Antiproliferative Modulation and Pro-Apoptotic Effect of BR2 Tumor-Penetrating Peptide Formulation 2-Aminoethyl Dihydrogen Phosphate in Triple-Negative Breast Cancer.

Breast cancer is the most common cancer in women, the so-called "Triple-Negative Breast Cancer" (TNBC) subtype remaining the most challenging to treat, with low tumor-free survival and poor clinical evolution. Therefore, there is a clear medical need for innovative and more efficient treatment options for TNBC. The aim of the present study was to evaluate the potential therapeutic interest of the association of the tumor-penetrating BR2 peptide with monophosphoester 2-aminoethyl dihydrogen phosphate (2-AEH2P), a monophosphoester involved in cell membrane turnover, in TNBC. For that purpose, viability, migration, proliferative capacity, and gene expression analysis of proteins involved in the control of proliferation and apoptosis were evaluated upon treatment of an array of TNBC cells with the BR2 peptide and 2-AEH2P, either separately or combined. Our data showed that, while possessing limited single-agent activity, the 2-AEH2P+BR2 association promoted significant cytotoxicity in TNBC cells but not in normal cells, with reduced proliferative potential and inhibition of cell migration. Mechanically, the 2-AEH2P+BR2 combination promoted an increase in cells expressing p53 caspase 3 and caspase 8, a reduction in cells expressing tumor progression and metastasis markers such as VEGF and PCNA, as well as a reduction in mitochondrial electrical potential. Our results indicate that the combination of the BR2 peptide with 2-AEH2P+BR2 may represent a promising therapeutic strategy in TNBC with potential use in clinical settings.

Effect from dinoponeratoxin M-PONTXDq3a arginine and lysine substituted analogues against Staphylococcus aureus strains.

The growing incidence of methicillin-resistant Staphylococcus aureus (MRSA) infections is associated with increased mortality rates, which has generated interest in the development of antimicrobial peptides (AMP), such as those found in the giant ant Dinoponera quadríceps. In order to improve the net positive charge and the antibacterial activity of the AMP, amino acids with positive side chain single substituted analogues have been proposed, mainly arginine or lysine. The present work aims to study the antimicrobial activity of the analogues of M-PONTX-Dq3a, a 23 amino acid AMP identified in the D. quadriceps venom. M-PONTX-Dq3a[1-15], a fragment containing the 15 central amino acids, and eight derivatives of single arginine or lysine substituted analogues were proposed. The antimicrobial activity of peptides was evaluated against Staphylococcus aureus ATCC 6538 P (MSSA) and ATCC 33591 (MRSA) strains, followed by minimum inhibitory concentration (MIC), minimum lethal concentration (MLC), and minimum biofilm inhibitory concentration (MBIC) measurement. The membrane permeability was then assessed via crystal violet assay and flow cytometry analysis. The effect of exposure time on microbial viability (Time-Kill) was evaluated. Finally, ultrastructural alterations were evaluated through scanning electron microscopy (SEM). Both arginine-substituted peptides [Arg]3M-PONTX-Dq3a[1-15] and [Arg]4M-PONTX-Dq3a[1-15], showed lowest MIC and MLC values (each 0.78 µM). In the biofilm formation assays, the peptide [Arg]3M-PONTX-Dq3a [1-15] showed MBIC of 3.12 µM against the two tested strains. Both peptides were able to alter the membrane permeability approximately by 80%. The treatment with MIC was able to eliminate bacteria after 2 h of contact on the other hand, treatment with half of the MIC, the population of both bacterial strains remained constant for up to 12 h, indicating a possible bacteriostatic effect. The SEM results showed that the treatment with the lowest concentration (0.78 µM) of both peptides caused disruption of the cell membrane, destabilization of the intercellular interaction and the CLM of [Arg]4M-PONTX-Dq3a [1-15] resulted in the complete eradication of the bacteria. Thus, this study describes two AMPs active against MSSA and MRSA and also inhibits the biofilm formation of these stains. This study finds [Arg]3M-PONTX-Dq3a[1-15] and [Arg]4M-PONTX-Dq3a[1-15] as alternative substances to treat resistant and/or biofilm-forming strains.

Antiproliferative mdulation and pro-apoptotic effect of BR2 tumor-penetrating peptide formulation 2-aminoethyl dihydrogen phosphate in triple-negative breast cancer

Breast cancer is the most common cancer in women, the so-called “Triple-Negative Breast Cancer” (TNBC) subtype remaining the most challenging to treat, with low tumor-free survival and poor clinical evolution. Therefore, there is a clear medical need for innovative and more efficient treatment options for TNBC. The aim of the present study was to evaluate the potential therapeutic interest of the association of the tumor-penetrating BR2 peptide with monophosphoester 2-aminoethyl dihydrogen phosphate (2-AEH2P), a monophosphoester involved in cell membrane turnover, in TNBC. For that purpose, viability, migration, proliferative capacity, and gene expression analysis of proteins involved in the control of proliferation and apoptosis were evaluated upon treatment of an array of TNBC cells with the BR2 peptide and 2-AEH2P, either separately or combined. Our data showed that, while possessing limited single-agent activity, the 2-AEH2P+BR2 association promoted significant cytotoxicity in TNBC cells but not in normal cells, with reduced proliferative potential and inhibition of cell migration. Mechanically, the 2-AEH2P+BR2 combination promoted an increase in cells expressing p53 caspase 3 and caspase 8, a reduction in cells expressing tumor progression and metastasis markers such as VEGF and PCNA, as well as a reduction in mitochondrial electrical potential. Our results indicate that the combination of the BR2 peptide with 2-AEH2P+BR2 may represent a promising therapeutic strategy in TNBC with potential use in clinical settings.

The BR2 peptide associated with 2-aminoethyl dihydrogen phosphate is a formulation with antiproliferative potential for a triple-negative breast cancer model.

Triple-negative breast cancer is the most aggressive subtype of breast cancer, with worse clinical evolution and tumor-free survival, leading to the need to develop new effective therapies for its control. The present study evaluated the action of tumor-penetrating peptide BR2 associated with 2-aminoethyl dihydrogen phosphate (2-AEH2P) on triple-negative breast tumor cells. Cell viability was evaluated by the MTT colorimetric method, mitochondrial electrical potential, and proteins involved in cell proliferation and death control were evaluated by flow cytometry and structural and morphological analysis by confocal microscopy. The results obtained showed that the peptide BR2 and the association 2-AEH2P + BR2 promoted significant cytotoxicity in tumor lines, compared to 2-AEH2P alone. In addition, the association 2-AEH2P + BR2 promoted tumor cells arrest in the G0/G1 phases. Interestingly, both treatments modulated the expression of markers CD44, CD34, CD24, cyclin D1, and Bcl-2, increased p21, Bax, and released cytochrome c. The association proved to be more effective, providing modulation of proteins involved in cell death and senescence, more pronounced cytotoxicity for tumor cells compared to normal cells, and the reduction of markers related to aggressiveness profile, progression, and tumor metastasis.

Rational design of a trypanocidal peptide derived from Dinoponera quadriceps venom.

Chagas disease is caused by the parasite Trypanosoma cruzi and affects millions of people worldwide, having no effective cure. The main sanitary emergency is related to patients with chronic infection, which accumulate comorbidities causing patient death. However, actual chemotherapeutic treatments do not effectively address the chronic forms of the disease. Invertebrates are a relevant source of antimicrobial peptides (AMPs) as part of the innate immune system for their protection. The AMP M-PONTX-Dq3a, isolated from the Dinoponera quadriceps ant venom, has shown very effective antimicrobial and trypanocidal activities. Although M-PONTX-Dq3a has better activity that the current therapies, the peptide length has limited its possibilities to reach clinical application. In this investigation, we aimed to dissect the trypanocidal effect of M-PONTX-Dq3a fragments and to study the activity of substituted analogs, to improve not only peptide trypanocidal activity and bioavailability, but also production costs. Our studies have led to the identification of two smaller peptides, M-PONTX-Dq3a [1-15] and [Lys]3-M-PONTX-Dq3a [3-153-15 with similar trypanocidal activities that the parent peptide has against the three forms of T. cruzi benznidazole-resistant Y strain. Both peptides represent promising candidates to develop novel and effective trypanocidal bio-therapeutic agents, opening new avenues for the treatment of chronic patients.

The BR2 peptide associated with 2-aminoethyl dihydrogen phosphate is a formulation with antiproliferative potential for a triple-negative breast cancer model

Triple-negative breast cancer is the most aggressive subtype of breast cancer, with worse clinical evolution and tumor-free survival, leading to the need to develop new effective therapies for its control. The present study evaluated the action of tumor-penetrating peptide BR2 associated with 2-aminoethyl dihydrogen phosphate (2-AEH2P) on triple-negative breast tumor cells. Cell viability was evaluated by the MTT colorimetric method, mitochondrial electrical potential, and proteins involved in cell proliferation and death control were evaluated by flow cytometry and structural and morphological analysis by confocal microscopy. The results obtained showed that the peptide BR2 and the association 2-AEH2P + BR2 promoted significant cytotoxicity in tumor lines, compared to 2-AEH2P alone. In addition, the association 2-AEH2P + BR2 promoted tumor cells arrest in the G0/G1 phases. Interestingly, both treatments modulated the expression of markers CD44, CD34, CD24, cyclin D1, and Bcl-2, increased p21, Bax, and released cytochrome c. The association proved to be more effective, providing modulation of proteins involved in cell death and senescence, more pronounced cytotoxicity for tumor cells compared to normal cells, and the reduction of markers related to aggressiveness profile, progression, and tumor metastasis.

Arg-substituted VmCT1 analogs reveals promising candidate for the development of new antichagasic agent.

VmCT1 is an antimicrobial peptide (AMP) isolated from the venom of the scorpion Vaejovis mexicanus with antimicrobial, anticancer and antimalarial activities, which the rational design with Arg-substitution has yielded AMPs with higher antimicrobial activity than VmCT1. Chagas is a neglected tropical disease, becoming the development of new antichagasic agents is urgent. Thus, we aimed to evaluate the antichagasic effect of VmCT1 and three Arg-substituted analogues, as well their action mechanism. Peptides were tested against the epimastigote, trypomastigote, amastigote forms of Trypanossoma cruzi Y strain and against LLC-MK2 mammalian cells. The mechanism of action of these peptides was evaluated by means of flow cytometry and scanning electron microscopy. VmCT1 presented activity against all three forms of T. cruzi, with EC50 against trypomastigote forms of 1.37 µmol L-1 and selectivity index (SI) of 58. [Arg]3-VmCT1, [Arg]7-VmCT1 and [Arg]11-VmCT1 also showed trypanocidal effect, but [Arg]11-VmCT1 had the best effect, being able to decrease the EC50 against trypomastigote forms to 0.8 µmol L-1 and increase SI to 175. Necrosis was cell death pathway of VmCT1, as well [Arg]7-VmCT1 and [Arg]11-VmCT1, such as observed by membrane damage in flow cytometry analyses and scanning-electron-microscopy. In conclusion, [Arg]11-VmCT1 revealed promising as a candidate for new antichagasic therapeutics.

Wasp venom peptide as a new antichagasic agent.

Chagas disease is caused by Trypanosoma cruzi and affects approximately 10 million people a year worldwide. The only two treatment options, benznidazole and nifurtimox, have low efficacy and high toxicity towards human cells. Mastoporan peptide (MP) a small cationic AMP from the venom of the wasp Polybia paulista has been reported as a potent trypanocidal agent. Thus, we evaluated the antichagasic effect of another AMP from the venom of the same wasp Polybia paulista, polybia-CP (ILGTILGLLSKL-NH2), and investigated its mechanism of action against different stages of the trypanosomal cells life cycle. Polybia-CP was tested against the epimastigote, trypomastigote and amastigote forms of the T. cruzi Y strain (benznidazole-resistant strain) and inhibited the development of these forms. We also assessed the selectivity of the AMP against mammalian cells by exposing LLC-MK2 cells to polybia-CP, the peptide presented a high selectivity index (>106). The mechanism of action of polybia-CP on trypanosomal cells was investigated by flow cytometry, scanning electron microscopy (SEM) and enzymatic assays with T. cruzi GAPDH (tcGAPDH), enzyme that catalyzes the sixth step of glycolysis. Polybia-CP induced phosphatidylserine exposure, it also increased the formation of reactive species of oxigen (ROS) and reduced the transmembrane mitochondrial potential. Polybia-CP also led to cell shrinkage, evidencing apoptotic cell death. We did not observe the inhibition of tcGAPDH or autophagy induction. Altogether, polybia-CP has shown the features of a promising template for the development of new antichagasic agents.