The strawberry (Fragaria x ananassa) was chosen as the model in this study for its high content of antioxidants and bioactive compounds, as well as for its high availability for food industry and fresh consumption13. In the last decades, growing interest has been focused on the antioxidant capacity of strawberries so that TAC is considered a quality parameter and an indicator of bioactive compounds present in the fruit. The antioxidant capacity of the strawberry indicates that its consumption could contribute to prevent and reduce oxidative reactions that cause negative effects on human health, playing different roles in the development of chronic diseases and cancers.

Moreover, even if the high TAC of strawberries has been proved, it has also been demonstrated that this parameter is strongly influenced by the strawberry genetic background and is strictly related to the presence of oxygen radical scavengers such as Vitamin C and polyphenols11,27,28,30,31. Among polyphenols, anthocyanins are quantitatively the most important phenolic compounds in strawberries13, so that more than 25 different anthocyanin pigments have been described from different varieties and selections32. Analyses of TPH, ACY and TFC in the “Alba” cultivar in the present study confirm the high content of phytochemicals of this strawberry.

Strawberries are important also for their high content of Vitamin C, which is even higher than that of citrus fruit. Vitamin C content is an essential parameter due to the high number of biological roles that it plays in humans, lowering the incidence of cardio-and cerebro-vascular diseases33, of several cancers34 and other health disorders such as lead toxicity35. When evaluating the Vitamin C content in strawberries, it is important to consider that the molecule is very labile and in adverse conditions undergoes oxidation, depending on several factors such as temperature, water and pH36.

The choice of using “Alba” strawberries in this study was also based on the very interesting results obtained in recent years using this cultivar, both in vitro37,38 and in vivo39,40.

As shown above, the “Alba” strawberry extract is able to strongly decrease cellular viability of the highly tumorigenic stromal cell line A17, which is characterized by mesenchymal features and metastasis formation ability26,41.

In particular, it is possible to distinguish two different biological behaviors: i) a cytostatic-like effect at low doses of PRSE and ii) an acute toxic effect at higher doses. The capacity of the strawberry extract to activate the apoptotic process, as well as its anti-cancer potential in other cancer models, was already characterized by other authors19. However, although these observations are in accordance with the increased hypodiploidy observed in this study at high PRSE doses, the analysis of DNA fragmentation by laddering assay did not show the induction of the apoptotic internucleosomic cleavage of DNA (data not shown). Interestingly, we observed an increased expression level of the precursor form of caspase-1 that, together with the monitored hypodiploidy, could suggest an activation of the apoptotic pathway at a very early stage and thus below the detection limit of the experimental approaches used. In addition, we found no evidence of caspase-3 modulation and/or activation (data not shown).

We hypothesized that sub-lethal doses of strawberry extract could be able to inhibit the known invasive potential of A17 breast cancer cells. Thus, the effect of PRSE on the cellular migration, which is known to play a pivotal role in invasion, was first analyzed at a biological level by wounding healing assay. The observation that low doses of “Alba” PRSE were sufficient to block cell migration gave us the rationale to extend the study at the molecular level. The subsequent characterization of the expression levels of genes involved in the cellular migration, adhesion and invasion processes allowed us to obtain an overall view of changes that take place in several pathways. In particular, the down-regulation of a subgroup of genes (Csf1, Mcam, Nr4a3 and Set) supports the anti-invasion effect of the PRSE extract because high expression levels of these genes are often associated to the invasive phenotype of cells. In the context of human breast cancer it has been already reported that: i) high expression of Csf1 is correlated to metastasis formation and thus has been proposed as negative prognostic factor; ii) up-regulation of human Mcam (named Muc18 in humans) promotes motility, invasiveness and tumorigenesis of human breast cancer cells; iii) high expression levels of Nr4a3 were found correlated with increased risk of developing distant metastasis in triple-negative breast cancer patients; iv) Set nuclear proto-oncogene (named also I2pp2a) is one of the genes down-regulated by the mushroom Ganoderma lucidum extract and is involved in the suppression of breast-to-lung cancer metastasis42,43,44,45. Interestingly, the modulation of Mcam and Nr4a3 was confirmed also at the protein level, further suggesting a role of these proteins in the effect induced by PRSE on A17 cells.

Likewise, we found two genes whose monitored up-regulation, as a consequence of PRSE treatment, can be consistent with an anti-invasion effect: i) Htatip2 (named also Tip30), a putative metastasis suppressor gene which is inversely correlated with lymph node metastasis in breast cancer patients and ii) Gpnmb (also named Osteoactivin/HGFIN), a gene with a controversial role in the metastatic process46,47.

Otherwise, it was not possible to find any correlation between the known activity of the remaining six genes found up-regulated by PRSE (Ccl7, Ctsl, Cxcr4, Itgb3, Mmp19, Mmp3) since their functions are generally associated to the acquisition of invasive features44,48,49,50,51.

As already stated, the role of strawberry bioactive compounds on cancer prevention seems to involve different mechanisms of action that are still unclear. Previous studies indeed indicate that the biological activity exerted by berries against cancer cells is probably mediated by the synergy between different compounds suggesting the involvement of several molecular pathways. To date, only few studies have tried to investigate the molecular basis of strawberry activity against breast cancer cells. For example, the role of p73 in triggering apoptosis of p53-null cells has been recently suggested19. Our study indicates the involvement of other genes known to play key roles in cellular invasion, adhesion and migration.

Notably, cell survival studies show that the effect of the extract is significantly higher in breast cancer cell lines and in particular A17 cells, with respect to normal cells, suggesting a therapeutic window for PRSE in vivo. This observation is in accordance with previous studies on mice that showed efficacy of strawberry methanolic extract against Ehrlich ascites carcinoma (EAC)19. We thus explored the efficacy of PRSE in inhibiting tumor formation in vivo exploiting the intriguing orthotopic breast cancer model generated by injection of A17 cells directly into the mammary gland of mice, finding a strong reduction of both tumor size and tumor volume in mice fed with PRSE from 4 weeks before tumor challenge.

Although the mechanism by which the strawberry extract exerts its biological effect is not completely understood at the molecular level, as well as which component plays the major antineoplastic role, our results suggest an interesting anti-invasive potential of “Alba” PRSE against breast cancer cells both in vitro and in vivo. Further studies will be necessary to unravel the pathways involved in the biological effects of PRSE and to elucidate the molecular basis of strawberry extract action, as well as to shed light on the possible introduction in the diet of “Alba” and, more in general, strawberries as a useful nutrient to limit (or prevent) tumor formation.