Abstract
5029
Background: Vitamin E consists of eight structurally similar compounds. Early research involving vitamin E considered α-tocopherol as the most important isoform due to its higher concentration in the plasma and its bioactivity in the gestation-resorption assays in rats deprived vitamin E. Recent data suggest that γ-tocopherol is more potent than α-tocopherol inhibiting cell proliferation in cancer cells and influencing carcinogenic biomarkers such as COX-2 and PPAR γ. These data suggest that vitamin E may be a signal transduction modulator as well as an antioxidant. Methods: To test this hypothesis we compared the gene modulation of α-tocopherol or γ-tocopherol (25 μM) treated HCT-116 cells with vehicle-treated HCT-116 cells for 24 hours by microarray analysis. Total RNA was isolated from HCT-116 cells using the Trizol protocol and an Agilent Bioanalyzer. We used the whole genome long oligo arrays produced by Agilent. A fluorescently labeled cDNA probe is made from 50-500 ng of total RNA starting material using the Low RNA input fluorescent amplification Kit (Agilent Technologies). In brief, a poly dT and a T7 polymerase promoter primer is annealed to the total RNA, then reverse transcriptase is added to synthesize the first and second strand cDNA (making double strand cDNA). T7 RNA polymerase and fluorescently-labeled CTPs are then added to synthesize cRNA while simultaneously incorporating cyanine 3- or cyanine 5-labeled CTP. Both labeled samples were combined and hybridized to the microarray. Arrays were scanned with a dual-laser based scanner. Agilent’s Feature Extraction software was used to link a feature to a design file and determine the relative fluorescence intensity between the two samples. Statistical analyses were carried out to identify the differentially expressed genes using the established programs previously described (Baggerly et al., 2001; Shmulevich and Zhang 2002). Results: A distinct subset of genes are regulated by both α-tocopherol and γ-tocopherol (8% of regulated genes are common). However, there is a unique gene set modulated by α-tocopherol that is not shared by γ-tocopherol and a unique set of genes modulated by γ-tocopherol that is not modulated by α-tocopherol. These unique gene sets have been classified by function and these functions are the same for both α- and γ-tocopherol even though the exact genes regulated are different. The functional groups include: angiogenesis, growth, cell cycle, apoptosis, oncogenes, tumor suppressor genes, immune response genes, and signaling genes. Conclusions: While there are similarities among functional groups between α- and γ-tocopherol treated cell lines, a significant percent of genes that are modulated by α- and γ-tocopherol are not shared. These findings are significant for vitamin E isoform-based cancer prevention (Funding source: MD Anderson Cancer Center).
[Proc Amer Assoc Cancer Res, Volume 47, 2006]