Inhibition of Enzymes Involved in Neurodegenerative Disorders and Aß1-40 Aggregation by Citrus limon Peel Polyphenol Extract.

Alzheimer's (AD) and Parkinson's diseases (PD) are multifactorial neurogenerative disorders of the Central Nervous System causing severe cognitive and motor deficits in elderly people. Because treatment of AD and PD by synthetic drugs alleviates the symptoms often inducing side effects, many studies have aimed to find neuroprotective properties of diet polyphenols, compounds known to act on different cell signaling pathways. In this article, we analyzed the effect of polyphenols obtained from the agro-food industry waste of Citrus limon peel (LPE) on key enzymes of cholinergic and aminergic neurotransmission, such as butyryl cholinesterase (BuChE) and monoamine oxidases (MAO)-A/B, on Aß1-40 aggregation and on superoxide dismutase (SOD) 1/2 that affect oxidative stress. In our in vitro assays, LPE acts as an enzyme inhibitor on BuChE (IC50 ~ 73 µM), MAO-A/B (IC50 ~ 80 µM), SOD 1/2 (IC50 ~ 10-20 µM) and interferes with Aß1-40 peptide aggregation (IC50 ~ 170 µM). These results demonstrate that LPE behaves as a multitargeting agent against key factors of AD and PD by inhibiting to various extents BuChE, MAOs, and SODs and reducing Aß-fibril aggregation. Therefore, LPE is a promising candidate for the prevention and management of AD and PD symptoms in combination with pharmacological therapies.

Inhibition of Interleukin-6-Induced Matrix Metalloproteinase-2 Expression and Invasive Ability of Lemon Peel Polyphenol Extract in Human Primary Colon Cancer Cells.

Among matrix metalloproteinases (MMPs), MMP-9/2 are key enzymes involved in the proteolysis of extracellular matrices in the inflammatory process and in cancer. Since MMP-9/2 expression levels, activity, and secretion is up-regulated during inflammation in response to pro-inflammatory cytokines, such as interleukin-6 (IL-6), many efforts have been devoted to identifying factors that could inhibit the IL-6-induced MMP-9/2 expression. Up to now, several reports indicated that polyphenols from fruits and vegetables are among the major components of health promotion for their antioxidant properties and also for their anti-inflammatory and anti-cancer agents. Among plant derived polyphenols, lemon (Citrus limon) peel extract (LPE) shows anti-cancer properties in various cancer types. In our previous work, we demonstrated that LPE can reduce IL-6-induced migration/invasiveness and MMP-9/2 up-regulation in some gastric cancer cell lines. This study aims to exploit the anti-cancer properties of LPE using an in vitro system model of inflammation, consisting of IL-6-exposed human primary colon cancer cells. We first analyzed the effect of LPE on IL-6-induced cell migration and invasiveness by wound healing and Boyden chamber assay, respectively. The MMP-2 mRNA expression levels and gelatinolytic activity in the cell culture media were determined by q-PCR analysis and gelatin zymography, respectively, and finally, the effects of LPE on IL-6-induced JAK2/STAT3 signaling pathways have been investigated by Western blotting analysis. Our results show that LPE is able to inhibit the IL-6-dependent cell migration and invasiveness associated with the up-regulation of MMP-2 expression levels and that these effects are correlated to the STAT3 phosphorylation in human primary T88 and T93 colon cancer cells.

Lemon Peel Polyphenol Extract Reduces Interleukin-6-Induced Cell Migration, Invasiveness, and Matrix Metalloproteinase-9/2 Expression in Human Gastric Adenocarcinoma MKN-28 and AGS Cell Lines.

Among plant polyphenols, lemon peels extract (LPE) from the residues of the industrial processing of lemon (Citruslimon) shows anti-proliferative properties in cancer cells and anticholinesterase activity. In this study, we analyze the anti-cancer properties of LPE on migration and invasiveness in MKN-28 and AGS human gastric cancer cell lines either in the absence or presence of the pro-inflammatory cytokine IL-6. We find that the pretreatment with non-cytotoxic concentrations (0.5-1 µg/ml of gallic acid equivalent) of LPE inhibits interleukin-6 (IL-6)-induced cell migration and invasiveness in MKN-28 and AGS cells, as analyzed by wound and matrigel assays. Pretreatment with LPE is able to prevent either IL-6-induced matrix metalloproteinases (MMP)-9/2 activity, as assessed by gel zymography, or mRNA and protein MMP-9/2 expression, as evaluated by qPCR and Western blotting analysis, respectively. These LPE effects are associated with an IL-6-dependent STAT3 signaling pathway in MKN-28 and AGS cells. Furthermore, LPE shows acetylcholinesterase inhibitory activity when assayed by the Ellman method. In conclusion, our results demonstrate that LPE reduces the invasiveness of gastric MKN-28 and AGS cancer cells through the reduction of IL-6-induced MMP-9/2 up-regulation. Therefore, these data suggest that LPE exerts a protective role against the metastatic process in gastric cancer.

Ageritin from poplar mushrooms: scale-up purification and cytotoxicity towards undifferentiated and differentiated SH-SY5Y cells.

Ageritin is the first reported ribotoxin-like protein from basidiomycetes fungi. It can induce ribosomal integrity damage and translation block, and interferes with mitochondrial redox activity of some glioma and neuroblastoma cell lines. Herein, Ageritin has been investigated as a valuable neurotoxin towards either undifferentiated or retinoic acid (RA)-differentiated SH-SY5Y neuroblastoma cells showing a selective cell toxicity against undifferentiated cells. MTT and sulforhodamine B (SRB) assays highlighted that Ageritin markedly decreases the mitochondrial redox activity and viability of undifferentiated cells, meanwhile inducing evident morphological changes eliciting neuronal-like appearance in these cells. Data from lactate dehydrogenase release assay, cytofluorimetric analysis and caspase-3 enzymatic activity measurement suggest that Ageritin promotes cell death through a caspase-dependent apoptotic pathway. The Z-VAD-FMK caspase inhibitor was able to prevent this apoptotic pathway activation. Based on the interesting behaviour of Ageritin vs. SH-SY5Y cells, the development of a scale-up procedure to obtain the purified protein in larger amounts (yield 2.5 mg per 100 g) has been optimized.