The Secreted Metabolome of Hela Cells under Effect of Crotamine, a Cell-Penetrating Peptide from a Rattlesnake Using NMR-Based Metabolomics Analyses

Sequestering and reprogramming of cellular metabolism represents one of the principal hallmarks of several cells. Antimicrobial peptides have been shown to exhibit selective anticancer activities. In this study, the secreted metabolome of HeLa cells under action of the antimicrobial peptide Crotamine from the venom of the South American rattlesnake Crotalus durissus terrificus was evaluated. Crotamine has been shown to be selective for highly proliferating cells and is able to extend the in vivo lifespan. The present study using a cell line of cervical cancer, HeLa cells, provide insights into how Crotamine acts in cell metabolism. NMR spectroscopy was used to identify and quantify relative metabolite levels, which are associated with Crotamine uptake. Statistical analysis reveals that Crotamine dramatically affects metabolites related to glycolysis, metabolism and biosynthesis of amino acids and pyruvate metabolism. The developed machine learning model is found to be robust by ROC curve analysis, suggesting that the metabolic state of HeLa cells treated with Crotamine is different from the control samples. To account for metabolite levels, it is suggested that Crotamine would have to act on glycolysis, which, in turn, affects several other metabolic pathways, such as, glutathione metabolism, TCA cycle and pyruvate metabolism. The observed metabolic changes shed light into the mode of Crotamine function

Ac2-26 mimetic peptide of annexin A1 inhibits local and systemic inflammatory processes induced by bothrops moojeni venom and the lys-49 phospholipase A2 in a rat model

Annexin A1 (AnxA1) is an endogenous glucocorticoid regulated protein that modulates anti-inflammatory process and its therapeutic potential has recently been recognized in a range of systemic inflammatory disorders. The effect of the N-terminal peptide Ac2-26 of AnxA1 on the toxic activities of Bothrops moojeni crude venom (CV) and its myotoxin II (MjTX-II) were evaluated using a peritonitis rat model. Peritonitis was induced by the intraperitoneal injection of either CV or MjTX-II, a Lys-49 phospholipase A2. Fifteen minutes after the injection, the rats were treated with either Ac2-26 or PBS. Four hours later, the CV and MjTX-II-induced peritonitis were characterized by neutrophilia (in the peritoneal exudate, blood and mesentery) and increased number of mesenteric degranulated mast cells and macrophages. At 24 hours post-injection, the local inflammatory response was attenuated in the CV-induced peritonitis while the MjTX-II group exhibited neutrophilia (peritoneal exudates and blood). Ac2-26 treatment prevented the influx of neutrophils in MjTX-II-induced peritonitis and diminished the proportion of mesenteric degranulated mast cells and macrophages in CV-induced peritonitis. Additionally, CV and MjTX-II promoted increased levels of IL-1β and IL-6 in the peritoneal exudates which were significantly reduced after Ac2-26 treatment. At 4 and 24 hours, the endogenous expression of AnxA1 was upregulated in the mesenteric neutrophils (CV and MjTX-II groups) and mast cells (CV group). In the kidneys, CV and MjTX-II administrations were associated with an increased number of macrophages and morphological alterations in the juxtamedullary nephrons in proximal and distal tubules. Ac2-26 promoted significant recovery of the juxtamedullary structures, decreased the number of macrophages and diminished the AnxA1 in epithelial cells from distal tubules and renal capsules. Our results show that Ac2-26 treatment significantly attenuates local and systemic inflammatory processes and indicate this peptide as a potential target for the development of new therapeutic strategies for the snakebite envenomation treatment.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Presence of renal macrophages after MjTX-II-induced peritonitis.

<p>An increased number of cells was observed in the glomeruli (arrows) and interstice (arrowheads) of the juxtamedullary region 24 h after peritonitis induced by MjTX-II (B), compared to the control group (A). Ac2-26 peptide post-treatment prevented the macrophages influx in renal tissue (C). Scale bars: 20 μm. Counterstain: Hematoxylin. The data represent the mean ± SEM of the number of interstitial (D) and glomerular (E) macrophages per 20 fields in the renal tissue (n = 5 animals/group). **P < 0.01 and ***P < 0.001 vs control; ^§P < 0.05 and ^§§P < 0.01 vs MjTX-II 24 h.</p

Expression of AnxA1 in the mesenteric neutrophils.

<p>Strong AnxA1 immunoreactivity in neutrophils (arrows) from CV (B, C) and MjTX-II (E, F) groups, after 4 and 24 h, compared with cells of control group (A). The absence of immunoreactivity in the negative control (D). Scale bars: 5 μm. Counterstain: hematoxylin. Densitometric analysis of mesenteric neutrophils immunostained for AnxA1 in the CV (G) and MjTX-II (H) groups. The values (arbitrary units) are expressed as mean ± SEM of the sections analyzed from 5 rats /group. **P < 0.01 and ***P < 0.001 vs control.</p

Histopathological analysis of kidneys after CV- and MjTX-II-induced peritonitis.

<p>(A) Normal histological condition of proximal (PT) and distal convuluted tubules (DT) in the renal control. (B-E) CV- and MjTX-II PLA₂-induced peritonitis provoked renal damage characterized by the presence of pyknotic nuclei (arrowheads), karyolisis (white arrow) and hyaline casts (asterisks) in proximal tubules. (F) Ac2-26 peptide restores the normal morphological aspect of juxtamedullary structures. Stain: Hematoxylin-Eosin. Scale bars: 20 μm.</p

Effect of Ac2-26 treatment on proinflammatory cytokine secretion.

<p>Peritonitis was induced in rats by i.p. injection of CV (100 μg) or MjTX-II (100 μg) in 0.5 mL of PBS. Control group received i.p. only PBS. Another set of animals were treated i.p. with 1 mg/kg of Ac2-26 peptide after 15 minutes of CV or MjTX-II injection. Peritoneal exudate was collected 4 and 24 hours after peritonitis inductions and cytokine dosages were performed by ELISA, as described in Methods. Values are expressed as the mean ± SEM of cytokine levels (n = 5 rats/group). ND < 31.25 pg/mL (not detected).</p><p>* P < 0.05 and</p><p>*** P < 0.001 vs control</p><p>^##P < 0.01 vs CV 4 h.</p><p>Effect of Ac2-26 treatment on proinflammatory cytokine secretion.</p

Quantitative analysis of degranulated mast cell and macrophages into mesentery.

<p>Peritonitis was induced in rats by i.p. injection of CV (100 μg) or MjTX-II (100 μg) in 0.5 mL of PBS. Control group received i.p. only PBS. Another set of animals were treated i.p. with 1 mg/kg of Ac2-26 peptide after 15 minutes of CV or MjTX-II injection. Mesentery was collected after 4 and 24 hours after peritonitis induction and cellular counts performed as described in Methods. Values are expressed as the mean ± SEM of the number of cells per mm² (n = 5 rats/group).</p><p>** P < 0.01 and</p><p>*** P < 0.001 vs control</p><p>^#P < 0.05 and</p><p>^###P < 0.001 vs CV 4 h.</p><p>Quantitative analysis of degranulated mast cell and macrophages into mesentery.</p

Analysis of AnxA1 expression in the juxtamedullary renal structures.

<p>AnxA1 immunostaining was detected in the epithelial cells under all of the experimental conditions (A, C, E, G, I), concentrated in the glomerular capsule (arrows) and distal convuluted tubules (asterisks). Ac2-26 post-treatment decreased the immunostaining for endogenous AnxA1 in the distal tubules and glomerular capsule (D, F, H, J) 4 and 24 h after CV- and MjTX-II-induced peritonitis. Negative control of the reaction (B). Counterstain: hematoxylin. Scale bars: 20 μm. Densitometric analysis of the epithelial cells from distal convuluted tubules (K-L) and glomerular capsule (M-N) immunostained for AnxA1. Values (arbitrary units) are expressed as the mean ± SEM of sections analyzed from 5 rats /group. **P < 0.01 and ***P < 0.001 vs control; ^###P < 0.001 vs CV groups; ^§§P < 0.01 and ^§§§P < 0.001 vs MjTX-II groups at the corresponding experimental time.</p

Model to summarize the effects of Ac2-26 treatment after either CV or MjTX-II induced peritonitis.

<p>Local inflammation in the peritoneal exudate augments the number of neutrophils and the pro-inflammatory cytokines, IL-1β and IL-6 (A). This inflammation is decreased after Ac2-26 treatment (B). In the mesentery, the inflammatory response promotes the influx of macrophages, and increases the number of neutrophils, degranulated mast cells and the levels of AnxA1 expression (C). The Ac2-26 treatment decreased the numbers of all the inflammatory cells (D). The systemic inflammation results in an increase of neutrophils in the bloodstream (E); these levels are restored after Ac2-26 post-treatment (F). In the kidney (G), MjTX-II augmented the infiltration by macrophages (H) and Ac2-26 prevented this influx (I). Histopathological analysis showed direct (casts) and indirect (pyknotic nuclei/karyolysis) effects of the envenoming (J), which were restored by the Ac2-26 treatment (K). Higher levels of AnxA1 were observed in the distal tubules and glomerular cells after the administration of either CV or MjTX-II (L), and these levels decreased with the anti-inflammatory treatment (M).</p