| Our laboratory has demonstrated the preventive effects of freeze-dried black raspberries (BRB) on tumor development in the rat esophagus and colon in vivo. One of the mechanisms by which BRB inhibit tumorigenesis in vivo is to reduce the growth rate of premalignant cells. In attempts to explain these inhibitory effects, we tested two BRB extracts and two component anthocyanins (cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside) in BRB for their effects on growth, apoptosis and gene expression in weakly tumorigenic (RE-149) and highly tumorigenic (RE-149 DHD) rat esophagus cell lines. The ethanol (EtOH) extract, at 100 mug/ml medium, produced 16% and 45% growth inhibition of RE-149 and RE-149 DHD cells, respectively. The water-soluble extract was less inhibitory for both cell lines than the EtOH extract, but retained selectivity for RE-149 DHD cells. The inhibitory effect does not appear to be due to the formation of hydrogen peroxide in the medium. The anthocyanins had a selective growth-inhibitory pattern similar to that of both extracts. The EtOH extract (100 mug/ml) and cyanidin-3-O-rutinoside (50 mug/ml) induced capsases 3 and 7 activities significantly in RE-149 DHD cells. The growth inhibitory and the pro-apoptotic effects were enhanced by the daily addition of the EtOH extract and the anthocyanins to the medium. Cyanidin-3-O-rutinoside and cyanidin-3-O-glucoside, each at 50 mug/ml medium, down-regulated COX-2 and iNOS significantly while the EtOH extract (100 mug/ml) up-regulated both genes non-significantly. Maximal uptake of the anthocyanins in the EtOH extract by RE-149 DHD cells occurred after 1 hour of extract treatment followed by a rapid loss of anthocyanins from the cells. The uptake of anthocyanins by RE-149 cells had the same pattern as RE-149 DHD cells, but the amount of anthocyanins taken up was 100 times lower. This finding might explain the difference in the inhibitory effect of EtOH extract in both cell lines. Using DNA microarray technique, the EtOH extract was found to down-regulate multiple genes associated with esophageal tumors including, but not limited to, fibroblast growth factor, integrin alpha 7, mitogen activated kinase 9 (MAPK 9), Wnts 1, 5 and 7, cyclin-dependent kinases (cdk2 and 5), cell division cycle (Cdc 25), and Met proto-oncogene. The matrix metalloproteinases were up-regulated at 6 hours, and down-regulated at the other time points. No other genes were up-regulated significantly at the 2-fold cutoff point. The calcium signaling pathway was a major pathway modulated by the extract since several genes were downregulated and all of them are linked to calcium, and all are involved in tumor development and progression in several organ sites. These genes include: calmodulin kinase II (CaMKII), protein kinase C iota (PKCliota), MAPK 9, and Cdc 25 a and b. The EtOH extract was found to down-regulate 16 genes consistently at all time points, one of these genes was SERCA2a which functions as a calcium pump in the endoplasmic reticulum, pointing to calcium regulation as being one of the mechanisms of action of the EtOH extract. In addition to SERCA2a, the EtOH extract consistently downregulated fibroblast growth factor receptor 2, insulin-like growth factor binding protein 5, secreted protein acidic and rich in cysteine (SPARC), and galanin, all of which were demonstrated to play crucial roles in tumor development in multiple organs including the esophagus. Additional studies should be done to measure the intracellular calcium level as well as the calcium calmodulin kinase activity in order to further establish a relationship between EtOH extract treatment, calcium level intracellularly, and down-regulation of calcium regulated genes that might explain the growth inhibitory effect of BRB. |
