Refinement of LC/ESR/MS for Studying Lipid Free Radicals in Colon Cancer Growth

Summary

Principal Investigator: Steven Qian
Abstract: DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.
Funding Period: 2009-07-01 - 2012-07-31
more information: NIH RePORT

Top Publications

  1. pmc An advanced Electron Spin Resonance (ESR) spin-trapping and LC/(ESR)/MS technique for the study of lipid peroxidation
    Yi Xu
    Department of Pharmaceutical Sciences, College of Pharmacy, Nursing and Allied Sciences, North Dakota State University, Fargo, ND 58108, USA
    Int J Mol Sci 13:14648-66. 2012
  2. pmc The first characterization of free radicals formed from cellular COX-catalyzed peroxidation
    Yan Gu
    Department of Pharmaceutical Sciences, College of Pharmacy, Nursing, and Allied Sciences, North Dakota State University, Fargo, ND 58105, USA
    Free Radic Biol Med 57:49-60. 2013
  3. pmc Characterization of free radicals formed from COX-catalyzed DGLA peroxidation
    Ying Xiao
    Department of Analytical Chemistry, China Pharmaceutical University, Nanjing 210009, China
    Free Radic Biol Med 50:1163-70. 2011
  4. pmc Antioxidant activity and phenolic compositions of lentil (Lens culinaris var. Morton) extract and its fractions
    Yanping Zou
    Department of Cereal and Food Science, College of Pharmacy, Nursing, and Allied Sciences, North Dakota State University, Fargo, North Dakota 58108 6050, United States
    J Agric Food Chem 59:2268-76. 2011
  5. pmc Docosahexaenoic acid (DHA) sensitizes brain tumor cells to etoposide-induced apoptosis
    F Wang
    Department of Pharmaceutical Sciences, North Dakota State University, Fargo, ND 58105, USA
    Curr Mol Med 11:503-11. 2011
  6. pmc Fluorescent liposomes for differential interactions with glycosaminoglycans
    Erin K Nyren-Erickson
    Department of Pharmaceutical Sciences, North Dakota State University, Fargo, North Dakota 58108 6050, United States
    Anal Chem 83:5989-95. 2011
  7. pmc LC/ESR/MS study of pH-dependent radical generation from 15-LOX-catalyzed DPA peroxidation
    Preeti Purwaha
    Department of Pharmaceutical Sciences, College of Pharmacy, Nursing, and Allied Sciences, North Dakota State University, Fargo, ND 58105, USA
    Free Radic Biol Med 51:1461-70. 2011

Detail Information

Publications7

  1. pmc An advanced Electron Spin Resonance (ESR) spin-trapping and LC/(ESR)/MS technique for the study of lipid peroxidation
    Yi Xu
    Department of Pharmaceutical Sciences, College of Pharmacy, Nursing and Allied Sciences, North Dakota State University, Fargo, ND 58108, USA
    Int J Mol Sci 13:14648-66. 2012
    ....
  2. pmc The first characterization of free radicals formed from cellular COX-catalyzed peroxidation
    Yan Gu
    Department of Pharmaceutical Sciences, College of Pharmacy, Nursing, and Allied Sciences, North Dakota State University, Fargo, ND 58105, USA
    Free Radic Biol Med 57:49-60. 2013
    ..Our results indicate that free radicals generated from the distinct radical reactions in COX-catalyzed peroxidation may represent the novel metabolites of AA and DGLA that correspond to their contrasting bioactivity...
  3. pmc Characterization of free radicals formed from COX-catalyzed DGLA peroxidation
    Ying Xiao
    Department of Analytical Chemistry, China Pharmaceutical University, Nanjing 210009, China
    Free Radic Biol Med 50:1163-70. 2011
    ..This new finding will be further investigated for its association with various bioactivities of PGs, with potential implications for inflammatory diseases...
  4. pmc Antioxidant activity and phenolic compositions of lentil (Lens culinaris var. Morton) extract and its fractions
    Yanping Zou
    Department of Cereal and Food Science, College of Pharmacy, Nursing, and Allied Sciences, North Dakota State University, Fargo, North Dakota 58108 6050, United States
    J Agric Food Chem 59:2268-76. 2011
    ..The results suggested that the extract of Morton lentils is a promising source of antioxidant phenolics and may be used as a dietary supplement for health promotion...
  5. pmc Docosahexaenoic acid (DHA) sensitizes brain tumor cells to etoposide-induced apoptosis
    F Wang
    Department of Pharmaceutical Sciences, North Dakota State University, Fargo, ND 58105, USA
    Curr Mol Med 11:503-11. 2011
    ....
  6. pmc Fluorescent liposomes for differential interactions with glycosaminoglycans
    Erin K Nyren-Erickson
    Department of Pharmaceutical Sciences, North Dakota State University, Fargo, North Dakota 58108 6050, United States
    Anal Chem 83:5989-95. 2011
    ....
  7. pmc LC/ESR/MS study of pH-dependent radical generation from 15-LOX-catalyzed DPA peroxidation
    Preeti Purwaha
    Department of Pharmaceutical Sciences, College of Pharmacy, Nursing, and Allied Sciences, North Dakota State University, Fargo, ND 58105, USA
    Free Radic Biol Med 51:1461-70. 2011
    ..This pH-dependent alteration of radical formation and oxygenation pattern may have significant implications and provide a basis for our ongoing investigations of LOXs as well as fatty acids in cancer biology...