The effect of combined vitamin E succinate and ascorbic acid supplementation on growth and cyclooxygenase expression in murine melanoma (BL6) cells
- Authors: Van Rooyen, Megan Lynne
- Date: 1999
- Subjects: Vitamin E , Vitamin C , Melanoma
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3982 , http://hdl.handle.net/10962/d1004041 , Vitamin E , Vitamin C , Melanoma
- Description: This thesis examines the effect of combined vitamin E succinate and Asc supplementation on the in vitro growth of a non-malignant monkey kidney (LLCMK) and a malignant melanoma (BL6) cell line, with nutritional concentration ranges of 5-20µg/ml and 25-50µg/ml respectively. Vitamin E and C are thought to interact synergistically to inhibit tumour cell growth by virtue of their antioxidant properties, whereby they quench free radicals and terminate lipid peroxidation. Furthermore vitamin E and C are thought to modulate the biosynthetic pathways in arachidonic acid metabolism at a number of different points. This may also offer a means of regulating tumour cell growth. It is well documented that vitamin E and C are distributed in the lipid and aqueous phases in the cell respectively. However, the cells need to obtain the vitamins from the environment in which they are found in order to exert a growth inhibitory effect. Supplementation of combined vitamin E succinate and Asc on BL6 and LLCMK cells resulted in a significant increase in LLCMK cell growth, and a significant decrease in cell growth was observed in BL6 cells. Vitamin E succinate in its esterified form cannot function as an antioxidant and requires the cleavage of the succinate to become an active antioxidant. The metabolism of vitamin E succinate to form free vitamin E in LLCMK and BL6 cells resulted in the cleavage of the succinate group from the vitamin E molecule in BL6 cells only, thus suggesting that an esterase may be present in BL6 cells. This would allow for a synergistic interaction between the two vitamins. The arachidonic acid cascade generates a family of bioactive lipids that modulate diverse physiological and pathological responses including tumour growth and promotion. The enzyme prostaglandin endoperoxide synthase (PGHS) or cyclooxygenase (Cox) is the key enzyme in the biosynthetic pathway leading to the formation of prostaglandins. Two enzyme isoforms of Cox have been identified, Cox 1 and Cox 2. Supplementation with vitamin E succinate and Asc at a combination 20:25µg/ml respectively resulted in a trend of increasing Cox activity over 12 hours suggesting that vitamin E and Asc have a stimulatory effect on Cox activity in BL6 cells. The inhibitors of Cox 2, dexamethasone, showed a decreasing trend in Cox activity at the 20:25µg/ml combination, while cycloheximide showed an initial stimulatory effect and then a gradual decrease in Cox activity. The elimination of the Cox activity by dexamethasone suggests that transcriptional regulation may be occurring in BL6 cells. We examined by Northern blot analysis whether combined supplementation of vitamin E succinate and Asc caused an elevation of Cox 2 RNA expression in BL6 cells. An inducible effect of Cox 2 was observed after 2 hours of supplementation with a combination of vitamin E succinate and Asc in BL6 cells, however the results are inconclusive and further studies are required to substantiate this finding.
- Full Text:
- Date Issued: 1999
- Authors: Van Rooyen, Megan Lynne
- Date: 1999
- Subjects: Vitamin E , Vitamin C , Melanoma
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3982 , http://hdl.handle.net/10962/d1004041 , Vitamin E , Vitamin C , Melanoma
- Description: This thesis examines the effect of combined vitamin E succinate and Asc supplementation on the in vitro growth of a non-malignant monkey kidney (LLCMK) and a malignant melanoma (BL6) cell line, with nutritional concentration ranges of 5-20µg/ml and 25-50µg/ml respectively. Vitamin E and C are thought to interact synergistically to inhibit tumour cell growth by virtue of their antioxidant properties, whereby they quench free radicals and terminate lipid peroxidation. Furthermore vitamin E and C are thought to modulate the biosynthetic pathways in arachidonic acid metabolism at a number of different points. This may also offer a means of regulating tumour cell growth. It is well documented that vitamin E and C are distributed in the lipid and aqueous phases in the cell respectively. However, the cells need to obtain the vitamins from the environment in which they are found in order to exert a growth inhibitory effect. Supplementation of combined vitamin E succinate and Asc on BL6 and LLCMK cells resulted in a significant increase in LLCMK cell growth, and a significant decrease in cell growth was observed in BL6 cells. Vitamin E succinate in its esterified form cannot function as an antioxidant and requires the cleavage of the succinate to become an active antioxidant. The metabolism of vitamin E succinate to form free vitamin E in LLCMK and BL6 cells resulted in the cleavage of the succinate group from the vitamin E molecule in BL6 cells only, thus suggesting that an esterase may be present in BL6 cells. This would allow for a synergistic interaction between the two vitamins. The arachidonic acid cascade generates a family of bioactive lipids that modulate diverse physiological and pathological responses including tumour growth and promotion. The enzyme prostaglandin endoperoxide synthase (PGHS) or cyclooxygenase (Cox) is the key enzyme in the biosynthetic pathway leading to the formation of prostaglandins. Two enzyme isoforms of Cox have been identified, Cox 1 and Cox 2. Supplementation with vitamin E succinate and Asc at a combination 20:25µg/ml respectively resulted in a trend of increasing Cox activity over 12 hours suggesting that vitamin E and Asc have a stimulatory effect on Cox activity in BL6 cells. The inhibitors of Cox 2, dexamethasone, showed a decreasing trend in Cox activity at the 20:25µg/ml combination, while cycloheximide showed an initial stimulatory effect and then a gradual decrease in Cox activity. The elimination of the Cox activity by dexamethasone suggests that transcriptional regulation may be occurring in BL6 cells. We examined by Northern blot analysis whether combined supplementation of vitamin E succinate and Asc caused an elevation of Cox 2 RNA expression in BL6 cells. An inducible effect of Cox 2 was observed after 2 hours of supplementation with a combination of vitamin E succinate and Asc in BL6 cells, however the results are inconclusive and further studies are required to substantiate this finding.
- Full Text:
- Date Issued: 1999
The role of vitamin E succinate in regulation of growth and cyclooxygenase expression in B16 murine melanoma cells
- Van der Merwe, Adele Shanette
- Authors: Van der Merwe, Adele Shanette
- Date: 1999
- Subjects: Melanoma , Vitamin E
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3988 , http://hdl.handle.net/10962/d1004047 , Melanoma , Vitamin E
- Description: 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.
- Full Text:
- Date Issued: 1999
- Authors: Van der Merwe, Adele Shanette
- Date: 1999
- Subjects: Melanoma , Vitamin E
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3988 , http://hdl.handle.net/10962/d1004047 , Melanoma , Vitamin E
- Description: 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.
- Full Text:
- Date Issued: 1999
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