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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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Effects of EVOO phenolics on breast cancer cell proliferation. SKBR3 cells were incubated with various concentrations of EVOO single phenols (left), fractions containing mainly EVOO lignans (middle) and fractions containing mainly EVOO secoiridoids (right) for 4 days. Cell proliferation, measured using a crystal violet assay as described in "Materials and methods", was expressed as % of untreated cells (dashed line = 100% cell proliferation). Results are means (columns) and 95% confidence intervals (bars) of three independent experiments made in triplicate. Statistically significant differences (one-factor ANOVA analysis) between experimental conditions and unsupplemented control cells are shown. All statistical tests were two-sided.
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Figure 3: Effects of EVOO phenolics on breast cancer cell proliferation. SKBR3 cells were incubated with various concentrations of EVOO single phenols (left), fractions containing mainly EVOO lignans (middle) and fractions containing mainly EVOO secoiridoids (right) for 4 days. Cell proliferation, measured using a crystal violet assay as described in "Materials and methods", was expressed as % of untreated cells (dashed line = 100% cell proliferation). Results are means (columns) and 95% confidence intervals (bars) of three independent experiments made in triplicate. Statistically significant differences (one-factor ANOVA analysis) between experimental conditions and unsupplemented control cells are shown. All statistical tests were two-sided.

Mentions: We initially assessed the metabolic status of HER2-overexpressing SKBR3 breast cancer cells following treatments with graded micromolar concentrations of EVOO polyphenols (6.25 → 100 μM). After 5 days of treatment, cell viability was measured using a tetrazolium salt-based (MTT) assay, and the IC50 value for each EVOO polyphenol was calculated as described in "Materials and methods". EVOO single phenols failed to significantly decrease SKBR3 cell viability. Thus, concentrations higher than 100 μM were needed to achieve cytotoxic responses in the presence of hydroxytyrosol, tyrosol, and elenolic acid (fractions 1, 2 and 3 respectively) (Figure 2). EVOO lignans exhibited significant cytotoxic activities against SKBR3 cells. Fraction 6, which mainly contained 1-(+)-acetoxypinoresinol was more effective than fraction 5, which mainly contained (+)-pinoresinol (IC50s = 51 ± 2 μM and 72 ± 5 μM, respectively) (Figure 2). Among the EVOO phenolic fractions tested, those containing the secoiridoids DAOA, oleuropein aglycone, and ligstroside aglycone were the most potent at decreasing breast cancer cell viability, with all of them exhibiting IC50 values lower than 50 μM (e.g. as low as 26 ± 6 μM for the fraction 8 mainly containing the secoiridoid ligstroside aglycone) (Figure 2). Equivalent pictures emerged when the dose-response effects of EVOO polyphenols on the proliferation rate of SKBR3 were assessed by photometry after crystal violet staining. As shown in Figure 3 (left panel), EVOO single phenols and polyphenol acids produced weak growth inhibitory effects in SKBR3 cells. By contrast, the fractions 5 and 6 containing EVOO lignans (Figure 3, middle panel) and the fractions 4, 7 and 8 containing EVOO secoiridoids (Figure 3, right panel) significantly inhibited SKBR3 cell proliferation in a concentration-dependent manner.


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)

Effects of EVOO phenolics on breast cancer cell proliferation. SKBR3 cells were incubated with various concentrations of EVOO single phenols (left), fractions containing mainly EVOO lignans (middle) and fractions containing mainly EVOO secoiridoids (right) for 4 days. Cell proliferation, measured using a crystal violet assay as described in "Materials and methods", was expressed as % of untreated cells (dashed line = 100% cell proliferation). Results are means (columns) and 95% confidence intervals (bars) of three independent experiments made in triplicate. Statistically significant differences (one-factor ANOVA analysis) between experimental conditions and unsupplemented control cells are shown. 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 3: Effects of EVOO phenolics on breast cancer cell proliferation. SKBR3 cells were incubated with various concentrations of EVOO single phenols (left), fractions containing mainly EVOO lignans (middle) and fractions containing mainly EVOO secoiridoids (right) for 4 days. Cell proliferation, measured using a crystal violet assay as described in "Materials and methods", was expressed as % of untreated cells (dashed line = 100% cell proliferation). Results are means (columns) and 95% confidence intervals (bars) of three independent experiments made in triplicate. Statistically significant differences (one-factor ANOVA analysis) between experimental conditions and unsupplemented control cells are shown. All statistical tests were two-sided.
Mentions: We initially assessed the metabolic status of HER2-overexpressing SKBR3 breast cancer cells following treatments with graded micromolar concentrations of EVOO polyphenols (6.25 → 100 μM). After 5 days of treatment, cell viability was measured using a tetrazolium salt-based (MTT) assay, and the IC50 value for each EVOO polyphenol was calculated as described in "Materials and methods". EVOO single phenols failed to significantly decrease SKBR3 cell viability. Thus, concentrations higher than 100 μM were needed to achieve cytotoxic responses in the presence of hydroxytyrosol, tyrosol, and elenolic acid (fractions 1, 2 and 3 respectively) (Figure 2). EVOO lignans exhibited significant cytotoxic activities against SKBR3 cells. Fraction 6, which mainly contained 1-(+)-acetoxypinoresinol was more effective than fraction 5, which mainly contained (+)-pinoresinol (IC50s = 51 ± 2 μM and 72 ± 5 μM, respectively) (Figure 2). Among the EVOO phenolic fractions tested, those containing the secoiridoids DAOA, oleuropein aglycone, and ligstroside aglycone were the most potent at decreasing breast cancer cell viability, with all of them exhibiting IC50 values lower than 50 μM (e.g. as low as 26 ± 6 μM for the fraction 8 mainly containing the secoiridoid ligstroside aglycone) (Figure 2). Equivalent pictures emerged when the dose-response effects of EVOO polyphenols on the proliferation rate of SKBR3 were assessed by photometry after crystal violet staining. As shown in Figure 3 (left panel), EVOO single phenols and polyphenol acids produced weak growth inhibitory effects in SKBR3 cells. By contrast, the fractions 5 and 6 containing EVOO lignans (Figure 3, middle panel) and the fractions 4, 7 and 8 containing EVOO secoiridoids (Figure 3, right panel) significantly inhibited SKBR3 cell proliferation in a concentration-dependent manner.

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