenols. ~5 × 103 cells (96-well plates) overnight serum-starved SKBR3 (left) and MCF-7/HER2 (right) cells were treated with 0.1 μM lapatinib before exposure to 25 μM 1-[+]-acetoxypinoresinol or 25 μM DAOA. The metabolic status of treated and untreated control cells was evaluated using a MTT-based cell viability assay and Interaction Indexes were calculated by dividing expected cytotoxicities (additive models: sum of cell toxicities induced by each agent alone) by those obtained experimentally in the actual combination (Interaction Indexes < 1, = 1, > 1 < 2, and > 2 denote a nature of the interaction synergistic, additive, antagonistic and protective, respectively). Results are means (dots) and 95% confidence intervals (bars) of three independent experiments made in triplicate.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2626601&req=5

Figure 10: Influence of the activation (phosphorylation) status of HER2 tyrosine kinase activity on the cytotoxic activity of EVOO polyphenols. ~5 × 103 cells (96-well plates) overnight serum-starved SKBR3 (left) and MCF-7/HER2 (right) cells were treated with 0.1 μM lapatinib before exposure to 25 μM 1-[+]-acetoxypinoresinol or 25 μM DAOA. The metabolic status of treated and untreated control cells was evaluated using a MTT-based cell viability assay and Interaction Indexes were calculated by dividing expected cytotoxicities (additive models: sum of cell toxicities induced by each agent alone) by those obtained experimentally in the actual combination (Interaction Indexes < 1, = 1, > 1 < 2, and > 2 denote a nature of the interaction synergistic, additive, antagonistic and protective, respectively). Results are means (dots) and 95% confidence intervals (bars) of three independent experiments made in triplicate.
Mentions: Since apigenin and luteolin, two naturally occurring flavonoids structurally related to EVOO polyphenols, have been found to inhibit tyrosine phosphorylation and deplete HER2 protein through HER2 protein degradation [41-43], we envisioned that an analogous posttranslational mechanism may also contribute to the anti-HER2 actions of EVOO polyphenols. First, to evaluate whether the biological activity of EVOO polyphenols in terms of cell viability not only related to HER2 protein overexpression but further required an enhanced tyrosine kinase activity of HER2 as a molecular "hallmark" in EVOO polyphenols-sensitive breast cancer cells, we used a pharmacological approach to determine the effect of "HER2 activity-dose" on the tumoricidal effects of EVOO polyphenols. Similarly to siRNA HER2-induced depletion of HER2 protein, specific blockade of HER2 constitutive hyperactivation (autophosphorylation at Tyr1248; Figure 2, Additional file 1) using the dual-HER1/HER2 tyrosine kinase inhibitor lapatinib (Tykerb™) was sufficient to preclude any further cytotoxic action of EVOO polyphenols. Thus, a combination of sequential lapatinib (0.1 μM) followed by treatment with sub-optimal doses of the 1-[+]-acetoxypinoresinol-rich fraction 6 and DAOA-rich fraction 4 demonstrated an antagonistic to protective nature in their cytotoxic interactions (Interaction Indexes > 2.0; Figure 10). To investigate the kinetics of inhibition/depletion of HER2 activity/expression, we treated SKBR3 and MCF-7/HER2 cells with EVOO polyphenols for different time periods and harvested them for ELISA-based analysis of HER2 protein expression. HER2 tyrosine autophosphorylation levels (i.e. activation status of HER2 Tyr1248) were measured by immunoblotting procedures. The HER2 protein levels decreased in a time-dependent manner following exposure to EVOO polyphenols, with significant HER2 depleting effects occurring as early as 6 hr after treatment with the fraction 6 containing mainly 1-[+]-acetoxypinoresinol (Figure 11). Fraction 6-decreased HER2 protein led to a coordinate decrease in HER2 autophosphorylation, which significantly declined after 6 hr and was barely detectable after 24 hr (Figure 11). Although less markedly than fraction 6, the fraction 4 containing mainly the EVOO secoiridoid DAOA also decreased HER2 protein expression and HER2 tyrosine kinase activity in a time-dependent manner, with the most significant anti-HER2 expression/activity effects occurring after 48 hr (Figure 11). These findings, altogether, strongly suggest that EVOO polyphenols could inhibit HER2 protein kinase activity by depleting the HER2 protein kinase itself.

Bottom Line: Among the fractions mainly containing the single phenols hydroxytyrosol and tyrosol, the polyphenol acid elenolic acid, the lignans (+)-pinoresinol and 1-(+)-acetoxypinoresinol, and the secoiridoids deacetoxy oleuropein aglycone, ligstroside aglycone, and oleuropein aglycone, all the major EVOO polyphenols (i.e. secoiridoids and lignans) were found to induce strong tumoricidal effects within a micromolar range by selectively triggering high levels of apoptotic cell death in HER2-overexpressors.EVOO polyphenols drastically depleted HER2 protein and reduced HER2 tyrosine autophosphorylation in a dose- and time-dependent manner.The ability of EVOO-derived polyphenols to inhibit HER2 activity by promoting the proteasomal degradation of the HER2 protein itself, together with the fact that humans have safely been ingesting secoiridoids and lignans as long as they have been consuming olives and OO, support the notion that the stereochemistry of these phytochemicals might provide an excellent and safe platform for the design of new HER2-targeting agents.

View Article: PubMed Central - HTML - PubMed

Affiliation: Catalan Institute of Oncology (ICO)-Health Services Division of Catalonia, Catalonia, Spain. jmenendez@ico.scs.es

ABSTRACT

Background: The effects of the olive oil-rich Mediterranean diet on breast cancer risk might be underestimated when HER2 (ERBB2) oncogene-positive and HER2-negative breast carcinomas are considered together. We here investigated the anti-HER2 effects of phenolic fractions directly extracted from Extra Virgin Olive Oil (EVOO) in cultured human breast cancer cell lines.

Methods: Solid phase extraction followed by semi-preparative high-performance liquid chromatography (HPLC) was used to isolate phenolic fractions from commercial EVOO. Analytical capillary electrophoresis coupled to mass spectrometry was performed to check for the composition and to confirm the identity of the isolated fractions. EVOO polyphenolic fractions were tested on their tumoricidal ability against HER2-negative and HER2-positive breast cancer in vitro models using MTT, crystal violet staining, and Cell Death ELISA assays. The effects of EVOO polyphenolic fractions on the expression and activation status of HER2 oncoprotein were evaluated using HER2-specific ELISAs and immunoblotting procedures, respectively.

Results: Among the fractions mainly containing the single phenols hydroxytyrosol and tyrosol, the polyphenol acid elenolic acid, the lignans (+)-pinoresinol and 1-(+)-acetoxypinoresinol, and the secoiridoids deacetoxy oleuropein aglycone, ligstroside aglycone, and oleuropein aglycone, all the major EVOO polyphenols (i.e. secoiridoids and lignans) were found to induce strong tumoricidal effects within a micromolar range by selectively triggering high levels of apoptotic cell death in HER2-overexpressors. Small interfering RNA-induced depletion of HER2 protein and lapatinib-induced blockade of HER2 tyrosine kinase activity both significantly prevented EVOO polyphenols-induced cytotoxicity. EVOO polyphenols drastically depleted HER2 protein and reduced HER2 tyrosine autophosphorylation in a dose- and time-dependent manner. EVOO polyphenols-induced HER2 downregulation occurred regardless the molecular mechanism contributing to HER2 overexpression (i.e. naturally by gene amplification and ectopically driven by a viral promoter). Pre-treatment with the proteasome inhibitor MG132 prevented EVOO polyphenols-induced HER2 depletion.

Conclusion: The ability of EVOO-derived polyphenols to inhibit HER2 activity by promoting the proteasomal degradation of the HER2 protein itself, together with the fact that humans have safely been ingesting secoiridoids and lignans as long as they have been consuming olives and OO, support the notion that the stereochemistry of these phytochemicals might provide an excellent and safe platform for the design of new HER2-targeting agents.

Show MeSH
Related in: MedlinePlus