Role of a polyphenol-enriched preparation on chemoprevention of mammary carcinoma through cancer stem cells and inflammatory pathways modulation

Abstract

Background: Naturally occurring polyphenolic compounds from fruits, particularly from blueberries, have been reported to be significantly involved in cancer chemoprevention and chemotherapy. Biotransformation of blueberry juice by Serratia vaccinii increases its polyphenolic content and endows it with anti-inflammatory properties.

Methods: This study evaluated the effect of a polyphenol-enriched blueberry preparation (PEBP) and its non-fermented counterpart (NBJ), on mammary cancer stem cell (CSC) development in in vitro, in vivo and ex vivo settings. Effects of PEBP on cell proliferation, mobility, invasion, and mammosphere formation were measured in vitro in three cell lines: murine 4T1 and human MCF7 and MDA-MB-231. Ex vivo mammosphere formation, tumor growth and metastasis observations were carried out in a BALB/c mouse model.

Results: Our research revealed that PEBP influence cellular signaling cascades of breast CSCs, regulating the activity of transcription factors and, consequently, inhibiting tumor growth in vivo by decreasing metastasis and controlling PI3K/AKT, MAPK/ERK, and STAT3 pathways, central nodes in CSC inflammatory signaling. PEBP significantly inhibited cell proliferation of 4T1, MCF-7 and MDA-MB-231. In all cell lines, PEBP reduced mammosphere formation, cell mobility and cell migration. In vivo, PEBP significantly reduced tumor development, inhibited the formation of ex vivo mammospheres, and significantly reduced lung metastasis.

Conclusions: This study showed that polyphenol enrichment of a blueberry preparation by fermentation increases its chemopreventive potential by protecting mice against tumor development, inhibiting the formation of cancer stem cells and reducing lung metastasis. Thus, PEBP may represent a novel complementary alternative medicine therapy and a source for novel therapeutic agents against breast cancer.

Fig. 1

PEBP suppressed the growth of mammary carcinoma cell lines. Proliferation of 4T1 (a), MDAMB-231 (b), and MCF-7 (c) cells after treatment with either 150 or 200 μM GAE (gallic acid equivalent) of either polyphenol-enriched blueberry preparation (PEBP) or normal blueberry juice (NBJ) for 24 h. All values are means of 3 separated experiments ±SEM. *Denotes statistical significance at p ≤ 0.05 vs. control. **Denotes p ≤ 0.01 vs. control

Fig. 2

PEBP decreased motility and invasiveness in gel invasion experiment. Cell mobility of 4T1 (a), MDAMB-231 (b), and MCF-7 (c) cells after treatment with 100 μM GAE (gallic acid equivalent) of either polyphenol-enriched blueberry preparation (PEBP) or normal blueberry juice (NBJ) for 24 or 48 h and cell invasion of 4T1 (d) and MDAMB-231 (e) cells after treatment with either 100 or 150 μM GAE of PEBP or NBJ for 24 h. Contrast was enhance to better show cell motility. All values are means of 3 separated experiments ±SEM. *Denotes statistical significance at p ≤ 0.05 vs. control

Fig. 3

PEBP and NBJ decreased the formation of mammospheres in cell culture. Mammospheres formation of 4T1 (a), MDAMB-231 (b), and MCF-7 (c) cells after treatment with either 100 or 150 μM GAE (gallic acid equivalent) of either polyphenol-enriched blueberry preparation (PEBP) or normal blueberry juice (NBJ) for 4–7 days and IL-6 production by 4T1 (d), MDAMB-231 (e), and MCF-7 (f) cells after treatment with 150 μM GAE of PEBP or NBJ for 6 and 24 h. All values are means of 4 separated experiments ±SEM. Bars that have no letter in common are significantly different from each other (p ≤ 0.05)

Fig. 4

PEBP inhibited STAT3/PI3K/Akt signaling pathway. Phosphorylation of STAT3, PI3K, Akt, and PTEN in 4T1 (a, d, g, j), MDAMB-231 (b, e, h, k), and MCF-7 (c, f, i, l) mammospheres after treatment with 150 μM GAE (gallic acid equivalent) of either polyphenol-enriched blueberry preparation (PEBP) or normal blueberry juice (NBJ) for 6 h. All values are means of 3 separated experiments ±SEM. Baseline represent the level of phosphorylation present in cells not exposed to the mammospheres forming medium. *Denotes statistical significance at p ≤ 0.05 vs. control at 6 h

Fig. 5

PEBP inhibited ERK1/2 but enhanced MAPKp38, and JNK signaling. Phosphorylation of ERK1/2, MAPK p38, and JNK in 4T1 (a, d, g), MDAMB-231 (b, e, h), and MCF-7 (c, f, i) mammospheres after treatment with 150 μM GAE (gallic acid equivalent) of either polyphenol-enriched blueberry preparation (PEBP) or normal blueberry juice (NBJ) for 2 h. All values are means of 3 separated experiments ±SEM. Baseline represent the level of phosphorylation present in cells not exposed to the mammospheres forming medium. * Denotes statistical significance at p ≤ 0.05 vs. control at 2 h

Fig. 6

Antitumoral effects of PEBP in BALB/c mice model with 4T1 cell challenge. Tumor volume (a), tumor weight (b), mammospheres formation from primary tumor cells (c) and metastasis present in lungs (d) of mice that received a 2-week pre-treatment and a 3-week post-inoculation treatment with either polyphenol-enriched blueberry preparation (PEBP) or normal blueberry juice (NBJ) incorporated in drinking water at concentrations of 12.5 % (NBJ 12.5 % and PEBP 12.5 %), 25 % (NBJ 25 % and PEBP 25 %), and 50 % (NBJ 50 % and PEBP 50 %). All values are means of 2 separated experiments ±SEM (n = 16). *Denotes statistical significance at p ≤ 0.05 vs. control