Evaluation of the antitumoral activity of Crotalicidin and NA-CATH-ATRA-1-ATRA-1 peptides in Breast cancer cell lines

Autores

Gallego-londoño, V. (UNIVERSIDAD DE ANTIOQUIA) ; Santa-gonzález, G.A. (INSTITUTO TECNOLÓGICO METROPOLITANO) ; Manrique-moreno, M. (UNIVERSIDAD DE ANTIOQUIA)

Resumo

Bioactive peptides (BAPs) are well-recognized for their broad spectrum of applications including antitumoral activity due to their high cytotoxic action, selectivity, and limited scope for drug resistance. In the present study, the antitumoral activity of the synthetic peptides Ctn and NA-CATH-ATRA-1ATRA-1 was evaluated against MCF-7 and MDA-MB-231 breast cancer cell lines. The flow cytometry experiments showed an increase of the DiOC6(3) uptake and a significant propidium iodide (PI) uptake at higher concentrations in both cell lines. Additionally, it was possible to determine that peptides exert their antitumoral activity by inducing cell cycle arrest. The results suggest that these peptides could be considered potential therapeutic molecules in the treatment of breast cancer.

Palavras chaves

Bioactive peptides (BAPs); antitumoral activity; breast cancer

Introdução

Bioactive peptides (BAPs) are considered part of the innate immune system of several organisms and participate in the first line of defense in response to the attack of pathogens (HANEY, MANSOUR, et al., 2017, TORNESELLO, BORRELLI, et al., 2020). They have been extensively studied as a promising alternative to antibiotics (HANCOCK, SAHL, 2006). However, their spectrum of activity extends to parasites, fungi, and in the last years cancer cells (TORNESELLO, BORRELLI, et al., 2020). The selectivity of these compounds for malignant over normal cells is based on the electrostatic attraction for the negatively charged molecules on cancer cell membranes such as phosphatidylserine phospholipids, O- glycosylated mucins, and sialic acid-derived gangliosides (BAXTER, LAY, et al., 2017). An interesting property of peptides is that most of them mediate anticancer effects irrespective of the genetic and epigenetic features of malignant cells, largely reflecting unique physiochemical properties that enable them to interact and disrupt lipid bilayers (VITALE, YAMAZAKI, et al., 2021). This is significantly important in a type of cancer with high intratumoral heterogeneity such as breast cancer (LÜÖND, TIEDE, et al., 2021). In this study, the Crotalicidin (Ctn) a peptide identified in Crotalus durissus (FALCAO, DE LA TORRE, et al., 2014) and NA-CATH-ATRA-1-ATRA-1, which is a modification of the NA-CATH peptide (DEAN, BISHOP, et al., 2011, ZHAO, GAN, et al., 2008) were evaluated against MCF-7 and MDA-MB-231 cancer cell lines. Both peptides showed promising activity against the cancer cell lines. As a control, the peptide LTX- 315 was used to compare the activity of the peptides.

Material e métodos

Peptides were synthesized by the solid-phase method (GUZMÁN, BARBERIS, et al., 2007), and purchased from GenScript (Piscataway Township, NJ, USA). The purity of the peptides was determined to be higher than 95% by analytical HPLC-UV, and the molecular weight (MW) was confirmed with LC-ESI-MS. The LTX-315 peptide was used as a positive control because it has been studied in the phase I trial in patients with advanced solid tumors including breast tumors and demonstrates being clinically active (SPICER, MARABELLE, et al., 2021). The cytotoxic activity of the peptides against MCF-7 (ATCC HTB-22) and MDA-MB-231 (ATCC CRM- HTB-26) cell lines was determined by a colorimetric 3-( 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assay after 24h of treatment. The antitumor activity of the peptides was evaluated at concentrations below the IC₅₀ value obtained. 1 x 10⁵cells were grown into a 24-well plate and incubated with different peptide concentrations for 6h. The following parameters were evaluated by flow cytometry: (a) disruption of cell membrane integrity by propidium iodide (PI) uptake; (b) change in mitochondrial membrane potential monitored through the lipophilic cationic fluorochrome lipophilic 3-3'-dihexyloxacarbocyanine iodide (DiOC₆(3)); and (c) distribution of cell cycle phases by staining of cells with PI. GraphPad Prism Software (Version 8.0.1, GraphPad Inc, CA, USA) was used to perform data analysis. A one-way/two-way analysis of variance (ANOVA) with post hoc comparisons via Fisher’s Least Significant Difference (LSD) test was used to evaluate the level of significance (expressed as the p- value).

Resultado e discussão

The physicochemical properties of the peptides are shown in Table 1. The results of the cytotoxic activity showed that Ctn and NA-CATH-ATRA-1-ATRA-1 are more active toward MDA-MB-231 cell line, with an IC₅₀ value approx. of 2.1 to 2.8 times lower that the concentration needed for MCF-7 cells. The higher cytotoxic activity of the peptides against MDA-MB-231 cell line could be associated with differences in the cell-membrane composition (VITALE, YAMAZAKI, et al., 2021). The flow cytometry results showed that both peptides induced an increase in the percentage of PI-positive cells at the highest concentration evaluated (Figure 1). However, when the peptides are used at lower concentrations than those required for their membranolytic effect, they can translocate to the cytosol and affect intracellular targets (VITALE, YAMAZAKI, et al., 2021). In this study, it was observed that at subtoxic concentrations of the peptides, induced an increase of the DiOC₆(3) mean fluorescence intensity (MFI) (Figure 1), which suggested that high trans-membrane potential, known as mitochondria hyperpolarization, was involved. Investigations in breast cell lines have suggested that cell cycle arrest is a common mechanistic marker for peptide anticancer activity (MANRIQUE-MORENO, SANTA-GONZÁLEZ, et al., 2021). All peptides in MCF-7 cells exerted an increase in the percentage of cells in the G0/G1 with increasing peptide concentrations, which is concomitant with a G2/M decrease (from 30.15% in untreated cells to 4.63-9.84% at the high concentration of peptides). In contrast, cell cycle distribution of MDA-MB-231 was characterized by the accumulation of the cell population in the S phase (from 33.87% in untreated cells to 45.03-69.88% at the highest concentration)

*Calculated with the ‘Peptide property calculator’ software (http://pepcal.com) +Molecular weight (MW) *+Calculated from http://heliquest.ipmc.cnrs.fr


PI-positive cells (%) on the left axis (blue line). Mitochondrial membrane polarization was evaluated with DiOC6(3)(MFI)on the right axis (red line)

Conclusões

Ctn and NA-CATH-ATRA-1-ATRA-1 peptides showed the highest cytotoxic activity towards MDA-MB-231 than MCF-7 cells. These peptides are more active than LTX-315 peptide control at MDA-MB-231 cell line. At lower peptide concentrations both cell lines displayed an increase in mitochondrial membrane potential and cell cycle arrest. The results suggest that Ctn and NA-CATH-ATRA-1-ATRA-1 are potential target-studied molecules for breast cancer treatment. More studies are needed to search the mechanism of action involved in their antitumoral effect.

Agradecimentos

This research was supported by MinCiencias Research Grant [Cod. 111584467189, RC 946-2019].

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