title: Pterostilbene induces mitochondrially derived apoptosis in breast cancer cells in vitro.
abstract: BACKGROUND
  The ability of a breast cancer cell to evade apoptosis has a key role in tumor progression and sensitivity to treatment. High levels of Bcl-2-associated X protein (Bax) in tumor cells have been found to promote apoptosis and sensitize cells to anti-cancer therapies. Bcl-2-associated X protein redistribution to the mitochondrial membrane results in the release of proapoptotic factors including cytochrome C, second-mitochondrial-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low PI (Smac/DIABLO), and Ca(2+). We aimed to explore this pathway in cancerous breast cell lines treated with the naturally occurring antioxidant 3,5-dimethoxy-4-hydroxystilbene (pterostilbene).
  
  
  METHODS
  We used whole cell lysates +/- Bax SiRNA from the cell lines MCF-7 and MDA-MB-231 in an enzyme-linked immunosorbent assay to quantify Bax, cytochrome C, Smac/DIABLO expression, and manganese superoxide dismutase (MnSOD) activity after treatment with pterostilbene. We quantified cell death using histone-related DNA complexes from cytosolic and mitochondrial fractions and used methylthiazol tetrazolium assay to analyze cell proliferation, in the presence of Bax-silencing or scrambled RNA. We measured changes in cytosolic calcium using the ratiometric calcium-sensitive dye fura-2-AM using an inverted ratiometric monochromator microscope.
  
  
  RESULTS
  Treatment of MCF-7 and MDA-MB-231 (MDA) cells with pterostilbene caused concentration-dependent increases in intracellular Bax at all doses tested. RNA silencing of Bax resulted in reduced rates of apoptosis in both cells types and increased cell survival when treated with pterostilbene. We observed an increase in cytochrome C in MDA cells after treatment with pterostilbene. The MCF-7 cells showed a net increase in cytosolic cytochrome C, with a corresponding reduction in mitochondrial cytochrome C after treatment with 50 and 75 μmol/L pterostilbene. We observed this again in Smac/DIABLO expression in both cell types. In MCF-7 cells, pterostilbene treatment caused an increase in cytosolic but a decrease in mitochondrial Smac/DIABLO protein concentrations. Pterostilbene significantly increase MnSOD activity in MDA-MB-231 cells. Finally, pterostilbene resulted in significant increases in cytosolic calcium concentrations.
  
  
  CONCLUSIONS
  The natural dietary compound pterostilbene has an anti-proliferative effect and induces apoptosis in breast cancer cells in vitro via Bax activation and overexpression, resulting in increased MnSOD, Smac/DIABLO, and cytochrome C activity and cytosolic Ca(2+) overload.
authors.affiliation: Division of Surgical Research, University of Vermont, 111 Colchester Avenue, Fletcher House 311, MCHV Campus, Burlington, Vermont.
authors.forename: Dora
authors.initials: D
authors.lastname: Moon
authors.forename: Denise
authors.initials: D
authors.lastname: McCormack
authors.forename: Debbie
authors.initials: D
authors.lastname: McDonald
authors.forename: David
authors.initials: D
authors.lastname: McFadden
mesh_terms: D000975:Antioxidants / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins; D001943:Breast Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology*; D002118:Calcium / Q000378:metabolism; D045744:Cell Line, Tumor; D045304:Cytochromes c / Q000378:metabolism; D005260:Female; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000032:analysis; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000032:analysis; D013267:Stilbenes / Q000494:pharmacology*; D013482:Superoxide Dismutase / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000032:analysis
chemical_list: D000975:Antioxidants; D051017:Apoptosis Regulatory Proteins; C494826:BAX protein, human; C412403:DIABLO protein, human; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; D013267:Stilbenes; D051028:bcl-2-Associated X Protein; C107773:pterostilbene; D045304:Cytochromes c; D013482:Superoxide Dismutase; D002118:Calcium