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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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Related in: MedlinePlus

A. Exogenous supplementation with oleuropein aglycone down-regulates HER2 expression and synergistically enhances trastuzumab-induced depletion of HER2 in trastuzumab-resistant SKBR3/Tzb100 cells. Overnight serum-starved SKBR3/Tzb100 cells were cultured in DMEM-0.1% FBS supplemented with increasing concentrations of oleuropein aglycone in the absence or presence of trastuzumab for 72 h. The Oncogene Science HER2 microtiter ELISA was used according to the manufacturer's instructions to compare HER2 concentrations in cell pellets. B. Oleuropein aglycone treatment does not affect EGFR (HER1) expression in trastuzumab-sensitive SKBR3 parental cell line (SKBR3/Wild Type) and in its derivative trastuzumab-resistant SKBR3/Tzb100 pool. Overnight serum-starved SKBR3/Tzb100 cells were cultured in DMEM-0.1% FBS supplemented with increasing concentrations of oleuropein aglycone in the absence or presence of trastuzumab for 72 h. The Oncogene Science HER1 (EGFR) microtiter ELISA was used according to the manufacturer's instructions to compare HER1 concentrations in cell pellets. Results in A and B 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 by asterisks (* P < .01, ** P < .001). All statistical tests were two-sided.
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Figure 7: A. Exogenous supplementation with oleuropein aglycone down-regulates HER2 expression and synergistically enhances trastuzumab-induced depletion of HER2 in trastuzumab-resistant SKBR3/Tzb100 cells. Overnight serum-starved SKBR3/Tzb100 cells were cultured in DMEM-0.1% FBS supplemented with increasing concentrations of oleuropein aglycone in the absence or presence of trastuzumab for 72 h. The Oncogene Science HER2 microtiter ELISA was used according to the manufacturer's instructions to compare HER2 concentrations in cell pellets. B. Oleuropein aglycone treatment does not affect EGFR (HER1) expression in trastuzumab-sensitive SKBR3 parental cell line (SKBR3/Wild Type) and in its derivative trastuzumab-resistant SKBR3/Tzb100 pool. Overnight serum-starved SKBR3/Tzb100 cells were cultured in DMEM-0.1% FBS supplemented with increasing concentrations of oleuropein aglycone in the absence or presence of trastuzumab for 72 h. The Oncogene Science HER1 (EGFR) microtiter ELISA was used according to the manufacturer's instructions to compare HER1 concentrations in cell pellets. Results in A and B 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 by asterisks (* P < .01, ** P < .001). All statistical tests were two-sided.

Mentions: The drawbacks to our approach (i.e., trastuzumab-resistant pool from once cell line) are that pools may contain cells with various degrees of resistance and that the pool developed during this study may harbor resistance acquired over time or may represent a selected subpopulation of cells with inherent resistance. However, pools also represent the alterations that are represented in the majority of tumor cell populations, whereas clones -another approach that would have been used to answer our research question- represent just one isolated alteration. Nonetheless, to eliminate the possibility of HER2-independent mechanisms for the sensitizing effects of oleuropein aglycone on trastuzumab-resistant breast cancer cells, we performed HER2-specific ELISAs to quantitatively determine the expression levels of HER2 in SKBR3/Tzb100 cells. This resistant pool, when maintained in the absence of trastuzumab for 72 h, exhibited HER2 levels notably higher to those found in trastuzumab-sensitive SKBR3 parental cells (~2-fold increase; Figure 7). This "HER2 super-expression", which has not been reported in earlier trastuzumab-resistant breast cancer models [58-60], decreased back and even below the baseline HER2 level found in trastuzumab-sensitive SKBR3 parental cells when SKBR3/Tzb100 cells were treated with oleuropein aglycone (up to 71% reduction; Figure 7A). Trastuzumab exposure was still capable to reduce HER2 by 21% in SKBR3/Tzb100 cells (a percentage comparable to that found in trastuzumab-sensitive SKBR3 parental cells). Interestingly, trastuzumab treatment drastically down-regulated HER2 expression by 83% in the presence of 50 μM oleuropein aglycone, which, as single agent, decreased HER2 "super-expression" by 43% in SKBR3/Tzb100 cells.


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)

A. Exogenous supplementation with oleuropein aglycone down-regulates HER2 expression and synergistically enhances trastuzumab-induced depletion of HER2 in trastuzumab-resistant SKBR3/Tzb100 cells. Overnight serum-starved SKBR3/Tzb100 cells were cultured in DMEM-0.1% FBS supplemented with increasing concentrations of oleuropein aglycone in the absence or presence of trastuzumab for 72 h. The Oncogene Science HER2 microtiter ELISA was used according to the manufacturer's instructions to compare HER2 concentrations in cell pellets. B. Oleuropein aglycone treatment does not affect EGFR (HER1) expression in trastuzumab-sensitive SKBR3 parental cell line (SKBR3/Wild Type) and in its derivative trastuzumab-resistant SKBR3/Tzb100 pool. Overnight serum-starved SKBR3/Tzb100 cells were cultured in DMEM-0.1% FBS supplemented with increasing concentrations of oleuropein aglycone in the absence or presence of trastuzumab for 72 h. The Oncogene Science HER1 (EGFR) microtiter ELISA was used according to the manufacturer's instructions to compare HER1 concentrations in cell pellets. Results in A and B 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 by asterisks (* P < .01, ** P < .001). 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 7: A. Exogenous supplementation with oleuropein aglycone down-regulates HER2 expression and synergistically enhances trastuzumab-induced depletion of HER2 in trastuzumab-resistant SKBR3/Tzb100 cells. Overnight serum-starved SKBR3/Tzb100 cells were cultured in DMEM-0.1% FBS supplemented with increasing concentrations of oleuropein aglycone in the absence or presence of trastuzumab for 72 h. The Oncogene Science HER2 microtiter ELISA was used according to the manufacturer's instructions to compare HER2 concentrations in cell pellets. B. Oleuropein aglycone treatment does not affect EGFR (HER1) expression in trastuzumab-sensitive SKBR3 parental cell line (SKBR3/Wild Type) and in its derivative trastuzumab-resistant SKBR3/Tzb100 pool. Overnight serum-starved SKBR3/Tzb100 cells were cultured in DMEM-0.1% FBS supplemented with increasing concentrations of oleuropein aglycone in the absence or presence of trastuzumab for 72 h. The Oncogene Science HER1 (EGFR) microtiter ELISA was used according to the manufacturer's instructions to compare HER1 concentrations in cell pellets. Results in A and B 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 by asterisks (* P < .01, ** P < .001). All statistical tests were two-sided.
Mentions: The drawbacks to our approach (i.e., trastuzumab-resistant pool from once cell line) are that pools may contain cells with various degrees of resistance and that the pool developed during this study may harbor resistance acquired over time or may represent a selected subpopulation of cells with inherent resistance. However, pools also represent the alterations that are represented in the majority of tumor cell populations, whereas clones -another approach that would have been used to answer our research question- represent just one isolated alteration. Nonetheless, to eliminate the possibility of HER2-independent mechanisms for the sensitizing effects of oleuropein aglycone on trastuzumab-resistant breast cancer cells, we performed HER2-specific ELISAs to quantitatively determine the expression levels of HER2 in SKBR3/Tzb100 cells. This resistant pool, when maintained in the absence of trastuzumab for 72 h, exhibited HER2 levels notably higher to those found in trastuzumab-sensitive SKBR3 parental cells (~2-fold increase; Figure 7). This "HER2 super-expression", which has not been reported in earlier trastuzumab-resistant breast cancer models [58-60], decreased back and even below the baseline HER2 level found in trastuzumab-sensitive SKBR3 parental cells when SKBR3/Tzb100 cells were treated with oleuropein aglycone (up to 71% reduction; Figure 7A). Trastuzumab exposure was still capable to reduce HER2 by 21% in SKBR3/Tzb100 cells (a percentage comparable to that found in trastuzumab-sensitive SKBR3 parental cells). Interestingly, trastuzumab treatment drastically down-regulated HER2 expression by 83% in the presence of 50 μM oleuropein aglycone, which, as single agent, decreased HER2 "super-expression" by 43% in SKBR3/Tzb100 cells.

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