Gamma (gamma) tocopherol upregulates peroxisome proliferator activated receptor (PPAR) gamma (gamma) expression in SW 480 human colon cancer cell lines

Abstract

Background: Tocopherols are lipid soluble antioxidants that exist as eight structurally different isoforms. The intake of gamma-tocopherol is higher than alpha-tocopherol in the average US diet. The clinical results of the effects of vitamin E as a cancer preventive agent have been inconsistent. All published clinical trials with vitamin E have used alpha-tocopherol. Recent epidemiological, experimental and molecular studies suggest that gamma-tocopherol may be a more potent chemopreventive form of vitamin E compared to the more-studied alpha-tocopherol. Gamma-tocopherol exhibits differences in its ability to detoxify nitrogen dioxide, growth inhibitory effects on selected cancer cell lines, inhibition of neoplastic transformation in embryonic fibroblasts, and inhibition of cyclooxygenase-2 (COX-2) activity in macrophages and epithelial cells. Peroxisome proliferator activator receptor gamma (PPARgamma) is a promising molecular target for colon cancer prevention. Upregulation of PPARgamma activity is anticarcinogenic through its effects on downstream genes that affect cellular proliferation and apoptosis. The thiazolidine class of drugs are powerful PPARgamma ligands. Vitamin E has structural similarity to the thiazolidine, troglitazone. In this investigation, we tested the effects of both alpha and gamma tocopherol on the expression of PPARgamma mRNA and protein in SW 480 colon cancer cell lines. We also measured the intracellular concentrations of vitamin E in SW 480 colon cancer cell lines.

Results: We have discovered that the alpha and gamma isoforms of vitamin E upregulate PPARgamma mRNA and protein expression in the SW480 colon cancer cell lines. gamma-Tocopherol is a better modulator of PPARgamma expression than alpha-tocopherol at the concentrations tested. Intracellular concentrations increased as the vitamin E concentration added to the media was increased. Further, gamma-tocopherol-treated cells have higher intracellular tocopherol concentrations than those treated with the same concentrations of alpha-tocopherol.

Conclusion: Our data suggest that both alpha and gamma tocopherol can upregulate the expression of PPARgamma which is considered an important molecular target for colon cancer chemoprevention. We show that the expression of PPARgamma mRNA and protein are increased and these effects are more pronounced with gamma-tocopherol. Gamma-tocopherol's ability to upregulate PPARgamma expression and achieve higher intracellular concentrations in the colonic tissue may be relevant to colon cancer prevention. We also show that the intracellular concentrations of gamma-tocopherol are several fold higher than alpha-tocopherol. Further work on other colon cancer cell lines are required to quantitate differences in the ability of these forms of vitamin E to induce apoptosis, suppress cell proliferation and act as PPAR ligands as well as determine their effects in conjunction with other chemopreventive agents. Upregulation of PPARgamma by the tocopherols and in particular by gamma-tocopherol may have relevance not only to cancer prevention but also to the management of inflammatory and cardiovascular disorders.

Figure 1

The structure comparison of the tocopherols A) with troglitazone B).

Figure 2

Tocopherol upregulation of PPARγ mRNA expression detected by QPCR after 24 hours of treatment with 5 μM tocopherol. A) Relative copy number of a representative QPCR AT 5 μM tocopherol concentration. B) Fold increase data of the representative QPCR data shown in figure 2A. C) Average fold increase data of three independent experiments at 5 μM tocopherol concentration. * shows sample statistical difference compared to the vehicle-treated.

Figure 3

Tocopherol upregulation of PPARγ mRNA expression detected by QPCR after 24 hours of treatment with 10 μM tocopherol. A) Relative copy number of a representative QPCR AT 10 μM tocopherol concentration. B) Fold increase data of the representative QPCR data shown in figure 3A. C) Average fold increase data of three independent experiments at 10 μM tocopherol concentration. * shows sample statistical difference compared to the vehicle-treated.

Figure 4

Tocopherol upregulation of PPARγ protein by Western Blot analysis of A) 24-hour tocopherol-treated SW480 nuclear extracts: lane 1 – 10 μM γ-tocopherol, lane 2 – 5 μM γ-tocopherol lane 3 – 10 μM α-tocopherol, lane 4 – 5 μM α-tocopherol, lane 5 – 100 μM troglitazone, lane 6 – blank, lane 7-ethanol carrier control.B) Bar graph of densitometries for Western Blot shown in figure 4A.

Figure 5

Tocopherol upregulation of PPARγ protein by Western Blot analysis of A)48-hour tocopherol-treated SW480 whole cell lysates: lane 1 – PPARγ control from transfected COS cells, lane 2 – negative control from untreated Jurkat cells, lane 3 – vehicle-treated control, lane 4 – 10 μM α-tocopherol, lane 5 – 10 μM γ-tocopherol, lane 6 – 100 μM troglitazone. B) Bar graph of densitometries for Western Blot shown in figure 5A.