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

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.

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.

Figure 2

The synergistic effect of CCM+DHA on SK-BR-3 proliferation. (A) Combination index (CI) calculated for the SK-BR-3 cell proliferation. (B) Direct comparisons of 30 μM treatments with CCM and DHA individually with 30 μM treatment using the 2:3 ratio CCM+DHA combination. P < 0.05 for three triplicate experiments.

Figure 3

Transcripts of functional importance relative to the synergistic antiproliferative effect of the curcumin/DHA combination. (A) All genes are labeled according to current HGNC symbols. Numbers to the right of gene symbols indicate references in a separate transcript annotation bibliography (see Additional file 8). Fold-change and associated P values corresponding to this figure can be found in Additional file 9. Heatmap values are log₂-transformed, normalized fluorescence ratios for untreated versus treated cells (see methods), with green indicating upregulation and red indicating downregulation relative to untreated SK-BR-3 cells. All responses shown for DHA+CCM were 2-fold or greater, p < 0.01, on three replicate arrays. For the purpose of visual comparison within a heatmap format, values for non-significant responses are mean normalized fluorescence ratios from three replicate arrays. For genes represented by more than one chip feature in the Agilent platform, mean normalized fluorescence ratio was retrieved for the feature producing the lowest mean P value. As such, some responses in this figure appear greater than 2-fold but were not significant according to the criterion p < 0.01. (B) RNA was isolated from SK-BR-3 cells treated with 30 μM DHA, μM CCM, or a mix of 12 μM CCM+18 μM DHA. RT-PCR was performed for selected genes in order to validate the microarray data. Results are representative of three separate experiments.

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.

Figure 5

Effects of CCM and DHA on p53 and PPARγ. SK-BR-3 (A) and MCF-7 (B) cells were treated with 30 μM DHA, CCM, or the CCM+DHA combination (12 μM CCM and 18 μM DHA) for 24 hours in 6 well plates. (C) SK-BR-3 cells were treated with escalating doses of CCM or DHA individually. Cells were lysed and analyzed by SDS-PAGE. Blots were probed for phosphorylated p53, overall p53, p21, PPARγ, and GAPDH as indicated. Data are representative of three separate duplicate experiments.

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.