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A synergistic antiproliferation effect of curcumin and docosahexaenoic acid in SK-BR-3 breast cancer cells: unique signaling not explained by the effects of either compound alone.

Altenburg JD, Bieberich AA, Terry C, Harvey KA, Vanhorn JF, Xu Z, Jo Davisson V, Siddiqui RA - BMC Cancer (2011)

Bottom Line: CCM+DHA had an antiproliferative effect in SK-BR-3, MDA-MB-231, MDA-MB-361, MCF7 and MCF10AT cells.The effect was synergistic for SK-BR-3 (ER⁻ PR⁻ Her2⁺) relative to the two compounds individually.DHA enhanced cellular uptake of CCM in SK-BR-3 cells without significantly enhancing CCM uptake in other cell lines.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cellular Biochemistry Laboratory, Methodist Research Institute, Indiana University Health, Indianapolis, Indiana, USA.

ABSTRACT

Background: Breast cancer is a collection of diseases in which molecular phenotypes can act as both indicators and mediators of therapeutic strategy. Therefore, candidate therapeutics must be assessed in the context of multiple cell lines with known molecular phenotypes. Docosahexaenoic acid (DHA) and curcumin (CCM) are dietary compounds known to antagonize breast cancer cell proliferation. We report that these compounds in combination exert a variable antiproliferative effect across multiple breast cell lines, which is synergistic in SK-BR-3 cells and triggers cell signaling events not predicted by the activity of either compound alone.

Methods: Dose response curves for CCM and DHA were generated for five breast cell lines. Effects of the DHA+ CCM combination on cell proliferation were evaluated using varying concentrations, at a fixed ratio, of CCM and DHA based on their individual ED₅₀. Detection of synergy was performed using nonlinear regression of a sigmoid dose response model and Combination Index approaches. Cell molecular network responses were investigated through whole genome microarray analysis of transcript level changes. Gene expression results were validated by RT-PCR, and western blot analysis was performed for potential signaling mediators. Cellular curcumin uptake, with and without DHA, was analyzed via flow cytometry and HPLC.

Results: CCM+DHA had an antiproliferative effect in SK-BR-3, MDA-MB-231, MDA-MB-361, MCF7 and MCF10AT cells. The effect was synergistic for SK-BR-3 (ER⁻ PR⁻ Her2⁺) relative to the two compounds individually. A whole genome microarray approach was used to investigate changes in gene expression for the synergistic effects of CCM+DHA in SK-BR-3 cells lines. CCM+DHA triggered transcript-level responses, in disease-relevant functional categories, that were largely non-overlapping with changes caused by CCM or DHA individually. Genes involved in cell cycle arrest, apoptosis, inhibition of metastasis, and cell adhesion were upregulated, whereas genes involved in cancer development and progression, metastasis, and cell cycle progression were downregulated. Cellular pools of PPARγ and phospho-p53 were increased by CCM+DHA relative to either compound alone. DHA enhanced cellular uptake of CCM in SK-BR-3 cells without significantly enhancing CCM uptake in other cell lines.

Conclusions: The combination of DHA and CCM is potentially a dietary supplemental treatment for some breast cancers, likely dependent upon molecular phenotype. DHA enhancement of cellular curcumin uptake is one potential mechanism for observed synergy in SK-BR-3 cells; however, transcriptomic data show that the antiproliferation synergy accompanies many signaling events unique to the combined presence of the two compounds.

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The effects of DHA and CCM on breast cancer cell line proliferation. SK-BR-3 (A), MDA-MB-231 (B), MDA-MB-361 (C), MCF7 (D), and MCF10AT (E) cell lines were treated for 24 hours with escalating doses of DHA (blue line), CCM (green line), or a 2:3 ratio of CCM+DHA (black line). A theoretical additive curve (red line (A)) was generated based on the curves for the individual compounds. Proliferation was measured with the WST-1 assay according to manufacturer protocol. Nonlinear regression of sigmoid dose-response model was performed with GraphPad Prism software. Results represent combinations of at least three triplicate experiments.
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Figure 1: The effects of DHA and CCM on breast cancer cell line proliferation. SK-BR-3 (A), MDA-MB-231 (B), MDA-MB-361 (C), MCF7 (D), and MCF10AT (E) cell lines were treated for 24 hours with escalating doses of DHA (blue line), CCM (green line), or a 2:3 ratio of CCM+DHA (black line). A theoretical additive curve (red line (A)) was generated based on the curves for the individual compounds. Proliferation was measured with the WST-1 assay according to manufacturer protocol. Nonlinear regression of sigmoid dose-response model was performed with GraphPad Prism software. Results represent combinations of at least three triplicate experiments.

Mentions: Initial cell proliferation assays were performed with dilution series (0-100 μM) of DHA and CCM using four breast cancer cell lines and one line (MCF10AT) representing 'premalignancy', each with a unique pattern of cellular receptor expression: SK-BR-3 (ER- PR- Her2+), MDA-MB-231 (ER- PR- Her2-), MDA-MB-361 (ER+ PR- Her2+), MCF7 (ER+ PR+ Her2-) and MCF10AT (ER+, PR isoform B but not A, Her2 variable). ED50 values were determined for DHA and CCM, for each cell line, and presented in Table 1. A combination of CCM+DHA (with proportions of DHA and CCM derived from their ED50 values) was used to measure the antiproliferation effect for each cell line at different doses (0-100 μM) as described by Tallarida [59]. The dose response curve for CCM+DHA was then compared to the theoretical additive dose response curve to determine if the combination of CCM+DHA resulted in a synergistic effect. It can be seen that the combination of CCM+DHA (2:3 ratio) when used below 50 μM exerted a synergistic effect only in the SK-BR-3 breast cancer cell line (Figure 1A). While there was no substantial difference in ED50 values, there was a significant difference in the hillslopes [(-7.6; 95% CI (-10.2, -5.1) for the theoretical additive curve and -1.9; 95% CI (-2.8, -1.0) for the actual mixture)]. This indicates that the span of doses where the actual combination of CCM+DHA is effective is much greater than expected. The synergism between DHA and CCM disappeared at higher doses, though a dose-dependent antiproliferative effect was still present. CCM+DHA also affected the MDA-MB-231, MDA-MB-361, MCF7, and MCF10AT cell lines, but subadditive to additive results were observed at all combinations tested (Figure 1B-E). Extending the treatments to 48 hours had no significant effect on the synergistic or subadditive status of each cell line (data not shown). The synergistic effect of CCM+DHA on SK-BR-3 cells was further confirmed using an alternative approach of calculating the Combination Index. The combination of CCM+DHA at concentrations below 50 μM had a Combination Index < 1, indicating synergism between two compounds (Figure 2A). We further experimentally compared antiproliferative effects of the combination with each compound individually using an optimal concentration (30 μM) and found that neither DHA or CCM were effective in inhibiting cell growth, whereas 30 μM of the combination (18 μM DHA + 12 μM CCM) significantly inhibited SK-BR-3 cell growth (Figure 2B).


A synergistic antiproliferation effect of curcumin and docosahexaenoic acid in SK-BR-3 breast cancer cells: unique signaling not explained by the effects of either compound alone.

Altenburg JD, Bieberich AA, Terry C, Harvey KA, Vanhorn JF, Xu Z, Jo Davisson V, Siddiqui RA - BMC Cancer (2011)

The effects of DHA and CCM on breast cancer cell line proliferation. SK-BR-3 (A), MDA-MB-231 (B), MDA-MB-361 (C), MCF7 (D), and MCF10AT (E) cell lines were treated for 24 hours with escalating doses of DHA (blue line), CCM (green line), or a 2:3 ratio of CCM+DHA (black line). A theoretical additive curve (red line (A)) was generated based on the curves for the individual compounds. Proliferation was measured with the WST-1 assay according to manufacturer protocol. Nonlinear regression of sigmoid dose-response model was performed with GraphPad Prism software. Results represent combinations of at least three triplicate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3111403&req=5

Figure 1: The effects of DHA and CCM on breast cancer cell line proliferation. SK-BR-3 (A), MDA-MB-231 (B), MDA-MB-361 (C), MCF7 (D), and MCF10AT (E) cell lines were treated for 24 hours with escalating doses of DHA (blue line), CCM (green line), or a 2:3 ratio of CCM+DHA (black line). A theoretical additive curve (red line (A)) was generated based on the curves for the individual compounds. Proliferation was measured with the WST-1 assay according to manufacturer protocol. Nonlinear regression of sigmoid dose-response model was performed with GraphPad Prism software. Results represent combinations of at least three triplicate experiments.
Mentions: Initial cell proliferation assays were performed with dilution series (0-100 μM) of DHA and CCM using four breast cancer cell lines and one line (MCF10AT) representing 'premalignancy', each with a unique pattern of cellular receptor expression: SK-BR-3 (ER- PR- Her2+), MDA-MB-231 (ER- PR- Her2-), MDA-MB-361 (ER+ PR- Her2+), MCF7 (ER+ PR+ Her2-) and MCF10AT (ER+, PR isoform B but not A, Her2 variable). ED50 values were determined for DHA and CCM, for each cell line, and presented in Table 1. A combination of CCM+DHA (with proportions of DHA and CCM derived from their ED50 values) was used to measure the antiproliferation effect for each cell line at different doses (0-100 μM) as described by Tallarida [59]. The dose response curve for CCM+DHA was then compared to the theoretical additive dose response curve to determine if the combination of CCM+DHA resulted in a synergistic effect. It can be seen that the combination of CCM+DHA (2:3 ratio) when used below 50 μM exerted a synergistic effect only in the SK-BR-3 breast cancer cell line (Figure 1A). While there was no substantial difference in ED50 values, there was a significant difference in the hillslopes [(-7.6; 95% CI (-10.2, -5.1) for the theoretical additive curve and -1.9; 95% CI (-2.8, -1.0) for the actual mixture)]. This indicates that the span of doses where the actual combination of CCM+DHA is effective is much greater than expected. The synergism between DHA and CCM disappeared at higher doses, though a dose-dependent antiproliferative effect was still present. CCM+DHA also affected the MDA-MB-231, MDA-MB-361, MCF7, and MCF10AT cell lines, but subadditive to additive results were observed at all combinations tested (Figure 1B-E). Extending the treatments to 48 hours had no significant effect on the synergistic or subadditive status of each cell line (data not shown). The synergistic effect of CCM+DHA on SK-BR-3 cells was further confirmed using an alternative approach of calculating the Combination Index. The combination of CCM+DHA at concentrations below 50 μM had a Combination Index < 1, indicating synergism between two compounds (Figure 2A). We further experimentally compared antiproliferative effects of the combination with each compound individually using an optimal concentration (30 μM) and found that neither DHA or CCM were effective in inhibiting cell growth, whereas 30 μM of the combination (18 μM DHA + 12 μM CCM) significantly inhibited SK-BR-3 cell growth (Figure 2B).

Bottom Line: CCM+DHA had an antiproliferative effect in SK-BR-3, MDA-MB-231, MDA-MB-361, MCF7 and MCF10AT cells.The effect was synergistic for SK-BR-3 (ER⁻ PR⁻ Her2⁺) relative to the two compounds individually.DHA enhanced cellular uptake of CCM in SK-BR-3 cells without significantly enhancing CCM uptake in other cell lines.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cellular Biochemistry Laboratory, Methodist Research Institute, Indiana University Health, Indianapolis, Indiana, USA.

ABSTRACT

Background: Breast cancer is a collection of diseases in which molecular phenotypes can act as both indicators and mediators of therapeutic strategy. Therefore, candidate therapeutics must be assessed in the context of multiple cell lines with known molecular phenotypes. Docosahexaenoic acid (DHA) and curcumin (CCM) are dietary compounds known to antagonize breast cancer cell proliferation. We report that these compounds in combination exert a variable antiproliferative effect across multiple breast cell lines, which is synergistic in SK-BR-3 cells and triggers cell signaling events not predicted by the activity of either compound alone.

Methods: Dose response curves for CCM and DHA were generated for five breast cell lines. Effects of the DHA+ CCM combination on cell proliferation were evaluated using varying concentrations, at a fixed ratio, of CCM and DHA based on their individual ED₅₀. Detection of synergy was performed using nonlinear regression of a sigmoid dose response model and Combination Index approaches. Cell molecular network responses were investigated through whole genome microarray analysis of transcript level changes. Gene expression results were validated by RT-PCR, and western blot analysis was performed for potential signaling mediators. Cellular curcumin uptake, with and without DHA, was analyzed via flow cytometry and HPLC.

Results: CCM+DHA had an antiproliferative effect in SK-BR-3, MDA-MB-231, MDA-MB-361, MCF7 and MCF10AT cells. The effect was synergistic for SK-BR-3 (ER⁻ PR⁻ Her2⁺) relative to the two compounds individually. A whole genome microarray approach was used to investigate changes in gene expression for the synergistic effects of CCM+DHA in SK-BR-3 cells lines. CCM+DHA triggered transcript-level responses, in disease-relevant functional categories, that were largely non-overlapping with changes caused by CCM or DHA individually. Genes involved in cell cycle arrest, apoptosis, inhibition of metastasis, and cell adhesion were upregulated, whereas genes involved in cancer development and progression, metastasis, and cell cycle progression were downregulated. Cellular pools of PPARγ and phospho-p53 were increased by CCM+DHA relative to either compound alone. DHA enhanced cellular uptake of CCM in SK-BR-3 cells without significantly enhancing CCM uptake in other cell lines.

Conclusions: The combination of DHA and CCM is potentially a dietary supplemental treatment for some breast cancers, likely dependent upon molecular phenotype. DHA enhancement of cellular curcumin uptake is one potential mechanism for observed synergy in SK-BR-3 cells; however, transcriptomic data show that the antiproliferation synergy accompanies many signaling events unique to the combined presence of the two compounds.

Show MeSH
Related in: MedlinePlus