Abstract
Berries and red fruits are important dietary sources of polyphenols [1]. In vitro and animal studies have demonstrated the bioavailability and the anti-proliferative and anticarcinogenic properties of these fruits or of their phenolic components [2, 3]. Consumption of berries may contribute to the reduction of colon cancer by mechanisms not yet understood. Gene expression analysis using microarrays allows for a more comprehensive study of the possible molecular mechanisms by which food or food components may prevent certain cancers of the gastrointestinal tract [4]. The aim of this research is to investigate the anti-proliferative effects of a polyphenol-rich berry juice on a human model of colon cancer cells and its association to transcriptional changes in relation to colon cancer.
Keywords: Colon cancer, Caco-2, Polyphenols, Chokeberry, Aronia Melanocarpa, Microarrays, Gene expression
Methodology
We investigated the effects of a commercial chokeberry (Aronia melanocarpa) juice on the human model of colon cancer Caco-2 cells. In vitro digested (pepsin + pancreatin) [5] chokeberry juice was added to the cells in the culture medium at a nontoxic dose (final pH 7.5 and osmolarity 325 miliosmoles L-1 in the culture medium) 2 h a day for a 4-day period. The concentration of phenolics in the medium at time 0 of the incubation period was ∼80 μM. Control cells were treated with an equivalent mix of enzymes and salts. Cells were counted using a hemacytometer and viability measured using Trypan blue dye exclusion. Results of proliferation and viability for both control and chokeberry treated Caco-2 cells are expressed as percentage of those values obtained for untreated cells. Gene expression changes were measured using microarrays (HG_U133A_2.0 human chips, Affymetrix). Transcripts that were 1.6-fold induced or repressed were selected. The changes in mRNA levels of several selected genes were further confirmed by RT-PCR.
Results and conclusions
Exposure of Caco-2 cells to pre-digested chokeberry juice resulted in inhibition of both cell proliferation (30–40%) and viability (∼20%) in comparison to untreated cells. A very low proportion of genes (0.44% of total transcripts represented in the chip) were found to change in response to the treatment and most changes were in the range 1.6–2.0-fold. Resulting altered genes were categorized into several functional groups based on gene ontology search (Fatigo, GEPAS 1.1, Bioinformatics Unit, CNIO; SOURCE, Genetics Department, Stanford University) and available literature (Fig.1): (1) DNA processing and transcription, (2) cell signalling and signal transduction, (3) apoptosis, cell growth, proliferation, (4) cell cytoskeleton (5) RNA processing, (6) translation and protein processing, (7) transporters, metabolism, (8) other markers and response to various stimuli. A group of genes were also categorized as “unknown” function. Among the responsive genes we detected changes in several genes that have been reported to be related to colon carcinogenesis, tumour migration and cell proliferation. Changes in the expression levels of some of these genes were further confirmed by RT-PCR (Table 1). In conclusion, polyphenol-rich chokeberry exhibited anti-proliferative effects in Caco-2 cancer colon cells. Inhibition of the cells proliferation by chokeberry juice may be associated to the modulation of transcription of specific genes such as: (1) upregulation of tumor suppressors (CEACAM1 [6] and BMP2 [7]) and (2) down-regulation of genes related to tumor invasion and metastasis (FGFR2 [8] and S100A4 [9]). We are currently further investigating the changes in these genes and proteins.
Fig. 1.
Diagram of the percentage of altered genes categorized in functional groups in Caco-2 colon cancer cells after treatment with a non-toxic recurrent dose of a chokeberry juice rich in polyphenols
Table 1.
List of genes related to colon cancer with altered expression levels after treatment with chokeberry juice
| Accession number | Gene name | Gene symbol | Affyxa | RT-PCRa | Biological process involved in |
|---|---|---|---|---|---|
| NM_001712 | Carcinoembryonic antigen-related cell adhesion molecule 1 | CEACAM1 | +2.1 | +2.6 | Reduced expression is a major event in colorectal cancer. Tumor suppressor involved in cell–cell adhesion that regulates apoptosis in colon epithelium |
| BC000478 | Heat shock 70 kDa protein 9B (mortalin 2) | HSPA9B | −2.0 | n.d. | Control of cell proliferation and cellular aging. Over-expressed in colorectal adenocarcinoma |
| NM_022975 | Fibroblast growth factor receptor 2 | FGFR2 | −4.6 | −1.8 | Receptor for fibroblast growth factor implicated in tumor growth and invasion (colon carcinoma) |
| NM_001200 | Bone morphogenetic protein 2 | BMP2 | +1.6 | +2.4 | Belongs to the transforming growth factor β family. It acts as tumor suppressor promoting apoptosis in mature colonic epithelial cells and inhibiting proliferation |
| NM_005228 | Epidermal growth factor receptor | EGFR | +1.8 | n.d. | Receptor of the EGF family members. Involved in the control of cell growth and differentiation of colon cancer |
| NM_002823 | Prothymosin, alpha (gene sequence 28) | PTMA | −1.8 | n.d. | Nuclear protein involved in cell proliferation. Highly expressed in human colorectal cancer |
| NM_005242 | Coagulation factor II (thrombin) receptor-like 1 | F2RL1 | +2.0 | n.d. | Protease activated receptor involved in the control proliferation in colon cancer. Transmembrane receptor coupled to G protein |
| D13889 | Inhibitor of DNA binding 1, dominant negative helix-loop-helix protein | ID1 | −1.7 | n.d. | Involved in cell growth regulation and tumorigenesis. Up-regulated in colorectal cancer |
| NM_002624 | Prefoldin 5 | PFDN5 | −1.6 | n.d. | Chaperone protein. It may target actin and tubulin. It represses transcritption of c-myc and it is related to colon cancer |
| NM_002961 | S100 calcium binding protein A4 (metastasin) | S100A4 | −2.5 | −1.9 | Associated with metastatic capacity of cancer cells. May function in motility, invasion and tubulin polymerisation. Significant pronostic marker of colorectal carcinoma |
| NM_000900 | Matrix Gla protein | MGP | +1.9 | n.d. | Extracellular matrix protein. It may be involved in cell differentiation and tumor progression. It is downregulated in colorectal cancer |
| NM_002203 | Integrin, alpha 2 (CD49B, alpha 2 subunit of VLA-2 receptor) | ITGA2 | +3.0 | +2.6 | Cell-surface protein that participates in cell adhesion (hemidesmosomes). Role in cell proliferation and migration. Involved in colorectal cancer |
| NM_004949 | Desmocollin 2 | DSC2 | −1.8 | n.d. | Required for cell adhesion and desmosome formation. May be involved in colorectal carcinoma metastasis |
| AJ224869 | Chemokine (C-X-C motif) receptor 4 | CXCR4 | +1.7 | n.d. | G-protein coupled receptor. May play a role in colon cancer and metastasis |
| NM_002184 | Interleukin 6 signal transducer (oncostatin M receptor) | IL6ST | +3.0 | −1.2 | Part of the cytokine receptor complex linked to signal transduction. May be involved in cell growth and proliferation. Expressed in colorectal cancer |
| NM_000846 | Glutathione S-transferase A2 | GSTA2 | −1.7 | n.d. | Detoxification of electrophilic compounds by conjugation with glutathione. Overexpression in colon cancer cells protects against cell cycle arrest and apoptosis |
n.d. not determined
aFold up-/down-regulation
Acknowledgments
This research was funded by the Fundación Séneca, Murcia, Spain (PB/18/FS/02; 00490/PPC/04) and by the CICYT, Spain (AGL2003-02195; AGL2004-03989).
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