Colon cancer remains a significant health concern as the third most prevalent cancer and the second leading cause of cancer deaths in the United States. Two lipid peroxidizing enzymes, e.g. lipoxygenase (15-LOXs) and cyclooxygenase (COXs), that metabolize dietary and membrane lipids (polyunsaturated fatty acids, or PUFAs) are particularly involved in colon cancer progression. The products generated from 15-LOX- and COX-catalyzed PUFA peroxidation have been of great interest due to their potent and diverse biological activities. However, the precise roles of COX-2 and 15-LOXs in colon cancer progression, as well as the effects of their individual lipid metabolites, are still unclear. In addition, PUFA-derived free radicals, the most reactive intermediates in 15-LOX and COX lipid peroxidation, have not yet been identified nor their bioactivities characterized in colon cancer progression due to the lack of appropriate methodology. Here, an innovative approach consisting of on-line Liquid Chromatography/Electron Spin Resonance (LC/ESR) and on-line Liquid Chromatography/Mass Spectrometry (LC/MS) will be used for the first time to identify novel free radicals from cellular 15-LOX- and COX-catalyzed peroxidation of I-6/I-3 PUFAs, and to characterize them individually and relative to each other in terms of their effects on colon cancer progression. The working hypotheses of this study are: (1) certain types of PUFA-derived radicals formed from 15-LOX and COXs are consistently correlated with colon cancer progression, e.g. cancer cell proliferation and cell apoptosis;and (2) the impaired balance between 15-LOX vs. COXs in metabolizing different PUFAs is the key event in colon cancer progression. The hypothesis will be tested by pursuing the following Specific Aims: (1) To structurally and quantitatively profile carbon-centered PUFA-derived free radicals in human colon cancer cells via LC/ESR and LC/MS;(2) To structurally and quantitatively profile oxygen-centered PUFA-derived free radicals in human colon cancer cells via LC/ESR and LC/MS;and (3) To assess the association among PUFA-derived radical metabolites, alteration of COX-2/15-LOX1 expression, and growth response (proliferation and apoptosis) in colon cancer cells.
These aims will be achieved using a variety of techniques, e.g., LC/ESR, LC/MS, real time RT-PCR, Western blotting, MTT, and TUNEL in conjunction with human colon (cancer) cell lines, HC116 and Caco-2, and their engineered variants (overexpression of 15-LOX via transfection of 15-LOX cDNA). Successful completion of these aims will open a new door into knowledge of PUFA peroxidation in cancer biology and will revolutionize how we use our knowledge of enzymatic peroxidation to control and prevent colon cancer growth.
The novel combined LC/ESR and LC/MS technique proposed in this study creates an opportunity to gain new insights into the mechanistic relevance of polyunsaturated fatty acid (PUFA) peroxidation in cancer research. For the first time, the most reactive intermediates in lipoxygenase (LOX)- and cyclooxygenase (COX)-catalyzed PUFA lipid peroxidation, e.g., both carbon- and oxygen-centered lipid-derived free radicals, will be studied for their potential effects on colon cancer progression. We anticipate that the outcome of our research will open a new door into knowledge of PUFA peroxidation in cancer biology and will revolutionize how we use our knowledge of enzymatic peroxidation to control colon cancer.
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