Cytotoxicity of a Lipid-Rich Extract from Native Mexican Avocado Seed (Persea americana var. drymifolia) on Canine Osteosarcoma D-17 Cells and Synergistic Activity with Cytostatic Drugs.

Osteosarcoma is the most common malignant bone tumor in both children and dogs. It is an aggressive and metastatic cancer with a poor prognosis for long-term survival. The search for new anti-cancer drugs with fewer side effects has become an essential goal for cancer chemotherapy; in this sense, the bioactive compounds from avocado have proved their efficacy as cytotoxic molecules. The objective of this study was to determine the cytotoxic and antiproliferative effect of a lipid-rich extract (LEAS) from Mexican native avocado seed (Persea americana var. drymifolia) on canine osteosarcoma D-17 cell line. Also, the combined activity with cytostatic drugs was evaluated. LEAS was cytotoxic to D-17 cells in a concentration-dependent manner with an IC50 = 15.5 µg/mL. Besides, LEAS induced caspase-dependent cell apoptosis by the extrinsic and intrinsic pathways. Moreover, LEAS induced a significant loss of mitochondrial membrane potential and increased superoxide anion production and mitochondrial ROS. Also, LEAS induced the arrest of the cell cycle in the G0/G1 phase. Finally, LEAS improved the cytotoxic activity of cisplatin, carboplatin, and in less extension, doxorubicin against the canine osteosarcoma cell line through a synergistic effect. In conclusion, avocado could be a potential source of bioactive molecules in the searching treatments for osteosarcoma.

Bioactive Molecules From Native Mexican Avocado Fruit (Persea americana var. drymifolia): A Review.

Avocado (Persea americana Mill.) is a tree native from central and eastern México that belongs to the Lauraceae family. Avocado has three botanical varieties known as Mexican (P. americana var. drymifolia), West Indian (P. americana var. americana), and Guatemalan (P. americana var. guatemalensis). It is an oil-rich fruit appreciated worldwide because of its nutritional value and the content of bioactive molecules. Several avocado molecules show attractive activities of interest in medicine. Avocado fatty acids have beneficial effects on cardiovascular disease risk factors. Besides, this fruit possesses a high content of carotenoids and phenolic compounds with possible antifungal, anti-cancer and antioxidant activities. Moreover, several metabolites have been reported with anti-inflammatory effects. Also, an unsaponifiable fraction of avocado in combination with soybean oil is used for the treatment of osteoarthritis. The Mexican variety is native from México and is characterized by the anise aroma in leaves and by small thin-skinned fruits of rich flavor and excellent quality. However, the study of the bioactive molecules of the fruit has not been addressed in detail. In this work, we achieved a literature review on the inflammatory, immunomodulatory and cytotoxic properties of long-chain fatty acids and derivatives from Mexican avocado seed. Also, the antioxidant and anti-inflammatory properties of the oil extracted from the avocado seed are referred. Finally, the antimicrobial, immunomodulatory, and cytotoxic activities of some antimicrobial peptides expressed in the fruit are reviewed.

Accelerated discovery of novel glycoside hydrolases using targeted functional profiling and selective pressure on the rumen microbiome.

BACKGROUND: Carbohydrate-active enzymes (CAZymes) form the most widespread and structurally diverse set of enzymes involved in the breakdown, biosynthesis, or modification of lignocellulose that can be found in living organisms. However, the structural diversity of CAZymes has rendered the targeted discovery of novel enzymes extremely challenging, as these proteins catalyze many different chemical reactions and are sourced by a vast array of microbes. Consequently, many uncharacterized members of CAZyme families of interest have been overlooked by current methodologies (e.g., metagenomic screening) used to discover lignocellulolytic enzymes. RESULTS: In the present study, we combined phenotype-based selective pressure on the rumen microbiota with targeted functional profiling to guide the discovery of unknown CAZymes. In this study, we found 61 families of glycoside hydrolases (GH) (out of 182 CAZymes) from protein sequences deposited in the CAZy database-currently associated with more than 20,324 microbial genomes. Phenotype-based selective pressure on the rumen microbiome showed that lignocellulolytic bacteria (e.g., Fibrobacter succinogenes, Butyrivibrio proteoclasticus) and three GH families (e.g., GH11, GH13, GH45) exhibited an increased relative abundance in the rumen of feed efficient cattle when compared to their inefficient counterparts. These results paved the way for the application of targeted functional profiling to screen members of the GH11 and GH45 families against a de novo protein reference database comprised of 1184 uncharacterized enzymes, which led to the identification of 18 putative xylanases (GH11) and three putative endoglucanases (GH45). The biochemical proof of the xylanolytic activity of the newly discovered enzyme validated the computational simulations and demonstrated the stability of the most abundant xylanase. CONCLUSIONS: These findings contribute to the discovery of novel enzymes for the breakdown, biosynthesis, or modification of lignocellulose and demonstrate that the rumen microbiome is a source of promising enzyme candidates for the biotechnology industry. The combined approaches conceptualized in this study can be adapted to any microbial environment, provided that the targeted microbiome is easy to manipulate and facilitates enrichment for the microbes of interest. Video Abstract.

Plant Defensin γ-Thionin Induces MAPKs and Activates Histone Deacetylases in Bovine Mammary Epithelial Cells Infected With Staphylococcus aureus.

Defensins are an important group of host defense peptides. They have immunomodulatory properties, which have been mainly described for mammal defensins, but similar effects for plant defensins remain unknown. Previously, we showed that the defensin γ-thionin (Capsicum chinense) reduces Staphylococcus aureus internalization into bovine mammary epithelial cells (bMECs) while inducing Toll-like receptor 2 (TLR2), modulating the inflammatory response. Here, we analyze the effect of γ-thionin on the TLR2 pathway in bMECs infected with S. aureus and determine if it modulates epigenetic marks. Pre-treated bMECs with γ-thionin (100 ng/ml) reduced the basal activation of p38 and ERK1/2 (~3-fold), but JNK was increased (~1.5-fold). Also, infected bMECs induced p38, but this effect was reversed by γ-thionin, whereas ERK1/2 was reduced by infection but stimulated by γ-thionin. Likewise, γ-thionin reduced the activation of Akt kinase ~50%. Furthermore, γ-thionin induced the activation of transcriptional factors of inflammatory response, highlighting EGR, E2F-1, AP-1, and MEF, which were turned off by bacteria. Also, γ-thionin induced the activation of histone deacetylases (HDACs, ~4-fold) at 24 h in infected bMECs and reduced LSD1 demethylase (HDMs, ~30%) activity. Altogether, these results demonstrated the first time that a plant defensin interferes with inflammatory signaling pathways in mammalian cells.

Lipid-Rich Extract from Mexican Avocado Seed (Persea americana var. drymifolia) Reduces Staphylococcus aureus Internalization and Regulates Innate Immune Response in Bovine Mammary Epithelial Cells.

Bovine mammary epithelial cells (bMECs) are capable of initiating an innate immune response (IIR) to invading bacteria. Staphylococcus aureus is not classically an intracellular pathogen, although it has been shown to be internalized into bMECs. S. aureus internalizes into nonprofessional phagocytes, which allows the evasion of the IIR and turns antimicrobial therapy unsuccessful. An alternative treatment to control this pathogen is the modulation of the innate immune response of the host. The Mexican avocado (Persea americana var. drymifolia) is a source of molecules with anti-inflammatory and immunomodulatory properties. Hence, we analyze the effect of a lipid-rich extract from avocado seed (LEAS) on S. aureus internalization into bMECs and their innate immunity response. The effects of LEAS (1-500 ng/ml) on the S. aureus growth and bMEC viability were assessed by turbidimetry and MTT assays, respectively. LEAS did not show neither antimicrobial nor cytotoxic effects. S. aureus internalization into bMECs was analyzed by gentamicin protection assays. Interestingly, LEAS (1-200 ng/ml) decreased bacterial internalization (60-80%) into bMECs. This effect correlated with NO production and the induction of the gene expression of IL-10, while the expression of the proinflammatory cytokine TNF-α was reduced. These effects could be related to the inhibition of MAPK p38 (∼60%) activation by LEAS. In conclusion, our results showed that LEAS inhibits the S. aureus internalization into bMECs and modulates the IIR, which indicates that avocado is a source of metabolites for control of mastitis pathogens.

Immunomodulatory effects of thionin Thi2.1 from Arabidopsis thaliana on bovine mammary epithelial cells.

Antimicrobial peptides (AMPs) are key elements of plant defense mechanisms, resembling conserved protection strategies also present in mammals. Among the AMPs, plant thionins are particularly interesting due that display antibacterial and antifungal activities. In Arabidopsis thaliana have been described four thionins: Thi2.1, Thi2.2, Thi2.3 and Thi2.4. Work from our group shows that Thi2.1 expressed by bovine endothelial cells has direct antibacterial activity against Staphylococcus aureus mastitis isolates, bacteria able to persist inside bovine mammary epithelial cells (bMECs). Thus, the objective of this work was to analyze the immunomodulatory effects of the AMP thionin Thi2.1 from A. thaliana on bMECs during S. aureus infection. According to the results, S. aureus internalization into bMECs was reduced in cells pre-treated with Thi2.1 at 5 and 10 µg/mL during 24 h, effect related to the participation of TLR2. In addition, bMECs pre-treated with Thi2.1 (24 h) significantly increased TNF-α (~2-fold) and IL-6 (~7-fold), whereas decreased IL-10 gene expression (~0.5-fold). Interestingly, Thi2.1 inhibits the up-regulation induced by S. aureus of TNF-α and IL-10 gene expression, as well as NO production. In addition, Thi2.1 (10 µg/mL) up-regulates the expression of the chemokine IL-8 (~3-fold) in infected bMECs. Some of these effects are related to TLR2 activation. In this sense, Thi2.1 also reduces S. aureus-induced TLR2 gene expression and membrane abundance. In conclusion, Thi2.1 from A. thaliana modulates bMEC innate immune response by inducing the production of pro- and anti-inflammatory molecules while inhibits S. aureus internalization. Some of these effects are mediated by TLR2.

The activation of the TLR2/p38 pathway by sodium butyrate in bovine mammary epithelial cells is involved in the reduction of Staphylococcus aureus internalization.

Staphylococcus aureus is an etiological agent of human and animal diseases, and it is able to internalize into non-professional phagocytic cells (i.e. bovine mammary epithelial cells, bMECs), which is an event that is related to chronic and recurrent infections. bMECs contribute to host innate immune responses (IIR) through TLR pathogen recognition, whereby TLR2 is the most relevant for S. aureus. In a previous report, we showed that sodium butyrate (NaB, 0.5mM), which is a short chain fatty acid (SCFA), reduced S. aureus internalization into bMECs by modulating their IIR. However, the molecular mechanism of this process has not been described, which was the aim of this study. The results showed that the TLR2 membrane abundance (MA) and mRNA expression were induced by 0.5mM NaB ∼1.6-fold and ∼1.7-fold, respectively. Additionally, 0.5mM NaB induced p38 phosphorylation, but not JNK1/2 or ERK1/2 phosphorylation in bMECs, which reached the baseline when the bMECs were S. aureus-challenged. Additionally, bMECs that were treated with 0.5mM NaB (24h) showed activation of 8 transcriptional factors (AP-1, E2F-1, FAST-1, MEF-1, EGR, PPAR, ER and CBF), which were partially reverted when the bMECs were S. aureus-challenged. Additionally, 0.5mM NaB (24h) up-regulated mRNA expression of the antimicrobial peptides, TAP (∼4.8-fold), BNBD5 (∼3.2-fold) and BNBD10 (∼2.6-fold). Notably, NaB-treated and S. aureus-challenged bMECs increased the mRNA expression of all of the antimicrobial peptides that were evaluated, and this was evident for LAP and BNBD5. In the NaB-treated bMECs, we did not detect significant expression changes for IL-1ß and IL-6 and only TNF-α, IL-10 and IL-8 were induced. Interestingly, the NaB-treated and S. aureus-challenged bMECs maintained the anti-inflammatory response that was induced by this SCFA. In conclusion, our results suggest that 0.5mM NaB activates bMECs via TLR2/p38, which leads to improved antimicrobial defense before/after pathogen invasion, and NaB may exert anti-inflammatory effects during infection.