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Induction of tumor-associated angiogenesis, production of high levels of reactive species, and sustained cellular proliferation are growth perturbations that often accompany malignant transformation. Our lab has recently shown that an ethanol extract of black raspberries (RO-ET) significantly suppresses proliferation of oral SCC cells without affecting cellular viability; implying RO-ET induces apoptotic and/or differentiation pathways. This study used oral SCC cell lines to investigate RO-ET’s abilities to suppress pathways (induction of angiogenesis, production of reactive species, sustained proliferation) that are known to facilitate oral SCC development. Accordingly, the ability of RO-ET (100 μg/ml) to suppress angiogenesis (VEGF ELISA), inhibit reactive species production [nitric oxide synthase (NOS) spectrophotometric assay], induce differentiation (transglutaminase spectrophotometric assay) and stimulate apoptosis (caspase-3 cleavage of DEVD-fluorimetric assay) was determined. Although baseline VEGF levels showed a large, cell line-associated range (206,000 to 1,197,000 pg/mg protein), RO-ET suppressed VEGF release in each cell line and in every experiment (n=8, p<0.005, Yates corrected Chi square test). Similarly, while baseline NOS activities showed a 10 fold variation (2.5 to 25 U/mg protein), RO-ET inclusion significantly suppressed NOS function (p<0.028, two tailed Mann Whitney U test). RO-ET treatment also increased activity of the differentiation-associated transglutaminase enzyme in all cell lines; with increases ranging from 8.5 to 260% relative to matched log growth controls. Finally, RO-ET activated the execution phase apoptotic enzyme caspase-3 in four out of five cell lines evaluated; with the single refractory cell line demonstrating a 119% fold increase in transglutaminase function. Studies to assess RO-ET’s effects on the apoptosis-inducing mitochondrial membrane permeability suggest that RO-ET perturbs mitochondrial membrane pore integrity. Additional ongoing studies imply that RO-ET reduces cyclooxygenase (both isoforms) function by a dual mechanism that entails suppression of gene expression as well as inhibition of enzyme function. Our data show that by its abilities to inhibit biochemical mechanisms whose activation facilitates malignant transformation while concurrently upregulating apoptotic and/or terminal differentiation pathways, RO-ET is a unique and promising chemopreventive agent.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]