title: 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.
authors.affiliation: Cellular Biochemistry Laboratory, Methodist Research Institute, Indiana University Health, Indianapolis, Indiana, USA.
authors.forename: Jeffrey D
authors.initials: JD
authors.lastname: Altenburg
authors.forename: Andrew A
authors.initials: AA
authors.lastname: Bieberich
authors.forename: Colin
authors.initials: C
authors.lastname: Terry
authors.forename: Kevin A
authors.initials: KA
authors.lastname: Harvey
authors.forename: Justin F
authors.initials: JF
authors.lastname: Vanhorn
authors.forename: Zhidong
authors.initials: Z
authors.lastname: Xu
authors.forename: V
authors.initials: V
authors.lastname: Jo Davisson
authors.forename: Rafat A
authors.initials: RA
authors.lastname: Siddiqui
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001943:Breast Neoplasms / Q000503:physiopathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D003474:Curcumin / Q000378:metabolism / Q000494:pharmacology*; D004281:Docosahexaenoic Acids / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D005260:Female; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D047495:PPAR gamma / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D047495:PPAR gamma; D016159:Tumor Suppressor Protein p53; D004281:Docosahexaenoic Acids; D003474:Curcumin