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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 EVOO phenolics on the levels of HER2 oncoprotein in MCF-7/HER2 breast cancer cells. Overnight serum-starved MCF-7/HER2 cells were cultured in DMEM medium-0.1% FBS in the absence or presence of increasing concentrations of EVOO phenolics for 48 h. The Oncogene Science HER2 microtiter ELISA was used according to the manufacturer's instructions to compare HER2 protein concentrations in whole cell lysates from EVOO polyphenols-treated and untreated control cells. Results are means (columns) and 95% confidence intervals (bars) of three independent experiments made in triplicate. Statistically significant differences between experimental conditions and unsupplemented control cells are shown (one-factor ANOVA analysis). All statistical tests were two-sided.
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Figure 9: Influence of EVOO phenolics on the levels of HER2 oncoprotein in MCF-7/HER2 breast cancer cells. Overnight serum-starved MCF-7/HER2 cells were cultured in DMEM medium-0.1% FBS in the absence or presence of increasing concentrations of EVOO phenolics for 48 h. The Oncogene Science HER2 microtiter ELISA was used according to the manufacturer's instructions to compare HER2 protein concentrations in whole cell lysates from EVOO polyphenols-treated and untreated control cells. Results are means (columns) and 95% confidence intervals (bars) of three independent experiments made in triplicate. Statistically significant differences between experimental conditions and unsupplemented control cells are shown (one-factor ANOVA analysis). All statistical tests were two-sided.

Mentions: The fact that depletion of endogenous HER2 significantly prevented the growth-inhibitory effects of EVOO polyphenols together with our earlier findings demonstrating that the down-regulatory effects of the secoiridoid oleuropein aglycone were specifically restricted to HER2 without affecting other key members of the oncogenic HER network such as HER1 (EGFR) [20], suggested that secoiridoids- and lignans-induced changes in cell viability might relate, at least in part, to changes in the expression of HER2 oncoprotein. To evaluate this hypothesis we examined the effects of long-term treatments (i.e. 48 hours) with graded concentrations of EVOO polyphenols on the naturally-occurring overexpression of HER2 protein in SKBR3 cells (Figure 8). At its highest concentration (i.e. 100 μM), the EVOO single phenol hydroxytyrosol slightly reduced HER2 protein expression by 35%. Remarkably, HER2 expression was drastically reduced by 68% and 86% in the presence of 100 μM of the fractions 5 and 6 containing mainly EVOO lignans (+)-pinoresinol and 1-(+)-acetoxypinoresinol, respectively. Similarly to EVOO lignans, EVOO secoiridoids were very effective at inhibiting HER2 expression, with inhibitory percentages ranging from 50%–60% in the presence of DAOA-rich fraction 4 and ligstroside aglycone-rich fraction 8 to 87% inhibition upon treatment with the oleuropein aglycone-rich fraction 7. We then evaluated if the growth inhibitory effects of EVOO polyphenols against MCF-7/HER2 cells were also accompanied with changes in the expression levels of HER2 oncoprotein (Figure 9). The EVOO single phenol hydroxytyrosol notably decreased HER2 protein expression by 41%, while the fraction 6 containing mainly the EVOO lignan 1-(+)-acetoxypinoresinol drastically reduced HER2 expression by 83%. All the EVOO secoiridoids significantly reduced HER2 expression, from ~30% inhibition in the presence of DAOA-rich fraction 4 to 56% inhibition in the presence of the oleuropein aglycone-rich fraction 7. These findings reveal for the first time that all the major complex phenols present in EVOO (i.e. secoiridoids and lignans) drastically suppress HER2 oncoprotein overexpression in human breast cancer cells. Neither secoiridoids nor lignans treatments caused detectable changes in HER1 (EGFR) expression in breast cancer cells (data not shown).


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 EVOO phenolics on the levels of HER2 oncoprotein in MCF-7/HER2 breast cancer cells. Overnight serum-starved MCF-7/HER2 cells were cultured in DMEM medium-0.1% FBS in the absence or presence of increasing concentrations of EVOO phenolics for 48 h. The Oncogene Science HER2 microtiter ELISA was used according to the manufacturer's instructions to compare HER2 protein concentrations in whole cell lysates from EVOO polyphenols-treated and untreated control cells. Results are means (columns) and 95% confidence intervals (bars) of three independent experiments made in triplicate. Statistically significant differences between experimental conditions and unsupplemented control cells are shown (one-factor ANOVA analysis). All statistical tests were two-sided.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 9: Influence of EVOO phenolics on the levels of HER2 oncoprotein in MCF-7/HER2 breast cancer cells. Overnight serum-starved MCF-7/HER2 cells were cultured in DMEM medium-0.1% FBS in the absence or presence of increasing concentrations of EVOO phenolics for 48 h. The Oncogene Science HER2 microtiter ELISA was used according to the manufacturer's instructions to compare HER2 protein concentrations in whole cell lysates from EVOO polyphenols-treated and untreated control cells. Results are means (columns) and 95% confidence intervals (bars) of three independent experiments made in triplicate. Statistically significant differences between experimental conditions and unsupplemented control cells are shown (one-factor ANOVA analysis). All statistical tests were two-sided.
Mentions: The fact that depletion of endogenous HER2 significantly prevented the growth-inhibitory effects of EVOO polyphenols together with our earlier findings demonstrating that the down-regulatory effects of the secoiridoid oleuropein aglycone were specifically restricted to HER2 without affecting other key members of the oncogenic HER network such as HER1 (EGFR) [20], suggested that secoiridoids- and lignans-induced changes in cell viability might relate, at least in part, to changes in the expression of HER2 oncoprotein. To evaluate this hypothesis we examined the effects of long-term treatments (i.e. 48 hours) with graded concentrations of EVOO polyphenols on the naturally-occurring overexpression of HER2 protein in SKBR3 cells (Figure 8). At its highest concentration (i.e. 100 μM), the EVOO single phenol hydroxytyrosol slightly reduced HER2 protein expression by 35%. Remarkably, HER2 expression was drastically reduced by 68% and 86% in the presence of 100 μM of the fractions 5 and 6 containing mainly EVOO lignans (+)-pinoresinol and 1-(+)-acetoxypinoresinol, respectively. Similarly to EVOO lignans, EVOO secoiridoids were very effective at inhibiting HER2 expression, with inhibitory percentages ranging from 50%–60% in the presence of DAOA-rich fraction 4 and ligstroside aglycone-rich fraction 8 to 87% inhibition upon treatment with the oleuropein aglycone-rich fraction 7. We then evaluated if the growth inhibitory effects of EVOO polyphenols against MCF-7/HER2 cells were also accompanied with changes in the expression levels of HER2 oncoprotein (Figure 9). The EVOO single phenol hydroxytyrosol notably decreased HER2 protein expression by 41%, while the fraction 6 containing mainly the EVOO lignan 1-(+)-acetoxypinoresinol drastically reduced HER2 expression by 83%. All the EVOO secoiridoids significantly reduced HER2 expression, from ~30% inhibition in the presence of DAOA-rich fraction 4 to 56% inhibition in the presence of the oleuropein aglycone-rich fraction 7. These findings reveal for the first time that all the major complex phenols present in EVOO (i.e. secoiridoids and lignans) drastically suppress HER2 oncoprotein overexpression in human breast cancer cells. Neither secoiridoids nor lignans treatments caused detectable changes in HER1 (EGFR) expression in breast cancer cells (data not shown).

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