Cytotoxic Activity and Antiproliferative Effects of Crude Skin Secretion from Physalaemus nattereri (Anura: Leptodactylidae) on in vitro Melanoma Cells

Anuran secretions are rich sources of bioactive molecules, including antimicrobial and antitumoral compounds. The aims of this study were to investigate the therapeutic potential of Physalaemus nattereri skin secretion against skin cancer cells, and to assess its cytotoxic action mechanisms on the murine melanoma cell line B16F10. Our results demonstrated that the crude secretion reduced the viability of B16F10 cells, causing changes in cell morphology (e.g., round shape and structure shrinkage), reduction in mitochondrial membrane potential, increase in phosphatidylserine exposure, and cell cycle arrest in S-phase. Together, these changes suggest that tumor cells die by apoptosis. This skin secretion was also subjected to chromatographic fractioning using RP-HPLC, and eluted fractions were assayed for antiproliferative and antibacterial activities. Three active fractions showed molecular mass components in a range compatible with peptides. Although the specific mechanisms causing the reduced cell viability and cytotoxicity after the treatment with crude secretion are still unknown, it may be considered that molecules, such as the peptides found in the secretion, are effective against B16F10 tumor cells. Considering the growing need for new anticancer drugs, data presented in this study strongly reinforce the validity of P. nattereri crude secretion as a rich source of new anticancer molecules

Histopathological Evaluation of the Exposure by Cyanobacteria Cultive Containing [d-Leu1]Microcystin-LR on Lithobates catesbeianus Tadpoles

This study evaluated the effects of [d-Leu1]Microcystin-LR variant by the exposure of Lithobates catesbeianus tadpole to unialgal culture Microcystis aeruginosa NPLJ-4 strain. The Tadpole was placed in aquariums and exposed to Microcystis aeruginosa culture or disrupted cells. For 16 days, 5 individuals were removed every 2 days, and tissue samples of liver, skeletal muscle, and intestinal tract were collected for histopathology and bioaccumulation analyses. After exposure, those surviving tadpoles were placed in clean water for 15 days to evaluate their recovery. A control without algae and toxins was maintained in the same conditions and exhibited normal histology and no tissue damage. In exposed tadpoles, samples were characterized by serious damages that similarly affected the different organs, such as loss of adhesion between cells, nucleus fragmentation, necrosis, and hemorrhage. Samples showed signs of recovery but severe damages were still observed. Neither HPLC-PDA nor mass spectrometry analysis showed any evidence of free Microcystins bioaccumulation

Molecular modeling of BPP-BrachyNH₂ and human ACE and <i>in silico</i> docking studies.

<p>(A) Theoretical model of BPP-BrachyNH₂ showing the structure with lower energy system; (B) theoretical model of ACE showing the structure with lower energy system; (C) Docking between BPP-BrachyNH₂/ACE; (D) binary complex relationship with zoom in and detailed interactions; and (E) interactions between the substrate and the catalytic triad His³⁸³, His³⁸⁷ and Glu⁴¹¹, indicating a probable block of the catalytic activity.</p

The inhibitory effect of BPP-BrachyNH₂ and captopril on rat serum ACE activity.

<p>Residual enzymatic activities are plotted against the corresponding inhibitor concentrations. IC₅₀ values were calculated from nonlinear regression analysis of obtained data using GraphPad 5.0 software (GraphPad Prism, San Diego, CA).</p

Vasodilator effect of BPP-BrachyNH₂ and captopril (10⁻⁹–3 × 10⁻⁵ M) on rat thoracic aorta.

<p>Aortic rings were pre-contracted with phenylephrine (3 × 10⁻⁷ M) and then cumulatively incubated with BPP-BrachyNH₂ (A) or captopril (B). Effect of L-NAME (100 μM) on BPP-BrachyNH₂-induced vasodilator effect (C). Respective comparisons among E_max (D) values were plotted. The results were expressed as means ± SEM (n = 6). Non-paired Student’s t test. ^**<i>p</i> < 0.01 and ^***<i>p</i> < 0.001 versus endothelium-intact (E+) preparations.</p

Sequencing of the proline-rich peptides (PROs) from the skin secretion of <i>B</i>. <i>ephippium</i>.

<p>(A) Mass spectra of BPP-Brachy, [M+H]⁺ = 907.37 and (B) BPP-BrachyNH₂, [M+H]⁺ = 906.36 acquired in an UltraFlex III MALDI-TOF/TOF operating under LIFT™ mode for MS/MS experiments. The observed fragments allowed complete assignment of the major y and b ion series. The peptide sequence using one-letter code following the y and b series orientation is shown on the top part of the spectra.</p