*3.3. Anti-Cancer Properties*

Peptides, due to their small size and chemical nature, can penetrate cell membranes without a build-up of toxic levels as seen with protein/antibodies. These compounds have shown high affinity and specificity while having low interactions with other medical treatments. However, there are limitations to overcome for their use, mainly related to the process of delivery of the peptide, as these compounds have regularly low bioavailability when taken orally, resulting in rapid clearance of the peptides. Peptides also have low levels of activity when compared to traditional drug treatments for cancer [85]. However, the peptide treatments have multiple problems mainly associated with the lack of specificity of the drugs that are not able to differentiate between carcinogenic and healthy cells. Moreover, when the chemotherapeutic agents are bound to a transport molecule, the breakage of these bounds can also reduce the efficacy of the peptides. Furthermore, the ability we have to currently treat multiple cancers is also dependent on the resistance of the cancer to the chemotherapeutic agents, which is a growing problem [86].

Limited studies are available on the anti-cancer properties of peptides derived from algae. Sheih, Fang, Wu and Lin [84] hydrolyzed protein by-products from the industrial processing of *C. vulgaris* and identified the peptide VECYGPNRPQF as an anti-proliferative. This peptide only had anti-proliferative effects on the human gastric cancer cell line AGS, but not on the other cell lines studied including human normal lung cell WI38, human colon adenocarcinoma cells C2BBel, human hepatoblastoma cell lines HepG2, human cervical epithelioid carcinoma cells Hela, and mouse BALB/c macrophage RAW 264.7 cells. The authors hypothesized that this peptide could have specific anti-cancer activity when treating certain tumor cells [84]. The peptide halted the cell cycle where the cell is given the chance to be either repaired by the TP53 mechanism or undergo apoptosis [87]. Moreover, the number of cells in the G1 phase decreased, while the Sub G1 phase category increased, indicating that the cells entered an apoptotic pathway following 48 h of incubation [84].

Anti-proliferation effects have been recorded from peptides produced by trypsin hydrolysis of proteins from *Porphyra haitanesis* [88]. The peptides generated were tested using five human cancer cell lines tested: MCF-7 (breast cancer), HepG2 (liver cancer cells), SGC-7901 (gastric cancer), A549 (lung cancer) and HT-29 (colon cancer), using the chemotherapeutic drug fluorouracil (5-FU) as a control. Four fractions (by size kDa) were obtained from the hydrolyzed peptides, and the peptide VPGTPKNLDSPR was reported as that with the highest antiproliferative activity, even significantly more potent than 5-FU in a HepG2 cell model [88]. Mechanically, this peptide appeared to interfere with the cell cycle and promoted apoptotic cell death in HepG2 and MCF7 cells [88].

The efficacy of several algal peptides in oncological treatments and their mechanisms of action have been elucidated. Kahalalides are an assortment of depsipeptides ranging in size from 31 carbon tripeptides to 75 carbon tridecapeptides. The peptide was firstly found in the mollusk *Elysia rufescens* and it was further discovered to be present in the algae *Bryopsis pennata* consumed by the mollusk and acting as a defense mechanism against predators [89]. Amongst all the Kahalalides, the one showing the most promise in cancer treatment is the largest peptide, Kahalalide F (KF) C75 H124 N14 O16 [90,91]. KF has shown its potential benefits for cancer treatment in both in vitro and in vivo preclinical trials. Suárez, González, Cuadrado, Berciano, Lafarga and Muñoz [90] studied the mechanism of action of KF to determine its cytotoxic action against neoplastic cells. The authors used prostate (PC3, DU145, LNCaP) and breast cancer (SKBR-3, MCF7, BT474, MDA-MB-231) cell lines. The IC50 of KF for all the cell lines was around 0.3 μM, except in the case of PC3 which was 0.07 μM. Moreover, the authors also showed that the cytotoxic response of KF appears quickly, within 15 min. KF's mechanism of action differs from that of other antineoplastic drugs as it does not cause apoptosis, but generated an ATP depletion and swelling of the cells or oncosis [90,92]. KF has a similar mechanism of action to that of maitotoxin, a peptide that causes oncosis as its action linked to the function of the calcium ion channels of the cells [92]. Although maitotoxin is one of the most potent marine peptides known to date, its action is non-selective, and it is responsible for a particular human intoxication syndrome, namely ciguatera fish poisoning [93]. In this regard, KF could be a better fit for anti-cancer treatments as it has displayed tumor-selective properties in testing and has low toxicity [94].

#### **4. Application of Novel Bioinformatic Tools**

Bioinformatic tools are routinely used in peptide and protein analysis. In the context of the production of peptides via protein hydrolysis, online peptide cutters can simulate the cleavage of peptide sequences by various enzymes [95]. Toxicological and allergenic properties may also be predicted, using tools such as ToxinPred [96] and AllerTOP [97], respectively. Moreover, the BIOPEP database [98] compiles the reported activities of various peptide sequences. In silico methods being used to identify bioactivity in peptides include the use of quantitative structure–activity relationships (QSAR) and molecular docking, aiming to identify the mechanism of action underlying the biological functions of these peptides.
