Approximately 10,000 sponges have been described worldwide and most of them live in marine environments [42,43]. A range of bioactive compounds has been found in about 11 sponge genera. Three of these genera (Haliclona, Petrosia, and Discodemia) produce influential anti-cancer and anti-inflammatory agents [44]. There are a number of research studies on bioactive peptides from sponges, mostly cyclodepsipeptides, which are secondary metabolites with unusual amino acids and non-amino acid moieties. These compounds possess a wide spectrum of biological activities; however, it is difficult to isolate them in sufficient quantity for pharmacological testing [30].

Jaspamide is a cyclic depsipeptide isolated from sponges of the genus Jaspis and Hemiastrella. It possess a 15-carbon macrocyclic ring containing three amino acid residues (Figure 2a) and has proved to be a bioactive compound inducing apoptosis in HL-60 human promyelocytic leukemia cell line [7,35,45], and Jurkat T cells [46]. Nine new cyclodepsipeptides, Homophymines, B–E, and A1–E1, isolated from the sponge Homophymia sp. have shown very potent cytotoxic activity with IC₅₀ values in the nM range. This activity has been reported against several human cancer cell lines [28,34] with moderate selectivity against human prostate (PC3) and ovarian (OV3) carcinoma. Homophymines A1–E1, which possesses the 4-amino-6-carbamoyl-2,3-dihydroxyhexanoic acid residue (Figure 2f), exerts stronger potency than the corresponding A–E compounds which possess the same residue present in its carboxy form [34].

Geodiamolide H (Figure 2b) isolated from a Brazilian sponge Geodia corticostylifera have demonstrated antiproliferative activity against breast cancer cells by altering the actin cytoskeleton [33]. Discodermins tetradecapeptides are another group of cytotoxic peptides obtained from sponges of the genus Discodermia sp. containing 13–14 known and rare amino acids as a chain, with a macrocyclic ring constituted by lactonization of a threonine unit with the carboxy terminal (Figure 2e). Discodermins A–H were tested against A549 human lung cell line and P388 murine leukemia cells, all showing cytotoxicity [3].

Arenastatin A (Figure 2c) is a cyclodepsipeptide isolated from Dysidia arenaria that have demonstrated a potent cytotoxicity against KB cells with an IC₅₀ of 5 pg/mL [3]. Papuamides A–D isolated from sponges of the genus Theonella, are the first marine-derived peptides reported to contain 3-hydroxyleucine and homoproline residues and a 2,3-dihydroxy-2,6,8-trimethyldeca-(4Z,6E)-dienoic acid moiety, N-linked to a terminal glycine residue (Figure 2g). It has also been discovered that Papuamides A and B inhibited the infection of human T-lymphoblastoid cells by HIV-1 in vitro [3,47].

Phakellistatins isolated from the Western Indian Ocean sponge Phalkellia carteri inhibit leukemia cell growth [39]. Another related compound, Phakellistatin 13 (Figure 2d) from sponge Phakellia fusca was cytotoxic against the human hepatoma BEL-7404 cell line with an ED₅₀ < 10⁻² μg/mL. Synthetic specimens of Phakellistatin were found to be chemically but not biologically (cancer cell lines) identical to the natural products. The reason might be a conformational difference, especially around the proline residue [30,48].

Bioactive peptides with novel structures have also been shown in ascidians. Sack-like sea squirts inhabiting the sea floor, produce a complex anti-tumor compound which is, hundreds to thousands of times more influential than any cancer concoction now in use [10]. One of these potent compounds is Didemnin, isolated at first from the Caribbean tunicate Trididemnum solidum but later obtained from other species of the same genus [3,49]. Among these compounds, Didemnin B (Figure 2h) has the most potent antitumor activity and also has showed antiproliferative activity against human prostatic cancer cell lines [3,31]. Didemnin B inhibits the synthesis of RNA, DNA and proteins [50]. Substantial evidence of activity in preclinical models with dose-dependent and tolerable toxicity profiles led to phase I clinical trials, making this peptide the first marine natural product to be evaluated in clinical trials [51,52]. The toxicity profile of Didemnin B was quite similar across the trials, with dose-dependent nausea and vomiting as the most commonly reported side effects. Phase II trials using Didemnin B at the recommended doses were inefficient, while trials using more aggressive regimens resulted in higher levels of toxicity, including cardiotoxicity [53,54,55].

Aplidine (Figure 2i) is a cyclodepsipeptide isolated from the tunicate Aplidium albicans, which has been shown to have anticancer activity against a variety of human cancer cell lines, such as breast, melanoma and lung cancers [28,56], which appear to be sensitive to low concentrations of this compound. Aplidine’s mode of action involves several pathways, including cell cycle arrest and inhibition of protein synthesis, thus inducing apoptosis of cancer cells [57]. Furthermore, Aplidine possesses a unique and differential mechanism of cytotoxicity which involves the inhibition of ornithine descarboxylase, an enzyme that is critical in the process of tumor formation and growth [24]. Aplidine also inhibits the expression of the vascular endothelial growth factor gene, having antiangiogenic effects [25]. Aplidine, was well tolerated with minor toxicity in finished Phase I clinical trials with the most common side effects being asthenia, nausea, vomiting and transient transaminitis, but not inducing hematological toxicity, mucositis or alopecia [56,58,59]. Neuromuscular toxicity with the elevation of creatine phosphokinase levels has been dose limited, but seemed to be readily reversible with oral carnitine [56]. Aplidine has shown antitumor activity in phase I trials [56,58], and has already undergone active phase II studies in solid tumors [60,61,62].

Tamandarins A (Figure 2j) and B are also cytotoxic depsipeptides from a marine ascidian of the family Didemnidae, which was evaluated against various human cancer cell lines [30,40]. Mollamide is a cyclodepsipeptide obtained from the ascidian Didemnum molle, and it has shown cytotoxicity against a range of cell lines with IC₅₀ values of 1 μg/mL toward P388 murine leukemia line and 2.5 μg/mL against A549 human lung carcinoma and HT29 human colon carcinoma [30,38]. Trunkamide A is a cyclopeptide with a tiazoline ring similar to Mollamide (Figure 2k,m) obtained from ascidians of the genus Lissoclinum, where antitumor activity under preclinical trials has been demonstrated [30,41].

Mollusks are species that have a wide range of uses in pharmacology. Sea hare, a shelled organism, produces bioactive metabolites used in the treatment of cancerous tumors [10]. Ziconotide is a 25 amino acid peptide with three disulphide bonds (Figure 2n); and is present in the venom of the predatory Indo-Pacific marine mollusk, Conus magus. It possesses remarkable analgesic activity, which has proved to be 1000 times more active than morphine in animal models of nociceptic pain [63]. Cone snails belonging to the genus Conus are a valuable source of active peptides named conotoxins. They consist of a mixture of peptides with short chains of amino acids (8–35) rich in disulfide. Studies have postulated that these peptides could be of interest in the treatment of cancer [3,64].

Dolastatins is a family of cytotoxic peptides isolated from the mollusk Dollabella auricularia, where the linear pentapeptide Dolastatin 10 (Figure 2o) and the depsipeptide Dolastatin 15 have had the most promising antiproliferative activity reported [65,66]. Dolastatin 10 is an antineoplastic substance proven against several cancer cell lines [67] and has been evaluated in various phase I clinical trials reporting good tolerability and identifying myelosuppression as the dose limiting toxicity. Other side effects observed were peripheral sensory neuropathies, pain, swelling, and erythema at the injection site [67,68]. Complexity and low yield of chemical synthesis of dolastatins, together with low water solubility, have been significant obstacles to broader clinical evaluation, triggering the development of analog compounds [69,70].

A 60-kDa protein, Bursatellanin-P, was purified from the purple ink of the sea hare Bursatella leachii showing anti-HIV activity [71]. Keenamide A is a cytotoxic cyclic hexapeptide isolated from the mollusk Pleurobranchus forskalii, which elicits antitumor activity via unknown mechanisms. This compound exhibited significant activity against the P388, A549, MEL-20 and HT-29 tumor cell lines [37].

Kahalalides is a family of peptides isolated from the sacoglossan mollusk Elysia rufescens. Among these, Kahalalide F is a dehydroamino-butyric acid-containing peptide (Figure 2p) which is known to exhibit interesting antitumor activity [72]. Kahalalide F has shown in vitro and in vivo selectivity for prostate-derived cell lines and tumors [73,74]. It has been observed that Kahalalide F induces disturbances in lysosomal function that might lead to intracellular acidification and cell death. These results suggest that cells with high lysosomal activity, such as prostate cancer cells, would be a suitable tumor type to use to explore the activity of this peptide [72]. In phase I clinical trials, Kahalalide F exhibited clinical benefits in treated patients and low toxicity with few side effects restricted to fatigue, headache, vomiting, and pruritus limited to the hands. Since hematological toxicities have not been observed, Kahalalide F results show suitability for trials in combination with other anticancer agents [75]. There is evidence that suggests that Kahalalide F may be active against other tumor types and deserves further clinical testing either as a single agent or in combination [75]. Currently, this agent is undergoing phase II clinical trials for the treatment of lung and prostate cancers, and melanoma [76].

In recent years, there has been a considerable amount of research focused on the liberation of bioactive peptides encrypted within food proteins, and towards the use of peptides as functional food ingredients that promote health maintenance or as potential drugs for the treatment of chronic diseases. Interestingly, within the parent protein sequence, the peptides are inactive and thus must be released to exert an effect. These bioactive peptides are usually 2–20 amino acid residues in length: however, some have been reported to be longer than 20 amino acid residues [77].

Protein hydrolysis is the method used to obtain peptides from food protein sources with different biological activities, such as antioxidant, antihypertensive, antimicrobial and antiproliferative. It consists of breaking the peptide bond and subsequent generation of smaller peptides or free amino acids, if an adequate control of the hydrolysis is achieved [78]. The protein hydrolysis method most commonly used is enzymatic hydrolysis, since alkaline hydrolysis is not frequently used due to the racemization or destruction of certain amino acids at high pH [79]. On the other hand, the acid method has the disadvantage that tryptophan is completely destroyed, while serine and threonine are destroyed by 5–10% and asparagine and glutamine are hydrolyzed to their corresponding acids [80].

Enzymatic hydrolysis is carried out under controlled pH and temperature conditions that reduce the formation of undesirable products [78]. Several enzymes are used to obtain hydrolysates, among which are the digestive and microbial proteases, including alcalase, trypsin, pepsin, chymotrypsin, pancreatin, pepsin, and thermolysin, among others [81]. Moreover, studies have demonstrated that enzymatic hydrolysis most likely increases the antioxidative activity of the resulting hydrolysate via the enhancement of radical scavenging activity [82].

Fish is an important source of protein worldwide; additionally, fish proteins offer huge potential as novel sources of bioactive peptides. Hydrolysates of several marine proteins have been assayed for various bioactivities. Peptides present in protein hydrolysates have biological activities depending on their molecular weights and amino acid sequences. Crude hydrolysates are subsequently fractionated to separate individual peptides using different techniques, mainly reverse phase high performance liquid chromatography (RP-HPLC) or gel permeation chromatography [4,13,83].

Enzymatic hydrolysis of food proteins is considered an efficient way to recover potent bioactive peptides, since several peptides obtained by this process have different bioactivities and this may represent a potential approach to anticancer drugs. Up to now, bioactive peptides with potential anticancer exhibiting antioxidant and antiproliferative effects have been found in the hydrolysates of marine proteins [84,85,86,87] (Table 2).

Antioxidants are known to be beneficial to human health as they may protect the body against molecules known as reactive oxygen species (ROS). ROS can attack membrane lipids, protein, and DNA. This consecutively can be a causative factor in many diseases such as cancer. As ROS are involved in cancer development, compounds with high ROS reduction activity are likely to be able to prevent cancer incidence, since the oxidative stress inhibition leads to reduced genetic alteration such as mutation and chromosomal rearrangements which play a vital role in the initiation of carcinogenesis [13].

Antioxidant peptides have been found in numerous foodstuffs including algae protein waste [102], milk [103] and enzymatically produced protein hydrolysates [84,86,104,105]. Among these, numerous fish protein hydrolysates (from sources such as Tilapia) have demonstrated antioxidant potential. They have significant ability to scavenge ROS and reduce ferric ions [106]. Hydrolysates from mackerel muscle obtained with Protease N, contain peptides with antioxidant activity in vitro. The antioxidant activity is measured by the peptide’s capacity to scavenge the free radical α,α-diphenyl-β-picrylhydrazil (DPPH) and reduce Fe³⁺ to Fe²⁺ [107]. This antioxidant potential is similar to that reported for protein hydrolysates from other sources, such as casein enzymatic hydrolysate, which exhibits significant ability to scavenge ROS [108]. Also soy and wheat protein hydrolysates showed strong capacity to scavenge DPPH [109].

Hydrolysates of several skin gelatins such as flying squid (Ommastrephes batramii) [90], tuna (Thunnus spp.) and jumbo flying squid (Dosidicus gigas) [82,110] have been shown to possess antioxidant activity. Gelatin peptides mainly contain hydrophobic amino acids and abundance of these amino acids favors a high emulsifying ability to hydrophilic-hydrophobic partitioning in the peptide sequence [84]. In addition, specific amino acid arrangements with their abundance of Gly, Pro and Hyp, merit special consideration, as the content of Pro residues has a scavenging effect on radicals and the percentage of hydroxylation seems to be related to the antioxidant properties as measured by FRAP [82]. Hence, marine gelatin derived peptides are expected to exert high antioxidant effects among other antioxidant peptide sequences [82,84]. Therefore, marine-derived bioactive peptides with antioxidative properties may have great potential for use as nutraceuticals and pharmaceuticals and as a substitute for synthetic antioxidants.

Muscle hydrolysates of horse mackerel (Magalapsis cordyla) and croaker (Otolithes ruber) [89], Nemipterus japonicas and Exocoetus volitans [14] have been shown to have an ability to scavenge free radicals and reactive oxygen species, showing antioxidant activity. Protein hydrolysates obtained from Channel catfish (Ictalurus punctatus) protein isolates [104] and from jellyfish (Rhopilema esculentum) umbrella collagen [105] have shown antioxidant activity as determined by different methods.

Flying squid gelatin hydrolysate (enzymatically obtained) showed high antioxidant ability. At concentrations of 16 and 12 mg/mL, the hydrolysate showed a superior ability to scavenge DPPH free radicals than BHA and α-tocopherol, respectively. This hydrolysate was found to be rich in antioxidant amino acids including tyrosine, histidine, proline, alanine, and leucine. Furthermore, it appeared that hydrolysate fractions having a molecular weight ranging from 383 to 1492 Da, might be responsible for its antioxidant activity. Moreover, the size (usually lower molecular weight) and the amino acid composition were found to be strongly correlated to their antioxidant activity [90]. The mechanisms of action of peptides as antioxidants is not clearly known, but its activity has been attributed to certain amino acid sequences, that include some aromatic amino acids and histidine [111]. High amounts of histidine and some hydrophobic amino acids are associated with antioxidant potency [112]. The activity of histidine-containing peptides is thought to be connected to a hydrogen-donating ability, lipid peroxyradical trapping, and/or the metal ion chelating ability of the imidazole group [113]. The addition of a leucine or proline residue to the N-terminus of a histidine–histidine dipeptide would enhance antioxidant activity. The hydrophobicity of the peptide also appears to be an important factor for its antioxidant activity due to an increased accessibility to hydrophobic targets [114].

Didemnin depsipeptides are cytotoxic to cancer cell lines by inhibiting protein synthesis in vitro [115]. It is suggested that protein synthesis may be inhibited by the binding of Didemnins to ribosome-EF-1α complex, since there is a correlation between inhibiting protein synthesis in cell lysates and in human adenocarcinoma MCF-7 cells [116]. Studies with Jaspamide in HL-60 human leukemia cell line revealed that nanomolar concentrations of this depsipeptide induced inhibition of cell proliferation and increased polynuclear cells [35].

Cryptophycin-52, a member of the family of the marine depsipeptides Cryptophycins, produced by total chemical synthesis, showed antitumor activity at picomolar concentrations. This compound was shown to inhibit cancer cell proliferation by stabilizing spindle microtubules, binding tightly and non-covalently to a single high-affinity site on tubulin, while also inducing a conformational change in the tubulin molecule [7,117].

Peptides and amino acids from several dietary proteins have been reported to show antitumor or antiproliferative activities, most of them from vegetal sources [118,119,120,121]. However, the antiproliferative activities of marine proteins have been barely studied. Hydrolysates from three blue whiting, three cod, three plaice and one salmon were identified as significant growth inhibitors on MCF-7/6 and MDA-MB-231 cell lines. Composition analysis evidenced they contained a complex mixture of free amino acids and peptides of various sizes ranging up to 7 kDa [85]. An enzymatic protein hydrolysate of oyster inhibited the growth of transplantable sarcoma-S180 in a dose-dependent manner in BALB/c mice, showing strong immunostimulating effects [4]. The antitumor drug cyclophosphamide, which possesses a high tumor inhibitory rate, was also shown to have a strong immunosuppressive effect [122]. In contrast, oyster hydrolysates inhibited tumor growth by improving the immune function in S108-bearing mice, which suggests a potential use in tumor therapy [4]. An enzymatic hydrolysate from jumbo squid skin gelatin showed cytotoxic effect against MCF-7 and U87 cell lines, with IC₅₀ values of 0.13 and 0.10 mg/mL, respectively [91]. Solitary tunicate hydrolysate exhibited strong antioxidant activity, including DPPH, ABTS, H₂O₂, and OH radical scavenging activities. Moreover, this hydrolysate also showed potent anticancer activity against AGS, DLD-1, and HeLa cancer cells. However, the anticancer activities of these fractions (IC₅₀ 577.1–1240.0 μg/mL) were much lower than that of commercial standards such as Paclitaxel (IC₅₀ 2.2–24.6 μg/mL) and 5-Fluorouracyl (IC₅₀ 3.4–34.5 μg/mL) [13].

Peptide fraction of Nemipterus japonicas and Exocoetus volitans hydrolysates exerted significant antiproliferative effect on human hepatocellular liver carcinoma cell lines (Hep G2) with IC₅₀ values 48.5 mg/mL and 21.6 mg/mL, respectively. Moreover, these fractions did not show any cytotoxicity effect for Vero (kidney epithelial cells of the African Green Monkey) cell lines [14]. Peptides isolated from enzymatic hydrolysate of tuna dark muscle by-product show a dose-dependent inhibition effect of the MCF-7 cells with IC₅₀ values of 8.1 and 8.8 μM [83]. These results showed that tuna dark muscle by-product might be a good source to produce antiproliferative peptides which may be useful in therapy as agents with high pharmaceutical value.

Isolation and identification of the specific peptide sequences of peptides that are responsible for the antioxidative and anticancer effects also should be carried out. It may be assumed that the low molecular weight peptides have greater molecular mobility and diffusivity than the high molecular weight peptides, which appears to improve interactions with cancer cell components and enhances anticancer activity [13]. Although a study on the mechanism of action revealed that modulation of hydrophobicity of peptides plays a crucial role against cancer cells [123]. However, studies on the effects of the antiproliferative peptides on cell cycle of normal and transformed cells, on the structure of the bioactive peptides, and in vivo studies of these activities, need to be further investigated.