There have been multiple cell studies with berry extracts produced as described by Xue, et al. [42
] to investigate the mechanisms of action of BRBs and to identify bioactive components. Only the alcohol fraction showed a dose-dependent decrease in ability of benzo[a]pyrene (B[a]P)-to induce transformation of Syrian hamster embryo (SHE) cells [42
]. Similarly, Han, et al. [43
] found that the dichloromethane and alcohol fractions, and two known components; namely, ß-sitosterol and ferulic acid, selectively inhibited the growth of premalignant and malignant human oral cavity cell lines but not normal oral cells. They further found reduced levels of cyclin A and cell division cycle gene 2 (cdc2) in the premalignant cells, and reduced levels of cyclin B1, D1 and cdc2 in the malignant cells. The alcohol fraction also increased the level of p21waf1/cip1
but did not influence cell cycle distribution. In contrast, treatment of premalignant and malignant oral cells with ferulic acid resulted in accumulation of the cells in the G2/M
phase of the cell cycle. Ferulic acid also induced cyclin B1 and cdc2 in both cell lines and p21waf1/cip1
in the malignant cell line. ß-sitosterol treatment led to accumulation of both premalignant and malignant cell lines in the G2/M and G0/G1
phases of the cell cycle, increased levels of p21waf1/cip1
and decreased levels of cyclin B1 and cdc2 genes in both cell lines. Rodrigo, et al. [44
] also evaluated the preventative effects of the alcohol fraction on human oral cancer cell lines and found inhibition of cell proliferation, VEGF production, nitric oxide synthase activity, apoptosis, and terminal cell differentiation. Lastly, preliminary data from our laboratory indicates that treatment of tumorigenic and non-tumorigenic rat esophageal epithelial cells with the alcohol fraction, or individual anthocyanins (cyanidin-3-O
-glucoside or cyanidin-3-O
-rutinoside) inhibits proliferation, induces apoptosis and modulates the expression of COX-2 and iNOS in tumorigenic cells as compared to non-tumorigenic cells [45
In initial studies with the JB-6 Cl 41 mouse epidermal cell model [40
] with the alcohol fraction showed inhibition of BPDE-induced AP-1 and NFκB transactivation. The inhibitory effects appeared to be mediated via inhibition of mitogen activated protein kinase activation and inhibitory subunit κB phosphorylation, respectively. Pretreatment of cells with this fraction did not result in an inhibition of BPDE binding to DNA; thus, this was not a mechanism of reduced AP-1 and NFκB activation. Interestingly, none of the tested fractions was found to affect p53-dependent transcription activity [40
]. The ability of BRBs to inhibit tumor development may be mediated by impairing signal transduction pathways leading to activation of AP-1 and NFκB. This supposition is supported by the demonstration that the alcohol fraction markedly inhibited activation of PI-3K, Akt, and p70 S6 kinase, suggesting that another mechanism for the chemopreventive activity may be through inhibition of the PI-3K/Akt/AP-1/VEGF pathway () [46
Fig. 3 Schematic illustration of known molecular mechanisms that may be involved in the chemopreventive activities of alcohol-soluble black raspberry fraction .
] fractionation studies point to cyanidin-3-O
-glucoside and cyanidin-3-O
-xylosylrutinoside as the active components of BRBs. However, another laboratory [47
] found that the ellagitannins in red raspberries were the most effective inhibitors of the growth of human cervical cancer (HeLa) cells in vitro.
Also, extracts of other berry types (bilberry, black currant, cloudberry, lingonberry, raspberry and strawberry) reduced the growth of HT-29 colon cancer cells [48
]. The bilberry extract was the most potent and the strawberry extract the least. Interestingly, even though having a low content of anthocyanins, the cloudberry extract caused a 14-fold induction of p21waf1
and induced BCL-2 associated X protein (Bax) leading to the overall conclusion that, in addition to the anthocyanins, other berry components are responsible for their chemopreventive effects.