Existing therapeutic strategies for breast cancer are limited by tumor recurrence and drug-resistance. Antioxidant plant-derived compounds such as flavonoids reduce adverse outcomes and have been identified as a potential source of antineoplastic agent with less undesirable side effects. Here, we describe the novel regulation of fatty-acid synthase (FASN), the key enzyme in de novo fatty-acid synthesis, whereby Vitis vinifera L. cv Vermentino leaf hydroalcoholic extract lowers its protein stability that is regulated by small ubiquitin-like modifier (SUMO)ylation. The phenolic compounds characterization was performed by liquid chromatography–mass spectrometry (LC–MS), whereas mass spectrometry (LC–MS/MS), Western blotting/co-immunoprecipitation (Co-IP) and RT-PCR, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), clonogenicity assays, and FACS analysis were used to measure the expression of targets and tumorigenicity. Vermentino extract exhibits antitumorigenic effects, and we went on to determine that FASN and ubiquitin-conjugating enzyme 9 (UBC9), the sole E2 enzyme required for SUMOylation, were significantly reduced. Moreover, FASN was found SUMOylated in human breast cancer tissues and cell lines, and lack of SUMOylation caused by SUMO2 silencing reduced FASN protein stability. These results suggest that SUMOylation protects FASN against proteasomal degradation and may exert oncogenic activity through alteration of lipid metabolism, whereas Vermentino extract inhibits these effects which supports the additional validation of the therapeutic value of this compound in breast cancer.

Background: Existing therapeutic strategies for breast cancer are limited by tumor recurrence and drug-resistance. Several epidemiological studies indicate that antioxidant plant-derived compounds such as flavonoids reduce adverse outcomes and have been identified as a potential source of antineoplastic agent with less undesirable side effects. Activation of lipid metabolism is an early event in carcinogenesis and a central hallmark in breast cancer. In fact, inhibition of fatty-acid synthesis in breast cancer results in cytotoxicity that triggers apoptosis. Here, we describe the novel regulation of lipid metabolism in breast cancer cells whereby the protein stability and degradation of fatty-acid synthase (FASN), the key enzyme in de novo fatty-acid synthesis, is regulated by SUMOylation. Methods: The phenolic characterization were analyzed by Liquid Chromatography-Mass Spectrometry (LCMS). 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 hydroalcoholic extract in dose and time course responses. 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 clonogenicity assays. Apoptotic induction was determined by Flow Cytometric assay using Annexin V-FITC and sorted by A FACSC analysis.Results: We first tested the potential antitumorigenic effects of Vitis vinifera L. cv. Vermentino leaf hydroalcoholic extract in MCF-7 and SKBR-3 breast cancer cell lines and found that this compound demonstrated cytotoxic effects. We went on to determine that FASN and UBC9, the sole E2 enzyme required for SUMOylation, were significantly reduced by treatment with the Vermentino extract.Moreover, we found that FASN was SUMOylated in human breast cancer tissues and cell lines. Finally, lack of SUMOylation caused by SUMO2 silencing reduced FASN protein stability.Conclusion: Altogether, these results suggest that SUMOylation protects FASN against proteasomal degradation and may exert oncogenic activity through alteration of lipid metabolism in breast cancer.Importantly, we found that these effects were significantly inhibited by treatment with Vermentino leaf extract, which supports the additional validation of the therapeutic value of this compound. BackgroundBreast cancer is the most common malignant cancer in females worldwide (1). The existing therapeutic strategies for breast cancer, which include surgery, endocrine therapy, and chemotherapy, are limited by tumor recurrence and drug-resistance (2). Therefore, novel approaches are needed to enhance the efficacy of existing therapeutic agents and to improve current clinical protocols.Adjuvant therapies often attempt to induce cytotoxicity in tumor cells. As tumor cells are known to rely on alternate metabolic processes, such as de novo fatty-acid synthesis, these pathways harbor many pote...

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 modu...

Propose Alcohol‐induced liver disease (ALD) is a major health concern of alcohol abuse and a leading cause of liver‐related morbidity and mortality. Alterations in mitochondrial morphology and function are a hallmark of ALD. Mitochondria synthesize most of the adenosine triphosphate (ATP) needed by mammalian cells and are intimately involved in the generation of and defense against reactive oxygen species (ROS). Evidence support the importance of ROS production and oxidative stress in the development of ALD. SUMOylation is a dynamic and reversible posttranscriptional modification involved in many cellular pathways including transcription, intracellular transport, DNA repair, replication, and cell signaling. We recently demonstrated that UBC9, the sole E2 protein required by SUMOylation machinery, is upregulated in Intragastric ethanol‐fed mouse model (IE) and cirrhotic tissues. We also found that UBC9 is phosphorylated and this is correlated with high level of SUMOylation in lipopolysaccharides‐activated Kupffer cells that lead inflammation development. In addition, we described a novel key role of SUMOylation inducing the enzymatic activity and protein stability of Cytochrome P450 2E1 (CYP2E1), that is the major component of the microsomal ethanol‐oxidizing system (MEOS), in NIAAA mouse model. This study aims to investigate whether ethanol induces mitochondrial dysfunction in ALD regulating the SUMOylation status of key respiratory chain proteins and explore the molecular mechanisms. Methods Primary mouse hepatocytes and livers from NIAAA mouse model were used. mRNA and protein levels were analyzed by Real‐Time PCR and Western Blotting, respectively. OCR (oxygen consumption rate) and Δψm (mitochondrial membrane potential) were measured by Seahorse mito stress test and JC‐1 staining by confocal microscopy, respectively. Results In order to explore the role of SUMOylation in ALD, Mass Spectrometry (MS) was performed to identify SUMOylated proteins in 10‐day ethanol‐feeding+1 Binge ethanol (NIAAA) model, where SUMOs binding columns were used to purify SUMOylated proteins from total livers. Interestingly, we found that ethanol induces changes in SUMOylation state of several mitochondrial proteins involved in ATP synthesis (ATP5B, SOD2, CLC25A5, HSPD1, FBP1, CYCS), ATP metabolism (ATP5A1, ATP5B, ATP5C1, ATP5F1, ATP5J2, ASPA8, AK3), and mitochondria disorder (UQCRC2, PC, HMGCS2, ECHS1, COX6B1, NDUFV3, COX6C). These finding could suggest a potential role of SUMOylation in oxidative phosphorylation, electron transport chain (ETC) and respiratory control ratio may be modulating the ability of these proteins to form the complex above and/or regulating their activity. Specifically, ethanol treatment decreases ATP production and maximal respiration in primary mouse hepatocytes, while UBC9 knockdown prevents the loss of respiration. Furthermore, UBC9 silencing prevents the drop of Δψm when ethanol is added. This finding clearly indicates that SUMOylation is involved in mitochondrial physiology. Conclusions these novel findin...