Evaluating the In Vitro Activity and Safety of Modified LfcinB Peptides as Potential Colon Anticancer Agents: Cell Line Studies and Insect-Based Toxicity Assessments.

Anticancer peptides are increasingly being considered as alternative treatments for cancer due to their potency, selectivity, and low toxicity. Previously, the peptide LfcinB (21-25)Pal showed in vitro anticancer effects against the Caco-2 colon cancer cell line (half-maximal inhibitory concentration (IC50): 86 µM). In this study, we developed modifications to the peptide sequence to increase its anticancer activity. Sequence modifications were made such as the inclusion of amino hexanoic acid (Ahx), N-terminal biotinylation, acetylation, and substitutions of Orn for Arg and/or d-Arg by l-Arg. The molecules were synthesized using manual solid-phase peptide synthesis (SPPS), and their synthetic feasibility (SAScore) ranged from 6.2 to 7.6. The chromatographic purities of the synthesized peptides were greater than 89%. We found that Ahx-RWQWRWQWR and RWQWRWQW-Orn showed activity against both Caco-2 and HT-29 cell lines and decreased IC50 values by approx. 50% in Caco-2 cells (IC50: 40 µM) when compared to the parent peptide RWQWRWQWR. Moreover, the modified peptides demonstrated lower hemolytic effects, with values <10% at 200 µg/mL. Toxicity was assessed using the Galleria mellonella model and the half-maximal lethal dose (LD50) for the best peptides was >100 mg/kg, indicating that their toxicity is classified as moderately toxic or lower. In contrast, cisplatin showed an LD50 of 13 mg/Kg. The designed anticancer peptides presented good in vitro activity and low toxicity, making them promising molecules for future drug development studies.

GPER: An Estrogen Receptor Key in Metastasis and Tumoral Microenvironments.

Estrogens and their role in cancer are well-studied, and some cancer types are classified in terms of their response to them. In recent years, a G protein-coupled estrogen receptor (GPER) has been described with relevance in cancer. GPER is a pleiotropic receptor with tissue-specific activity; in normal tissues, its activation is related to correct development and homeostasis, while in cancer cells, it can be pro- or anti-tumorigenic. Also, GPER replaces estrogen responsiveness in estrogen receptor alpha (ERα)-lacking cancer cell lines. One of the most outstanding activities of GPER is its role in epithelial-mesenchymal transition (EMT), which is relevant for metastasis development. In addition, the presence of this receptor in tumor microenvironment cells contributes to the phenotypic plasticity required for the dissemination and maintenance of tumors. These characteristics suggest that GPER could be a promising therapeutic target for regulating cancer development. This review focuses on the role of GPER in EMT in tumorigenic and associated cells, highlighting its role in relation to the main hallmarks of cancer and possible therapeutic options.

The Antimicrobial Peptide γ-Thionin from Habanero Chile (Capsicum chinense) Induces Caspase-Independent Apoptosis on Human K562 Chronic Myeloid Leukemia Cells and Regulates Epigenetic Marks.

Cancer is a relevant health problem worldwide. In 2020, leukemias represented the 13th most commonly reported cancer cases worldwide but the 10th most likely to cause deaths. There has been a progressive increase in the efficacy of treatments for leukemias; however, these still generate important side effects, so it is imperative to search for new alternatives. Defensins are a group of antimicrobial peptides with activity against cancer cells. However, the cytotoxic mechanism of these peptides has been described mainly for animal defensins. This study shows that defensin γ-thionin (Capsicum chinense) is cytotoxic to the K562 leukemia cells with an IC50 = 290 µg/mL (50.26 µM) but not for human peripheral blood mononuclear cells. Results showed that γ-thionin did not affect the membrane potential; however, the peptide modified the mitochondrial membrane potential (ΔΨm) and the intracellular calcium release. In addition, γ-thionin induced apoptosis in K562 cells, but the activation of caspases 8 and 9 was not detected. Moreover, the activation of calpains was detected at one hour of treatment, suggesting that γ-thionin activates the caspase-independent apoptosis. Furthermore, the γ-thionin induced epigenetic modifications on histone 3 in K562 cells, increased global acetylation (~2-fold), and specific acetylation marks at lysine 9 (H3K9Ac) (~1.5-fold). In addition, γ-thionin increased the lysine 9 methylation (H3K9me) and dimethylation marks (H3K9me2) (~2-fold), as well as the trimethylation mark (H3K9me3) (~2-fold). To our knowledge, this is the first report of a defensin that triggers caspase-independent apoptosis in cancer cells via calpains and regulating chromatin remodelation, a novel property for a plant defensin.

Biological Activities of Organic Extracts of the Genus Aristolochia: A Review from 2005 to 2021.

Different ethnomedicinal studies have investigated the relationship between various phytochemicals as well as organic extracts and their bioactive aspects. Studies on biological effects are attributed to secondary metabolites such as alkaloids, phenolic compounds, and terpenes. Since there have been no reviews in the literature on the traditional, phytochemical, and ethnomedicinal uses of the genus Aristolochia so far, this article systematically reviews 141 published studies that analyze the associations between secondary metabolites present in organic extracts and their beneficial effects. Most studies found associations between individual secondary metabolites and beneficial effects such as anticancer activity, antibacterial, antioxidant activity, snake anti-venom and anti-inflammatory activity. The aim of this review was to analyze studies carried out in the period 2005-2021 to update the existing knowledge on different species of the genus Aristolochia for ethnomedicinal uses, as well as pharmacological aspects and therapeutic uses.

PaDef (Persea americana var. drymifolia), a Plant Antimicrobial Peptide, Triggers Apoptosis, and Induces Global Epigenetic Modifications on Histone 3 in an Acute Lymphoid Leukemia Cell Line.

In recent years, it has been recognized that epigenetic alterations play an important role in the development and maintenance of cancer, including leukemias. Furthermore, it is known that these alterations are involved in the emergence of resistance to conventional chemotherapeutics. Consequently, molecules with an anticancer activity whose activity is ruled by epigenetic modifications are attractive to search for new therapies against cancer. The plant antimicrobial peptides have been widely evaluated as molecules with anticancer activity; however, the analysis of the epigenetic regulation induced by these molecules associated with this activity is scarce and still is an unexplored field. In this work, we show that the PaDef defensin, a plant antimicrobial peptide from Mexican avocado fruit (Persea americana var. drymifolia) is cytotoxic for Jurkat cell line from acute lymphoid leukemia cells, through an apoptotic process. PaDef inhibited cell viability in a concentration-dependent manner, with an IC50 = 47.3 µM. Treatment of Jurkat cells with PaDef (IC50) induced cell death by apoptosis dependent on caspases 8 and 9; besides, it was related to an increase in the production of reactive oxygen species and the loss of mitochondrial membrane potential. Interestingly, the inhibition of caspase activation by inhibitors of caspases 8 and 9 does not revert the reduction in viability, suggesting that other mechanisms, in addition to caspase activity, could be participating in the PaDef cytotoxic effect. Also, the modifications in the histone 3 tails induced by PaDef in Jurkat cells were evaluated, specifically acetylation and methylation. PaDef increased global histone 3 acetylation and lysine 9 specific marks (2-fold and up to 4-fold, respectively). These effects correlated with the reduction of the Histone Deacetylase activity (HDAC, ∼50%). Based on methylation marks, PaDef treatment increased lysine 9 di- and tri-methylation tags (2-fold in both cases). The epigenetic modulation induced by PaDef on Jurkat cells could be related to the chromatin compaction-decompaction promoting gene expression or repression; however, further studies are necessary to correlate these marks with the transcription of specific genes. Therefore, the study of new molecules that may have anticancer activity through epigenetic modulation is interesting.

In vitro cytotoxic potential of extracts from Aristolochia foetida Kunth against MCF-7 and bMECs cell lines.

The aim of this study was to evaluate the cytotoxic potential of Aristolochia foetida Kunth. Stems and leaves of A. foetida Kunth (Aristolochiaceae) have never been investigated pharmacologically. Recent studies of species of the Aristolochiaceae family found significant cytotoxic activities. Hexane, dichloromethane, ethyl acetate and methanol extracts were analyzed by 1H NMR and GC-MS to know the metabolites in each extract. In GC-MS analysis, the main compounds were methyl hexadecanoate (3); hexadecanoic acid (4); 2-butoxyethyl dodecanoate (9); ethyl hexadecanoate (20); methyl octadeca-9,12,15-trienoate (28) and (9Z,12Z,15Z)-octadeca-9,12,15-trienoic acid (40). The results showed a significant reduction in cell viability of the MCF-7 (breast cancer) cell line caused by organic extracts in a dose-dependent manner. The cytotoxicity activity of the dichloromethane extract from the stems (DSE) showed IC50 values of 45.9 µg/mL and the dichloromethane extract of the leaves (DLE) showed IC50 values of 47.3 µg/mL. DSE and DLE had the highest cytotoxic potential in an in vitro study against the MCF-7 cell line and non-tumor cells obtained from the bovine mammary epithelial (bMECs). DSE and DLE induced a loss in mitochondrial membrane potential (ΔΨm) and can cause cell death by apoptosis through the intrinsic pathway in the MCF-7 cell line. DSE and DLE are cytotoxic in cancer cells and cause late apoptosis. Higher concentrations of DSE and DLE are required to induce a cytotoxic effect in healthy mammary epithelial cells. This is the first report of the dichloromethane extract of A. foetida Kunth that induces late apoptosis in MCF-7 cancer cells and may be a candidate for pharmacological study against breast cancer.

Defensin c-thionin from Capsicum chinense improves butyrate cytotoxicity on human colon adenocarcinoma cell line Caco-2

BACKGROUND: Butyrate is a histone deacetylase inhibitor that induces apoptosis and inhibits cell proliferation of colorectal cancer cells. To improve its anticancer activity, butyrate has been evaluated mixed with drugs and different molecules. Plant antimicrobial peptides are attractive anticancer alternative molecules because they show selective cytotoxic activity against different cancer cell lines. In this work, we explore if the plant defensin c-thionin (Capsicum chinense) can improve butyrate activity on Caco-2 cell line and we also determined the mechanism of death activated. RESULTS: The combined treatment of c-thionin (3.5 mM) and butyrate (50 mM) showed higher cytotoxicity on Caco-2 cells with respect to single treatments. Also, the combined treatment reduced cell proliferation and exhibited a higher rate of apoptosis than single treatments. Combined treatment induced caspases 8 and 9 activation to an extent comparable with that of butyrate while c-thionin did not activate caspases. Additionally, reactive oxygen species generation preceded the onset of apoptosis, and superoxide anion production was higher in cells treated with the combined treatment. CONCLUSIONS: The c-thionin from Habanero chili pepper improved the butyrate cytotoxicity on Caco-2 cells. This effect occurred through apoptosis induction associated with reactive oxygen species production. Therefore, the combination of butyrate with cytotoxic antimicrobial peptides could be an attractive strategy for cancer therapy.

Molecular characterization of bacterial microbiota associated with infectious bovine keratoconjunctivitis in Michoacán, Mexico / Caracterización molecular de la microbiota bacteriana asociada con queratoconjuntivitis infecciosa bovina en Michoacán, México / Caracterização molecular da microbiota bacteriana associada à ceratoconjuntivite bovina infecciosa em Michoacán, México

Abstract Background: The most common ocular disease affecting cattle worldwide is infectious bovine keratoconjunctivitis (IBK), which has been associated with Moraxella bovis bacterium. Objective: To report the molecular characterization of the ocular bacterial microbiota and its relation to IBK in cattle in two dairy regions in Michoacán, Mexico. Methods: A total population of 761 bovines were evaluated, of which 17 (2.23%) showed symptoms of IBK. Thirty-eight bacterial isolates from ocular samples of bovines with IBK were characterized by Gram-staining and antimicrobial sensitivity. In addition, isolates were identified by sequence comparisons of the 16S ribosomal gene. Results: The genus Moraxella was one of the most abundant bacteria and M. bovoculi was the most predominant species. Conclusion: The bacterial isolates identified in eye lesions of cattle and associated to IBK are diverse. To the author´s knowledge, this is the first study on the subject in Mexico; therefore, more research is needed to estimate the incidence of IBK and determine its associated microbiota.

Resumen Antecedentes: la enfermedad ocular más común que afecta al ganado en todo el mundo es la queratoconjuntivitis infecciosa bovina (IBK), que se ha asociado con la bacteria Moraxella bovis. Objetivo: reportar la caracterización molecular de la microbiota bacteriana ocular y su relación con IBK en ganado de dos regiones lecheras en Michoacán, México. Métodos: se evaluó una población total de 761 bovinos de los cuales 17 (2,23%) mostraron síntomas de IBK. Se obtuvieron treinta y ocho aislamientos bacterianos de muestras oculares de bovinos con IBK, los cuales se caracterizaron por tinción de Gram y sensibilidad antimicrobiana. Además, los aislamientos se identificaron mediante comparaciones de secuencias del gen ribosomal 16S. Resultados: el género Moraxella fue una de las bacterias más abundantes y M. bovoculi fue la especie más predominante. Conclusión: los aislamientos bacterianos identificados en lesiones oculares de bovinos y asociados a IBK son diversos. Hasta donde sabemos, este es el primer estudio sobre el tema realizado en México; por lo tanto, es necesario ampliar esta investigación para estimar la incidencia de IBK y determinar la microbiota asociada con la misma.

Resumo Antecedentes: a doença ocular mais comum que afeta o gado no mundo é a ceratoconjuntivite bovina (IBK), que tem sido associada à bactéria Moraxella bovis. Objetivo: relatar a caracterização molecular da microbiota bacteriana ocular e sua relação com a IBK em bovinos de duas regiões leiteiras de Michoacán, México. Métodos: foi avaliada uma população total de 761 bovinos, más apenas 17 (2,23%) apresentaram sintomas de IBK. Trinta e oito isolados bacterianos de amostras de olho bovino com IBK foram caracterizados por coloração de Gram e sensibilidade antimicrobiana. Além disso, os isolados foram identificados por comparação de sequências do gene ribossômico 16S. Resultados: a microbiota bacteriana associada à IBK foi diversa, sendo o gênero Moraxella uma das mais abundantes e M. bovoculi a espécie predominante. Conclusão: de acordo com o conhecimento dos autores, este é o primeiro estudo sobre o tema no México até o momento, portanto é necessário expandir essa pesquisa para estimar a incidência de IBK e determinar a microbiota associada à mesma.

Analysis of virulence traits of Staphylococcus aureus isolated from bovine mastitis in semi-intensive and family dairy farms.

BACKGROUND: Staphylococcus aureus is one of the main microorganisms that causes bovine mastitis, and its well-known virulence characteristics and interactions with the environment are used to aid the design of more efficient therapies. OBJECTIVES: To determine whether the virulence traits, such as antibiotic resistance and biofilm-forming and internalization abilities, of S. aureus isolated from bovine mastitis are related to dairy production system types. METHODS: The study was performed in the Mexican states of Guanajuato and Michoacan. Semi-intensive dairy farms (SIDFs) and family dairy farms (FDFs) (454 and 363 cows, respectively) were included. The 194 milk samples from mastitis affected quarters were collected and 92 strains of S. aureus were isolated and identified by biochemical and molecular tests. Antibiotic resistance, biofilm and internalization assays were performed on 30 randomly selected isolated strains to determine virulence traits, and these strains were equally allocated to the 2 dairy production systems. RESULTS: All 30 selected strains displayed a high degree of resistance (50%-91.7%) to the antibiotics tested, but no significant difference was found between SIDF and FDF isolates. S. aureus strains from SIDFs had an average biofilm forming capacity of up to 36% (18.9%-53.1%), while S. aureus strains from FDFs registered an average of up to 53% (31.5%-77.8%) (p > 0.05). Internalization assays revealed a higher frequency of internalization capacity for strains isolated from FDFs (33.3%) than for those isolated from SIDFs (6.7%) (p > 0.05). fnbpA gen was detected in 46.6% of FDF strains and 33.3% of SIDF strains, and this difference was significant (p < 0.05). CONCLUSIONS: Our findings show that the virulence traits of S. aureus isolates analyzed in this study, depend significantly on several factors, such as phenotype, genotype, and environmental conditions, which are significantly related to dairy production system type and daily management practices.

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.

Peptides Derived from (RRWQWRMKKLG)2-K-Ahx Induce Selective Cellular Death in Breast Cancer Cell Lines through Apoptotic Pathway.

The effect on the cytotoxicity against breast cancer cell lines of the substitution of 26Met residue in the sequence of the Bovine Lactoferricin-derived dimeric peptide LfcinB (20-30)2: (20RRWQWRMKKLG30)2-K-Ahx with amino acids of different polarity was evaluated. The process of the synthesis of the LfcinB (20-30)2 analog peptides was similar to the original peptide. The cytotoxic assays showed that some analog peptides exhibited a significant cytotoxic effect against breast cancer cell lines HTB-132 and MCF-7, suggesting that the substitution of the Met with amino acids of a hydrophobic nature drastically enhances its cytotoxicity against HTB-132 and MCF-7 cells, reaching IC50 values up to 6 µM. In addition, these peptides have a selective effect, since they exhibit a lower cytotoxic effect on the non-tumorigenic cell line MCF-12. Interestingly, the cytotoxic effect is fast (90 min) and is maintained for up to 48 h. Additionally, through flow cytometry, it was found that the obtained dimeric peptides generate cell death through the apoptosis pathway and do not compromise the integrity of the cytoplasmic membrane, and there are intrinsic apoptotic events involved. These results show that the obtained peptides are extremely promising molecules for the future development of drugs for use against breast cancer.

Production and characterization of a monoclonal antibody against the sialidase of Gardnerella vaginalis using a synthetic peptide in a MAP8 format.

Bacterial vaginosis is one of the most frequent vaginal infections. Its main etiological agent is Gardnerella vaginalis, which produces several virulence factors involved in vaginal infection and colonization, in particular, sialidase (SLD), a potential clinical biomarker that participates in immune response modulation and mucus degradation. The main objective of this work was the production and evaluation of a monoclonal antibody against G. vaginalis sialidase and its validation in immunoassays. For immunization of mice, a synthetic multiantigenic peptide was used, and hybridomas were generated. After fusion, hybridomas were evaluated for antibody production and cloned by limited dilution. One clone producing IgG1 was selected and characterized by indirect ELISA, dot blot, and Western blot, and we also tested clinical isolates and HeLa cells infected with G. vaginalis. The results showed that the anti-SLD antibody recognized a single protein of ~90 kDa that correlated with the estimated molecular weight of SLD. In addition, anti-SLD antibody recognized SLD from complete bacteria and from culture supernatants of infected Hela cells. In conclusion, our results showed that the anti-SLD antibody recognized SLD from different sources and could be considered a new tool for the diagnosis of bacterial vaginosis. KEY POINTS: • Anti-sialidase mAb was generated using a synthetic peptide • The mAb recognizes synthetic peptide and intact protein from multiple sources • The antibody was characterized by several immunological methods.

Immunomodulatory molecules regulate adhesin gene expression in Staphylococcus aureus: Effect on bacterial internalization into bovine mammary epithelial cells.

Staphylococcus aureus is a major pathogen of subclinical bovine mastitis that usually is chronic and recurrent, which has been related to its ability to internalize into bovine mammary epithelial cells (bMECs). Previously, we reported that short and medium fatty acids and cholecalciferol reduce S. aureus internalization into pretreated-bMECs with these molecules suggesting a role as immunomodulatory agents. Hence, we assessed the role of sodium butyrate (NaB), sodium octanoate (NaO) and cholecalciferol on S. aureus adhesin expression and its internalization into bMECs. S. aureus pre-treated 2 h with 0.5 mM or 2 mM NaB showed a reduction in internalization into bMECs (∼35% and ∼55%; respectively), which coincided with a down-regulated expression of clumping factor B (ClfB). Also, the S. aureus internalization reduction by 2 mM NaB (2 h) agreed with a down-regulated expression of sdrC. Moreover, the 2 mM NaB (24 h) pre-treatment induced bacterial internalization (∼3-fold), which was related with an up-regulation of spa, clfB and sdrC genes. Also, NaO (0.25 mM and 1 mM) only reduced S. aureus internalization when bacteria were grown 2 h with this molecule but there was no relationship with adhesin expression. In addition, cholecalciferol (50 nM) reduced bacteria internalization at similar levels (∼50%) when bacteria were grown 2 and 24 h in broth supplemented with this compound, which correlated with spa and sdrC mRNA expression down-regulated at 2 h, and fnba and clfB mRNA expression decreased at 24 h. In conclusion, our data support the fact that fatty acids and cholecalciferol regulate adhesin gene expression as well as bacteria internalization in nonprofessional phagocytic cells, which may lead to development of anti-virulence agents for control of pathogens.

Immunomodulatory Effects of 17ß-Estradiol on Epithelial Cells during Bacterial Infections.

The innate immune system can function under hormonal control. 17ß-Estradiol (E2) is an important sexual hormone for the reproductive cycle of mammals, and it has immunomodulatory effects on epithelial cells, which are the first line of defense against incoming bacteria. E2 regulates various pathophysiological processes, including the response to infection in epithelial cells, and its effects involve the regulation of innate immune signaling pathways, which are mediated through estrogen receptors (ERs). E2 modulates the expression of inflammatory and antimicrobial elements such as cytokines and antimicrobial peptides. The E2 effects on epithelial cells during bacterial infections are characterized by an increase in the production of antimicrobial peptides and by the diminution of the inflammatory response to abrogate proinflammatory cytokine induction by bacteria. Here, we review several novel molecular mechanisms through which E2 regulates the innate immune response of epithelial cells against bacterial infections.

PaDef defensin from avocado (Persea americana var. drymifolia) is cytotoxic to K562 chronic myeloid leukemia cells through extrinsic apoptosis.

Plant defensins, a group of antimicrobial peptides, show selective cytotoxicity toward cancer cells. However, their mechanisms of action remain poorly understood. Here, we evaluated the cytotoxicity of PaDef defensin from avocado (Persea americana var. drymifolia) on K562 chronic myeloid leukemia cells and analyzed the pathway involved in the induction of cell death. The defensin PaDef was not cytotoxic against human PBMCs; however, it was cytotoxic for K562 cell line (IC50 = 97.3 µg/ml) activating apoptosis at 12 h. PaDef did not affect the mitochondrial membrane potential (ΔΨm), neither the transmembranal potential or the release of intracellular calcium. Also, PaDef induced gene expression of caspase 8 (∼2 fold), TNF-α (∼4 fold) and TNFR1 (∼10 fold). In addition, the activation of caspase 8 was detected at 24 h, whereas caspase 9 activity was not modified, suggesting that the extrinsic apoptosis pathway could be activated. In conclusion, PaDef induces apoptosis on K562 cells, which is related to the activation of caspase 8 and involves the participation of TNF-α, which is a novel property for a plant defensin.

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.

Sodium Octanoate Modulates the Innate Immune Response of Bovine Mammary Epithelial Cells through the TLR2/P38/JNK/ERK1/2 Pathway: Implications during Staphylococcus aureus Internalization.

Bovine mammary epithelial cells (bMECs) contribute to mammary gland defense against invading pathogens, such as Staphylococcus aureus (intracellular facultative), which is recognized by TLR2. In a previous report, we showed that sodium octanoate [NaO, a medium chain fatty acid (C8)] induces (0.25 mM) or inhibits (1 mM) S. aureus internalization into bMECs and differentially regulates the innate immune response (IIR). However, the molecular mechanisms have not been described, which was the aim of this study. The results showed that α5ß1 integrin membrane abundance (MA) was increased in 0.25 mM NaO-treated cells, but TLR2 or CD36 MA was not modified. When these receptors were blocked individually, 0.25 mM NaO-increased S. aureus internalization was notably reduced. Interestingly, in this condition, the IIR of the bMECs was impaired because MAPK (p38, JNK, and ERK1/2) phosphorylation and the activation of transcription factors related to these pathways were decreased. In addition, the 1 mM NaO treatment induced TLR2 MA, but neither the integrin nor CD36 MA was modified. The reduction in S. aureus internalization induced by 1 mM NaO was increased further when TLR2 was blocked. In addition, the phosphorylation levels of the MAPKs increased, and 13 transcriptional factors related to the IIR were slightly activated (CBF, CDP, c-Myb, AP-1, Ets-1/Pea-3, FAST-1, GAS/ISRE, AP-2, NFAT-1, OCT-1, RAR/DR-5, RXR/DR-1, and Stat-3). Moreover, the 1 mM NaO treatment up-regulated gene expression of IL-8 and RANTES and secretion of IL-1ß. Notably, when 1 mM NaO-treated bMECs were challenged with S. aureus, the gene expression of IL-8 and IL-10 increased, while IL-1ß secretion was reduced. In conclusion, our results showed that α5ß1 integrin, TLR2 and CD36 are involved in 0.25 mM NaO-increased S. aureus internalization in bMECs. In addition, 1 mM NaO activates bMECs via the TLR2 signaling pathways (p38, JNK, and ERK1/2), which improves IIR before S. aureus invasion. Additionally, NaO (1 mM) might exert anti-inflammatory effects after bacterial internalization.

The defensin from avocado (Persea americana var. drymifolia) PaDef induces apoptosis in the human breast cancer cell line MCF-7.

Antimicrobial peptides (AMPs) are cytotoxic to cancer cells; however, mainly the effects of AMPs from animals have been evaluated. In this work, we assessed the cytotoxicity of PaDef defensin from avocado (Persea americana var. drymifolia) on the MCF-7 cancer cell line (a breast cancer cell line) and evaluated its mechanism of action. PaDef inhibited the viability of MCF-7 cells in a concentration-dependent manner, with an IC50=141.62µg/ml. The viability of normal peripheral blood mononuclear cells was unaffected by this AMP. Additionally, PaDef induced apoptosis in MCF-7 cells in a time-dependent manner, but did not affect the membrane potential or calcium flow. In addition, PaDef IC50 induced the expression of cytochrome c, Apaf-1, and the caspase 7 and 9 genes. Likewise, this defensin induced the loss of mitochondrial Δψm and increased the phosphorylation of MAPK p38, which may lead to MCF-7 apoptosis by the intrinsic pathway. This is the first report of an avocado defensin inducing intrinsic apoptosis in cancer cells, which suggests that it could be a potential therapeutic molecule in the treatment of cancer.

Anti-Inflammatory and Antimicrobial Effects of Estradiol in Bovine Mammary Epithelial Cells during Staphylococcus aureus Internalization.

17ß-Estradiol (E2), the predominant sexual hormone in females, is associated with the modulation of the innate immune response (IIR), and changes in its levels at parturition are related to intramammary infections, such as mastitis. In bovine mammary epithelial cells (bMECs), E2 regulates differentiation and proliferation, but its immunomodulatory functions have not been explored. Staphylococcus aureus is the predominant pathogen causing mastitis, which can persist intracellularly in bMECs. The aim of this work was to analyze whether E2 modulates the IIR of bMECs during S. aureus internalization. bMECs treated with E2 (50 pg/mL, 24 h) reduced bacteria internalization (~50%). The host receptors α5ß1 and TLR2 do not participate in this reduction. However, E2 activates ERα and modulates the IIR reducing the S. aureus induced-mRNA expression of TNF-α (~50%) and IL-1ß (90%). E2 also decreased the secretion of these cytokines as well as IL-6 production; however, in infected bMECs, E2 induced the secretion of IL-1ß. Furthermore, E2 upregulates the expression of the antimicrobial peptides DEFB1, BNBD5, and psoriasin S100A7 (~5-, 3-, and 6-fold, resp.). In addition, E2 induced the production of antimicrobial compounds in bMEC culture medium, which, together with the modulation of the IIR, could be related to the reduction of S. aureus internalization.