Comparative effects of RRR-alpha- and RRR-gamma-tocopherol on proliferation and apoptosis in human colon cancer cell lines

Differences in the incidence of colorectal cancer throughout the world suggest that diet has a role in colorectal carcinogenesis [1]. Mediterranean diets, rich in vitamin E isoforms, are associated with a lower incidence of colon cancer [2, 3]. Vitamin E refers to any of four tocopherols or tocotrienols (α, β, δ, and γ) isoforms. All of the tocopherol isoforms found in nature are the RRR-forms since all three chiral carbons on the side chain attached to the chroman head group have the “R-” configuration rather then the “S-” configuration. Synthetic vitamin E (typically found in dietary supplements) almost always refers to all-rac-alpha-tocopherol which is a racemic mixture containing eight stereo isomers, one eighth of which is the biologically active RRR-isoform. The isoform found in highest concentration in the serum is the RRR-α-tocopherol. The primary form of vitamin E found in the North American diet is RRR-γ-tocopherol, which is present at levels 2-4 times higher than that of RRR-α-tocopherol [4].

While most clinical studies with vitamin E have used all-rac-α-tocopherol, recent studies show that RRR-γ-tocopherol also may play a unique role in preventing colon cancer [5]. Epidemiological evidence shows that RRR-γ-tocopherol levels in plasma correlate with a reduced risk for both colon and prostate cancer [6-8]. Animal studies measuring direct end-points of cancer (i.e., survival studies, tumor reduction, tumor prevention) with RRR-γ-tocopherol are lacking. Vitamin E was demonstrated a chemopreventive in animal models of chemically induced colon cancer [9], but these studies tested only α-tocopherol or α-tocopherol acetate (RRR- or all-rac- not specified). Animal studies measuring the potential chemopreventive mechanisms of RRR-γ-tocopherol have used surrogate biomarkers of carcinogenesis. For example, RRR-γ-tocopherol can suppress the expression of ras p-21 in rat colonocytes in vivo [10]. Using Wistar rats, Jiang has demonstrated that RRR-γ-tocopherol, but not α-tocopherol (RRR- or all-rac- not specified) decreases the proinflammatory eicosanoid PGE2 (which is known to play a role in the progression of colorectal cancer through inflammation). Further, hemodialysis patients administered RRR-γ-enriched tocopherols showed a consistently lower level of C-reactive protein (a biomarker of inflammation) while the administration of α-enriched tocopherols (RRR- or all-rac- not specified) did not [11].

Cooney et al. found that RRR-γ-tocopherol was a much more potent inhibitor of neoplastic transformation in 3-methycholanthrene-treated C3H/H10T/1/2 murine fibroblasts than α-tocopherol (RRR- or all-rac- not specified) [12]. In addition, RRR-γ-tocopherol is a potent inhibitor of COX-2 activity and inhibits human cancer cell cycle progression and cell proliferation by down-regulation of cyclins [13, 14]. Stone et al. have found that RRR-γ-tocopherol is taken up by RAW 264.7 macrophages to a much greater extent than RRR-α-tocopherol [15].

Vitamin E isoforms (and metabolites) have had varying effectiveness at inhibiting cell growth and inducing apoptosis. RRR-γ-Tocopherol is superior to all-rac-α-tocopherol at cell growth inhibition in vitro in human prostate cancer cells [16]. Vitamin E succinate (VES) and tocotrienols demonstrate potent apoptotic inducing properties [17]. Zu et al. [18] have shown differential synergistic effects of selenium with vitamin E isoforms on cell growth and apoptosis in PC-3 human prostate cancer cells. VES was the most effective form tested and synergizes with selenium, while RRR-α-tocopherol and RRR-α-tocopheryl acetate were weaker in their effects on suppressing growth and inducing apoptosis in PC-3 prostate cancer cells. α-Tocopherol (RRR or all-rac not specified) is a poor inducer of apoptosis in the colon cancer xenograft nude mouse model, while the synthetic form, α-tocopheryl succinate (a redox inactive analogue of vitamin E) is a strong inducer of apoptosis [19]. In addition, carboxyethyl hydroxychromans (CEHC) metabolites of γ-tocopherol are powerful inducers of apoptosis and inhibit cell growth and down regulate cyclin expression in PC-3 prostate cancer cell lines [20]. The performance of vitamin E derivatives and analogues with regard to apoptosis also vary among tissue types. VES and a vitamin E analogue, 2,5,7,8-tetramethyl-2R-(4R,8R,12-trimethyltridecyl)chroman-6-yloxy acetic acid (α-TEA) can induce human breast, prostate, colon, lung, cervical, and endometrial tumor cells, but the apoptotic-inducing effects of α-TEA are greater in human ovarian and cervical cancer cells [21]. To date, no study has compared the anti-proliferative and apoptotic effects of α- and γ-tocopherol on colon cancer cell lines with differences in molecular features. Gysin et al. demonstrated the inhibition of cell proliferation by RRR-γ-tocopherol treatment at 25 μM on CaCo-2 (colon carcinoma), DU-145 and LNCap (prostate carcinoma) and SaOs-2 (osteosarcoma) cells, but demonstrated no apoptosis in any of the cell lines tested.

In this study, we compared the anti-proliferative and apoptotic effects of RRR-α-tocopherol and RRR-γ-tocopherol in four colon cancer cell lines with varying molecular characteristics, SW480 (APC, type I truncation and COX-2 deficient), HCT-15 (COX-2 deficient), HCT-116 (APC, wild-type and COX-2 inducible), and HT-29 (APC, type II truncation and COX-2 constitutive expression) and normal untransformed colon cells (CCD-12CoN). Proliferation studies demonstrated that RRR-γ-tocopherol is able to inhibit proliferation and cause cell death in all four cancer cell lines, but not in the normal cells. In the colon cancer cell lines tested for apoptosis, we have shown that RRR-γ-tocopherol activates cleavage of PARP and caspase 3, 7 and 8, but not caspase 9. Dietary RRR-γ-tocopherol may be an effective colon cancer preventive agent while pharmacological concentrations of RRR-γ-tocopherol may be useful as an adjuvant chemotherapy agent resulting lower dose of agents that are cytotoxic to normal cells.