Marine Animal-Derived Compounds and Autophagy Modulation in Breast Cancer Cells

Home , Aaptos, Fascaplysinopsis, Haliclona caerulea, Klyxum, Paracentrotus, Verongiida

Review Marine Animal-Derived Compounds and Autophagy Modulation in Breast Cancer Cells

Claudio Luparello

Department of Biological, Chemical and Pharmaceutical Sciences and Technologies, University of Palermo, Viale delle Scienze, Ediﬁcio 16, 90128 Palermo, Italy; [email protected]

Abstract: It is known that in breast cancer biology, autophagy mainly plays a cytoprotective and anti- apoptotic role in vitro, being conceivably responsible for cell resistance to drug exposure and a higher metastatic attitude in vivo. Thus, the development of novel autophagy-targeting agents represents a valuable strategy to improve the efficacy of anticancer interventions. It is widely acknowledged that the enormous biodiversity of marine organisms represents a highly promising reserve for the isolation of bioactive primary and secondary metabolites targeting one or several specific molecular pathways and displaying active pharmacological properties against a variety of diseases. The aim of this review is to pick up selected studies that report the extraction and identification of marine animal-derived extracts or isolated compounds which exert a modulatory effect on the autophagic process in breast cancer cells and list them with respect to the taxonomical hierarchy of the producing species. Where available, the molecular and biochemical aspects associated with the molecules or extracts under discussion will be also summarized.

Keywords: marine invertebrates; breast cancer; autophagy; anticancer compounds; apoptosis; cytotoxicity; demosponges; cnidarians; mollusks; echinoderms

Citation: Luparello, C. Marine Animal-Derived Compounds and Autophagy Modulation in Breast 1. A Brief Insight into Autophagy and Breast Cancer Cells Cancer Cells. Foundations 2021, 1, The term “macroautophagy”, hereafter referred as “autophagy”, relates to a ubiquitous 3–20. https://doi.org/10.3390/ cellular function that encompasses the engulﬁng of selected cytoplasmic components by foundations1010002 double-membrane structures called autophagosomes, as well as their subsequent delivery to lysosomes and degradation by resident hydrolases. Such a process guarantees the Academic Editor: Martin Schröder removal and turnover of damaged and aged cytoplasmic organelles or protein aggregates and the recycling of breakdown products for reutilization and energy production, which Received: 2 April 2021 bring the autophagic ﬂux to completion. Thus, autophagy ensures the maintenance of Accepted: 6 May 2021 Published: 17 May 2021 cellular homeostasis and, upon upregulation, the active response to different cellular stresses such as starvation, cancer, infection, and ischemia [1,2]. The machinery and

Publisher’s Note: MDPI stays neutral signalization process of autophagy, which is an evolutionarily conserved, genetically with regard to jurisdictional claims in programmed mechanism, is regulated by the expression of more than 30 autophagy- published maps and institutional affil- related genes (ATGs) in concert with other genes with multiple functions, which also iations. include autophagy modulation. ATGs are traditionally classified in functional groups according to whether they operate in the initiation of autophagosome formation, the elongation of phagophores, maturation of autophagosomes, or fusion with late endosomes or lysosomes. Their expression undergoes upstream control by an intricate signalization network which involves AKT/mechanistic targeting of rapamycin (mTOR)-dependent and Copyright: © 2021 by the author. ++ Licensee MDPI, Basel, Switzerland. independent (e.g., mucolipin Ca channel-mediated) pathways, whose understanding This article is an open access article is still incomplete [3,4]. Research on autophagy is a field in constant acceleration due to distributed under the terms and its involvement in the whole aspect of life and medical sciences, and therefore, updated conditions of the Creative Commons guidelines for standardizing experimentation and monitoring the autophagic process in Attribution (CC BY) license (https:// diverse organisms and conditions are published on a regular basis up to this day [5]. creativecommons.org/licenses/by/ The role played by autophagy in cancer biology is complex since it exhibits a dual 4.0/). nature, being involved both in oncogenesis and in oncosuppression in different conditions,

Foundations 2021, 1, 3–20. https://doi.org/10.3390/foundations1010002 https://www.mdpi.com/journal/foundations Foundations 2021, 1 4

which take a variety of cell types, cycle phase distributions, genetic makeups, and also microenvironmental scenarios (e.g., inflammation and hypoxia) into account. Autophagy may help cancer cell survival and tumor promotion by fulfilling the elevated metabolic and energetic demands of actively proliferating and migrating cancer cells, thereby allowing them to overcome oxygen and nutrient deprivation stresses. On the other hand, autophagy may inhibit neoplastic growth, as demonstrated by the correlation between the depletion of key autophagy genes and the increased proliferation of various cancer cytotypes [6]. Concerning breast tumor biology and the autophagic flux, the literature data sug- gest that in each cancer cell subtype (e.g., luminal, HER2-enriched, and above all, the aggressive triple-negative which exhibits the highest level of basal autophagy), this process mainly plays a cytoprotective and anti-apoptotic role in vitro, being conceivably responsible for cell resistance to drug exposure and a higher metastatic attitude in vivo. It is highly indicative that expression of the LC3B autophagosome marker was found to be higher in node-positive vs. node-negative primary breast tumors and associated with an increased nuclear grade and shortened survival, and that higher expression of the autophagy-promoting factor Beclin-1 in triple-negative breast cancers (TNBCs) was corre- lated with an increase of lymph node and distant metastases in patients [7,8]. On the other hand, a number of autophagy-restraining isolated compounds and secretomes, including the histone deacetylase inhibitors and conditioned media which were examined in [9–12], have been shown to restore the inhibition of survival, the impairment of cell cycle progression, and the induction of cell death programs in breast cancer cell cultures [13–15]. Thus, with the warning to take the specific breast cancer subtype and treatment used into consideration, nevertheless, the cumulative preclinical data obtained prompt the development of novel autophagy-targeting agents as a valuable strategy to improve the efficacy of anticancer interventions.

2. The Marine Animal Species as a Source of Bioactive Molecules The marine environment, which occurs in three-quarters of the globe’s surface in seas and oceans, is the largest habitat on earth, featuring an enormous level of biodiversity with many still unknown species, and it is not yet exploited extensively in terms of bioactive natural products. Marine biotechnology and pharmacology represent key ever-evolving issues based on the utilization of marine natural resources [16,17]. In fact, animal adaptation processes for the different and sometimes hostile aquatic environments have led to the promotion and accumulation of a remarkable genetic and biochemical variability, with the consequence that the compounds produced often display prominent differences with respect to those from terrestrial species. Within this context, for example, biotic factors such as the need for interindividual signalization and defense against predators, infective agents, and UV radiations prompted sessile species to develop complex strategies via chemical communication through very disparate metabolites, which is known to encompass different taxonomic lineages and even kingdoms. The study of these unique marine species-derived metabolites, whose peculiar chemical scaffolds can also be utilized for the design of analogs with increased bioavailability and efficacy and less toxicity, offering great potential for the development of new classes of molecules aimed at various applications, encompassing health management, biomaterial engineering, and environmental remediation [18,19]. Thus, to cite just a few examples, anticancer compounds have been found among the primary and secondary metabolites of starfishes and Mediterranean ascidians [20,21], and extracts or isolated molecules obtained from marine invertebrates have been proven to exert a modulatory effect on collective cell migration, a process at the basis of different biological events such as neoplastic cell metastasization and wound repair [22]. Chemicals with histone deacetylase inhibitory properties, whose wide range of potential biomedical applications is generally acknowledged, have also been extracted and identified in preparations from marine invertebrates [23]. The contribution of marine vertebrates to the food, biomedical, and pharmaceutical sectors has been mainly provided by fish oils, containing long-chain ω−3 polyunsaturated fatty acids, vitamin E, essential aminoacids, and bioactive Foundations 2021, 1, FOR PEER REVIEW 3

applications is generally acknowledged, have also been extracted and identified in preparations from marine invertebrates [23]. The contribution of marine vertebrates to the food, biomedical, and pharmaceutical sectors has been mainly provided by fish oils, containing long-chain ω−3 polyunsaturated fatty acids, vitamin E, essential aminoacids, and Foundations 2021, 1 bioactive peptides exerting antioxidant, anti-allergic, and angiotensin-I-converting en- 5 zyme (ACE) inhibitory effects. On the other hand, the processing of by-products of fish, shrimps, crabs, and squids have also been proven to be abundant sources of chitin, lipids, and proteins, granting a number of beneficial properties such as being antimicrobial, an- ticancerpeptides, and exerting anticoagulant antioxidant,, among anti-allergic, others [24,25 and]. angiotensin-I-converting enzyme (ACE) inhibitoryIt is well effects.-known On that the invertebrates other hand, theare processingendowed with of by-products a remarkable of fish,ability shrimps, to regen- crabs, erateand tissue squids and have organs also, and been that proven the efficiency to be abundant of this process sources is of guaranteed chitin, lipids, by andan appro- proteins, priategranting regulation a number of autophagy of beneficial. In addition properties, autophagy such as beingis a survival antimicrobial, mechanism anticancer, allowing and aquaticanticoagulant, organisms among to withstand others [different24,25]. stress conditions, such as hyperthermia and exposure toIt istoxic well-known compounds that invertebrates[26,27]. By considering are endowed the with things a remarkable mentioned, ability the aim to regenerate of this tissue and organs, and that the efficiency of this process is guaranteed by an appropriate review is to pick up selected studies that report the extraction and identification of marine regulation of autophagy. In addition, autophagy is a survival mechanism allowing aquatic animal-derived mixed fractions or isolated compounds which exert a modulatory effect organisms to withstand different stress conditions, such as hyperthermia and exposure to on the autophagic process in breast cancer cells and list them with respect to the taxonom- toxic compounds [26,27]. By considering the things mentioned, the aim of this review is ical hierarchy of the producing species. Where available, the molecular and biochemical to pick up selected studies that report the extraction and identification of marine animal- aspects associated with the molecules or extracts under discussion will be also summa- derived mixed fractions or isolated compounds which exert a modulatory effect on the rized. autophagic process in breast cancer cells and list them with respect to the taxonomical hierarchy of the producing species. Where available, the molecular and biochemical aspects 3. Autophagy Modulators from Porifera associated with the molecules or extracts under discussion will be also summarized. Most of the information available in the literature on compounds targeting autoph- agy3. in Autophagy breast tumor Modulators cells come from from Porifera studies performed on demosponges (“siliceous sponges”)Most. These of the species information belong available to the phylum in the literature Porifera, on the compounds oldest me targetingtazoan group autophagy on Earth,in breast to which tumor sessile cells come organisms from studiesbelong, performed displaying on a demosponges simple structure (“siliceous and being sponges”). en- dowedThese with species specialized belong cells to the that phylum are not Porifera,organized the into oldest tissue metazoans and organs group. The on cells Earth, lin- to ingwhich the internal sessile system organisms of canals belong, and displaying chambersa (“choanocytes”) simple structure ens andure being filtering endowed activity, with whereasspecialized the rest cells of that the arebody not is organized filled with into a tissuescollagenous and organs. matrix The(“mesohyl”) cells lining harboring the internal cells,system sponging of canals fibers and, and chambers skeletal (“choanocytes”) spicules and covered ensure filteringby a skin activity, made whereasof T-shaped the rest or of flatthe pinacocytes body is filled [28]. with The adata collagenous on the autophagy matrix (“mesohyl”) modulators harboring obtained cells,from spongingthese organ- fibers, ismsand will skeletal be presented spicules and and discussed covered by in achronological skin made of order. T-shaped or flat pinacocytes [28]. The dataThe on genus the autophagy Haliclona modulators(Grant, 1841 obtained; Demospongiae, from these Haplosclerida: organisms will Chalinidae be presented) in- and cludesdiscussed demosponges in chronological widely distributed order. around the world and, in particular, populating the NorthThe Sea, genus the wHaliclonaestern Mediterranean(Grant, 1841; S Demospongiae,ea and the Atlantic Haplosclerida: Ocean. They Chalinidae)show a rela- in- tivelycludes smooth demosponges surface with widely several distributed pores and aroundcan be found the world as encrusting and, inparticular, species growing populat- overing coral the rubbles North Sea, [29]. the In western2013, Yamazaki Mediterranean et al. [30 Sea] reported and the the Atlantic cytotoxic Ocean. activity They of the show pentacyclica relatively alkaloid smooth papuamine surface with (1S,2 severalE,4E,6S,7 poresR,12S and,14R can,20R be,22 foundS,27R)- as 15,19 encrusting-diazapenta- species cyclgrowingo[18.7.0.0 over6,14.07,12 coral.022,27 rubbles]heptacosa [29].-2,4 In-diene, 2013, Figure Yamazaki 1), the et al. major [30] constituentreported the of cytotoxic these sponges,activity in of MCF the pentacyclic-7 breast cancer alkaloid cells. papuamine (1S,2E,4E,6S,7R,12S,14R,20R,22S,27R)- 15,19- diazapentacyclo[18.7.0.06,14.07,12.022,27]heptacosa-2,4-diene, Figure1), the major constituent of these sponges, in MCF-7 breast cancer cells.

Figure 1. 2D structure of papuamine. (https://pubchem.ncbi.nlm.nih.gov/compound/Papuamine, accessed on 26 March 2021).

A more detailed molecular examination of the effect exerted on MCF-7 cells by the alka-

loid at concentrations at or greater than 5 µM demonstrated the expression of LC3, a central component of the autophagy process, as revealed by confocal microscopic observations of the time-dependent increase of LC3-associated ﬂuorescence intensity from 4 h of exposure onward. In addition, the accumulation of the lipidated LC3-II form, a standard marker for autophagosomes [5], from early stages of cell incubation with papuamine was observed Foundations 2021, 1, FOR PEER REVIEW 4

Figure 1. 2D structure of papuamine. (https://pubchem.ncbi.nlm.nih.gov/compound/Papuamine, accessed on 26 March 2021).

A more detailed molecular examination of the effect exerted on MCF-7 cells by the alkaloid at concentrations at or greater than 5 μM demonstrated the expression of LC3, a central component of the autophagy process, as revealed by confocal microscopic observations of the time-dependent increase of LC3-associated fluorescence intensity from 4 h Foundations 2021, 1 6 of exposure onward. In addition, the accumulation of the lipidated LC3-II form, a standard marker for autophagosomes [5], from early stages of cell incubation with papuamine was observed through Western blot experiments by Kanno et al. [31]. According to their throughhypothesis Western, which blot was experiments also advanced by Kannoin the light et al. of [31 the]. collateral According assays to their performed, hypothesis, pa- whichpuamine was could also advanced target mitochondria in the light,of inducing the collateral their dysfunction assays performed, and membrane papuamine depolari- could targetzation mitochondria, and leading to inducing their sequestration their dysfunction into autophagosomes and membrane depolarization (“mitophagy”) and [32 leading]. Then, tothe their subsequent sequestration JNK activation into autophagosomes and release of (“mitophagy”)cytochrome C would [32]. Then,conceivably the subsequent trigger the JNKdecrease activation of cell and survival release ofand cytochrome the promotion C would of apoptotic conceivably cell trigger death. the Interestingly, decrease of cell pa- survivalpuamine and was the shown promotion to exhibit of apoptotic synergy cell when death. coadministered Interestingly, with papuamine doxorubicin was shown to MCF to - exhibit7 cells, synergy probably when on the coadministered basis of a shared with doxorubicinactivation of toJNK MCF-7 phosphorylation cells, probably [33 on], thus the basissuggesting of a shared the introduction activation of of JNK the phosphorylation alkaloid in a combined [33], thus chemotherapy suggesting the regime introduction for the oftreatment the alkaloid of breast in a combined cancer. The chemotherapy anticancer effect regime of papuamine for the treatment on non of-small breast cell cancer. lung Thecan- anticancercer cells w effectas also of reported papuamine [34]. on non-small cell lung cancer cells was also reported [34]. HaliclonaHaliclona caerulea caerulea(Hechtel, (Hechtel, 1965; 1965; Demospongiae, Demospongiae, Haplosclerida: Haplosclerida: Chalinidae) Chalinidae) is is a a blue blue spongesponge populatingpopulating thethe shallow waters, waters, dredged dredged channels channels,, and and artificial artiﬁcial lagoons lagoons of ofthe the Pa- Paciﬁccific and and W Westernestern Central Central Atlantic Atlantic oceans oceans.. It is It charac is characterizedterized by a byskin a skinstudded studded with withthick- thick-walled,walled, volcano volcano-shaped-shaped oscul osculaa protruding protruding from from its smooth its smooth surface surface (Figure (Figure 2) [352)[]. 35].

FigureFigure 2. 2. AA specimen of HH.. caerulea caerulea demosponge.demosponge. Author: Author: Chaloklum Chaloklum Diving Diving (CC (CC BY BY3.0); 3.0); http: //eol.org/data_objects/31317401http://eol.org/data_objects/31317401 (accessed (accessed on 26on March 26 March 2021). 2021).

FromFrom this this sponge, sponge, Carneiro Carneiro et et al. al. [ 36[36]] isolated isolated halilectin-3, halilectin-3, an an N-acetyl N-acetyl group-binding group-binding heterotrimericheterotrimeric lectin lectin classiﬁed classified as as a a pigment pigment protein protein since since it it interacts interacts with with the the hydrophobic hydrophobic blueblue chromophore-597.chromophore-597. Subsequently, when when Nascimento Nascimento-Neto-Neto et et al. al. [37 [37] examined] examined the the ef- effectfect of of the the molecule molecule on on MCF MCF-7-7 breast breast cancer cancer cells cells,, they they found found that that cell cell adhesion adhesion was was im- impaired,paired, and and cell cell cycle cycle arrest arrest at at the the G G1 phase,1 phase, apoptosis apoptosis,, and and autophagy autophagy were were induced. induced. In Inparticular, particular, the the latter latter represented represented the the earliest earliest effect of halilectin-3halilectin-3 treatment,treatment, whichwhich was was provenproven to to upregulate upregulate MAP1LC3B MAP1LC3B gene gene expression expression and and accumulate accumulate LC3-II LC3-II vs. vs. unlipidated unlipidated LC3-ILC3-I molecules molecules already already at at 6 h6 from h from administration. administration. Conceivably, Conceivably, autophagy autophagy was triggered was trig- bygered lectin-induced by lectin-induced downregulation downregulation of BCL2 of and BCL2 upregulation and upregulatio of TP53n of genes, TP53 the genes, latter the also lat- likelyter also being likely responsible being responsible for cell cycle for impairment.cell cycle impairment On the other. On hand,the other although hand, the although exact mechanismthe exact mechanism of apoptosis of promotion apoptosis waspromotion not clariﬁed, was not in light clarified, of the in increased light of expressionthe increased of bothexpression caspase-8 of both and -9caspase observed,-8 and it was-9 observed speculated, it was that sp theeculated impaired that adhesion-dependent the impaired adhe- anoikission-dependent and some anoikis signalization and some by potentialsignalization lectin by binding potential to thelectin surface binding receptors to the surface might inducereceptors both might the intrinsic induce both and extrinsicthe intrinsic apoptosis and extrinsic pathway. apoptosis pathway. Cliona celata (Grant, 1826; Demospongiae, Clionaida: Clionaidae) is a cosmopolitan organism showing a body composed of a large network of inhalant sieve-like openings (Figure3). It is a perforating sponge which creates holes in limestones or other calcareous surfaces, such as oysters’ shells, which they live on. Foundations 2021, 1, FOR PEER REVIEW 5

Cliona celata (Grant, 1826; Demospongiae, Clionaida: Clionaidae) is a cosmopolitan Foundations 2021, 1, FOR PEER REVIEW 5 organism showing a body composed of a large network of inhalant sieve-like openings (Figure 3). It is a perforating sponge which creates holes in limestones or other calcareous surfaces, such as oysters’ shells, which they live on. Cliona celata (Grant, 1826; Demospongiae, Clionaida: Clionaidae) is a cosmopolitan Foundations 2021, 1 organism showing a body composed of a large network of inhalant sieve-like openings7 (Figure 3). It is a perforating sponge which creates holes in limestones or other calcareous surfaces, such as oysters’ shells, which they live on.

Figure 3. A specimen of C. celata demosponge (licensed under the Creative Commons Attribution- ShareAlike 4.0 International License) [38].

From methanolic extracts of this organism, Keyzers et al. [39] isolated four amino- steroidsFigureFigure 3. 3(.A clionamineA specimen specimen of of AC. C–D.celata celata), whichdemosponge demosponge appear (licenseded(licensed to be under underable thethe to induceCreativeCreative autophagosome CommonsCommons Attribution-Attribution for-- ShareAlike 4.0 International License) [38]. mationShareAlike in MCF 4.0 International-7 breast cancer License) cells [38, as]. revealed by the accumulation of cytoplasmic green fluorescent protein (GFP)-LC3 puncta, that being clionamine A ((1R,2FromSFrom,4S,7 methanolicSmethanolic,8R,9S,12S,13 extractsextractsS,16S) of-of16 this -thisamino organism, organism,-7-[(E)-3,4 Keyzers Keyzers-dimethylpent et et al. al. [ 39[39-]1 isolated]- enyl]isolated-7- fourhydroxy four aminos- amino-- 9,13teroidssteroids-dimethyl (clionamine (clionamine-5-oxapentacycl A–D), A–D which), o[1which appeared0.8.0.0 appear2,9.0 to4,8ed be.0 able13,18to ]icosan be to able induce- 6 to-one; autophagosome induce Figure autophagosome 4A) formation, the most for- in powerfulMCF-7mation breast in autophagy MCF cancer-7 breast cells,stimulator cancer as revealed. cellsSubsequently,, as by revealed the accumulation total by synthesisthe accumulation of cytoplasmic of one of of the cytoplasmic green aminosteroid ﬂuorescent greens (proteinclionaminefluorescent (GFP)-LC3 B) ((1proteinR puncta,,2S,4 S,7S that (GFP),8R,9 beingS-LC3,12S clionamine,13S,16 punctaS,18S,A )-16 ((1that-Ramino,2S ,4S-7,7being-Shydroxy,8R ,9S,12 clionamine-9,13S,13-dimethylS,16S )-16-- A 7amino-7-[(-((1(4-Rmethylpentyl),2S,4SE,7)-3,4-dimethylpent-1-enyl]-7-hydroxy-9,13-dimethyl-5-oxapentacycloicosan-6-S,8R,9-S5,12-oxapentacyclo[10.8.0.0S,13S,16S)-16-amino-2,97.0-[(4,8E.0)-13,183,4-]icosandimethylpent-6-one; -Figure1-enyl] -47B)-hydroxy was ob-- tainedone;9,13- Figuredimethyl for the4A), first-5 the-oxapentacycl time most, starting powerfulo[1 from 0.8.0.0 autophagy the2,9 plant.04,8.0 stimulator. 13,18sapogenin]icosan-6 Subsequently,tigogenin,-one; Figure and total4 theA), synthesissyntheticthe most compoundofpowerful one of the autophagywas aminosteroids proven stimulator to strongly (clionamine. Subsequently, promote B) ((1 autophagyR,2S total,4S,7 Ssynthesis ,8inR MCF,9S,12- 7Sof ,13cells oneS,16 withofS the,18 aS aminosteroidpotency)-16-amino-7- sim- s ilarhydroxy-9,13-dimethyl-7-(4-methylpentyl)-5-oxapentacyclo[10.8.0.0(clionamine to that of clionamine B) ((1R,2S ,4AS [,740S].,8 R,9S,12S,13S,16S,18S)-16-amino-7-hydroxy2,9.04,8.0-9,1313,18-]icosan-6-dimethyl- one;7-(4- Figuremethylpentyl)4B) was obtained-5-oxapentacyclo[10.8.0.0 for the ﬁrst time,2,9 starting.04,8.013,18 from]icosan the- plant6-one; sapogenin Figure 4B) tigogenin, was ob- andtained the for synthetic the first compound time, starting was provenfrom the to plant strongly sapogenin promote tigogenin, autophagy and in MCF-7the synthetic cells withcompound a potency was similar proven to to that strongly of clionamine promote Aautophagy [40]. in MCF-7 cells with a potency similar to that of clionamine A [40].

Figure 4. 2D structures of (A) clionamine A (https://pubchem.ncbi.nlm.nih.gov/compound/Cliona mine-A#section=Structures, accessed on 26 March 2021) and (B) clionamine B (https://pubchem.nc Figurebi.nlm.nih.gov/compound/Clionamine-B#section=2D-Structure 4. 2D structures of (A) clionamine A (https://pubchem.ncbi.nlm.nih.gov/compound/Cliona-, accessed on 26 March 2021). mine-A#section=Structures, accessed on 26 March 2021) and (B) clionamine B (https://pubchem.ncbi.nlm.nih.gov/compound/ClionamineThe sponges of the genus Psammaphysilla-B#section=2D, and currently-StructurePseudoceratina, accessed on(Carter, 26 March 1885; 2021Demospongiae,). Verongiida: Pseudoceratinidae), are distributed across the Red Sea and the Indo-PaciﬁcFigure 4. 2D oceanstructures and of display (A) clionamine a smooth, A ( conulose,https://pubchem.ncbi.nlm.nih.gov/compound/Cliona- or tuberculate surface and a hard body withmineThe a-A#section=Structures dense sponges collagenous of the genus,matrix accessed Psammaphysilla containing on 26 March sparse, and 2021 currently skeletal) and ﬁbers Pseudoceratina (B) clionamine (Figure5)[ (Carter,41B ]. (https://pub- 1885; Demospongiae,chem.ncbi.nlm.nih.gov/compound/Clionamine Verongiida: Pseudoceratinidae)-B#section=2D, are distributed-Structure across, accessed the Red on 2Sea6 Marchand 2021).

The sponges of the genus Psammaphysilla, and currently Pseudoceratina (Carter, 1885; Demospongiae, Verongiida: Pseudoceratinidae), are distributed across the Red Sea and

Foundations 2021, 1, FOR PEER REVIEW 6

the Indo-Pacific ocean and display a smooth, conulose, or tuberculate surface and a hard body with a dense collagenous matrix containing sparse skeletal fibers (Figure 5) [41].

Foundations 2021, 1, FOR PEER REVIEW 6

Foundations 2021, 1 the Indo-Pacific ocean and display a smooth, conulose, or tuberculate surface and a hard 8 body with a dense collagenous matrix containing sparse skeletal fibers (Figure 5) [41].

Figure 5. A specimen of the Psammaphysilla kelleri demosponge (YPM 086046), courtesy of the Yale Peabody Museum of Natural History. Photo by D. Drew, 2017 (http://peabody.yale.edu, accessed on 26 March 2021).

PsammaplinFigure 5. A specimen A ((2E of)- the3-(3Psammaphysilla-bromo-4- kellerihydroxyphenyl)demosponge (YPM-N 086046),-[2-[2 courtesy-[[(2E) of-3 the-(3 Yale-bromo-4-hy- Figure 5. A specimen of the Psammaphysilla kelleri demosponge (YPM 086046), courtesy of the Yale Peabody Museum of Natural History. Photo by D. Drew, 2017 (http://peabody.yale.edu, accessed on droxyphenyl)Peabody -Museum2-hydrox of Naturalyiminopropanoyl]amino]ethyldisulfanyl]ethyl] History. Photo by D. Drew, 2017 (http://peabody.yale.edu, accessed-2-hydroxy- 26 March 2021). iminopropanamide;on 26 March 2021) .Figure 6), the first one of the phenolic compounds isolated from these Porifera spongesPsammaplin, is a brominated A ((2E)-3-(3-bromo-4-hydroxyphenyl)-, tyrosine-derived disulfideN-[2-[2-[[(2 dimerE)-3-(3-bromo-4- initially described as Psammaplin A ((2E)-3-(3-bromo-4-hydroxyphenyl)-N-[2-[2-[[(2E)-3-(3-bromo-4-hy- an antimicrobialhydroxyphenyl)-2-hydroxyiminopropanoyl]amino]ethyldisulfanyl]ethyl]-2-hydroxyimino- and antifungal compound and subsequently proven to act as an enzy- droxyphenyl)propanamide;-2-hydrox Figureyiminopropanoyl]amino]ethyldisulfanyl]ethyl]6), the ﬁrst one of the phenolic compounds- isolated2-hydroxy- from these matic inhibitoriminopropanamide;Porifera sponges, against isFigure atopoisomerase, brominated, 6), the first tyrosine-derived one of farnesyl the phenolic disulﬁde protein compounds dimer transferase, initially isolated described from chitinase, these as an histone deacetylasesPoriferaantimicrobial, and sponges DNA and, is a antifungal methyltransferases,brominated compound, tyrosine and-derived subsequently thereby disulfide also proven dimer demonstrating toinitially act as andescribed enzymatic anticancer as in- activ- an antimicrobial and antifungal compound and subsequently proven to act as an enzy- ity [23,42–hibitor45]. against topoisomerase, farnesyl protein transferase, chitinase, histone deacetylases, maticand inhibitor DNA methyltransferases, against topoisomerase, thereby farnesyl also demonstrating protein transferase, anticancer chitinase, activity [ 23 histone,42–45 ]. deacetylases, and DNA methyltransferases, thereby also demonstrating anticancer activity [23,42–45].

Figure 6. 2D structure of psammaplin A (https://pubchem.ncbi.nlm.nih.gov/compound/Psammaplin-A#section=2D-Structure, accessed on 26 March 2021). Figure 6. 2D structure of psammaplin A (https://pubchem.ncbi.nlm.nih.gov/compound/Psamma Figure 6. 2Dplin-A#section=2D-Structure Instructure 2015, Kim of et psammaplin al. [46] investigated, accessed A ( onhttps://pubchem.ncbi.nlm.nih.gov/com- 26the March antitumor 2021). effect of psammaplin A as an in- pound/Psammaplinhibitor of sirtuin-A#section=2D-1 (SIRT1) histone-Structure deacetylase, accessed on doxorubicin on 26 March-resistant 2021 MCF). -7/adr human breastIn 2015, cancer Kim cell ets, al.which [46] are investigated strongly susceptible the antitumor to the effect cytotoxic of psammaplin effect of the A marine as an inhibitor of sirtuin-1 (SIRT1) histone deacetylase on doxorubicin-resistant MCF-7/adr human In compound.2015, Kim Among et al. the[46 data] investigated collected, psammaplin the antitumor inhibition effect of SIRT1 of waspsammaplin proven to A as an in- promotebreast autophagy cancer cells,, as which revealed are by strongly the increased susceptible intracellular to the cytotoxic formation effect of acidic of the vacuo- marine hibitor oflar compound.organellessirtuin-1 and Among(SIRT1 the increased the) histone data expression collected, deacetylase psammaplinlevel of LC3 on, inhibitionATGdoxorubicin-3, -5, of-7, SIRT1and-resistant -12 was, as provenwell MCF as to -7/adr hu- promote autophagy, as revealed by the increased intracellular formation of acidic vacuolar man breastbeclin cancer-1, the latter cell sbeing, which a core are component strongly of susceptible the class III phosphatidylinositol to the cytotoxic- 3effect kinase of the marine organelles and the increased expression level of LC3, ATG-3, -5, -7, and -12, as well as (PI3K) complex required for autophagosome formation [47]. As a further confirmation of compound.beclin-1, Among the latter the being data a collected, core component psammaplin of the class III inhibition phosphatidylinositol-3 of SIRT1 kinase was proven to promote autophagy(PI3K) complex, as required revealed for autophagosome by the increased formation intracellular [47]. As a further formation conﬁrmation of acidic of vacuolar organellespsammaplin and the A-promoted increased autophagic expression ﬂux, cell level exposure of LC3 to the, ATG compound-3, -5, also -7, induced and - the12, as well as downregulation of the p62/SQSTM1 protein, an event linked to autophagosome degrada- beclin-1, the latter being a core component of the class III phosphatidylinositol-3 kinase (PI3K) complex required for autophagosome formation [47]. As a further confirmation of

Foundations 2021, 1, FOR PEER REVIEW 7

Foundations 2021, 1 9

psammaplin A-promoted autophagic flux, cell exposure to the compound also induced the downregulation of the p62/SQSTM1 protein, an event linked to autophagosome deg- tionradation [5]. Both [5]. Both knockdown knockdown and transfectionand transfection experiments experiment demonstrateds demonstrated that that induction induction of autophagyof autophagy was was dependent dependent upon upon the SIRT1the SIRT1 inhibition inhibition pathway. pathway. Of note, Of note, SIRT1 SIRT1 inhibition inhibi- determinedtion determined the nuclear the nuclear accumulation accumulation of acetylated of acetylated p53 and p53 the and upregulation the upregulation of its target of its genestargetDRAM, genes codeDRAM for, thecod damage-regulatede for the damage- autophagyregulated a modulatorutophagy involvedmodulator in involved autophagy in initiationautophagy events initiation [48], andeventsCDKN1A [48], and, code CDKN1A for p21/WAF1,, code for p21/WAF1 whose role, whose played role in the played onset in ofthe autophagy onset of autophagy is acknowledged is acknowledged [49]. The [ anti-breast49]. The anti cancer-breast effect cancer of effect psammaplin of psammaplin A was alsoA was demonstrated also demonstrated in a xenograft in a xenograft model, model indicating, indicating that the that compound the compound has apotential has a po- therapeutic role for clinical use. tential therapeutic role for clinical use. Xestospongia exigua (Kirkpatrick, 1900; Demospongiae, Haplosclerida, Petrosiidae) Xestospongia exigua (Kirkpatrick, 1900; Demospongiae, Haplosclerida, Petrosiidae) populates the shallow lagoons and the fringing reefs of the Indo-Paciﬁc area and displays a populates the shallow lagoons and the fringing reefs of the Indo-Pacific area and displays spreading, irregular surface endowed with branches, ﬁngers, blades, and scattered oscula a spreading, irregular surface endowed with branches, fingers, blades, and scattered os- (Figure7)[50]. cula (Figure 7) [50].

Figure 7. A specimen of the X. exigua demosponge (YPM 086991), courtesy of the Yale Peabody Figure 7. A specimen of the X. exigua demosponge (YPM 086991), courtesy of the Yale Peabody Museum of Natural History. Photo by D. Drew, 2018 (http://peabody.yale.edu, accessed on 26 Museum of Natural History. Photo by D. Drew, 2018 (http://peabody.yale.edu, accessed on 26 March 2021). March 2021).

ThisThis organism organism proved proved to to be be a a good good source source of of araguspongine/xestospongine araguspongine/xestosponginealkaloids, alkaloids, dimericdimeric 2,9- 2,9- disubstituted disubstituted 1-oxaquinolizidines 1-oxaquinolizidines that that have have been been shown shown to to possess possess a a variety variety ofof pharmacological pharmacological activities activities [ 51[51,52,52].]. In In 2015, 2015, Akl Akl et et al. al. [ 53[53]] reported reported that that araguspongine araguspongine 8,11 10,15 25,29 CC ((1 R ((1,8RR,8,10R,10S,15SR,15,22RR,22,29RS,29)-9,30-dioxa-11,25-diazapentacyclo[20.6.2.2S)-9,30-dioxa-11,25-diazapentacyclo[20.6.2.28,11.010,15.0 .025,29.0 ]dotria-]dotri- contane-1,15-diol;acontane-1,15-diol; Figure Figure8) 8 exerted) exerted an an antiproliferative antiproliferative effect effec ont on a panela panel of of breast breast cancer cancer cellcell lineslines inin culture.culture. More More detailed detailed analyses analyses aimed aimed at at identify identifyinging the the cause cause of ofthe the accumu- accu- mulationlation of vacuoles of vacuoles in BT in BT-474-474 breast breast tumor tumor cells cells revealed revealed the thedose dose-dependent-dependent ability ability of ara- of araguspongineguspongine C to C tostimulate stimulate autophagy autophagy,, as as reveal revealeded by by the the increase increase in in the the accumulation accumulation of offluorescent ﬂuorescent autophagosomes autophagosomes and and the the upregulation upregulation of ofthe the total total protein protein levels levels of LC3, of LC3, Be- Beclin-1,clin-1, and and A ATG-5,TG-5, -7, -7, and and - -16L1.16L1. Molecular Molecular studies studies on the putative mechanism ofof thethe ara-araguspongineguspongine C C promotion promotion of of autophagy autophagy demonstrated demonstrated that that the the compound compound directly directly inhibited inhib- theited expression the expression and activation and activation of c-Met of c and-Met HER2 and HER2 tyrosine tyrosine kinase kinase receptors receptors which, which, in turn, in suppressedturn, suppressed the downstream the downstream signalization signalization by the PI3K/Akt/mTORby the PI3K/Akt/mTOR pathway. pathway. Concurrently, Concur- aragusponginerently, araguspongine C determined C determined the downregulation the downregulation of the of IP3 the receptor IP3 receptor by breast by breast cancer can- cells.cer cells. Taken Taken together, together, these these effects effects of the of exposurethe exposure of araguspongine of araguspongine C were C were conceivably conceiva- responsiblebly responsible for the for activation the activation of autophagy of autophagy and subsequent and subsequent inhibition inhibition of BT-474 of cell BT- growth474 cell andgrowth proliferation. and proliferation. Interestingly, Interestingly, araguspongine araguspongine C recently C recently showed showed promising promising results inre- primarysults in andprimary secondary and secondary angiogenesis angiogenesis screening modules,screening thereby modules also, thereby representing also represent- a future anti-angiogenicing a future anti drug-angiogenic candidate drug [54 candidate]. [54].

Foundations 2021, 1, FOR PEER REVIEW 8

Foundations 2021, 1 10

FigureFigure 8. 8.2D 2D structure structure of of araguspongine araguspongine C C (https://pubchem.ncbi.nlm.nih.gov/compound/Aragus (https://pubchem.ncbi.nlm.nih.gov/compound/Ara- pongine-C#section=Structuresguspongine-C#section=Structures, accessed, accessed on 26 on March 26 March 2021). 2021). Figure 8. 2D structure of araguspongine C (https://pubchem.ncbi.nlm.nih.gov/compound/Ara- guspongineFascaplysinopsisFascaplysinopsis-C#section=Structures(Demospongiae, (Demospongiae,, accessed on Dictyoceratida: 26 March 2021). Thorectidae) Thorectidae) is is a rare a rare monotypic monotypic ge- genusnus as as it it contains contains only one species,species,F. F. reticulata reticulata(Hentschel, (Hentschel, 1912; 1912 Figure; Figure9), a9) hermaphroditic, a hermaphro- demospongediticFascaplysinopsis demosponge distributed (Demospongiae, distributed in the Westernin Dictyoceratida:the Western Central PaciﬁcCentral Thorectidae) area Pacific and isarea endowed a rare and monotypic endowed with a membra- ge- with a nusnous asmembranous it andcontains opaque onlyand variform opaqueone species, surfacevariform F. reticulata with surface large (Hentschel, with conules large and conul 1912 ridges;es Figure and [55 ridges]. 9), a hermaphro- [55]. ditic demosponge distributed in the Western Central Pacific area and endowed with a membranous and opaque variform surface with large conules and ridges [55].

Figure 9. A specimen of the F. reticulata demosponge. Author: Hooper, John Figure 9. A specimen of the F. reticulata demosponge. Author: Hooper, John (http://www.marine (http://www.marinespecies.org/aphia.php?p=image&tid=165315&pic=142739 , accessed on 26 species.org/aphia.php?p=image&tid=165315&pic=142739, accessed on 26 March 2021). Licensed FigureMarch 9. A specimen2021). Licensed of the underF. reticulata the Creative demosponge. Commons Author: Attribution Hooper,-Noncommercial John -Share Alike 4.0 (http://www.marinespecies.org/aphia.php?p=image&tid=165315&pic=142739underLicense. the Creative Commons Attribution-Noncommercial-Share Alike 4.0, accessed License. on 26 March 2021). Licensed under the Creative Commons Attribution-Noncommercial-Share Alike 4.0 Extracts of this sponge are rich in the indole alkaloid fascaplysin (3-aza-13-azoniapent- License. Extracts of this sponge are rich in the indole alkaloid fascaplysin (3-aza-13-azoni- acyclo[11.7.0.02,10.04,9.014,19]icosa-1(13),2(10),4,6,8,11,14,16,18-nonaen-20-one; Figure 10) apentacyclo[11.7.0.02,10.04,9.014,19]icosa-1(13),2(10),4,6,8,11,14,16,18-nonaen-20-one; Figure and related compounds endowed with selective cyclin-dependent kinase 4 inhibitory 10Extracts) and related of this compounds sponge are endowed rich in the with indole selective alkaloid cyclin fascaplysin-dependent (3 kinase-aza-13 4-azoni- inhibitory and DNA intercalation properties [56], which are the basis of the anti-angiogenic and apentacyclo[11.7.0.0and DNA intercalation2,10.04,9.0 14,19properties]icosa-1(13),2(10),4,6,8,11,14,16,18 [56], which are the basis of-nonaen the anti-20-angiogenic-one; Figure and an- anticancer properties of both the parental molecule and its derivatives [57–59]. In 2017, 10) andticancer related properties compounds of bothendowed the parental with selective molecule cyclin and-dependent its derivatives kinase [57 4 inhibitory–59]. In 2017 , Sharma et al. [60] studied the molecular mechanism of the anti-breast tumor effect ex- and DNASharma intercalation et al. [60] studied properties the molecular [56], which mechanism are the bas ofis theof the anti anti-breast-angiogenic tumor effect and an-exerted erted by the analog 4-chloro fascaplysin on MDA-MB231 cells and, besides other results ticancer properties of both the parental molecule and its derivatives [57–59]. In 2017, indicatingby the analog the onset 4-chloro of mitochondrial fascaplysin on dysfunction MDA-MB231 and cells apoptosis, and, besides found other that the results alkaloid indi- Sharma et al. [60] studied the molecular mechanism of the anti-breast tumor effect exerted determinedcating the onset the increase of mitochondrial in the number dysfunction of ﬂuorescent and apoptosis acidic vacuolar, found that organelles the alkaloid and the de- by the analog 4-chloro fascaplysin on MDA-MB231 cells and, besides other results indi- upregulationtermined the ofincrease LC3-II, in as the revealed number by of immunoblotting. fluorescent acidic Their vacuolar cumulative organelles data and depicted the up- cating the onset of mitochondrial dysfunction and apoptosis, found that the alkaloid de- theregulation 4-chloro of fascaplysin-triggered LC3-II, as revealed growthby immunoblotting. inhibition of MDA-MB231Their cumulative cells data as being depicted a result the termined the increase in the number of fluorescent acidic vacuolar organelles and the up- of4- thechloro simultaneous fascaplysin induction-triggered ofgrowth autophagy inhibition and of apoptosis, MDA-MB231 the latter cells stimulatedas being a result by the of regulation of LC3-II, as revealed by immunoblotting. Their cumulative data depicted the lossthe ofsimultaneous mitochondrial induction transmembrane of autophagy potential. and Bothapoptosis, events the converged latter stimulated in the activation by the loss of 4-chloro fascaplysin-triggered growth inhibition of MDA-MB231 cells as being a result of caspase-3,of mitochondrial and in this transmembrane case, the Akt/mTOR potential. pathway Both events was also converged severely in abrogated, the activation which of the simultaneous induction of autophagy and apoptosis, the latter stimulated by the loss contributedcaspase-3, and to autophagy in this case promotion., the Akt/mTOR Interestingly, pathway the wasin vitro alsodata severely were abrogated corroborated, which by of mitochondrial transmembrane potential. Both events converged in the activation of incontributed vivo evidence to autophagy of solid tumor promotion. growth Interestingly, inhibition in murinethe in vitro models, data therebywere corroborated representing by caspase-3, and in this case, the Akt/mTOR pathway was also severely abrogated, which a preclinical rationale for the development of anti-tumoral therapeutic agents, also in the contributedlight of the to autophagy very good ADMETpromotion. proﬁle Interestingly, shown by the 4-chloro in vitro fascaplysin. data were corroborated by

Foundations 2021, 1, FOR PEER REVIEW 9

in vivo evidence of solid tumor growth inhibition in murine models, thereby representing a preclinical rationale for the development of anti-tumoral therapeutic agents, also in the in vivolight evidence of the very of solid good tumor ADMET growth profile inhibition shown in by murine 4-chloro models fascaplysin, thereby. representing a preclinical rationale for the development of anti-tumoral therapeutic agents, also in the light of the very good ADMET profile shown by 4-chloro fascaplysin. Foundations 2021, 1 11

Figure 10. 2D structure of fascaplysin (https://pubchem.ncbi.nlm.nih.gov/compound/Fascaplysin# Figure 10. 2D structure of fascaplysin (https://pubchem.ncbi.nlm.nih.gov/com- section=2D-Structure, accessed on 26 March 2021). pound/Fascaplysin#section=2D-Structure , accessed on 26 March 2021). Figure 10The. 2D speciesstructure of ofthe fascaplysin genus Aaptos (https://pubchem.ncbi.nlm.nih.gov/com-(Gray, 1867; Demospongiae, Suberitida: Suberitidae) The species of the genus Aaptos (Gray, 1867; Demospongiae, Suberitida: Suberitidae) pound/Fascaplysin#section=2Dare tropical and subtropical-Structure organisms, accessed mainly on 26 distributed March 2021 in). the Western Central Atlantic are tropical and subtropical organisms mainly distributed in the Western Central Atlantic and Mediterranean areas. They show a spherical or lobate shape and a characteristic radial and Mediterranean areas. They show a spherical or lobate shape and a characteristic radial skeletonThe species that containsof the genus “strongyloxeas”, Aaptos (Gray, rhabds 1867; Demospongiae, which are rounded Suberitida: at one end Suberitidae) and pointed skeleton that contains “strongyloxeas”, rhabds which are rounded at one end and pointed are attropical the other and (Figuresubtropical 11). organisms mainly distributed in the Western Central Atlantic and Mediterraneanat the other (Figure areas 1. 1They). show a spherical or lobate shape and a characteristic radial skeleton that contains “strongyloxeas”, rhabds which are rounded at one end and pointed at the other (Figure 11).

FigureFigure 11. 11.A A specimen specimen of ofthe theAaptos Aaptos pernucleatapernucleata demosponge.demosponge. Photographer: C. C. G. G. Messing, Messing, Diaz, Diaz, M.C., Kohler, K.E., Reed, J.K., Ruetzler, K., Van Soest, R.W.M., Wulff, J, and Zea, S. 2010. South M.C., Kohler, K.E., Reed, J.K., Ruetzler, K., Van Soest, R.W.M., Wulff, J, and Zea, S. 2010. South Florida. Florida. Publisher: Freeman, Chris (http://porifera.myspecies.info/taxonomy/term/22534, accessed FigurPublisher:e 11. A specimen Freeman, of Chris the Aaptos (http://porifera.myspecies.info/taxonomy/term/22534 pernucleata demosponge. Photographer: C. G. Messing,, accessed Diaz, on 26 on 26 March 2021 (CC BY-NC 3.0)). M.C.,March Kohler, 2021 K.E., (CC Reed, BY-NC J.K., 3.0)). Ruetzler, K., Van Soest, R.W.M., Wulff, J, and Zea, S. 2010. South Florida. Publisher: Freeman, Chris (http://porifera.myspecies.info/taxonomy/term/22534, accessed Bioactivity-guided fractionation of the methanolic extracts obtained from these or- on 26 MarchBioactivity-guided 2021 (CC BY-NC fractionation 3.0)). of the methanolic extracts obtained from these organisms ganisms resulted in the isolation of aaptamine alkaloids, among which isoaaptamine (11- resulted in the isolation of aaptamine alkaloids, among which isoaaptamine (11-methoxy-2- methoxy-2-methyl-2,6-diazatricyclo[7.3.1.05,13]trideca-1(13),3,5,7,9,11-hexaen-12-ol; Fig- methyl-2,6-diazatricyclo[7.3.1.0Bioactivity-guided fractionation5,13 ]trideca-1(13),3,5,7,9,11-hexaen-12-ol;of the methanolic extracts obtained Figurefrom these 12) was or- the ure 12) was the most prominent in the active fraction and showed the most potent cyto- ganismsmost resulted prominent in the in theisolation active of fraction aaptamine and showedalkaloids, the among most which potent isoaaptamine cytotoxic activity (11- on methoxyT47-Dtoxic- 2activity breast-methyl cancer -on2,6 T47-diazatricyclo[7.3.1.0 cells,-D breast as revealed cancer by5,13cells,]trideca a short-term as revealed-1(13),3,5,7,9,11 MTT by assaya short-hexaen and-term long-term-12 MTT-ol; assayFig- colony and ure formation1long2) was-term the assay. colony most The prominent formation anti-proliferative assayin the. activeThe activity anti fraction-proliferative of the and alkaloid showed activity on the different of most the alkaloid tumorpotent cell cyto-on differ- lines, toxicincludingent activity tumor on MDA-MB cell T47 lines,-D breast 231,including had cancer alreadyMDA cells,-M been Bas 231, revealed reported had already by by a Dyshlovoyshort been- termreported MTT et al. by assay [ 61Dyshlovoy]. Furtherand et longand-al.term [ more61 colony]. Further detailed formation and studies more assay on detailed the. The T47-D anti studies- cellproliferative model on the delineated T47 activity-D cell of that themodel the alkaloid inhibitorydelineated on differ- effect that ofthe ent isoaaptaminetumor cell lines, was including based upon MDA the-M inductionB 231, had of already both apoptosis been reported and autophagy, by Dyshlovoy the latteret al. [revealed61]. Further by theand dose- more and detailed time-dependent studies on increasethe T47- ofD acridinecell model orange-positive delineated that vacuoles, the upregulation of LC3-II and p62/SQSTM1, and downregulation of mTOR. In addition, a signiﬁcant increase in the generation of reactive oxygen species (ROS), as well as the disruption of the mitochondrial transmembrane potential, was also demonstrated. Thus,

the cumulative data allowed for the conclusion that isoaaptamine-promoted T-47D cell apoptosis and autophagy was accomplished via cellular Nrf1/Keap1 antioxidant depletion and p62-regulated ROS accumulation [62]. Foundations 2021, 1, FOR PEER REVIEW 10

inhibitory effect of isoaaptamine was based upon the induction of both apoptosis and autophagy, the latter revealed by the dose- and time-dependent increase of acridine orange- positive vacuoles, upregulation of LC3-II and p62/SQSTM1, and downregulation of mTOR. In addition, a significant increase in the generation of reactive oxygen species (ROS), as well as the disruption of the mitochondrial transmembrane potential, was also demonstrated. Thus, the cumulative data allowed for the conclusion that isoaaptamine- Foundations 2021, 1 promoted T-47D cell apoptosis and autophagy was accomplished via cellular Nrf1/Keap1 12 antioxidant depletion and p62-regulated ROS accumulation [62].

Figure 12. 2D structure of isoaaptamine (https://pubchem.ncbi.nlm.nih.gov/compound/Isoaapta mine#section=2D-Structure, accessed on 26 March 2021). Figure 12. 2D structure of isoaaptamine (https://pubchem.ncbi.nlm.nih.gov/compound/Isoaapta- mine#section=2D4. Autophagy-Structure Modulators, accessed from on 26 CnidariaMarch 2021). Cnidaria, also called coelenterates, are animals that share the radial symmetry of the 4. Autophagy Modulators from Cnidaria body, the lack of cephalization, and the organization of cells in tissues, although they lack Cnidaria,organs. In also particular, called coelenterates, cnidarians possess are animals two cell that layers, share thethe ectodermradial symmetry and the of endoderm, the body, separatedthe lack of by cephalization the mesoglea,, and an the elastic organization jelly-like substance of cells in in tissues which, although a network they of supporting lack organs.fibers In particular, is present. cnidaria Membersns possess of the two group, cell layers, the polyps, the ectoderm secrete externaland the endoderm, skeletons made separatedof chitin by the or mesoglea, calcium carbonate, an elastic jelly whereas-like substance the alcyoniarians in which area network endowed of supporting with an internal fibers skeleton.is present. Only Members one member of the ofgroup this phylum, the polyps, has been secrete reported external toproduce skeletons compounds made of able chitin toor modulatecalcium carbonate, autophagy whereas in breast the tumor alcyon cells.iarians are endowed with an internal skeleton. Only Klyxumone member flaccidum of this, originally phylum namedhas beenAlcyonium reportedflaccidum to produce(Tixier-Durivault, compounds able 1966; to An- modulatethozoa, autophagy Alcyonacea: in breast Alcyoniidae), tumor cells. a coral populating the Western Central Pacific area and Klyxumthe coasts flaccidum of Madagascar,, originally forms named lobate Alcyonium and quite flaccidum small colonies.(Tixier-Durivault, The non-retractile 1966; An- polyps thozoa,are Alcyonacea: clustered onAlcyoniidae) the lobes,, anda coral the populating sclerite-containing the Western mesoglea Central (thePacific “coenenchyme”) area and the coastsform of cone-shaped Madagascar, prominences forms lobate and (Figure quite 13 s)[mall63]. colonies. In 2018, The Weng non- etretractile al. [64] polyps submitted a are clusteredsmall library on the oflobes purified, and the marine sclerite natural-containing molecules mesoglea to a (the screening “coenenchyme”) aimed at identifying form cone-shapedpotential prominences activators of (Figure peroxisome 13) [63 proliferator-activated]. In 2018, Weng et al. receptor [64] submittedγ(PPARγ a), small a therapeutic library targetof purified for anticancer marine natural therapy molecules [65] in MCF-7 to a screening breast tumor aimed cells, at identify and identifieding potential the sterol activators3β,11-dihydroxy-9,11-secogorgost-5-en-9-one of peroxisome proliferator-activated receptor obtained γ(PPAR fromγK.), flacciduma therapeutic[66] astarget a receptor for anticanceractivator therapy through [65 a luciferase] in MCF-7 reporter breast tumor assay. cells, The sterol and identified was proven the to sterol exert 3 aβ,11 cytotoxic- dihydroxyeffect-9,11 on the-secogorgost neoplastic-5 cells-en-9 via-one the obtained induction from of K. caspase flaccidum 3-dependent [66] as a receptor apoptosis acti- and au- vator throughtophagy. a In luciferase particular, reporter it determined assay. The the sterol ROS-mediated was proven DNA to exert damage a cytotoxic and the effect decrease of cyclin D1, cyclin-dependent kinase 6, Bcl-2, phospho-ERK, and phospho-p38, which are Foundations 2021, 1, FOR PEERon theREVIEW neoplastic cells via the induction of caspase 3-dependent apoptosis and autophagy.11 In particular,responsible it determined for the impairment the ROS- ofmediated cell cycle DNA progression damage andand onset the decrease of apoptosis. of cyclin D1, cyclin-dependent kinase 6, Bcl-2, phospho-ERK, and phospho-p38, which are responsible for the impairment of cell cycle progression and onset of apoptosis.

Figure 13. A specimen of K. flaccidum coral, taken from the Dongsha Bafang Coral Ecological Illus- Figure 13. A specimen of K. ﬂaccidum coral, taken from the Dongsha Bafang Coral Ecological Illustrated trated Book, 2017. Authors: Dai Changfeng and Qin Qixiang. Marine National Park Management BookOffice, 2017. (CC Authors:BY-NC). Dai Changfeng and Qin Qixiang. Marine National Park Management Ofﬁce (CC BY-NC). The promotion of autophagy was revealed by the dose-dependent accumulation of acidic vesicular organelles in the cytoplasm and the upregulation of LC3-II and p62, the latter also being confirmed in sterol-treated MDA-MB231 tumor cells. The cumulative data obtained suggested the translational potential of the compound in breast cancer therapy.

5. Autophagy Modulators from Mollusca Mollusca are the largest marine phylum and are characterized by a bilateral simmetry, the absence of segmentation, and the appearance of organs, such as the characteristic “radula” used for feeding, and a nervous system. Their dorsal body wall (“mantle”) covers the visceral mass and, in many species, is responsible for the formation of a shell made of calcium carbonate and conchiolin. In addition, in the case of this phylum, only one member has been reported to produce compounds able to modulate autophagy in breast tumor cells. Crassostrea virginica (Gmelin, 1791; Bivalvia, Ostreida: Ostreidae; Figure 14), known as the eastern oyster, is a bivalve mollusk endowed with a calcite shell that can be found naturally in a great diversity of habitats along the coasts of the western Atlantic Ocean from Canada to Argentina. It is known that oysters are rich sources of the sphingolipid ceramide N-methylaminoethylphosphonate (CMAEPn; Figure 15), which is concentrated in their adductor, mantle, gills, and viscera [67,68]. On the other hand, ceramide and other sphingolipid mediators are involved in the mechanism of signalization that regulates cancer progression, and ceramide has been proven to play a key role in cancer chemotherapy metabolism and efficacy, thus emerging as a potential strategy for restraining carcinogen- esis [69]. Chintalapati et al. [70] studied the biological bases of the cell death effect exerted by CMAEPn isolated from C. virginica on hormone-dependent MCF-7 and independent MDA-MB435 breast cancer cells.

Foundations 2021, 1 13

The promotion of autophagy was revealed by the dose-dependent accumulation of acidic vesicular organelles in the cytoplasm and the upregulation of LC3-II and p62, the latter also being conﬁrmed in sterol-treated MDA-MB231 tumor cells. The cumulative data obtained suggested the translational potential of the compound in breast cancer therapy.

5. Autophagy Modulators from Mollusca Mollusca are the largest marine phylum and are characterized by a bilateral simmetry, the absence of segmentation, and the appearance of organs, such as the characteristic “radula” used for feeding, and a nervous system. Their dorsal body wall (“mantle”) covers the visceral mass and, in many species, is responsible for the formation of a shell made of calcium carbonate and conchiolin. In addition, in the case of this phylum, only one member has been reported to produce compounds able to modulate autophagy in breast tumor cells. Crassostrea virginica (Gmelin, 1791; Bivalvia, Ostreida: Ostreidae; Figure 14), known as the eastern oyster, is a bivalve mollusk endowed with a calcite shell that can be found naturally in a great diversity of habitats along the coasts of the western Atlantic Ocean from Canada to Argentina. It is known that oysters are rich sources of the sphingolipid ceramide N-methylaminoethylphosphonate (CMAEPn; Figure 15), which is concentrated in their adductor, mantle, gills, and viscera [67,68]. On the other hand, ceramide and other sphingolipid mediators are involved in the mechanism of signalization that regulates cancer progression, and ceramide has been proven to play a key role in cancer chemotherapy metabolism and efﬁcacy, thus emerging as a potential strategy for restraining carcinogene- sis [69]. Chintalapati et al. [70] studied the biological bases of the cell death effect exerted Foundations 2021, 1, FOR PEER REVIEW 12 by CMAEPn isolated from C. virginica on hormone-dependent MCF-7 and independent MDA-MB435 breast cancer cells.

FigureFigure 14. 14A. A specimen specimen of the of C.the virginica C. virginicaoyster. oyster.© johnbotany © johnbotany (https://www.inaturalist.org/photos (https://www.inaturalist.org/photos/ /117869887117869887, accessed, accessed on on 26 26 March March 2021 2021 (CC (CC BY-NC)). BY-NC)).

Figure 15. 2D structure of ceramide N-methylaminoethylphosphonate (https://pubchem.ncbi.nlm.nih.gov/compound/6444126, accessed on 26 March 2021).

Their results demonstrated that oyster ceramide down-regulated VEGF, EGF, and PI3K, which control some of the main signalization pathways associated with breast tumor progression, and inhibited the phosphorylation and degradation of Iκb, which blocks NfκB nuclear translocation by masking its nuclear localization sequence [71]. In line with the published data [72–74], these molecular events appeared to stimulate autophagy in exposed cells, as revealed by the accumulation of fluorescent autophagosomes and the increase of the beclin-1 protein level. It is noteworthy that CMAEPn was also able to inhibit angiogenesis in vitro and in vivo, thereby resulting in it being a promising tool rele- vant to counteracting breast tumorigenesis.

6. Autophagy Modulators from Echinodermata Echinodermata are invertebrates characterized by the presence of a hard, spiny cov- ering or skin, a mesodermal skeleton endowed with calcareous plates or ossicle, and, apart from a few exceptions, a pentaradial symmetry. This phylum represents the second-largest grouping of deuterostomes after the chordate, in light of the roughly 7000 extant species contained. Two species of echinoderms have been reported to produce compounds able to modulate autophagy in breast tumor cells. Arbacia lixula (Linnaeus, 1758; Echinoidea, Diadematoida: Arbaciidae, Figure 16) is a medium-sized deep black sea urchin populating the shallow waters of the Mediterranean area and the coasts of the Azores, Madeira, and Canary Islands.

Foundations 2021, 1, FOR PEER REVIEW 12

Foundations 2021, 1 Figure 14. A specimen of the C. virginica oyster. © johnbotany (https://www.inaturalist.org/photos/14 117869887, accessed on 26 March 2021 (CC BY-NC)).

Figure 15. 2D structure of ceramide N-methylaminoethylphosphonate (https://pubchem.ncbi.nlm.nih.gov/compound/64 Figure 15. 2D structure of ceramide N-methylaminoethylphosphonate (https://pubchem.ncbi.nlm.nih.gov/com- 44126, accessed on 26 March 2021). pound/6444126, accessed on 26 March 2021). Their results demonstrated that oyster ceramide down-regulated VEGF, EGF, and Their results demonstrated that oyster ceramide down-regulated VEGF, EGF, and PI3K, which control some of the main signalization pathways associated with breast tumor PI3K, which control some of the main signalization pathways associated with breast tu- progression, and inhibited the phosphorylation and degradation of Iκb, which blocks mor progression, and inhibited the phosphorylation and degradation of Iκb, which blocks NfκB nuclear translocation by masking its nuclear localization sequence [71]. In line with NfκB nuclear translocation by masking its nuclear localization sequence [71]. In line with the published data [72–74], these molecular events appeared to stimulate autophagy in theexposed published cells, data as revealed [72–74], by these the molecular accumulation events of ﬂuorescentappeared to autophagosomes stimulate autophagy and the in exposedincrease ofcells, the as beclin-1 revealed protein by the level. accumulation It is noteworthy of fluorescent that CMAEPn autophagosomes was also able toand inhibit the increaseangiogenesis of thein beclin vitro -and1 proteinin vivo level., thereby It is resultingnoteworthy in itthat being CMAEPn a promising was also tool relevantable to in- to hibitcounteracting angiogenesis breast in tumorigenesis.vitro and in vivo, thereby resulting in it being a promising tool rele- vant to counteracting breast tumorigenesis. 6. Autophagy Modulators from Echinodermata 6. AutophagyEchinodermata Modulators are invertebrates from Echinodermata characterized by the presence of a hard, spiny cover- ing orEchinodermata skin, a mesodermal are invertebrates skeleton endowed characterized with calcareous by the presence plates of or a ossicle, hard, spiny and, apartcov- eringfrom or a few skin exceptions,, a mesodermal a pentaradial skeleton symmetry.endowed with This calcareous phylum represents plates or ossicle, the second-largest and, apart fromgrouping a few of exceptions, deuterostomes a pentaradial after the symmetry. chordate, in This light phylum of the roughlyrepresent 7000s the extant second species-larg- estcontained. grouping Two of deuterostomes species of echinoderms after the havechordate, been reportedin light of to the produce roughly compounds 7000 extant able spe- to ciesmodulate contained. autophagy Two species in breast of tumorechinoderms cells. have been reported to produce compounds able toArbacia modulate lixula autophagy(Linnaeus, in 1758; breast Echinoidea, tumor cells. Diadematoida: Arbaciidae, Figure 16) is a Foundations 2021, 1, FOR PEER REVIEWmedium-sized Arbacia lixula deep (Linnaeus, black sea 1758; urchin Echinoidea, populating Diadematoida: the shallow waters Arbaciidae of the, MediterraneanFigure 16) is a13

mediumarea and-sized the coasts deep ofblack the Azores,sea urchin Madeira, populating and Canarythe shallow Islands. waters of the Mediterranean area and the coasts of the Azores, Madeira, and Canary Islands.

Figure 16. A specimen of the A. lixula sea urchin. Taken from [75]. Figure 16. A specimen of the A. lixula sea urchin. Taken from [75].

HolothuriaHolothuria tubulosa tubulosa(Gmelin, (Gmelin, 1788; 1788; Holothuroidea, Holothuroidea, Aspidochirotida: Aspidochirotida: Holothuridae, Holothuridae, FigureFigure 17 1)7 is) is a a roughly roughly cylindrical cylindrical brownish brownish sea sea cucumber cucumber abundant abundant in in the the sandy sandy or or rocky rocky substratessubstrates of of the the eastern eastern Atlantic Atlantic Ocean Ocean and and Mediterranean Mediterranean Sea. Sea. This This benthic benthic echinoderm, echinoderm, whichwhich displays displays a tougha tough tegument tegument covered covered with with many many dark-colored dark-colored papillae, papillae, represents represents an excellentan excellent animal animal system system for the for study the ofstudy the morphometricof the morphometric and humoral and humoral responses responses during tissueduring repair tissue [76 repair,77]. [76,77]. Both organisms have been widely used as models to examine the effects of anthro- pogenic chemical and noise pollution [78–80].

Figure 17. A specimen of the H. tubulosa sea cucumber. Taken from [81].

On the basis of the acknowledged diverse biological and pharmacological properties shown by the compounds and extracts from sea urchins and holothurians [82–85], in search of novel potential anti-breast cancer preparations, crude and <10 kDa-filtered fractions of cell-free aqueous extracts of the coelomic fluids from A. lixula and H. tubulosa were administered to MDA-MB231 TNBC cells, and the biological aspects of the observed cytotoxic effects were investigated [75,81]. Concerning A. lixula’s preparations, the sole filtered extract was proven to promote autophagic cell activity, as revealed by the accumulation of acidic vesicular organelles and the increase of the beclin-1, LC3-II, and total LC3 protein levels. Of note, autophagy upregulation occurred in parallel with ROS overproduction and the blocking of the cell cycle at the S-phase with no evidence of dissipation of the mitochondrial transmembrane potential and the onset of apoptosis. The obtained results allowed for the speculation that (1) the increase of the endogenous ROS levels could be a consequence of cellular metabolic stress and impairment of the antioxidant system in the absence of mitochondrial potential dysfunction; (2) autophagy upregulation could be a result of intracellular signalization switched on by ROS, acting as redox signaling molecules; and (3) autophagy might be responsible for the blocking of the cell cycle at the S-phase, analogous with the data pro-

Foundations 2021, 1, FOR PEER REVIEW 13

Figure 16. A specimen of the A. lixula sea urchin. Taken from [75].

Holothuria tubulosa (Gmelin, 1788; Holothuroidea, Aspidochirotida: Holothuridae, Figure 17) is a roughly cylindrical brownish sea cucumber abundant in the sandy or rocky Foundations 2021, 1 substrates of the eastern Atlantic Ocean and Mediterranean Sea. This benthic echinoderm15 , which displays a tough tegument covered with many dark-colored papillae, represents an excellent animal system for the study of the morphometric and humoral responses during tissue repair [76,77]. BothBoth organisms organisms have have been been widely widely used used as as models models to to examine examine the the effects effects of of anthro- anthro- pogenicpogenic chemical chemical and and noise noise pollution pollution [78 [78–80–80].].

Figure 17. A specimen of the H. tubulosa sea cucumber. Taken from [81]. Figure 17. A specimen of the H. tubulosa sea cucumber. Taken from [81].

OnOn the the basis basis of of the the acknowledged acknowledged diverse diverse biological biological and and pharmacological pharmacological properties properties shownshown by by the the compounds compounds and and extracts extracts from from sea urchins sea urchins and holothurians and holothurians [82–85], [82 in search–85], in ofsearch novel of potential novel potential anti-breast anti- cancerbreast preparations,cancer preparations crude, andcrude <10 and kDa-filtered <10 kDa-filtered fractions frac- oftions cell-free of cell aqueous-free aqueous extracts extracts of the of coelomic the coelomic fluids fluid froms fromA. lixulaA. lixulaand andH. H. tubulosa tubulosawere were administeredadministered to to MDA-MB231MDA-MB231 TNBCTNBC cells cells,, and and the the biological biological aspects aspects of of the the observed observed cy- cytotoxictotoxic effect effectss were were investigated investigated [75 [75,81,81].]. ConcerningConcerningA. A. lixula lixula’s’s preparations, preparations, the the sole sole filtered filtered extract extract was was proven proven to to promote promote autophagicautophagic cell cell activity,activity, asas revealed by by the the accumulation accumulation of of acidic acidic vesicular vesicular organelles organelles and andthe theincrease increase of the ofthe beclin beclin-1,-1, LC3 LC3-II,-II, and and total total LC3 LC3 protein protein levels. levels. Of Ofnote, note, autophagy autophagy up- upregulationregulation occurred occurred in inparallel parallel with with ROS ROS overproduction overproduction and and the theblock blockinging of the of cell the cycle cell cycleat the at S the-phase S-phase with with no evidence no evidence of dissipation of dissipation of the ofthe mitochondrial mitochondrial transmembrane transmembrane po- potentialtential and and the the onset onset of of apoptosis. apoptosis. T Thehe obtained resultsresults allowedallowed forfor the the speculation speculation that that (1)(1 the) the increase increase of of the the endogenous endogenous ROS ROS levels levels could could be be a a consequence consequence of of cellular cellular metabolic metabolic stressstress and and impairment impairment of of the the antioxidant antioxidant system system in in the the absence absence of of mitochondrial mitochondrial potential potential dysfunction;dysfunction; (2) (2) autophagy autophagy upregulation upregulation could could be be a a result result of of intracellular intracellular signalization signalization switchedswitched on on by by ROS, ROS, acting acting as as redox redox signaling signaling molecules; molecules and; and (3) (3 autophagy) autophagy might might be be responsibleresponsible for for thethe blockingblocking of the the cell cell cycle cycle at at the the S- S-phase,phase, analog analogousous with with the thedata data pro- produced in [86]. As an alternative, the upregulation of the autophagic activity was interpreted as a cell repair mechanism aimed at counteracting ROS-induced necrotic death but ineffective at ensuring cell survival. Concerning H. tubulosa’s extracts, by evaluating the same endpoints as for A. lixula’s preparations, both the crude and filtered samples were proven to promote autophagy, although the latter ones appeared more powerful in inducing an early stimulation at 24 h from exposure. At this time point, autophagy upregulation appeared to parallel mitochondrial transmembrane potential dissipation, whereas ROS overproduction was undetectable and remained unchanged thereafter. Such evidence suggested that the autophagic program activated by the extracts was of the “mitophagic” type given the absence of apoptotic death, likely due to the elimination of damaged mitochondria that reduced the intracytoplas- mic release of cytochrome C, a known inductor of intrinsic apoptosis [87]. On the other hand, the autophagic clearance of the damaged mitochondria might be responsible for the inability to detect increased ROS accumulation [88]. The above-discussed results represent a good starting point for the identification of the water-soluble bioactive component(s) present in the extracts, with the aim to develop novel prevention or treatment agents efficacious against highly metastatic breast carcinomas.

7. Conclusions In 2020, the World Health Organization reported the occurrence of 2.3 million women diagnosed with breast tumors and 685,000 deaths globally, thereby conﬁrming that this Foundations 2021, 1 16

neoplastic histotype has surpassed lung cancer as the most commonly diagnosed one and represents the world’s most prevalent neoplasia [89,90]. Since the currently available treat- ments are not fully working with most breast cancer patients, the procurement of alternative prevention or therapeutic agents appears to be imperative, including nanomaterials [91,92] to improve both medication and the quality of life of patients. It is widely acknowledged that the enormous biodiversity of marine organisms represents a highly promising reserve for the isolation of bioactive primary and secondary metabolites targeting one or several specific molecular pathways and displaying active pharmacological properties against a variety of diseases. Among the anticancer compounds searched in extracts from marine organisms, in this review, a focus was put on modulators that control and modify autophagy in breast cancer cells. Table1 summarizes, in a synoptic way, the invertebrate species of origin, the substances, and the autophagy modulatory effects discussed in the text. Attention on the autophagic process has increased over time due to its critical role in dictating life or death cellular decisions, leading to a wide range of biomedical applications (e.g., [93,94]). In addition, the role of marine natural products in the induction and inhibition of autophagy and its outcome in tumorigenesis have been widely acknowledged [95]. Marine invertebrates have contributed to this field, with a number of compounds displaying autophagy-regulating properties toward breast tumor cell models, and demosponges have been the most investigated ones to date. Nevertheless, despite the extent of the marine environment’s biomedical chest, current research on this topic is quite limited, and further study efforts are needed to expand the list of tested bioactives discovered across the different taxonomic groups, as well as in vivo work and human trials to ensure the effective chemotherapeutic efficacy of marine compounds as treatment options for breast cancer.

Table 1. Autophagy-modulating substances from marine species.

Organism of Origin Substance Autophagy-Related Effects Accumulation of total LC3 and LC3-II; Haliclona genus (Porifera) Papuamine possible mitophagy Upregulation of MAP1LC3B gene H. caerulea (Porifera) Halilectin-3 expression; accumulation of LC3-II increase of cytoplasmic green C. celata (Porifera) Clionamine A-D fluorescent protein (GFP)-LC3 puncta Increase of acidic vacuolar organelles; upregulation of Psammaphysilla genus Psammaplin A LC3, ATG-3, -5, -7, -12, and beclin-1; downregulation (Porifera) of p62/SQSTM1 protein Increase of fluorescent autophagosomes; upregulation X. esigua (Porifera) Araguspongine C of total LC3, Beclin-1, ATG-5, -7, and -16L1 Increase of acidic vacuolar organelles; upregulation of F. reticulata (Porifera) Fascaplysin LC3-II (by 4-chloro-fascaplysin derivative) Increase of acridine orange-positive Aaptos genus (Porifera) Isoaaptamine vacuoles; upregulation of LC3-II and p62/SQSTM1; downregulation of mTOR 3β,11-dihydroxy-9,11- Increase of acidic vesicular organelles; upregulation of K. flaccidum (Cnidaria) secogorgost-5-en-9-one LC3-II and p62 N-methylaminoethyl Increase of fluorescent autophagosomes; upregulation C. virginica (Mollusca) phosphonate of beclin-1 A. lixula Cell-free aqueous extracts of the Increase of acidic vesicular organelles; upregulation of (Echinodermata) coelomic fluid beclin-1, LC3-II, and total LC3 H. tubulosa Cell-free aqueous extracts of the Increase of acidic vesicular organelles; upregulation of (Echinodermata) coelomic fluid beclin-1, LC3-II, and total LC3; possible mitophagy Foundations 2021, 1 17

Funding: This research was funded by the University of Palermo (Italy), grants FFR 2019 and 2020. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Conﬂicts of Interest: The authors declare no conﬂict of interest.

References 1. Chun, Y.; Kim, J. Autophagy: An Essential Degradation Program for Cellular Homeostasis and Life. Cells 2018, 7, 278. [CrossRef][PubMed] 2. Caradonna, F.; Cruciata, I.; Luparello, C. Nutrigenetics, nutrigenomics and phenotypic outcomes of dietary low-dose alcohol consumption in the suppression and induction of cancer development: Evidence from in vitro studies. Crit. Rev. Food Sci. Nutr. 2020, 8, 1–32, epub ahead of print. [CrossRef][PubMed] 3. Ravikumar, B.; Sarkar, S.; Davies, J.E.; Futter, M.; Garcia-Arencibia, M.; Green-Thompson, Z.W.; Jimenez-Sanchez, M.; Korolchuk, V.I.; Lichtenberg, M.; Luo, S.; et al. Regulation of mammalian autophagy in physiology and pathophysiology. Physiol. Rev. 2010, 90, 1383–1435. [CrossRef] 4. Al-Bari, M.A.A.; Xu, P. Molecular regulation of autophagy machinery by mTOR-dependent and -independent pathways. Ann. N. Y. Acad. Sci. 2020, 1467, 3–20. [CrossRef][PubMed] 5. Klionsky, D.J.; Abdel-Aziz, A.K.; Abdelfatah, S.; Abdellatif, M.; Abdoli, A.; Abel, S.; Abeliovich, H.; Abildgaard, M.H.; Abudu, Y.P.; Acevedo-Arozena, A.; et al. Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition). Autophagy 2021, 8, 1–382. [CrossRef] 6. Yun, C.W.; Lee, S.H. The Roles of Autophagy in Cancer. Int. J. Mol. Sci. 2018, 19, 3466. [CrossRef] 7. Lazova, R.; Camp, R.L.; Klump, V.; Siddiqui, S.F.; Amaravadi, R.K.; Pawelek, J.M. Punctate LC3B expression is a common feature of solid tumors and associated with proliferation, metastasis, and poor outcome. Clin. Cancer Res. 2012, 18, 370–379. [CrossRef] 8. Wang, M.C.; Wu, A.G.; Huang, Y.Z.; Shao, G.L.; Ji, S.F.; Wang, R.W.; Yuan, H.J.; Fan, X.L.; Zheng, L.H.; Jiao, Q.L. Autophagic regulation of cell growth by altered expression of Beclin 1 in triple-negative breast cancer. Int. J. Clin. Exp. Med. 2015, 8, 7049–7058. [PubMed] 9. Librizzi, M.; Longo, A.; Chiarelli, R.; Amin, J.; Spencer, J.; Luparello, C. Cytotoxic effects of Jay Amin hydroxamic acid (JAHA), a ferrocene-based class I histone deacetylase inhibitor, on triple-negative MDA-MB231 breast cancer cells. Chem. Res. Toxicol. 2012, 25, 2608–2616. [CrossRef] 10. Librizzi, M.; Tobiasch, E.; Luparello, C. The conditioned medium from osteo-differentiating human mesenchymal stem cells affects the viability of triple negative MDA-MB231 breast cancer cells. Cell Biochem. Funct. 2016, 34, 7–15. [CrossRef][PubMed] 11. Librizzi, M.; Spencer, J.; Luparello, C. Biological Effect of a Hybrid Anticancer Agent Based on Kinase and Histone Deacetylase Inhibitors on Triple-Negative (MDA-MB231) Breast Cancer Cells. Int. J. Mol. Sci. 2016, 17, 1235. [CrossRef] 12. Luparello, C.; Asaro, D.M.L.; Cruciata, I.; Hassell-Hart, S.; Sansook, S.; Spencer, J.; Caradonna, F. Cytotoxic Activity of the Histone Deacetylase 3-Selective Inhibitor Pojamide on MDA-MB-231 Triple-Negative Breast Cancer Cells. Int. J. Mol. Sci. 2019, 20, 804. [CrossRef][PubMed] 13. Maycotte, P.; Thorburn, A. Targeting autophagy in breast cancer. World J. Clin. Oncol. 2014, 5, 224–240. [CrossRef][PubMed] 14. Folkerts, H.; Hilgendorf, S.; Vellenga, E.; Bremer, E.; Wiersma, V.R. The multifaceted role of autophagy in cancer and the microenvironment. Med. Res. Rev. 2019, 39, 517–560. [CrossRef][PubMed] 15. Cocco, S.; Leone, A.; Piezzo, M.; Caputo, R.; Di Lauro, V.; Di Rella, F.; Fusco, G.; Capozzi, M.; Gioia, G.d.; Budillon, A.; et al. Targeting Autophagy in Breast Cancer. Int. J. Mol. Sci. 2020, 21, 7836. [CrossRef] 16. Malve, H. Exploring the ocean for new drug developments: Marine pharmacology. J. Pharm. Bioallied Sci. 2016, 8, 83–91. [CrossRef][PubMed] 17. Barbosa, A.J.M.; Roque, A.C.A. Free Marine Natural Products Databases for Biotechnology and Bioengineering. Biotechnol. J. 2019, 14, e1800607. [CrossRef][PubMed] 18. Mayekar, T.S.; Salgaonkar, A.A.; Koli, J.M.; Patil, P.; Chaudhari, A.; Murkar, A.; Salvi, S.; Surve, D.; Jadhav, R.; Kazi, T. Marine Biotechnology: Bioactive Natural Products and their Applications. Available online: http://aquaﬁnd.com/articles/Marine-Biot echnology.php (accessed on 19 March 2021). 19. Saha, M.; Berdalet, E.; Carotenuto, Y.; Fink, P.; Harder, T.; John, U.; Not, F.; Pohnert, G.; Potin, P.; Selander, E.; et al. Using chemical language to shape future marine health. Front. Ecol. Environ. 2019, 17, 530–537. [CrossRef] 20. Lazzara, V.; Arizza, V.; Luparello, C.; Mauro, M.; Vazzana, M. Bright Spots in the Darkness of Cancer: A Review of Starﬁshes- Derived Compounds and Their Anti-Tumor Action. Mar. Drugs 2019, 17, 617. [CrossRef] 21. Mauro, M.; Lazzara, V.; Punginelli, D.; Arizza, V.; Vazzana, M. Antitumoral compounds from vertebrate sister group: A review of Mediterranean ascidians. Dev. Comp. Immunol. 2020, 108, 103669. [CrossRef] 22. Luparello, C.; Mauro, M.; Lazzara, V.; Vazzana, M. Collective Locomotion of Human Cells, Wound Healing and Their Control by Extracts and Isolated Compounds from Marine Invertebrates. Molecules 2020, 25, 2471. [CrossRef][PubMed] 23. Luparello, C.; Mauro, M.; Arizza, V.; Vazzana, M. Histone Deacetylase Inhibitors from Marine Invertebrates. Biology 2020, 9, 429. [CrossRef][PubMed] Foundations 2021, 1 18

24. Wang, C.-H.; Doan, C.T.; Nguyen, V.B.; Nguyen, A.D.; Wang, S.-L. Reclamation of Fishery Processing Waste: A Mini-Review. Molecules 2019, 24, 2234. [CrossRef] 25. Šimat, V.; Elabed, N.; Kulawik, P.; Ceylan, Z.; Jamroz, E.; Yazgan, H.; Cagalj,ˇ M.; Regenstein, J.M.; Özogul, F. Recent Advances in Marine-Based Nutraceuticals and Their Health Benefits. Mar. Drugs 2020, 18, 627. [CrossRef][PubMed] 26. Chiarelli, R.; Martino, C.; Agnello, M.; Bosco, L.; Roccheri, M.C. Autophagy as a defense strategy against stress: Focus on Paracentrotus lividus sea urchin embryos exposed to cadmium. Cell Stress Chaperones 2016, 21, 19–27. [CrossRef] 27. Song, Q.; Liu, H.; Zhen, H.; Zhao, B. Autophagy and its role in regeneration and remodeling within invertebrate. Cell Biosci. 2020, 10, 111. [CrossRef] 28. Van Soest, R.W.; Boury-Esnault, N.; Vacelet, J.; Dohrmann, M.; Erpenbeck, D.; De Voogd, N.J.; Santodomingo, N.; Vanhoorne, B.; Kelly, M.; Hooper, J.N. Global diversity of sponges (Porifera). PLoS ONE 2012, 7, e35105. [CrossRef] 29. Invertebrates of the Coral Sea. Haliclona sp. Claudia Tay. 2012. Available online: https://www.gbri.org.au/Classes/Holothuriaat ra%7CEmilyPurton/Haliclonasp%7CClaudiaTay.aspx (accessed on 23 March 2021). 30. Yamazaki, H.; Wewengkang, D.S.; Kanno, S.; Ishikawa, M.; Rotinsulu, H.; Mangindaan, R.E.; Namikoshi, M. Papuamine and haliclonadiamine, obtained from an Indonesian sponge Haliclona sp., inhibited cell proliferation of human cancer cell lines. Nat. Prod. Res. 2013, 27, 1012–1015. [CrossRef] 31. Kanno, S.; Yomogida, S.; Tomizawa, A.; Yamazaki, H.; Ukai, K.; Mangindaan, R.E.; Namikoshi, M.; Ishikawa, M. Papuamine causes autophagy following the reduction of cell survival through mitochondrial damage and JNK activation in MCF-7 human breast cancer cells. Int. J. Oncol. 2013, 43, 1413–1419. [CrossRef][PubMed] 32. Killackey, S.A.; Philpott, D.J.; Girardin, S.E. Mitophagy pathways in health and disease. J. Cell Biol. 2020, 219, e202004029. [CrossRef] 33. Kanno, S.I.; Yomogida, S.; Tomizawa, A.; Yamazaki, H.; Ukai, K.; Mangindaan, R.E.; Namikoshi, M.; Ishikawa, M. Combined effect of papuamine and doxorubicin in human breast cancer MCF-7 cells. Oncol. Lett. 2014, 8, 547–550. [CrossRef] 34. Min, H.Y.; Jung, Y.; Park, K.H.; Lee, H.Y. Papuamine Inhibits Viability of Non-small Cell Lung Cancer Cells by Inducing Mitochondrial Dysfunction. Anticancer Res. 2020, 40, 323–333. [CrossRef] 35. Haliclona caerulea (Hechtel, 1965) Blue Caribbean Sponge. Available online: https://www.sealifebase.ca/summary/Haliclona-c aerulea.html# (accessed on 24 March 2021). 36. Carneiro, R.F.; de Melo, A.A.; de Almeida, A.S.; Moura Rda, M.; Chaves, R.P.; de Sousa, B.L.; do Nascimento, K.S.; Sampaio, S.S.; Lima, J.P.; Cavada, B.S.; et al. H-3, a new lectin from the marine sponge Haliclona caerulea: Purification and mass spectrometric characterization. Int. J. Biochem. Cell Biol. 2013, 45, 2864–2873. [CrossRef] 37. do Nascimento-Neto, L.G.; Cabral, M.G.; Carneiro, R.F.; Silva, Z.; Arruda, F.V.S.; Nagano, C.S.; Fernandes, A.R.; Sampaio, A.H.; Teixeira, E.H.; Videira, P.A. Halilectin-3, a Lectin from the Marine Sponge Haliclona caerulea, Induces Apoptosis and Autophagy in Human Breast Cancer MCF7 Cells Through Caspase-9 Pathway and LC3-II Protein Expression. Anti-cancer Agents Med. Chem. 2018, 18, 521–528. [CrossRef] 38. Bijukumar, A.; Nair, A.S. (Eds.) Marine Biodiversity Informatics for Kerala. Available online: http://www.keralamarinelife.in (accessed on 25 March 2021). 39. Keyzers, R.A.; Daoust, J.; Davies-Coleman, M.T.; Van Soest, R.; Balgi, A.; Donohue, E.; Roberge, M.; Andersen, R.J. Autophagy- modulating aminosteroids isolated from the sponge Cliona celata. Org. Lett. 2008, 10, 2959–2962. [CrossRef][PubMed] 40. Forestieri, R.; Donohue, E.; Balgi, A.; Roberge, M.; Andersen, R.J. Synthesis of clionamine B, an autophagy stimulating aminosteroid isolated from the sponge Cliona celata. Org. Lett. 2013, 15, 3918–3921. [CrossRef][PubMed] 41. Bergquist, P.R.; de Cook, S.C. Family Pseudoceratinidae Carter, 1885. In Systema Porifera: A Guide to the Classification of Sponges; Hooper, J.N.A., Van Soest, R.W.M., Eds.; Kluwer Academic/Plenum Publishers: New York, NY, USA, 2002; pp. 1086–1088. 42. Kim, D.; Lee, I.S.; Jung, J.H.; Yang, S.I. Psammaplin A, a natural bromotyrosine derivative from a sponge, possesses the antibacterial activity against methicillin-resistant Staphylococcus aureus and the DNA gyrase-inhibitory activity. Arch. Pharm. Res. 1999, 22, 25–29. [CrossRef][PubMed] 43. Tabudravu, J.N.; Eijsink, V.G.; Gooday, G.W.; Jaspars, M.; Komander, D.; Legg, M.; Synstad, B.; van Aalten, D.M. Psammaplin A, a chitinase inhibitor isolated from the Fijian marine sponge Aplysinella rhax. Bioorg. Med. Chem. 2002, 10, 1123–1128. [CrossRef] 44. Kim, D.H.; Shin, J.; Kwon, H.J. Psammaplin A is a natural prodrug that inhibits class I histone deacetylase. Exp. Mol. Med. 2007, 39, 47–55. [CrossRef][PubMed] 45. Piña, I.C.; Gautschi, J.T.; Wang, G.Y.; Sanders, M.L.; Schmitz, F.J.; France, D.; Cornell-Kennon, S.; Sambucetti, L.C.; Remiszewski, S.W.; Perez, L.B.; et al. Psammaplins from the sponge Pseudoceratina purpurea: Inhibition of both histone deacetylase and DNA methyltransferase. J. Org. Chem. 2003, 68, 3866–3873. [CrossRef] 46. Kim, T.H.; Kim, H.S.; Kang, Y.J.; Yoon, S.; Lee, J.; Choi, W.S.; Jung, J.H.; Kim, H.S. Psammaplin A induces Sirtuin 1-dependent autophagic cell death in doxorubicin-resistant MCF-7/adr human breast cancer cells and xenografts. Biochim. Biophys. Acta 2015, 1850, 401–410. [CrossRef] 47. DeVorkin, L.; Choutka, C.; Gorski, S.M. The Interplay between Autophagy and Apoptosis. In Autophagy: Cancer, Other Pathologies, Inflammation, Immunity, Infection, and Aging; Hayat, M.A., Ed.; Academic Press: Amsterdam, The Netherlands, 2014; pp. 369–383. 48. Hu, W.; Chen, S.; Thorne, R.F.; Wu, M. TP53, TP53 Target Genes (DRAM, TIGAR), and Autophagy. Adv. Exp. Med. Biol. 2019, 1206, 127–149. [CrossRef][PubMed] 49. Kreis, N.-N.; Louwen, F.; Yuan, J. The Multifaceted p21 (Cip1/Waf1/CDKN1A) in Cell Differentiation, Migration and Cancer Therapy. Cancers 2019, 11, 1220. [CrossRef] Foundations 2021, 1 19

50. Richmond, M. (Ed.) A Guide to the Seashores of Eastern Africa and the Western Indian Ocean Islands; The Swedish International Development Co-operation: Stockholm, Sweden, 1997. 51. Kobayashi, M.; Kawazoe, K.; Kitagawa, I. Araguspongines B, C, D, E, F, G, H, and J, new vasodilative bis-1-oxaquinolizidine alkaloids from an okinawan marine sponge, Xestospongia sp. Chem. Pharm. Bull. 1989, 37, 1676–1678. [CrossRef][PubMed] 52. Orabi, K.Y.; El Sayed, K.A.; Hamann, M.T.; Dunbar, D.C.; Al-Said, M.S.; Higa, T.; Kelly, M. Araguspongines K and L, new bioactive bis-1-oxaquinolizidine N-oxide alkaloids from Red Sea specimens of Xestospongia exigua. J. Nat. Prod. 2002, 65, 1782–1785. [CrossRef][PubMed] 53. Akl, M.R.; Ayoub, N.M.; Ebrahim, H.Y.; Mohyeldin, M.M.; Orabi, K.Y.; Foudah, A.I.; El Sayed, K.A. Araguspongine C induces autophagic death in breast cancer cells through suppression of c-Met and HER2 receptor tyrosine kinase signaling. Mar. Drugs 2015, 13, 288–311. [CrossRef] 54. Ebrahim, H.Y.; El Sayed, K.A. Discovery of Novel Antiangiogenic Marine Natural Product Scaffolds. Mar. Drugs 2016, 14, 57. [CrossRef][PubMed] 55. Wang, Q.; Tang, X.L.; Luo, X.C.; de Voog, N.J.; Li, P.L.; Li, G.Q. Aplysinopsin-type and Bromotyrosine-derived Alkaloids from the South China Sea Sponge Fascaplysinopsis reticulata. Sci. Rep. 2019, 9, 2248. [CrossRef] 56. Segraves, N.L.; Robinson, S.J.; Garcia, D.; Said, S.A.; Fu, X.; Schmitz, F.J.; Pietraszkiewicz, H.; Valeriote, F.A.; Crews, P. Comparison of fascaplysin and related alkaloids: A study of structures, cytotoxicities, and sources. J. Nat. Prod. 2004, 67, 783–792. [CrossRef] 57. Zheng, Y.L.; Lu, X.L.; Lin, J.; Chen, H.M.; Yan, X.J.; Wang, F.; Xu, W.F. Direct effects of fascaplysin on human umbilical vein endothelial cells attributing the anti-angiogenesis activity. Biomed. Pharmacother. 2010, 64, 527–533. [CrossRef] 58. Chen, S.; Guan, X.; Wang, L.L.; Li, B.; Sang, X.B.; Liu, Y.; Zhao, Y. Fascaplysin inhibit ovarian cancer cell proliferation and metastasis through inhibiting CDK4. Gene 2017, 635, 3–8. [CrossRef][PubMed] 59. Rath, B.; Hochmair, M.; Plangger, A.; Hamilton, G. Anticancer Activity of Fascaplysin against Lung Cancer Cell and Small Cell Lung Cancer Circulating Tumor Cell Lines. Mar. Drugs 2018, 16, 383. [CrossRef] 60. Sharma, S.; Guru, S.K.; Manda, S.; Kumar, A.; Mintoo, M.J.; Prasad, V.D.; Sharma, P.R.; Mondhe, D.M.; Bharate, S.B.; Bhushan, S. A marine sponge alkaloid derivative 4-chloro fascaplysin inhibits tumor growth and VEGF mediated angiogenesis by disrupting PI3K/Akt/mTOR signaling cascade. Chem. Biol. Interact. 2017, 275, 47–60. [CrossRef] 61. Dyshlovoy, S.A.; Fedorov, S.N.; Shubina, L.K.; Kuzmich, A.S.; Bokemeyer, C.; Keller-von Amsberg, G.; Honecker, F. Aaptamines from the marine sponge Aaptos sp. display anticancer activities in human cancer cell lines and modulate AP-1-, NF-κB-, and p53-dependent transcriptional activity in mouse JB6 Cl41 cells. Biomed. Res. Int. 2014, 2014, 469309. [CrossRef] 62. Wu, C.F.; Lee, M.G.; El-Shazly, M.; Lai, K.H.; Ke, S.C.; Su, C.W.; Shih, S.P.; Sung, P.J.; Hong, M.C.; Wen, Z.H.; et al. Isoaaptamine Induces T-47D Cells Apoptosis and Autophagy via Oxidative Stress. Mar. Drugs 2018, 16, 18. [CrossRef][PubMed] 63. Alderslade, P. Four new genera of soft corals (Coelenterata: Octocorallia), with notes on the classification of some established taxa. Zool. Med. Leiden. 2000, 74, 237–249. 64. Weng, J.R.; Chiu, C.F.; Hu, J.L.; Feng, C.H.; Huang, C.Y.; Bai, L.Y.; Sheu, J.H. A Sterol from Soft Coral Induces Apoptosis and Autophagy in MCF-7 Breast Cancer Cells. Mar. Drugs 2018, 16, 238. [CrossRef][PubMed] 65. Kotta-Loizou, I.; Giaginis, C.; Theocharis, S. The role of peroxisome proliferator-activated receptor-γ in breast cancer. Anticancer Agents Med. Chem. 2012, 12, 1025–1044. [CrossRef][PubMed] 66. Tsai, C.R.; Huang, C.Y.; Chen, B.W.; Tsai, Y.Y.; Shih, S.P.; Hwang, T.L.; Dai, C.F.; Wang, S.Y.; Sheu, J.H. New bioactive steroids from the soft coral Klyxum flaccidum. RSC Adv. 2015, 5, 12546–12554. [CrossRef] 67. Matsubara, T.; Hayashi, A. Identification of molecular species of ceramide aminoethylphosphonate from oyster adductor by gas-liquid chromatography-mass spectrometry. Biochim. Biophys. Acta 1973, 296, 171–178. 68. Matsubara, T. The structure and distribution of ceramide aminoethylphosphonates in the oyster (Ostrea gigas). Biochim. Biophys. Acta 1975, 388, 353–360. [PubMed] 69. Moro, K.; Nagahashi, M.; Gabriel, E.; Takabe, K.; Wakai, T. Clinical application of ceramide in cancer treatment. Breast Cancer 2019, 26, 407–415. [CrossRef][PubMed] 70. Chintalapati, M.; Truax, R.; Stout, R.; Portier, R.; Losso, J.N. In vitro and in vivo anti-angiogenic activities and inhibition of hormone-dependent and -independent breast cancer cells by ceramide methylaminoethylphosphonate. J. Agric. Food Chem. 2009, 57, 5201–5210. [CrossRef] 71. Beg, A.A.; Ruben, S.M.; Scheinman, R.I.; Haskill, S.; Rosen, C.A.; Baldwin, A.S., Jr. I kappa B interacts with the nuclear localization sequences of the subunits of NF-kappa B: A mechanism for cytoplasmic retention. Genes Dev. 1992, 6, 1899–1913. [CrossRef][PubMed] 72. Monkkonen, T.; Debnath, J. Inflammatory signaling cascades and autophagy in cancer. Autophagy 2018, 14, 190–198. [CrossRef] 73. Cui, J.; Shen, H.-M.; Lim, L.H.K. The Role of Autophagy in Liver Cancer: Crosstalk in Signaling Pathways and Potential Therapeutic Targets. Pharmaceuticals 2020, 13, 432. [CrossRef] 74. Kma, L.; Baruah, T.J. The interplay of ROS and the PI3K/Akt pathway in autophagy regulation. Biotechnol. Appl. Biochem. 2021. epub ahead of print. [CrossRef] 75. Luparello, C.; Ragona, D.; Asaro, D.M.L.; Lazzara, V.; Affranchi, F.; Arizza, V.; Vazzana, M. Cell-Free Coelomic Fluid Extracts of the Sea Urchin Arbacia lixula Impair Mitochondrial Potential and Cell Cycle Distribution and Stimulate Reactive Oxygen Species Production and Autophagic Activity in Triple-Negative MDA-MB231 Breast Cancer Cells. J. Mar. Sci. Eng. 2020, 8, 261. [CrossRef] Foundations 2021, 1 20

76. Vazzana, M.; Siragusa, T.; Arizza, V.; Buscaino, G.; Celi, M. Cellular responses and HSP70 expression during wound healing in Holothuria tubulosa (Gmelin, 1788). Fish Shellfish Immunol. 2015, 42, 306–315. [CrossRef] 77. Mauro, M.; Queiroz, V.; Arizza, V.; Campobello, D.; Custódio, M.R.; Chiaramonte, M.; Vazzana, M. Humoral responses during wound healing in Holothuria tubulosa (Gmelin, 1788). Comp. Biochem. Physiol. B Biochem. Mol. Biol. 2021, 253, 110550. [CrossRef] 78. Martino, C.; Bonaventura, R.; Byrne, M.; Roccheri, M.; Matranga, V. Effects of exposure to gadolinium on the development of geographically and phylogenetically distant sea urchins species. Mar. Environ. Res. 2017, 128, 98–106. [CrossRef] 79. Martín, J.; Hidalgo, F.; García-Corcoles, M.T.; Ibáñez-Yuste, A.J.; Alonso, E.; Vilchez, J.L.; Zafra-Gómez, A. Bioaccumulation of perfluoroalkyl substances in marine echinoderms: Results of laboratory-scale experiments with Holothuria tubulosa Gmelin, 1791. Chemosphere 2019, 215, 261–271. [CrossRef][PubMed] 80. Vazzana, M.; Mauro, M.; Ceraulo, M.; Dioguardi, M.; Papale, E.; Mazzola, S.; Arizza, V.; Beltrame, F.; Inguglia, L.; Buscaino, G. Underwater high frequency noise: Biological responses in sea urchin Arbacia lixula (Linnaeus, 1758). Comp. Biochem. Physiol. A Mol. Integr. Physiol. 2020, 242, 110650. [CrossRef][PubMed] 81. Luparello, C.; Ragona, D.; Asaro, D.M.L.; Lazzara, V.; Affranchi, F.; Celi, M.; Arizza, V.; Vazzana, M. Cytotoxic Potential of the Coelomic Fluid Extracted from the Sea Cucumber Holothuria tubulosa against Triple-Negative MDA-MB231 Breast Cancer Cells. Biology 2019, 8, 76. [CrossRef][PubMed] 82. Khotimchenko, Y. Pharmacological Potential of Sea Cucumbers. Int. J. Mol. Sci. 2018, 19, 1342. [CrossRef] 83. Abdelkarem, F.M.; Desoky, E.K.; Nafady, A.M.; Allam, A.E.; Mahdy, A.; Ashour, A.; Shimizu, K. Diadema setosum: Isolation of bioactive secondary metabolites with cytotoxic activity toward human cervical cancer. Nat. Prod. Res. 2020, 4, 1–4. [CrossRef] 84. Yurasakpong, L.; Apisawetakan, S.; Pranweerapaiboon, K.; Sobhon, P.; Chaithirayanon, K. Holothuria scabra Extract Induces Cell Apoptosis and Suppresses Warburg Effect by Down-Regulating Akt/mTOR/HIF-1 Axis in MDA-MB-231 Breast Cancer Cells. Nutr. Cancer 2020, 3, 1–12. [CrossRef][PubMed] 85. Mohamed, A.S. Echinochrome Exhibits Antitumor Activity against Ehrlich Ascites Carcinoma in Swiss Albino Mice. Nutr. Cancer 2021, 73, 124–132. [CrossRef] 86. Tasdemir, E.; Maiuri, M.C.; Tajeddine, N.; Vitale, I.; Criollo, A.; Vicencio, J.M.; Hickman, J.A.; Geneste, O.; Kroemer, G. Cell cycle-dependent induction of autophagy, mitophagy and reticulophagy. Cell Cycle 2007, 6, 2263–2267. [CrossRef] 87. Guo, W.; Wang, Y.; Wang, Z.; Wang, Y.P.; Zheng, H. Inhibiting autophagy increases epirubicin’s cytotoxicity in breast cancer cells. Cancer Sci. 2016, 107, 1610–1621. [CrossRef] 88. Poole, L.P.; Macleod, K.F. Mitophagy in tumorigenesis and metastasis. Cell. Mol. Life Sci. 2021.[CrossRef][PubMed] 89. Breast Cancer. Available online: https://www.who.int/news-room/fact-sheets/detail/breast-cancer#:~:text=In%202020%2C% 20there%20were%202.3,the%20world’s%20most%20prevalent%20cancer (accessed on 28 April 2021). 90. Sung, H.; Ferlay, J.; Siegel, R.L.; Laversanne, M.; Soerjomataram, I.; Jemal, A.; Bray, F. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin. 2021.[CrossRef][PubMed] 91. Barabadi, H.; Mahjoub, M.A.; Tajani, B.; Ahmadi, A.; Junejo, J.; Saravanan, M. Emerging Theranostic Biogenic Silver Nanomaterials for Breast Cancer: A Systematic Review. J. Cluster Sci. 2019, 30, 259–279. [CrossRef] 92. Saravanan, M.; Vahidi, H.; Medina Cruz, D.; Vernet-Crua, A.; Mostafavi, E.; Stelmach, R.; Webster, T.J.; Mahjoub, M.A.; Rashedi, M.; Barabadi, H. Emerging Antineoplastic Biogenic Gold Nanomaterials for Breast Cancer Therapeutics: A Systematic Review. Int. J. Nanomed. 2020, 15, 3577–3595. [CrossRef][PubMed] 93. Deretic, V. Autophagy in inflammation, infection, and immunometabolism. Immunity 2021, 54, 437–453. [CrossRef] 94. Kouroumalis, E.; Voumvouraki, A.; Augoustaki, A.; Samonakis, D.N. Autophagy in liver diseases. World J. Hepatol. 2021, 13, 6–65. [CrossRef][PubMed] 95. Wargasetia, T.L.; Widodo, N. The Link of Marine Products with Autophagy-Associated Cell Death in Cancer Cell. Curr. Pharmacol. Rep. 2019, 5, 35–42. [CrossRef]