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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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Effect of DHA on CCM uptake. Cells were treated with 20 μM CCM for 24 hours. CCM uptake was quantified by flow cytometry (A) in comparison with HPLC (B) as described in Materials and Methods. (C) SK-BR-3, MDA-MB-231, MDA-MB-361, MCF7, and MCF10AT cell lines were treated with escalating doses of CCM in the presence or absence of 10 μM DHA and analyzed by flow cytometry. Fold changes (A, C) were compared to respective cell line controls (without CCM or DHA). *P < 0.05 for Student's t-tests comparing the treatments with DHA to the treatments without DHA in three duplicate assays.
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Figure 6: Effect of DHA on CCM uptake. Cells were treated with 20 μM CCM for 24 hours. CCM uptake was quantified by flow cytometry (A) in comparison with HPLC (B) as described in Materials and Methods. (C) SK-BR-3, MDA-MB-231, MDA-MB-361, MCF7, and MCF10AT cell lines were treated with escalating doses of CCM in the presence or absence of 10 μM DHA and analyzed by flow cytometry. Fold changes (A, C) were compared to respective cell line controls (without CCM or DHA). *P < 0.05 for Student's t-tests comparing the treatments with DHA to the treatments without DHA in three duplicate assays.

Mentions: While the mechanism of CCM entry into cells is unknown, we have analyzed the differences among four breast cancer cell lines for ability to absorb CCM. We quantified CCM uptake by flow cytometry as CCM is known to fluoresce in the green band [63,64]. In order to validate the flow cytometry results, HPLC analysis was also performed concurrently with initial flow cytometry experiments (Figure 6A-B). The flow cytometry data match trends seen in the HPLC results, but with smaller dynamic range. SK-BR-3 cells treated with 20 μM CCM absorbed four-fold greater levels of CCM than the MCF7 cells and 10-fold greater levels than the MDA-MB-231 and MDA-MB-361 cells (Figure 6C). Combining DHA with CCM further enhances the CCM absorption of SK-BR-3 cells without significantly enhancing CCM uptake in the other cell lines (Figure 6C). This suggests that DHA enhancement of cellular permissiveness for CCM absorption is a potential mechanism for the increased antiproliferative effects. It should be noted that both HPLC and flow cytometry do not distinguish CCM taken into the cell from CCM that is bound to the cell surface. However, it is clear that the SK-BR-3 cells exhibit a higher level of CCM fluorescence than the other cell lines and that the fluorescence is increased in the presence of DHA. When viewed with the data describing antiproliferative effect and molecular level changes, it is unlikely that these data reflect only CCM on cell surfaces.


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)

Effect of DHA on CCM uptake. Cells were treated with 20 μM CCM for 24 hours. CCM uptake was quantified by flow cytometry (A) in comparison with HPLC (B) as described in Materials and Methods. (C) SK-BR-3, MDA-MB-231, MDA-MB-361, MCF7, and MCF10AT cell lines were treated with escalating doses of CCM in the presence or absence of 10 μM DHA and analyzed by flow cytometry. Fold changes (A, C) were compared to respective cell line controls (without CCM or DHA). *P < 0.05 for Student's t-tests comparing the treatments with DHA to the treatments without DHA in three duplicate assays.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Effect of DHA on CCM uptake. Cells were treated with 20 μM CCM for 24 hours. CCM uptake was quantified by flow cytometry (A) in comparison with HPLC (B) as described in Materials and Methods. (C) SK-BR-3, MDA-MB-231, MDA-MB-361, MCF7, and MCF10AT cell lines were treated with escalating doses of CCM in the presence or absence of 10 μM DHA and analyzed by flow cytometry. Fold changes (A, C) were compared to respective cell line controls (without CCM or DHA). *P < 0.05 for Student's t-tests comparing the treatments with DHA to the treatments without DHA in three duplicate assays.
Mentions: While the mechanism of CCM entry into cells is unknown, we have analyzed the differences among four breast cancer cell lines for ability to absorb CCM. We quantified CCM uptake by flow cytometry as CCM is known to fluoresce in the green band [63,64]. In order to validate the flow cytometry results, HPLC analysis was also performed concurrently with initial flow cytometry experiments (Figure 6A-B). The flow cytometry data match trends seen in the HPLC results, but with smaller dynamic range. SK-BR-3 cells treated with 20 μM CCM absorbed four-fold greater levels of CCM than the MCF7 cells and 10-fold greater levels than the MDA-MB-231 and MDA-MB-361 cells (Figure 6C). Combining DHA with CCM further enhances the CCM absorption of SK-BR-3 cells without significantly enhancing CCM uptake in the other cell lines (Figure 6C). This suggests that DHA enhancement of cellular permissiveness for CCM absorption is a potential mechanism for the increased antiproliferative effects. It should be noted that both HPLC and flow cytometry do not distinguish CCM taken into the cell from CCM that is bound to the cell surface. However, it is clear that the SK-BR-3 cells exhibit a higher level of CCM fluorescence than the other cell lines and that the fluorescence is increased in the presence of DHA. When viewed with the data describing antiproliferative effect and molecular level changes, it is unlikely that these data reflect only CCM on cell surfaces.

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