Vermentino Leaf Extracts Inhibit Tumorigenicity via FASN Downregulation in Breast Cancer Cell Lines

Background Existing therapeutic strategies for breast cancer include surgery, endocrine therapy, and chemotherapy are limited by tumor recurrence and drug‐resistance, and novel approaches need to improve current clinical protocols. A number of epidemiological studies indicate that antioxidant plant‐derived compounds such as flavonoids reduce adverse outcomes of reactive species and have been identified as a potential source of antineoplastic and cytotoxic agents with less undesirable side effects. It is well known that in breast cancer the inhibition of fatty acid synthesis promotes apoptosis and produces cytotoxicity which is likely to trigger cell death. A key enzyme in de novo fatty acid synthesis is fatty acid synthase (FASN). FASN catalyzes Acetyl‐CoA and Malonyl‐CoA, forming Palmitate and a 16‐carbon fatty acid. The present investigation chose Vitis vinifera L. cv. Vermentino , since it is representative of the productive activities of the Sardinian territory and because of the peculiarity of its phenolic profile. The aim of this study is to explore for the first time the potential cytotoxicity of the Vermentino leaf water and hydroalcoholic extracts and its property as a FASN inhibitor. Methods The phenolic characterization were analyzed by LCMS (Liquid Chromatography‐Mass Spectrometry) . Profile protein contents was evaluated by Mass Spectrometry (LC‐MS/MS). The experiments were performed using MCF7, SKBR‐3 human carcinoma cell lines and MCF‐12A breast epithelial cell line treated with Vermentino water and hydroalcoholic extract at an increasing concentration of (100, 200 and 400μg/ml) in a time course of 16 and 24 hours. Protein and mRNA levels were analyzed by western blotting/Co‐immunoprecipitation (Co‐IP) and RT‐PCR, respectively. The number of viable cells and the cell‐surviving has been detected by MTT and clonogenic assays, respectively. Apoptotic induction was determined by Flow Cytometric assay using Annexin V‐FITC and sorted by A FACSCalibur™ analyzer ( BD Biosciences ). Results We found that Vermentino extracts treatment selectively shows cytotoxic activity against the breast cancer cell line, but not against the normal epithelial breast cell. Vermentino extracts lower cell viability and clonogenic survival in breast cancer cell and drives them toward late apoptosis and necrosis by caspase‐3 and caspase‐9 activation. Vermentino treatment resulted in FASN downregulation and an increase in AKT‐phosphorylation in a dose‐dependent and time‐dependent manner. We used the Co‐IP to demonstrate that Vermentino leaf extracts increase the ubiquitination status of FASN, which causes an increase of affinity with proteasomal 20s subunit and an increase of FASN degradation by proteasomal Caspase‐like activity. Combined, these data suggest a level of crosstalk between the fatty acid synthase and proteasome pathways in which there is a reduction of FASN mediated by increased ubiquitination. Conclusion This is the first report demonstrating that Vermentino leaves contain a mixture of bioactive compounds able to modulate the ubiquitination status of protein FASN driving it toward proteasomal degradation in breast cancer cell lines. Support or Funding Information NIA/NIAAA R21AA027325