A relationship between reactive oxygen species and several effectors of apoptosis has been reported. Although the mechanism by which oxidants induce apoptosis is unknown, it is likely some signaling factor is generated. Since polyunsaturated fatty acids are highly susceptible to oxidation, oxidized products that are known to induce apoptosis are reasonable candidates. Lipoxygenase enzymes are a possible source of such products and various inhibitors have been used to implicate these enzymes in apoptosis. However, several of these inhibitors, including MK886 which blocks 5-lipoxygenase activating protein (FLAP), induce apoptosis even in cells lacking LOX. We have also shown that NM886 induces apoptosis independent of FLAP. Nevertheless, a link between FLAP and the BCL family of anti-apoptotic proteins is suggested by our findings that FL5.12 cells overexpressing bcl-xL have diminished levels of FLAP and there is a rapid loss of FLAP from such cells upon withdrawal of IL-3. There is also a rapid loss of bcl-xL and bcl-2 protein in cells treated with mK886. The overall goal of this project is to enhance our understanding of the mechanisms associated with apoptosis by determining the apoptotic mechanism of MK886. A related goal will be to clarify the relationship between FLAP and bcl-XL, and the induction of apoptosis. The hypotheses to be tested are that: 1) the inhibition of peroxisome proliferator activated receptors (PPAR) by MK886 plays a role in the induced apoptosis, 2) MK886 blocks the binding of unsaturated fatty acids increasing their cellular content. These fatty acids then activate apoptotic signaling pathways either directly or following conversion to other species, and 3) disrupting fatty acid signaling modulates the expression of bcl-xL. Preliminary data show that MK886 is a potent inhibitor of PPARalpha. This unique effect will be studied in more detail and any link to apoptosis investigated by up- and down-regulating PPAR and determining the apoptotic potency of MK886. The 2nd hypothesis will be studied by determining fatty acid oxidation after treatment with MK886, comparing the effects of MK886 to those of exogenous fatty acids including oxidized species, and examining whether glutathione can affect MK886-induced apoptosis. Any relationship between FLAP and bcl-XL will be determined by measuring the effect of bcl-XL overexpression on FLAP expression in other cell lines, assessing FLAP and bcl-XL protein and mRNA levels in bcl-XL overexpressing cells following withdrawal of IL-3 or treatment with MK886, and assessing the role of various proteolytic pathways in the loss of bcl-XL and FLAP. The results of these studies will improve our understanding of fatty acid signaling and apoptotic death in conjunction with FLAP and bcl proteins.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
1R01CA083701-01A1
Application #
6194812
Study Section
Alcohol and Toxicology Subcommittee 4 (ALTX)
Program Officer
Yang, Shen K
Project Start
2000-06-01
Project End
2005-05-31
Budget Start
2000-06-01
Budget End
2001-05-31
Support Year
1
Fiscal Year
2000
Total Cost
$265,500
Indirect Cost
Name
University of Texas Austin
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
City
Austin
State
TX
Country
United States
Zip Code
78712
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