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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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Related in: MedlinePlus

CCM+DHA inhibit proliferation through a caspase-mediated process. SK-BR-3 cells were pretreated for one hour with 20 μM Z-VAD-FMK, a pan-caspase inhibitor, prior to the addition of escalating doses of CCM+DHA (2:3 ratios). After 24 hour incubation, proliferation was analyzed with WST-1 reagent according to manufacturer protocol. Results are representative of three separate triplicate experiments. *P < 0.05 for Student's t-tests comparing the treatments with or without the pan-caspase inhibitor.
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Figure 4: CCM+DHA inhibit proliferation through a caspase-mediated process. SK-BR-3 cells were pretreated for one hour with 20 μM Z-VAD-FMK, a pan-caspase inhibitor, prior to the addition of escalating doses of CCM+DHA (2:3 ratios). After 24 hour incubation, proliferation was analyzed with WST-1 reagent according to manufacturer protocol. Results are representative of three separate triplicate experiments. *P < 0.05 for Student's t-tests comparing the treatments with or without the pan-caspase inhibitor.

Mentions: As shown above, our data clearly indicated an induction of genes involved in apoptosis. In order to further validate contribution of apoptosis to the reduction of cell proliferation during CCM+DHA treatment, we used a pan-caspase inhibitor (Z-VAD-FMK) to inhibit initiation of the apoptotic process. Data shown in Figure 4 indicate that CCM and DHA, in varying concentrations of a fixed 2:3 ratio, inhibited cell proliferation in a dose dependent manner (20-90 μM), and the pan-caspase inhibitor significantly reduced the effect of CCM+DHA at all concentrations tested.


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)

CCM+DHA inhibit proliferation through a caspase-mediated process. SK-BR-3 cells were pretreated for one hour with 20 μM Z-VAD-FMK, a pan-caspase inhibitor, prior to the addition of escalating doses of CCM+DHA (2:3 ratios). After 24 hour incubation, proliferation was analyzed with WST-1 reagent according to manufacturer protocol. Results are representative of three separate triplicate experiments. *P < 0.05 for Student's t-tests comparing the treatments with or without the pan-caspase inhibitor.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: CCM+DHA inhibit proliferation through a caspase-mediated process. SK-BR-3 cells were pretreated for one hour with 20 μM Z-VAD-FMK, a pan-caspase inhibitor, prior to the addition of escalating doses of CCM+DHA (2:3 ratios). After 24 hour incubation, proliferation was analyzed with WST-1 reagent according to manufacturer protocol. Results are representative of three separate triplicate experiments. *P < 0.05 for Student's t-tests comparing the treatments with or without the pan-caspase inhibitor.
Mentions: As shown above, our data clearly indicated an induction of genes involved in apoptosis. In order to further validate contribution of apoptosis to the reduction of cell proliferation during CCM+DHA treatment, we used a pan-caspase inhibitor (Z-VAD-FMK) to inhibit initiation of the apoptotic process. Data shown in Figure 4 indicate that CCM and DHA, in varying concentrations of a fixed 2:3 ratio, inhibited cell proliferation in a dose dependent manner (20-90 μM), and the pan-caspase inhibitor significantly reduced the effect of CCM+DHA at all concentrations tested.

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