*4.2. Dermaseptins and Anticancer Properties*

The first two anticancer DRSs peptides were isolated from the South American Amazonian tree frog, *Phyllomedusa bicolor*. These molecules, DRSs-B2 and DRS-B3, were tested in vitro against a human prostatic adenocarcinoma PC-3 cell line, showing an antiproliferative effect with an EC50 around 2–3 μM and demonstrating the inhibition of proliferation of more than 90% [83]. In addition, these two peptides also inhibited PC-3 cell colony formation in soft agar and the proliferation, differentiation and capillary formation of endothelial cells [83,84]. Furthermore, DRS-B2 blocks the proliferation and colony formation of several human tumor cell types, such as prostatic adenocarcinoma LNCAP, prostatic carcinoma DU145, mammary carcinoma (MDA-MB2318) cell lines and B-lymphoma lines [83]. These effects were also confirmed in vivo by a cell line PC3 murine xenograft model, showing that DRS-B2 inhibits tumor growth [83]. The anticancer mechanism of action of DRS-B2 was demonstrated by in vitro experiments with tumor PC3 cells. This peptide interacted with tumor cell surface, aggregating and penetrating the cells. Furthermore, it induced the release of cytosolic lactate dehydrogenase, a marker of cytotoxicity and necrosis, but no effects were observed on mitochondrial membrane potential and caspase 3 activations for apoptotic involvement [83]. Concerning the mechanisms of action of DRS-B2, confocal microscopy studies revealed that this peptide rapidly accumulates to cytoplasmic membranes, packed in vesicles and into the nucleus [85]. These effects were also partially mediated by glycosaminoglycans' interaction with DRS-B2 and the consequent structural modification of the peptide with the α-helical domain [85]. Recently, a synthetic hormonotoxin molecule composed of dermaseptin-B2 associated with luteinizing hormone-releasing hormone (LHRH) was tested to improve the peptide's antitumor activity, reducing its peripheral toxicity and lethality. This hormonotoxin displayed an anticancer effect very similar to DRS-B2 both in vitro and in vivo [86]. The LHRH addition to dermaseptin-B2 does not alter the peptide's secondary structure and biological function [86]. On the other hand, double staining flow cytometry analysis showed that this hormonotoxin induced apoptosis instead of a necrotic process caused by DRS-B2 [86]. This different anticancer mechanism of action explains better tolerance and the lower toxicity of the hormonotoxin compared to dermaseptin-B2 [86]. In addition, other biochemical approaches have been used to increase the antitumor activity of DRS, delivering these agents in tumor cells, as seen in DRS-DStomo01 peptide [87]. DRS-DStomo01 was entrapped in chitosan nanoparticles, and the antitumor activity was tested in vitro against HeLa cells. The peptide induces DNA fragmentation and mitochondrial hyperpolarization with consequent cytotoxicity for cancer cells [87]. However, when used in chitosan nanoparticles, DRS-DStomo01 was more active than free peptides [87]. In 2016, two novel members of DRSs family were identified in the skin secretion of the frog *Pachymedusa dacnicolor* and called DRS-PD 1 and 2 [88]. Both peptides were reported to be active against many microorganisms, such as *E. coli*, *S. aureus*, *P. aeruginosa* and *C. albicans*, but with no lytic effects on mammalian red cells [88]. DRS-PD 2 displayed anti-proliferative effects against cancer cell lines, such as H157, PC-3 and U251-MG, within the concentration range of 10−<sup>9</sup> to 10−<sup>4</sup> M [88]. This property was also reported for DRS-PD 1 but only for human neuronal glioblastoma U251MG cell line [88]. In addition, these peptides could also inhibit the proliferation of human microvessel endothelial cells with the same concentration range for anticancer activity [88]. Other peptides from the South American orange-legged leaf frog (*Phyllomedusa hypochondrialis*) were identified and called DRS-PH. This peptide was active against several pathogens,

such as *E. coli*, *P. aeruginosa*, *S. aureus* and its methicillin-resistant strain (MRSA), *E. faecalis* and *C. albicans*, in a concentration range from 1 μM to 512 μM [89]. Once again, this DRS displayed a broad spectrum of anticancer properties against different cancer cell lines, including MCF-7, H157, U251MG, MDA-MB-435S and PC-3 [89]. Very recently, different studies identified, from the skin of *Phyllomedusa sauvagei*, DRSs-PS type 1, 3 and 4, characterizing their anticancer properties [90–92]. In 2019, Long and coworkers demonstrated that DRS-PS1 has antimicrobial effects against *S. aureus*, *E. coli* and *C. albicans* [90]. Interestingly, DRS-PS 1 showed anti-proliferative effects on human glioblastoma U-251 MG, perturbing cell membrane integrity at the concentration of 0.1 μM [90]. Furthermore, the anticancer action with lower concentrations involves apoptosis activation by mitochondrial-related signal involvement [90]. DRS-PS 3 showed a broad spectrum of antimicrobial activities against several pathogens, such as *S. aureus*, *E. coli* and *C. albicans*, at high concentrations but with reduced cytotoxicity for erythrocytes [91]. However, the synthetic, more cationic and hydrophobic analogues created by replacing acidic amino acids D and E at 5 and 17, respectively, of the DRS-PS3 sequence by lysines (K5/D5, K17/E17-DRS-PS 3) or by replacing two neutral amino acids A10 and G11 with the hydrophobic amino acid leucine (L10/A10, L11/G11-DRS-PS 3) strongly increased their antimicrobial activities against the same pathogens with MIC values of 8 μM or less [91]. On the other hand, both artificial analogues exhibit a more significant hemolytic effect on red blood cells than DRS-PS 3 [91]. Furthermore, these peptides showed anticancer activities against H157, PC3 and HMEC-1 cell lines in the micromolar range but the most active was L10/A10, L11/G11-DRS-PS 3 [91]. Additionally, DRS-PS 4 displayed antimicrobial effects with many pathogens, such as *S. aureus* and MRSA, *E. faecalis*, *E. coli*, *P. aeruginosa* and *C. albicans*, in a range of concentrations from 1 μM to 32 μM, with biofilms eradicating properties of these microorganisms [92]. The antimicrobial mechanism of action is based on the ability of this peptide to permeabilize the bacterial cell membrane [92]. However, the hemolysis activity of DRS-PS 4 was tested using horse red blood cells showing slight effects at antimicrobial concentrations [92]. In addition, the anticancer activity of DRS-PS 4 was also evaluated on several human cell lines, including U251MG, MDA-MB-435S, H157, PC-3 and MCF-7, displaying a dose-dependent inhibitory activity with high cytotoxicity in a concentration range from 10−<sup>9</sup> to 10−<sup>4</sup> M [92]. On the other hand, it presents a slight suppressing effect on human microvascular endothelial cells [92]. Very recently, Dong et al. discovered the DRS-PP from frog *Phyllomedusa palliata*. This peptide was active at 2 μM against *E. coli*, *S. aureus* and MRSA, *C. albicans*, *P. aeruginosa*, *E. faecalis* and *K. pneumoniae* [93]. It is of great interest that DRS-PP showed anti-proliferative effects with cytotoxic activities on different cancer cells, such as H157, MCF-7, PC-3 and U251 MG, but no effects on human microvascular endothelial cells [93]. In vivo studies confirmed the anticancer property of this agent; in fact, DRS-PP was tested on a subcutaneous H157 tumor model of nude mice showing significant anti-tumor activity in a dose-dependent manner without hepatopulmonary and toxic side effects [93]. These effects are mediated via disruptive membrane action but exert pro-apoptotic effects induced by mitochondrial and death receptor pathways [93]. Finally, in 2021, DRS-TO was identified in the tiger-striped Leaf Frog, *Phyllomedesa tomopterna*, showing that this peptide was active against *S. aureus* and MRSA, *E. faecalis*, *E. coli*, and *C. albicans* [94]. Additionally, no hemolytic effect was observed on red blood cells, but DRS-TO showed anticancer activity against U251MG, H157 and PC-3 cancer cell lines at higher concentrations [94]. All these data report the great potential of DRSs as anticancer agents and their mechanism of action targeting membrane but also inducing pro-apoptotic effects by mitochondrial dysfunction and death receptor pathways (Figure 3).

**Figure 3. Dermaseptins and Cancer**. Dermaseptins act as an anti-proliferative agent against several cancer cells in vitro and in vivo. The anticancer mechanism of action is based on the ability of these MF-AMPs to accumulate in cancer cells, inducing cell death and blocking tumor vascularization.
