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Olive oil's bitter principle reverses acquired autoresistance to trastuzumab (Herceptin) in HER2-overexpressing breast cancer cells.

Menendez JA, Vazquez-Martin A, Colomer R, Brunet J, Carrasco-Pancorbo A, Garcia-Villalba R, Fernandez-Gutierrez A, Segura-Carretero A - BMC Cancer (2007)

Bottom Line: Both the anti-proliferative and the pro-apoptotic effects of EVOO phenolics were evaluated by using MTT-based quantification of metabolically viable cells and ELISA-based detection of histone-associated DNA fragments, respectively.Indeed, the nature of the interaction between oleuropein aglycone and trastuzumab was found to be strongly synergistic in Tzb-resistant SKBR3/Tzb100 cells.Mechanistically, oleuropein aglycone treatment significantly reduced HER2 ECD cleavage and subsequent HER2 auto-phosphorylation, while it dramatically enhanced Tzb-induced down-regulation of HER2 expression.

View Article: PubMed Central - HTML - PubMed

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

ABSTRACT

Background: A low incidence of breast cancer in the Mediterranean basin suggests that a high consumption of Extra Virgin Olive Oil (EVOO) might confer this benefit. While the anti-HER2 oncogene effects of the main omega-9 fatty acid present in EVOO triacylglycerols (i.e., oleic acid) have been recently described, the anti-breast cancer activities of EVOO non-glyceridic constituents--which consist of at least 30 phenolic compounds--remained to be evaluated.

Methods: Semi-preparative HPLC was used to isolate EVOO polyphenols (i.e., tyrosol, hydroxytyrosol, oleuropein). Both the anti-proliferative and the pro-apoptotic effects of EVOO phenolics were evaluated by using MTT-based quantification of metabolically viable cells and ELISA-based detection of histone-associated DNA fragments, respectively. The nature of the interaction between oleuropein aglycone and the anti-HER2 monoclonal antibody trastuzumab (Herceptin) was mathematically evaluated by the dose-oriented isobologram technique. HER2-specific ELISAs were employed to quantitatively assess both the basal cleavage of the HER2 extracellular domain (ECD) and the expression level of total HER2. The activation status of HER2 was evaluated by immunoblotting procedures using a monoclonal antibody specifically recognizing the tyrosine phosphorylated (Phosphor-Tyr1248) form of HER2.

Results: Among EVOO polyphenols tested, oleuropein aglycone was the most potent EVOO phenolic in decreasing breast cancer cell viability. HER2 gene-amplified SKBR3 cells were ~5-times more sensitive to oleuropein aglycone than HER2-negative MCF-7 cells. Retroviral infection of the HER2 oncogene in MCF-7 cells resulted in a "SKBR3-assimilated" phenotype of hypersensitivity to oleuropein aglycone. An up to 50-fold increase in the efficacy of trastuzumab occurred in the presence of oleuropein aglycone. A preclinical model of acquired autoresistance to trastuzumab (SKBR3/Tzb100 cells) completely recovered trastuzumab sensitivity (> 1,000-fold sensitization) when co-cultured in the presence of oleuropein aglycone. Indeed, the nature of the interaction between oleuropein aglycone and trastuzumab was found to be strongly synergistic in Tzb-resistant SKBR3/Tzb100 cells. Mechanistically, oleuropein aglycone treatment significantly reduced HER2 ECD cleavage and subsequent HER2 auto-phosphorylation, while it dramatically enhanced Tzb-induced down-regulation of HER2 expression.

Conclusion: Olive oil's bitter principle (i.e., oleuropein aglycone) is among the first examples of how selected nutrients from an EVOO-rich "Mediterranean diet" directly regulate HER2-driven breast cancer disease.

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Effects of oleuropein aglycone on breast cancer apoptotic cell death. Quantification of apoptosis-related cell death in MCF-7, MCF-7/(pBABE)HER2 and SKBR3 cells treated with increasing concentrations of oleuropein aglycone was determined by Cell Death ELISA as described in "Material and methods". The enrichment of histone-DNA fragments in oleuropein aglycone-treated cells was expressed as fold-increase in absorbance by comparing with control (vehicle-treated) cells using the following formula: [A405 – A490]TREATED/[A405-A490]UNTREATED. Data are the mean (columns) and 95% confidence intervals (bars) of three independent experiments performed in duplicate. One-factor ANOVA was used to analyze differences in the percentage of apoptosis between the various treatment groups and the control group. * P < .01; ** P < .001; N.S.: Not statistically significant (one-factor analysis of variance). All statistical tests were two-sided.
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Figure 2: Effects of oleuropein aglycone on breast cancer apoptotic cell death. Quantification of apoptosis-related cell death in MCF-7, MCF-7/(pBABE)HER2 and SKBR3 cells treated with increasing concentrations of oleuropein aglycone was determined by Cell Death ELISA as described in "Material and methods". The enrichment of histone-DNA fragments in oleuropein aglycone-treated cells was expressed as fold-increase in absorbance by comparing with control (vehicle-treated) cells using the following formula: [A405 – A490]TREATED/[A405-A490]UNTREATED. Data are the mean (columns) and 95% confidence intervals (bars) of three independent experiments performed in duplicate. One-factor ANOVA was used to analyze differences in the percentage of apoptosis between the various treatment groups and the control group. * P < .01; ** P < .001; N.S.: Not statistically significant (one-factor analysis of variance). All statistical tests were two-sided.

Mentions: We speculated that the increased sensitivity to oleuropein aglycone that occurs in HER2-overexpressing breast cancer cells was not simply the result of changes in cell proliferation, but it might actually be due to changes in apoptotic cell death. To address this question, cells were exposed to increasing concentrations of oleuropein aglycone, apoptotic cell death was measured by a Cell Death ELISA that detects apoptosis-induced DNA-histone fragmentation, and the x-fold increase in apoptosis was calculated by comparing the ELISA optical density readings of treated samples, with the values of untreated cells as 1.0. HER2-negative MCF-7 cells exhibited the lowest degree of apoptosis upon treatment with oleuropein aglycone (up to 1.8-fold increase versus 1.0-fold in untreated control MCF-7 cells; Figure 2, left panel). Interestingly, forced expression of HER2 in MCF-7 cells increased by ~2-fold the apoptotic effects of oleuropein aglycone. Thus, treatment with 50 μM oleuropein aglycone increased apoptotic cell death by 3.2 times in MCF-7/(pBABE)HER2 cells (Figure 2, middle panel). HER2-overexpressing SKBR3 cells were exquisitely sensitive to oleuropein aglycone-induced apoptosis. They exhibited the highest degree of apoptotic cell death following exposure to oleuropein aglycone (up to ~6.0-fold increase versus 1.0-fold in untreated control SKBR3 cells; Figure 2, right panel). The results support the notion that HER2 overexpression associates with an increased sensitivity to oleuropein aglycone-induced breast cancer cell damage (Figure 3).


Olive oil's bitter principle reverses acquired autoresistance to trastuzumab (Herceptin) in HER2-overexpressing breast cancer cells.

Menendez JA, Vazquez-Martin A, Colomer R, Brunet J, Carrasco-Pancorbo A, Garcia-Villalba R, Fernandez-Gutierrez A, Segura-Carretero A - BMC Cancer (2007)

Effects of oleuropein aglycone on breast cancer apoptotic cell death. Quantification of apoptosis-related cell death in MCF-7, MCF-7/(pBABE)HER2 and SKBR3 cells treated with increasing concentrations of oleuropein aglycone was determined by Cell Death ELISA as described in "Material and methods". The enrichment of histone-DNA fragments in oleuropein aglycone-treated cells was expressed as fold-increase in absorbance by comparing with control (vehicle-treated) cells using the following formula: [A405 – A490]TREATED/[A405-A490]UNTREATED. Data are the mean (columns) and 95% confidence intervals (bars) of three independent experiments performed in duplicate. One-factor ANOVA was used to analyze differences in the percentage of apoptosis between the various treatment groups and the control group. * P < .01; ** P < .001; N.S.: Not statistically significant (one-factor analysis of variance). All statistical tests were two-sided.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1878493&req=5

Figure 2: Effects of oleuropein aglycone on breast cancer apoptotic cell death. Quantification of apoptosis-related cell death in MCF-7, MCF-7/(pBABE)HER2 and SKBR3 cells treated with increasing concentrations of oleuropein aglycone was determined by Cell Death ELISA as described in "Material and methods". The enrichment of histone-DNA fragments in oleuropein aglycone-treated cells was expressed as fold-increase in absorbance by comparing with control (vehicle-treated) cells using the following formula: [A405 – A490]TREATED/[A405-A490]UNTREATED. Data are the mean (columns) and 95% confidence intervals (bars) of three independent experiments performed in duplicate. One-factor ANOVA was used to analyze differences in the percentage of apoptosis between the various treatment groups and the control group. * P < .01; ** P < .001; N.S.: Not statistically significant (one-factor analysis of variance). All statistical tests were two-sided.
Mentions: We speculated that the increased sensitivity to oleuropein aglycone that occurs in HER2-overexpressing breast cancer cells was not simply the result of changes in cell proliferation, but it might actually be due to changes in apoptotic cell death. To address this question, cells were exposed to increasing concentrations of oleuropein aglycone, apoptotic cell death was measured by a Cell Death ELISA that detects apoptosis-induced DNA-histone fragmentation, and the x-fold increase in apoptosis was calculated by comparing the ELISA optical density readings of treated samples, with the values of untreated cells as 1.0. HER2-negative MCF-7 cells exhibited the lowest degree of apoptosis upon treatment with oleuropein aglycone (up to 1.8-fold increase versus 1.0-fold in untreated control MCF-7 cells; Figure 2, left panel). Interestingly, forced expression of HER2 in MCF-7 cells increased by ~2-fold the apoptotic effects of oleuropein aglycone. Thus, treatment with 50 μM oleuropein aglycone increased apoptotic cell death by 3.2 times in MCF-7/(pBABE)HER2 cells (Figure 2, middle panel). HER2-overexpressing SKBR3 cells were exquisitely sensitive to oleuropein aglycone-induced apoptosis. They exhibited the highest degree of apoptotic cell death following exposure to oleuropein aglycone (up to ~6.0-fold increase versus 1.0-fold in untreated control SKBR3 cells; Figure 2, right panel). The results support the notion that HER2 overexpression associates with an increased sensitivity to oleuropein aglycone-induced breast cancer cell damage (Figure 3).

Bottom Line: Both the anti-proliferative and the pro-apoptotic effects of EVOO phenolics were evaluated by using MTT-based quantification of metabolically viable cells and ELISA-based detection of histone-associated DNA fragments, respectively.Indeed, the nature of the interaction between oleuropein aglycone and trastuzumab was found to be strongly synergistic in Tzb-resistant SKBR3/Tzb100 cells.Mechanistically, oleuropein aglycone treatment significantly reduced HER2 ECD cleavage and subsequent HER2 auto-phosphorylation, while it dramatically enhanced Tzb-induced down-regulation of HER2 expression.

View Article: PubMed Central - HTML - PubMed

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

ABSTRACT

Background: A low incidence of breast cancer in the Mediterranean basin suggests that a high consumption of Extra Virgin Olive Oil (EVOO) might confer this benefit. While the anti-HER2 oncogene effects of the main omega-9 fatty acid present in EVOO triacylglycerols (i.e., oleic acid) have been recently described, the anti-breast cancer activities of EVOO non-glyceridic constituents--which consist of at least 30 phenolic compounds--remained to be evaluated.

Methods: Semi-preparative HPLC was used to isolate EVOO polyphenols (i.e., tyrosol, hydroxytyrosol, oleuropein). Both the anti-proliferative and the pro-apoptotic effects of EVOO phenolics were evaluated by using MTT-based quantification of metabolically viable cells and ELISA-based detection of histone-associated DNA fragments, respectively. The nature of the interaction between oleuropein aglycone and the anti-HER2 monoclonal antibody trastuzumab (Herceptin) was mathematically evaluated by the dose-oriented isobologram technique. HER2-specific ELISAs were employed to quantitatively assess both the basal cleavage of the HER2 extracellular domain (ECD) and the expression level of total HER2. The activation status of HER2 was evaluated by immunoblotting procedures using a monoclonal antibody specifically recognizing the tyrosine phosphorylated (Phosphor-Tyr1248) form of HER2.

Results: Among EVOO polyphenols tested, oleuropein aglycone was the most potent EVOO phenolic in decreasing breast cancer cell viability. HER2 gene-amplified SKBR3 cells were ~5-times more sensitive to oleuropein aglycone than HER2-negative MCF-7 cells. Retroviral infection of the HER2 oncogene in MCF-7 cells resulted in a "SKBR3-assimilated" phenotype of hypersensitivity to oleuropein aglycone. An up to 50-fold increase in the efficacy of trastuzumab occurred in the presence of oleuropein aglycone. A preclinical model of acquired autoresistance to trastuzumab (SKBR3/Tzb100 cells) completely recovered trastuzumab sensitivity (> 1,000-fold sensitization) when co-cultured in the presence of oleuropein aglycone. Indeed, the nature of the interaction between oleuropein aglycone and trastuzumab was found to be strongly synergistic in Tzb-resistant SKBR3/Tzb100 cells. Mechanistically, oleuropein aglycone treatment significantly reduced HER2 ECD cleavage and subsequent HER2 auto-phosphorylation, while it dramatically enhanced Tzb-induced down-regulation of HER2 expression.

Conclusion: Olive oil's bitter principle (i.e., oleuropein aglycone) is among the first examples of how selected nutrients from an EVOO-rich "Mediterranean diet" directly regulate HER2-driven breast cancer disease.

Show MeSH
Related in: MedlinePlus