Limits...
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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3111403&req=5

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.

Mentions: The most striking initial result was that CCM+DHA caused far fewer significant transcript level changes in SK-BR-3 cells than CCM alone, even though CCM+DHA had a greater antiproliferative effect. A cursory examination of the data also showed that some of the transcripts responding to CCM+DHA did not appear as significant responders in either of the other two treatments. Therefore, our strategy for an initial informatic analysis of these data was to rank CCM+DHA responders by magnitude of change, place a minimum cutoff at 5-fold (up or down), and annotate genes appearing within this set according to known disease-relevant functions. In addition, CCM+DHA responders below 5-fold were scanned for genes of known relevance to antiproliferation processes, such as caspases. Figure 3 illustrates the results in the form of a heatmap divided into functional blocks. This strategy recovered an interesting picture showing 31 functionally diverse transcript regulatory responses, many of which are unique to the CCM+DHA combination. We observed that genes involved in tumor progression/growth, cell cycle progression, metastasis, and anti-apoptosis/survival were synergistically downregulated by the combination, while genes involved in apoptosis, tumor suppression, and inhibition of metastasis were synergistically upregulated. Interestingly, we also found a group of cytochrome p450 genes (1A1, 1A2, 1B1), which are involved in enhancement of anti-cancer effects of small molecules, including some dietary compounds (see discussion below). Results from the microarray transcript analysis were further validated by RT-PCR for selected genes, and our results (Figure 3B) have validated changes observed within the microarray data. RT-PCR also confirmed that CYP1A1/CYP1A2 and CXCR4 did not respond to DHA or CCM alone, but their expression was stimulated or reduced, respectively, by combined DHA+CCM treatment (Figure 3B). Similarly, RT-PCR data also verified other directional changes indicating that the CCM effect on CYP1B1 was reversed by CCM+DHA, whereas SERPINB5 expression was enhanced following CCM+DHA treatment. While it is important to confirm expression levels of proteins directly before concluding specific signaling mechanisms, the genome-wide study of transcript and protein level correlation by Shankavaram et al. demonstrates predictive power of transcripts for protein levels across the NCI60 cell line panel[65]. Therefore, as a hypothesis building tool, transcript screens serve as valuable indicators of relevant network areas to study further.


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)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

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.
Mentions: The most striking initial result was that CCM+DHA caused far fewer significant transcript level changes in SK-BR-3 cells than CCM alone, even though CCM+DHA had a greater antiproliferative effect. A cursory examination of the data also showed that some of the transcripts responding to CCM+DHA did not appear as significant responders in either of the other two treatments. Therefore, our strategy for an initial informatic analysis of these data was to rank CCM+DHA responders by magnitude of change, place a minimum cutoff at 5-fold (up or down), and annotate genes appearing within this set according to known disease-relevant functions. In addition, CCM+DHA responders below 5-fold were scanned for genes of known relevance to antiproliferation processes, such as caspases. Figure 3 illustrates the results in the form of a heatmap divided into functional blocks. This strategy recovered an interesting picture showing 31 functionally diverse transcript regulatory responses, many of which are unique to the CCM+DHA combination. We observed that genes involved in tumor progression/growth, cell cycle progression, metastasis, and anti-apoptosis/survival were synergistically downregulated by the combination, while genes involved in apoptosis, tumor suppression, and inhibition of metastasis were synergistically upregulated. Interestingly, we also found a group of cytochrome p450 genes (1A1, 1A2, 1B1), which are involved in enhancement of anti-cancer effects of small molecules, including some dietary compounds (see discussion below). Results from the microarray transcript analysis were further validated by RT-PCR for selected genes, and our results (Figure 3B) have validated changes observed within the microarray data. RT-PCR also confirmed that CYP1A1/CYP1A2 and CXCR4 did not respond to DHA or CCM alone, but their expression was stimulated or reduced, respectively, by combined DHA+CCM treatment (Figure 3B). Similarly, RT-PCR data also verified other directional changes indicating that the CCM effect on CYP1B1 was reversed by CCM+DHA, whereas SERPINB5 expression was enhanced following CCM+DHA treatment. While it is important to confirm expression levels of proteins directly before concluding specific signaling mechanisms, the genome-wide study of transcript and protein level correlation by Shankavaram et al. demonstrates predictive power of transcripts for protein levels across the NCI60 cell line panel[65]. Therefore, as a hypothesis building tool, transcript screens serve as valuable indicators of relevant network areas to study further.

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