Polyunsaturated fatty acids and induction ofcell death in leukemia/lymphoma cell lines

Hilde Heimli

Dissertation for the degree of Doctor Philosophiae

Institute for Nutrition Research, Institute of Basic Medical Sciences, Faculty of Medicine

University of Oslo , Norway 2002

Purpose of the study

The aim of the study was to explore molecular mechanisms behind the effects of n-3 polyunsaturated fatty acids on proliferation and death of different leukemic and lymphoid cell lines, summarized as follows:

• a) to investigate how fatty acids in general, and eicosapentaenoic acid (EPA) in particular affect cell proliferation and viability of leukemic/lymphoma cell lines.
• b) to elucidate the effect of different antioxidants on the EPA-induced necrosis and apoptosis in the leukemic/lymphoma cell lines Raji and Ramos, respectively.
• c) To study molecular mechanisms responsible for inhibition of cell multiplication and induction of apoptosis by EPA in Ramos cells.
• d) To examine the role of acyl-CoA synthetase (ACS) for EPA-induced apoptosis in Ramos cells.

The results were presented in several papers which were summarized as follows:

1) Multiplication and death-type of leukemia/lymphoma cell-lines exposed to very long-chain polyunsaturated fatty acids.

We investigated 14 different leukemia/lymphoma cell-lines regarding sensitivity to fatty acids. We found that cell number was reduced in 10 cell-lines when incubated with 30-120 uM AA, EPA, or DHA, whereas four cell-lines were resitant to these PUFAs. The sensitivity to fatty acids was specific for PUFAs as none of the other cell lines was sensitive to oleic or stearic acid. Because EPA had the strongest effect we further studied the effects of this fatty acid. EPA induced necrosis in Raji cells and apoptosis in Ramos cells. The EPA-induced necrosis in Raji cells was counteracted by 50uM vitamine E, whereas the EPA-induced apoptosis in Ramos cells was unaffected. Our data demonstrate that the majority of the leukemia/lymphoma cell-lines investigated were sensitive to PUFA, and that EPA may induce apoptosis as well as necrosis in different cell-lines.

2) Necrosis and apoptosis in lymphoma cell linesexposed to eicosapentaenoic acid and antioxidants.

In this work we focused on the role of oxidative stress in EPA-induced cell death in Raji and Ramos cells.To further investigate the differences in death modes induced by EPA, we included both lipid-soluble and water-soluble antioxidants in combination with EPA. We found that the necrotic cell death induced by EPA was reducible when antioxidants were introduced. On the contrary, no opposing effects of antioxidants were observed on apoptosis induced by Ramos cells. In Ramos cells, some of the investigated antioxidants (N-acetyl cysteine (NAC) and vitamin C) even enhanced the apoptosis induced by EPA. To assess if ROS was involved in the necrotic and apoptotic cell death, we measured the formation of superoxide anion. When vitamin E was given in combination with EPA, we observed that the EPA-induced accumulation of superoxide anion in Raji cells was reduced, whereas the level of EPA-induced superoxide anion in Ramos cells further increased. Our findings suggest that EPA promotes oxidative stress in Raji cells eventually leading to necrosis. The EPA-induced apoptosis in Ramos cells may be initiated by other factors than oxidative stress and the observed level of superoxide anion might be a result of the apoptotic process itself, as described by Hampton and coworkers. Alternatively, EPA may lead to an irreversible threshold of ROS that initiate the apoptotic process in Ramos cells. We found that low concentrations (nano-molar range) as well as preincubation of vitamin E opposed necrotic cell death in Raji cells. Our results indicate that the EPA-induced necrosis is a slow structural or metabolic reversible damage, sensitive to antioxidants.

3) Eicosapentaenoic acid promotes apoptosis in Ramos cells via activation of caspase-3 and -9.

EPA has been shown to promote apoptosis in Ramos cells, and this study was focused on a possible cell cycle arrest and the pathway by which apoptosis was induced. By incorporation of 3H thymidine and 3H valine we showed that synthesis of DNA as well as protein, was reduced after incubation of Ramos cells with EPA already after 6 h. We monitored cell cycle distribution by BrdU-staining in flow-cytometry, and observed no cell cycle arrest in EPA-incubated cells. Incubation of cells with EPA caused phosphatidylserine (PS)-flipping, as demonstrated by annexin V (AnV)-binding (flow cytometry), but we did not find translocation of PS to be an earlier event than the DNA-fragmentation in Ramos cells reported previously (I). Cleavage of PARP was detected by Western after 8 h incubation of EPA. Apoptosis may proceed along the intrinsic (mitochondrial) or extrinsic (death receptor) pathway, which are mediated via different caspases. We observed activity of caspase-3 and -9 at 4 h incubation with EPA, and the activity of these caspases was markedly above background for at least 12 h. No significant activity of caspase-8 was observed up to 24 h. Whereas inhibitos of caspase-3 and -9 reduced EPA-induced apoptosis, inhibition of caspase-8 had no effect. These findings suggest that EPA promotes apoptosis via the intrinsic pathway in Ramos cells. Thus, the reduction in cell number can be explained by a direct apoptotic effect of EPA rather than via cell cycle arrest.

4) Eicosapentaenoic acid-induced apoptosis depends on acyl CoA-synthetase.

We investigated the incorporation of ACS in EPA-induced apoptosis and we therefore incubated Ramos cells with triacsin C, an inhibitor of ACS. This caused a 70% reduction in the amount of cell-associated EPA and diminished activation of EPA. In addition, triacsin C reduced EPA-induced apoptosis by 90%. Several different approaches were tried to overexpress ACS4 in EPA-insensitive lymphoma cell lines, but we did not obtain viable cells with high expression of acyl activation activity. However, we found that overexpression of ACS4 in the more robust COS-1 cells caused up to 5-fold increase in activation of EPA and 67% increase in the amount of cellular uptake of radiolabelled EPA. Furthermore, we observed 28% elevated cellular level of TAG in EPA-incubated COS-1 cells overexpressing ACS4. We suggest that ACS is an important enzyme for cellular uptake of EPA and EPA-induced apoptosis in Ramos cells.

Conclusion:

From the present study the following can be concluded:

1. PUFAs inhibit cell multiplication in 10 out of 14 leukemic/lymphoma cell lines. The reduction in cell number could be ascribed to effects on the cell proliferative process and/or induction of cell death.

2. EPA induced elevated level of superoxide anion in both Raji and Ramos cells. Lipid-soluble as well as water-soluble antioxidants prevented the EPA-induced necrosis in Raji cells, whereas no such opposing effect was observed in Ramos cells.

3. EPA induced apoptosis via the intrinsic rather than the extrinsic apoptotic pathway in Ramos cells.

4. ACS is involved in cellular uptake and activation of EPA, and is crucial for EPA-induced apoptosis in Ramos cells. Overexpression of ACS4 in COS-1 cells increased both cellular uptake and activation of EPA.