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Breast cancer is a major health problem that threatens the lives of millions of women worldwide each year. Most of the chemotherapeutic agents that are currently used to treat this complex disease are highly toxic with long-term side effects. Therefore, novel generation of anti-cancer drugs with higher efficiency and specificity are urgently needed.

Breast cancer cell lines were treated with eugenol and cytotoxicity was measured using the WST-1 reagent, while propidium iodide/annexinV associated with flow cytometry was utilized in order to determine the induced cell death pathway. The effect of eugenol on apoptotic and pro-carcinogenic proteins, both in vitro and in tumor xenografts was assessed by immunoblotting. While RT-PCR was used to determine eugenol effect on the E2F1 and survivin mRNA levels. In addition, we tested the effect of eugenol on cell proliferation using the real-time cell electronic sensing system.

Eugenol at low dose (2 muM) has specific toxicity against different breast cancer cells. This killing effect was mediated mainly through inducing the internal apoptotic pathway and strong down-regulation of E2F1 and its downstream antiapoptosis target survivin, independently of the status of p53 and ERalpha. Eugenol inhibited also several other breast cancer related oncogenes, such as NF-kappaB and cyclin D1. Moreover, eugenol up-regulated the versatile cyclin-dependent kinase inhibitor p21WAF1 protein, and inhibited the proliferation of breast cancer cells in a p53-independent manner. Importantly, these anti-proliferative and pro-apoptotic effects were also observed in vivo in xenografted human breast tumors.

Eugenol exhibits anti-breast cancer properties both in vitro and in vivo, indicating that it could be used to consolidate the adjuvant treatment of breast cancer through targeting the E2F1/survivin pathway, especially for the less responsive triple-negative subtype of the disease.

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