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404 Nω-HYDROXY L-ARGININE-INDUCED APOPTOSIS IN HUMAN BREAST CANCER CELLS: PROTEOMIC APPROACH FOR THE IDENTIFICATION OF KEY TARGET PROTEINS.
  1. R. Singh1,
  2. S. Pervin2,
  3. N. K. Avliyakulov3,
  4. M. Haykinson3,
  5. T. Rajavashisth1,
  6. G. Chaudhuri2
  1. 1Division of Endocrinology,and RCMI Molecular Medicine Research Core at Charles R. Drew University, Los Angeles, CA
  2. 2Division of Obstetrics and Gynecology
  3. 3Functional Proteomics Laboratory, David Geffen School of Medicine at UCLA, Los Angeles, CA

Abstract

NΩ-Hydroxy l-arginine (NOHA) induces apoptosis in human breast cancer MDA-MB-468 cells by caspase 8-mediated cleavage of proapoptotic Bid molecule, leading to the release of cytochrome c and activation of caspase 3. This apoptotic action was antagonized by exogenous l-ornithine (Singh et al. Cancer Research 2000; Singh et al. J Biol Chem 2002).

Objective To identify key proteins involved during NOHA-induced apoptosis and understand precise mechanisms by which l-ornithine inhibit NOHA-induced apoptosis.

Methods and Results We transfected MDA-MB-468 cells with full-length Bcl2 and selected stable cells line overexpressing Bcl2. NOHA-induced cytochrome c release, activation of caspase 3, and inhibition of cell proliferation were blocked in these cells; however, it did not blocked cleavage of Bid or caspase-8 activation, suggesting that mitochondria may be the primary site of NOHA action. We performed proteomic analysis of control cells and cells treated with NOHA (1 mM) alone or in combination with l-ornithine (0.5 mM). Protein samples obtained from all three groups were labeled with Cy2-, Cy3-, and Cy5-based fluorescent dyes and analyzed using methods of difference gel electrophoresis (DIGE). Samples were subjected to two-dimensional electrophoresis and fluorescent images were obtained. More than 100 spots have been identified as having protein expression changes of 1.5 times or more after NOHA treatment compared with a control group. The majority of proteins after simultaneous treatment with l-ornithine and NOHA had very similar protein expression profiles as a control group. Proteins of interest are currently being excised from gels, digested with trypsin, and identified using a MALDI TOF-TOF mass spectrometer.

Significance Macrophages and myoepithelial cells at the site of breast cancer express nitric oxide synthase (NOS) and therefore have the capacity to generate both NOHA and NO. Proteomic analysis leading to the identification of key mitochondrial proteins targeted by these two signaling molecules, during macrophage activation, as a result of various inflammatory diseases including breast cancer, will help our overall understanding of the process required for better therapeutic interventions.

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