Can transcriptomics provide insight into the chemopreventive mechanisms of complex mixtures of phytochemicals in humans?

Abstract

Blueberries contain relatively large amounts of different phytochemicals, which are suggested to have chemopreventive properties, but little information is available on the underlying molecular modes of action. This study investigates whole genome gene expression changes in lymphocytes of 143 humans after a 4-week blueberry-apple juice dietary intervention. Differentially expressed genes and genes correlating with the extent of antioxidant protection were identified in four subgroups. The magnitude of the preventive effect after the intervention differed between these four subgroups. Furthermore, subjects in two groups carried genetic polymorphisms that were previously found to influence the chemopreventive response. Pathway analysis of the identified genes showed strong but complex gene expression changes in pathways signaling for apoptosis, immune response, cell adhesion, and lipid metabolism. These pathways indicate increased apoptosis, upgraded growth control, induced immunity, reduced platelet aggregation and activation, blood glucose homeostasis, and regulation of fatty acid metabolism. Based on these observations, we hypothesize that combining transcriptomic data with phenotypic markers of oxidative stress may provide insight into the relevant cellular processes and genetic pathways, which contribute to the antioxidant response of complex mixtures of phytochemicals, such as found in blueberry-apple juice.

FIG. 1.

Venn diagrams created using Venny (5) of GeneGo cellular processes after (A) pairwise t-test analyses, and (B) correlation analyses, as found by MetaCore analyses of significantly modulated genes. Analyses included the following: 1. all subjects; 2. comet group tail moment (TM)<−2: subjects demonstrating a significant reduction in oxidative DNA damage upon ex vivo challenge of lymphocytes as indicated by difference in mean TM<−2; 3. GSTT1 wild types: subjects carrying the GSTT1 wild-type variant; and 4. XRCC1 wild types: subjects carrying the XRCC1 wild-type variant. Numbers indicate the amount of cellular processes and characters indicate the type of cellular processes. A: Immune response; B: Development; C: Apoptosis; D: Neurophysiological processes; E: G-protein signaling; F: Transport; G: Muscle contraction; H: Transcription; I: Carbohydrate metabolism; J: Lipid metabolism; K: Translation; L: Cystic fibrosis; M: Cell adhesion; N: Neoplastic processes; O: Proteolysis; P: Cytoskeleton remodeling development; Q: Nucleotide metabolism; R: Cell cycle; S: Amino acid metabolism; T: Reproduction; U: Vitamin and cofactor metabolism; V: Oxidative stress. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 2.

Shortest path network of genes involved in apoptosis related pathways developed in MetaCore including the affected biological processes. (A) Network description: Shortest path network prefiltered on a subcellular level indicating the localization of gene expression of a selection of genes modulated in apoptosis-related pathways. Those genes for which the gene product eventually will result in a cellular effect were defined as target genes and grouped at the base of the network. The color of the upper right small circle at each network object indicates whether the gene was upregulated (red) or downregulated (blue). Detailed information on the symbols can be found at www.genego.com/pdf/MC_legend.pdf. Gene Symbols, full names and information on the function of the genes, including their full name can be found at EntrezGene (www.ncbi.nlm.nih.gov/gene/). The expression of a number of ligands was increased, that is, IL1B, IFN, and FASLG. FASLG can bind to both the death receptor TNFRSF1A and FAS, which were both upregulated. In addition, more cell type-specific genes encoding for membrane receptors were modulated, such as TLR4 (upregulated), beta-2 adrenergic receptor (upregulated), IGF1R (downregulated), and IFNGR1 (upregulated). Upon binding of ligands to these extracellular receptors, several signaling cascades are initiated, such as PI3K/AKT and G-protein signaling, but also activation of downstream targets by MAP kinases, TRAFs, MTOR, PKC, and calmodulin. Activation of FAS and TNFRSF1A results in the recruitment and activation of the initiator CASP8. Active CASP8 subsequently cleaves and activates CASP7, CASP6, and CASP3. Activation of CASP3 leads to the ultimate death of the cell. Inhibition of the PI3K/AKT signaling cascades results in induction of apoptosis, cell survival, and cell cycle progression. Genes involved in this cascade were modulated, that is, GNAS2, GNAO1, GNG2, RACK1, and GNB5 (all encoding G-proteins), and PIK3CA, PIK3R1, and PKA (all downregulated). The PI3Ks can also be activated by FYN, which in turn is activated through binding of nicotine to the CHRNA. The downstream effector protein AKT acts via inhibition of FKHR or via activation of p70 S6 kinase 1 (RPS6KB1), thereby initiating apoptosis and downregulating cell survival. In addition to the extrinsic death receptor pathway, the intrinsic mitochondrial pathway was modulated. Associated with mitochondrial regulation of apoptosis are the members of the apoptosis regulator Bcl-2 family. The antiapoptotic genes BCL2 and MCL1 were downregulated, thereby abolishing their inhibiting effects on apoptosis. Furthermore, BAK1 and BAX were upregulated, which are proapoptotic members of the Bcl-2 gene family involved in initiating apoptosis. (B) Affected biological processes based on the interpretation of gene annotation and function retrieved from EntrezGene (www.ncbi.nlm.nih.gov/gene/) and MetaCore. The color of the square indicates whether the process will be activated (red) or deactivated (green). To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars