Düsseldorf, Universiẗat, Diss., 2000.

München, Techn. Universiẗat, Diss., 2001.

Braunschweig, Techn. Universiẗat, Diss., 2002.

The present study was undertaken to determine the effects and possible mechanism of action of vitamin E succinate on malignant murine melanoma (BL6) and non-malignant monkey kidney (LLCMK) cell growth in vitro. Studies revealed that supplementation of 5, 7 and lOJLg/ml vitamin E succinate significantly inhibited BL6 cell growth, while in LLCMK cells no significant increase or decrease in growth was observed. The actual mechanism by which vitamin E succinate inhibits BL6 cell growth is at present unclear. Studies have suggested a radical or oxidant involvement in a number of degenerative diseases such as cancer, and that supplementation of antioxidant vitamins such as vitamin E may function to reduce cancer cell growth by quenching free radical species and preventing lipid peroxidation. In addition to its antioxidant role in a cell, vitamin E is believed to modulate the activities of various enzymes and metabolites in the eicosanoid pathway. Hence, this study investigated the effects of vitamin E succinate supplementation on free radical and lipid peroxidation levels, as well as the activities of various enzymes and metabolites ill the eicosanoid pathway. Throughout this study, emphasis was placed on BL6 melanoma cells since the magnitude of the relationship between LLCMK growth and the levels of various enzymes and metabolites in the eicosanoid pathway varied considerably from one experiment to another and did not show the consistent trend found with the BL6 cells. A decrease in cell growth was found to be accompanied by a concomitant increase rather than a decrease in the levels of free radicals and lipid peroxidation, suggesting that the growth inhibitory effects of vitamin E succinate on BL6 cells in vitro was not due to its antioxidant properties associated with the vitamin E component, but rather due to one or more of its other potential roles within the cell. This proposal was further strengthened by findings that vitamin E succinate, a non-physiological antioxidant in its esterified form, did not undergo significant cleavage to free vitamin E in the BL6 cells. Vitamin E succinate is believed to modulate membrane bound enzyme activities through physicochemical interactions with membrane lipids and changes in membrane fluidity. Hence, this study investigated the role of vitamin E succinate in modulating the activity of various enzymes and secondary messengers in the eicosanoid pathway. Supplementation of l-lOjLg/ml vitamin E succinate resulted in an overall increase in phospholipase A2 activity while cyclooxygenase and adenyl ate cyclase activities were found to be significantly increased at vitamin E succinate concentrations of 7 and WjLg/ml respectively. A significant increase in" 5-LOX activity was observed a! 10jLg/mi supplementation. The suggestion that vitamin E succinate modulates membrane bound enzyme activities was further strengthened by uptake and cellular distribution studies, which showed significantly higher levels of vitamin E succinate in membrane fractions of BL6 cells when compared with stroma fractions. Another factor which could account for elevated PLA2,-5-LOX and COX activities in BL6 cells as a result of vitamin E succinate supplementation, was that of intracellular calcium levels. Supplementation of BL6 cells with 1-7 jLg/ml vitamin E succinate resulted in an overall increase in intracellular calcium levels. These changes in calcium levels however were positively correlated with changes in PLA2 activity only. Since the rate of prostaglandin synthesis is controlled by phospholipase A2 activity, and net prostagiandin production is dependant on cyclooxygenase activity, the effects of vitamin E succinate supplementation on prostaglandin levels in BL6 cells was determined. Vitamin E succinate supplementation resulted in a significant decrease in prostaglandin D2 levels at vitamin E succinate concentrations of 3, 5, 7 and lOjLg/ml respectively, while prostaglandin F2a levels were significantly decreased at 1-10jLg/ml vitamin E succinate. The increases in prostaglandin E2 and 12 levels were inversely related to BL6 cell growth suggesting that both prostaglandins may act as negative regulators of BL6 cell growth. When comparing prostaglandin E2 levels to prostaglandin 12 levels in BL6 cells, significantly higher levels of prostaglandin E2 were found, suggesting that vitamin E succinate effects were mediated primarily through an increase in prostaglandin E2 levels. Furthermore, prostaglandin E2 levels are believed to modulate adenylate cyclase activity. It is therefore reasonable to conclude that the increased adenyl ate cyclase activity found in BL6 cells was dependant on prostaglandin E2 levels, since increases in prostaglandin E2 levels at 7 and lOjLg/ml vitamin E succinate correlated with an increase in adenylate cyclase activity and cyclic adenosine monophosphate levels. Thus it appeared that the observed inhibitory effects of vitamin E succinate supplementation on BL6 cell growth was not due to the antioxidant properties associated with the vitamin E component of the vitamin E succinate molecule, but was rather mediated in part through a cascade effect initiated by phospholipase A2 activation and archidonic acid release. This initial effect then appeared to result in an increase in cyclooxygenase activity and activation of a prostaglandin E2-adenylate cyclase-cyclic adenosine monophosphate linked system, ultimately altering cyclic adenosine monophosphate levels and inhibiting BL6 cell growth. This was confirmed when BL6 cells were supplemented with indomethacin, a cyclooxygenase inhibitor. Supplementation with the inhibitor resulted in vitamin E succinate having no inhibitory effects on BL6 cell growth. Furthermore, when comparing the levels of prostaglandin ~, adenylate cyclase activity and cyclIC adenosine monophosphate in the indomethacin treated cultures to non-indomethacin treated cultures, markedly lower levels of these metabolites were found in the indomethacin treated cultures. The cause of the increase in free radical and lipid peroxidation levels in BL6 cells following vitamin E succinate supplementation was further investigated. Cyclooxygenase enzymes are believed to generate free radical species and contribute to lipid peroxidation levels during catalytic activity. Markedly lower levels of free radicals and lipid peroxidation in indomethacin treated cultures were found when compared with vitamin E succinate treated cultures alone, suggesting that the increases in free radical and lipid peroxidation levels in BL6 cells supplemented with vitamin E succinate were indirectly due to an increase in cyclooxygenase activity in these cells.

This study was undertaken to determine the effects and possible mechanism of action of vitamin E succinate supplementation on B16 murine melanoma cell growth in vitro. Studies revealed that supplementation of 5, 7 and 10µg/ml of this vitamin significantly inhibited growth of B16 cells. Non-malignant LLCMK cells supplemented with the same concentrations of vitamin E succinate resulted in similar inhibition of cell growth. The actual mechanism by which vitamin E succinate inhibits B16 cell growth is unclear, though there has been much speculation about its possible role as an antioxidant. Vitamin E succinate is not a physiological antioxidant and for this ester to behave as an antioxidant, cleavage of the ester bond must occur, releasing the antioxidant vitamin E part of the molecule. To determine whether the observed inhibitory effects on B16 cell growth were due to the intact vitamin E succinate or the vitamin E cleavage product, cleavage studies were undertaken. Results from these studies revealed that in B16 cells vitamin E succinate cleavage did not occur suggesting that the observed inhibitory effects of vitamin E succinate on B16 cells were due to the intact compound. In contrast vitamin E succinate cleavage was shown to occur in LLCMK cells, suggesting that these cells may contain an esterase capable of liberating succinic acid and vitamin E. Further studies focussed on the possible role of vitamin E succinate in regulation of cyclooxygenase activity in B16 cells as vitamin E succinate was found to effect the activity of various enzymes involved in the arachidonic acid cascade, notably cyclooxygenase, the rate-limiting enzyme in prostaglandin synthesis. Time course studies were used to determine when the cyclooxygenase protein was being produced, thus allowing an estimation of when the gene was being 'switched on'. These studies revealed that vitamin E succinate does not significantly effect cyclooxygenase activity in B16 cells over a period of 2 to 12 hours as compared to the OE control cultures. Further studies using RNA techniques investigated whether vitamin E succinate was having an effect on cyclooxygenase activity at a molecular level. These investigations were unsuccessful for the 6 day supplementation for a number of possible reasons, the main reason being RNA stability. Subsequent studies revealed an increase in COX mRNA after 2 hours, suggesting that the gene was 'switched on' soon after supplementation with vitamin E succinate, and further increases in COX mRNA were observed after 8 to 12 hours. The molecular studies were, however, inconclusive. Previous studies suggested that vitamin E succinate was indirectly causing growth inhibition of B16 cells via regulation of cyclooxygenase activity, however, this study does not support these findings and it would seem unlikely that regulation of cyclooxygenase expression in B16 cells by vitamin E succinate has a role to play in the mechanism by which vitamin E succinate inhibits growth in B16 cells.

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