JASON J. EVERLY, PHARM.D.
University of Arizona, College of Pharmacy
Pfizer – AFPE Clinical Pharmacy Post Pharm.D. Fellowship in the Biomedical Research Sciences

INTERIM PROGRESS REPORT

TITLE(S) OF RESEARCH: Expression patterns for 5-, 12-, and 15-lipoxygenase enzymes in human non-small cell lung adenocarcinoma cells lines with both smoking (NCI-H1395 and NCI-H1437) and nonsmoking (NCI-H2126) histories.

Effects of preferential inhibition of 5-, 12-, and 15-lipoxygenases in human non-small cell lung adenocarcinoma cells lines with both smoking (NCI-H1395 and NCI-H1437) and nonsmoking (NCI-H2126) histories.

5-, 12-, and 15-lipoxygenase expression in progressive clinical stages of human non-small cell lung adenocarcinoma cells (NCI-H1437 – Stage 1, NCI-H1395 – Stage 2, NCI-H647 – Stage 3A, NCI-H1792 – Stage 4).

Effects of pharmacological manipulation of 5-, 12-, and 15-lipoxygenases in progressive clinical stages of human non-small cell lung adenocarcinoma cells (NCI-H1437 – Stage 1, NCI-H1395 – Stage 2, NCI-H647 – Stage 3A, NCI-H1792 – Stage 4).

AIMS: Specific Aim 1: To define the expression patterns for 5-, 12-, and 15-lipoxygenase enzymes in human non-small cell lung adenocarcinoma cells lines with both smoking (NCI-H1395 and NCI-H1437) and nonsmoking (NCI-H2126) histories.

Specific Aim 2: To define the expression patterns for 5-, 12-, and 15-lipoxygenase enzymes in progressive clinical stages of human non-small cell lung adenocarcinoma cells (NCI-H1437-Stage 1, NCI-H1395-Stage 2, NCI-H647-Stage 3A, and NCI-H1792-Stage 4).

Specific Aim 3: To treat selected cell lines with selected chemopreventative agents with the overall objective of identifying the most effective lipoxygenase expression pattern for inhibition of lung tumor growth.

STUDIES AND RESULTS:
1st Year Studies and Results:
Specific Aim 1 & 2
Over the past year, each cell line (NCI-H1395, NCI-H1437, and NCI-H2126) has been characterized by measuring lipoxygenase metabolites (LTB4; cysteinyl leukotrienes (LTC4, LTD4, and LTE4); 5(S)-HETE; 12(S)-HETE, 15(S)-HETE; 13(S)-HODE). Two approaches were used to characterize baseline expression patterns: enzyme-linked immunosorbent assay (ELISA) and western blotting. Specifically, ELISA was used for the quantization of LTB4, cysteinyl leukotrienes, 12(S)HETE, and 15(S)HETE in cell culture lysates. ELISA data was analyzed utilizing AssayZap 3.0 (Biosoft, U.K). Because 5-, 12-, and 15- lipoxygenase enzymes as well as 5(S)-HETE cannot be quantified by ELISA, western blotting was used. Quantification of western blotting results was preformed on NIH ImageJ (NIH Image Processing and Analysis in Java). Additionally, Western blotting was used to validate the results observed in ELISA methodologies previously mentioned. Two additional cell lines (NCI-H647 and NCI-H1792) have been characterized using these approaches. The additional cell lines allow for the investigation of lipoxygenase baseline expression patterns in various clinical stages of human non-small cell lung adenocarcinoma: (NCI-H1437 – Stage 1, NCI-H1395 – Stage 2, NCI-H647 – Stage 3A, NCI-H1792 – Stage 4).

Specific Aim 3
Lipoxygenases are non-heme iron-containing dioxygenases that catalyze the addition of molecular oxygen to fatty acids containing a cis,cis-1,4-pentadiene system. The initial product of this reaction is a 4-hydroperoxy cis-trans-1,3-conjugated pentadienyl moiety within the unsaturated fatty acid. The three main LO enzymes are designated 5-, 12-, and 15- Lipoxygenase based on the position of the introduced hydroperoxide. Linoleate and arachidonate are the common substrates for lipoxygenases in plants and animals. Determination of the IC50 of the drug treatment groups (Table 1) was preformed by detecting and measures the hydroperoxides produced in the lipoxygenation reaction using a purified lipoxygenase. The detection reaction is equally sensitive to hydroperoxides at various positions within the fatty acid and will work with fatty acids of any carbon length. These results are being used to establish a dose to correlate baseline lipoxygenase expression and metabolite formation to growth inhibition.

TABLE 1: DRUG TREATMENT GROUPS
DRUG CLASSIFICATION DOSE RANGE (µM) TREATMENT DURATION
NDGA 5-, 12-, 15-LO inhibitor 2 to 50 5 minutes
REV 5901 Cysteinyl-leukotriene receptor antagonist 2 to 50 5 minutes
LY255283 Competitive BLT2 receptor antagonist 2 to 50 5 minutes
NDGA + REV 5901 (1:1) 5-, 12-, 15-LO inhibitor + cysteinyl-leukotriene receptor antagonist 2 to 50 5 minutes
NDGA + LY255283 (1:1) 5-, 12-, 15-LO inhibitor + competitive BLT2 receptor antagonist 2 to 50 5 minutes

The IC50 values of the drug treatment groups measured via 5-, 12-, or 15-lipoxygenase expression are listed in Tables 2, 3, and 4, respectively. Of note, a direct relationship was observed between the clinical stage of the human non-small cell lung adenocarcinoma cell line and 5-, 12-, or 15-lipoxygenase expression. Addition of REV 5901 or LY255283 to NDGA increases the IC50 values of 5- and 12-lipoxyengase expression compared to NDGA monotherapy. In contrast, addition of REV 5901 or LY255283 to NDGA decreases the IC50 values of 15-lipoxyengase expression compared to NDGA monotherapy. The direct relationship between the clinical stage of the human non-small cell lung adenocarcinoma cell line and 5-, 12-, or 15-lipoxygenase expression was not affected by the addition of REV 5901 or LY255283 to NDGA. Differences in lipoxygenase expression were determined by ANOVA (significance determined by p < 0.05).

2nd Year Studies:
The second year of the fellowship will involve the exploration of the therapeutic impact the drug treatment groups have on lipoxygenase modulation with the overall objective of identifying the most effective lipoxygenase expression pattern for inhibition of lung tumor growth. This will involve extensive training in cellular signaling that will support my transition into a career in chemopreventative drug development research. Specifically, second year investigations will involve death receptor signaling (Table 5) and mitochondrial control of apoptosis (Table 6) evaluated via western blotting. The results of these experiments will lead to further investigations of signal transduction events that occurred as a result of lipoxygenase modulation.

SIGNIFICANCE:
Significance of Specific Aim 1: Lipoxygenase expression and metabolite formation data is available for only two non-small cell lung adenocarcinoma cells lines, A549 and NCI-H1264, and focuses only on one specific lipoxygenase enzyme. This aim would provide data on (1) differences and similarities of lipoxygenase expression and metabolite formation between smokers and nonsmokers (2) the sum effect that lipoxygenase enzymes and metabolites have in terms of tumor growth and apoptosis.

Significance of Specific Aim 2: Lipoxygenase expression and metabolite formation data have never been evaluated by clinical stage of non-small cell lung adenocarcinoma. This aim would provide data on (1) differences and similarities of lipoxygenase expression and metabolite formation related to clinical stage of non-small cell lung adenocarcinoma and (2) the sum effect that lipoxygenase enzymes and metabolites have in terms of tumor growth and apoptosis.

Significance of Specific Aim 3: A comparison of these cell lines treated with or without specific or nonspecific lipoxygenase inhibition can provide valuable information as to which lipoxygenase or combination of lipoxygenases supports and inhibits tumor growth.

TRAINING ACTIVITIES:
1st Year Training Activities: Over the last year, I have gained extensive experience in a variety of laboratory skills (i.e., working with cancer cell lines, a mouse lung adenocarcinoma model, various aspects of drug treatment, protein identification and quantification, etc.). To augment my fellowship experience, I have begun formal clinical research training through the Arizona Clinical Research Training Program (AzCRTP) offered through the Mel and Enid Zuckerman Arizona College of Public Health. The AzCRTP objectives are to educate program scholars in the principles, methods, and techniques used in clinical research and to provide the tools necessary to become independently funded clinical researchers. Individuals are awarded an AzCRTP Certificate of Completion at the end of the two year program. Table 7 lists the courses I have completed in the program along with my cumulative grade point average.

2nd Year Training Activities: During my second year, I will continue to expand my laboratory and analytical skills to enable me to transition into a career in chemopreventative drug development research. I will complete the remaining coursework (Table 8) in the AzCRTP program so I will be eligible for the certificate of completion at the end of the program. In addition, I will be working with undergraduate students and other post-doctorial fellows in the lab.

PUBLICATIONS: I plan to submit my preliminary results (abstracts/posters) to the 2007 Annual Meeting of The American Association for Cancer Research (AACR) in Los Angeles, CA. Upon completion of the studies, I plan to submit my final manuscript to a leading cancer research journal.