The aims of this study were to determine the anti-tumor activity of a whole blueberry preparation against MDA-MB-231 cells in a xenograft mouse model and to investigate the modulation of the metastatic process in vitro. Preliminary results showed that among a panel of whole food preparations, blueberry exhibited the highest antiproliferative effect against MDA-MB-231 cells while having no effect on the non-transformed MCF-10A cell line. One aim in developing chemopreventive agents is to identify those that are easily accessible and non-toxic, therefore, blueberry was chosen for further study.
The failure of the isolated blueberry fractions to equal or exceed the antiproliferative activity of the whole blueberry extract is similar to results seen with the fractionation of cranberry juice. In that study, whole cranberry juice had higher antiproliferative activity than isolated anthocyanin and proanthocyanidins fractions (33
). Because the isolated compounds from blueberry and cranberry are phenolic in nature, they are prone to oxidation/decomposition when purified and taken out of the matrix of the whole fruit/juice. Therefore, we conclude that the isolated fractions are less active than whole blueberry due to their separation from the whole, and whole blueberry extract was utilized for further investigations.
experiments showed no adverse effects of blueberry when ingested by mice. In addition, oral activity was observed through decreased growth of MDA-MB-231-derived tumors in a xenograft mouse model. The decreased tumor size was attributed to decreased cell proliferation and increased apoptosis, as measured by Ki-67 and caspase-3 immunohistochemical staining of tumor tissues. A recent study by Aiyer et al. showed comparable results in rats, where oral administration of whole blueberry powder decreased breast tumor volume by 40% compared to untreated control animals (34
). Similar studies showed that oral ingestion of blueberry extract decreased hemangioendothelioma tumor size in mice (35
). In contrast, studies by Stoner et al. showed that diets containing 5 and 10% of whole blueberry powder did not inhibit N-nitrosomethylbenzylamine-induced esophageal tumors in rats (36
). The differing inhibitory effects on breast and esophageal tumor growth observed in these two studies are likely due to the different tumor models used. Overall, studies suggest that the oral intake of blueberries may convey chemopreventive benefits in vivo
Inhibition of invasive potential is important to the prevention of tumor reoccurrence. In vitro
experiments demonstrated that treatment with blueberry extract inhibited HGF-induced migration of MDA-MB-231 cells and decreased MMP-9 activity. A large number of natural products, including berries, have been shown to inhibit metastatic potential in cancer cell lines including prostate, lung, breast and fibrosarcoma. These products were found to target key proteins including MMP-2 and 9, uPA and the MMP inhibitors TIMP-1 and PAI. In addition, the AKT, NFκB, AP-1, JNK and ERK signaling pathways were also regulated in a number of these studies, illustrating the importance of these signaling pathways. (18
). The secretion of metastasis-related proteins has been shown to be, at least in part, under the control of the above-mentioned cell signaling pathways, although their involvement can vary by tissue/cell type. For example, our in vitro
experiments showed that blueberry treatment of MDA-MB-231 cells had no effect on the activation of PKC and ERK, where it inhibited the activation of the PI3K/AKT/NFκB pathway. Therefore, in further studies on the effects of blueberry on metastasis related proteins, we focused on the role of this pathway.
In our experiments with blueberry, the modulation of metastatic proteins was shown to be through a decrease in uPA secretion and increased secretion of the MMP inhibitor PAI, the expression of which can be controlled by the PI3K/AKT/NFκB pathway. These cell signaling proteins are also important to the proliferation and survival of tumor cells (42
). Therefore, one mechanism by which blueberry may alter tumor growth and metastatic potential of MDA-MB-231 cells is through modulation of the PI3K/AKT/NFκB pathway. In contrast, Huang et al. reported that a methanol fraction of blueberry failed to inhibit UVB-induced activation of NFκB and AP-1 in mouse epidermal cells (30
). The authors speculated that these observations may be due to the different anthocyanin profile of blueberries as compared to other berries. The Huang study induced skin carcinogenesis in mice via exposure to UV radiation. In addition, the authors treated the mice with a methanol fraction of blueberry. Therefore, the different cancer type, route of induction and blueberry treatment (our study utilized blueberry juice which contains both water and lipid-soluble compounds that could work synergistically to inhibit NFκB activity) likely accounts for the differing inhibitory effect of blueberry on NFκB between the Huang study and ours.
The results of our studies suggest that the oral intake of blueberries could be a key component of long-term breast cancer prevention strategies. We showed not only that blueberry ingestion in mice decreased tumor growth but also the activity of AKT and NFκB, which are markers for metastatic potential in breast tumors. Several studies of tissues from breast cancer patients have shown that the activity of PI3K and AKT are significantly increased in the triple-negative subset of breast cancers (44
). This illustrates the importance of these signaling pathways to the pathology of this disease.
A limitation of this study is that in vitro studies cannot be extrapolated to possible activity in vivo. The role of in vitro studies is to screen for activity against biomarkers that can then be tested in vivo using animal studies. Our intent with the in vitro studies was to evaluate mechanisms through which blueberry may modulate the metastatic potential of MDA-MB-231 cells, which are now under investigation in vivo.
It was suggested that the antiproliferative activity of fruit extracts against cancer cell lines is due to the production of hydrogen peroxide (H2
) and resultant oxidative stress (47
). However, Liu et al showed that H2
was not produced after addition of apple extracts to medium, there is no correlation between phenolic content and antiproliferative activity, and suggested that the previous results were due to incorrect measurement methods (48
). Therefore, the results of our proliferation studies are likely a consequence of the effects of the phytochemicals in blueberries, not the production of H2
in the cell medium.
The dose of blueberry in our in vivo
study is equal to a fresh blueberry intake of 25 grams/kg. With a conversion to human dose (based on surface area) (49
), this is equal to 2.03 g/kg human or 122 grams (4.3 oz) of fresh blueberries/day for a 60 kg person. A single serving of fresh blueberries is 6 oz. which is an attainable intake for the average person. Therefore, blueberry intake could be an important part of dietary cancer prevention strategies.