Bioactivity Screening of Antarctic Sponges Reveals Anticancer Activity and Potential Cell Death via Ferroptosis by Mycalols

Sponges are known to produce a series of compounds with bioactivities useful for human health. This study was conducted on four sponges collected in the framework of the XXXIV Italian National Antarctic Research Program (PNRA) in November-December 2018, i.e., Mycale (Oxymycale) acerata, Haliclona (Rhizoniera) dancoi, Hemimycale topsenti, and Hemigellius pilosus. Sponge extracts were fractioned and tested against hepatocellular carcinoma (HepG2), lung carcinoma (A549), and melanoma cells (A2058), in order to screen for antiproliferative or cytotoxic activity. Two different chemical classes of compounds, belonging to mycalols and suberitenones, were identified in the active fractions. Mycalols were the most active compounds, and their mechanism of action was also investigated at the gene and protein levels in HepG2 cells. Of the differentially expressed genes, ULK1 and GALNT5 were the most down-regulated genes, while MAPK8 was one of the most up-regulated genes. These genes were previously associated with ferroptosis, a programmed cell death triggered by iron-dependent lipid peroxidation, confirmed at the protein level by the down-regulation of GPX4, a key regulator of ferroptosis, and the up-regulation of NCOA4, involved in iron homeostasis. These data suggest, for the first time, that mycalols act by triggering ferroptosis in HepG2 cells.


Introduction
Marine organisms represent an excellent source of natural products with bioactivities useful for the treatment and prevention of human pathologies, such as cancer, inflammation, and infections. In recent years, the chemistry of natural products derived from marine organisms has received growing interest in the scientific community. In particular, there are fourteen approved pharmaceutical products in clinical use and more than 20 marine natural products in various stages of clinical development (i.e., four compounds in Phase III, twelve in Phase II, and seven in Phase I clinical trials), especially in the field of oncology (https:// www.midwestern.edu/departments/marinepharmacology/clinical-pipeline.xml). These  (2) and mycalols, and the main results on the mechanism of action of mycalols, which were identified as the most active compounds in the current study.

Species Identification of Specimen C7
The C7 specimen belongs to the species Hemimycale topsenti (Burton, 1929) due to the characteristic areolated surface-presence of a plumose skeleton formed by columns of subtilostyles characterized by oval elongated heads.

Chemical Fractionation and Cell Viability on Cancer Cell Lines
Different concentrations (1, 10, and 100 μg mL −1 ) of total MeOH extract (TE) of sampled Antarctic sponges, and their enriched extracts B-E, obtained by solid-phase extraction (SPE) fractionation [22], were screened for their capability to affect the viability of the human cancer cell lines HepG2 (hepatocellular carcinoma), A549 (lung adenocarcinoma) and A2058 (melanoma), and the normal lung cell line MRC5 ( Figure S2).
Bioactivity screening on A2058 cells showed that fraction C, D, and E of the sponge M. a. were active only at 100 μg/mL (p < 0.1 for fraction C and p < 0.001 for fraction D and E), as well as fraction C and D of H. d. (p < 0.05 for fraction C and p < 0.001 for fraction D). For H. t., fraction C was active at both 10 and 100 μg/mL (p < 0.001 for both) while total extract and fraction D were active only at 100 μg/mL (p < 0.001 for both) ( Figure S2). For H. p., total extract, fraction C and fraction E were active only at 100 μg/mL (p < 0.01 for total extract and fraction C, while p < 0.05 for fraction E; Figure S2). (2) and mycalols, and the main results on the mechanism of action of mycalols, which were identified as the most active compounds in the current study.

Species Identification of Specimen C7
The C7 specimen belongs to the species Hemimycale topsenti (Burton, 1929) due to the characteristic areolated surface-presence of a plumose skeleton formed by columns of subtilostyles characterized by oval elongated heads.

Chemical Fractionation and Cell Viability on Cancer Cell Lines
Different concentrations (1, 10, and 100 µg mL −1 ) of total MeOH extract (TE) of sampled Antarctic sponges, and their enriched extracts B-E, obtained by solid-phase extraction (SPE) fractionation [22], were screened for their capability to affect the viability of the human cancer cell lines HepG2 (hepatocellular carcinoma), A549 (lung adenocarcinoma) and A2058 (melanoma), and the normal lung cell line MRC5 ( Figure S2).
Bioactivity screening on A2058 cells showed that fraction C, D, and E of the sponge M. a. were active only at 100 µg/mL (p < 0.1 for fraction C and p < 0.001 for fraction D and E), as well as fraction C and D of H. d. (p < 0.05 for fraction C and p < 0.001 for fraction D). For H. t., fraction C was active at both 10 and 100 µg/mL (p < 0.001 for both) while total extract and fraction D were active only at 100 µg/mL (p < 0.001 for both) ( Figure S2). For H. p., total extract, fraction C and fraction E were active only at 100 µg/mL (p < 0.01 for total extract and fraction C, while p < 0.05 for fraction E; Figure S2).
Regarding A549 cells, M. a. fraction C, D, and E were able to significantly reduce A549 cell proliferation only at 100 µg/mL (p < 0.05 for fraction C and p < 0.001 for fraction D and E,). For H. d., total extract and fraction D were active at 100 µg/mL (p < 0.01 for total extract and p < 0.01 for fraction D), while fraction C was active both at 10 and 100 µg/mL (p < 0.05 and p < 0.001, respectively). For H. t., total extract, fraction C and fraction D were active at 100 µg/mL (p < 0.001 for all). For H. p., fraction E was the only one active at 100 µg/mL (p < 0.001) ( Figure S2).
Bioactivity on HepG2 cells showed that fraction D and E of M. a. were active at 100 µg/mL (p < 0.01 and p < 0.001, respectively). Fraction C of H. d. was active at both 10 and 100 µg/mL (p < 0.001 for both), total extract and fraction D were active only at 100 µg/mL (p < 0.001 for both). For H. t., total extract, fraction C, D and E were active only at 100µg/mL (p < 0.001 for all). H. p. total extract and fraction D were active at 100 µg/mL (p, <, 0001 and p < 0.01, respectively).

Identification of Compounds in Most Active Fractions
Preliminary 1 H NMR of the active fraction D of the sponge H. t. ( Figure S3) showed the methyl pattern that clearly indicated the presence of a sesterterpene. Further purification on silica column led to the isolation of two known compounds, suberitenone A (1) and B (2) (Figure 2), whose identification was obtained by comparing the 1 H NMR data ( Figures S4 and S5) with the literature [23], and confirmed by ESI + MS analysis ( Figures S6 and S7).
Regarding A549 cells, M. a. fraction C, D, and E were able to significantly reduce A549 cell proliferation only at 100 μg/mL (p < 0.05 for fraction C and p < 0.001 for fraction D and E,). For H. d., total extract and fraction D were active at 100 μg/mL (p < 0.01 for total extract and p < 0.01 for fraction D), while fraction C was active both at 10 and 100 μg/mL (p < 0.05 and p < 0.001, respectively). For H. t., total extract, fraction C and fraction D were active at 100 μg/mL (p < 0.001 for all). For H. p., fraction E was the only one active at 100 μg/mL (p < 0.001) ( Figure S2).
Bioactivity on HepG2 cells showed that fraction D and E of M. a. were active at 100 μg/mL (p < 0.01 and p < 0.001, respectively). Fraction C of H. d. was active at both 10 and 100 μg/mL (p < 0.001 for both), total extract and fraction D were active only at 100 μg/mL (p < 0.001 for both). For H. t., total extract, fraction C, D and E were active only at 100μg/mL (p < 0.001 for all). H. p. total extract and fraction D were active at 100 μg/mL (p < 0.001 and p < 0.01, respectively).

Identification of Compounds in Most Active Fractions
Preliminary 1 H NMR of the active fraction D of the sponge H. t. (Figure S3) showed the methyl pattern that clearly indicated the presence of a sesterterpene. Further purification on silica column led to the isolation of two known compounds, suberitenone A (1) and B (2) (Figure 2), whose identification was obtained by comparing the 1 H NMR data ( Figures S4 and S5) with the literature [23], and confirmed by ESI + MS analysis ( Figure  S6 and S7).

Bioactivity of Suberitenones A and B from Hemimycale topsenti and Mycalols from Haliclona (Rhizoniera) dancoi on Cancer Cell Lines
The mixture of mycalols, and suberitenones A and B purified from the most active fractions were tested on cancer cell lines (A549, A2058 and HepG2) and a normal cell line (MRC5) to confirm the cytotoxic activity. In order to evaluate the IC 50 values, the compounds were challenged at different concentrations (0.05, 0.10, 0.19, 0.39, 0.78, 1.56, 3.12, 6.25, 12.5, 25, 50, 100 µM) (Figure 3 for mycalols and Figure S11 for suberitenone A and B).

Bioactivity of Suberitenones A and B from Hemimycale Topsenti and Mycalols from Haliclona (Rhizoniera) Dancoi on Cancer Cell Lines
The mixture of mycalols, and suberitenones A and B purified from the most active fractions were tested on cancer cell lines (A549, A2058 and HepG2) and a normal cell line (MRC5) to confirm the cytotoxic activity. In order to evaluate the IC50 values, the compounds were challenged at different concentrations (0.05, 0.10, 0.19, 0.39, 0.78, 1.56, 3.12, 6.25, 12.5, 25, 50, 100 μM) (Figure 3 for mycalols and Figure S11 for suberitenone A and B).  (Table 2), which were roughly in line with the activity reported for the pure compounds on anaplastic thyroid carcinoma-derived FRO cells [21]. IC50 values of suberitenones A (1) on A549, A2058, HepG2, and MRC5 cells were 28.5, 10.2, 17.6, and 7.4 μM, respectively. IC50 values of suberitenones B (2) on A549, A2058, HepG2, and MRC5 were 80.7, 14.6, 19.2, and 8.5 μM, respectively (Table 2, Figure  S10). The mycalols showed higher activity against cancer cell lines with respect to normal cell lines, and, in particular, they had the lowest IC50 when tested against HepG2 cells. On the contrary, 1 and 2 showed lower activity against cancer cell lines with respect to the normal ones (Table 2).   (Table 2), which were roughly in line with the activity reported for the pure compounds on anaplastic thyroid carcinoma-derived FRO cells [21]. IC 50 values of suberitenones A (1) on A549, A2058, HepG2, and MRC5 cells were 28.5, 10.2, 17.6, and 7.4 µM, respectively. IC 50 values of suberitenones B (2) on A549, A2058, HepG2, and MRC5 were 80.7, 14.6, 19.2, and 8.5 µM, respectively (Table 2, Figure S10). The mycalols showed higher activity against cancer cell lines with respect to normal cell lines, and, in particular, they had the lowest IC 50 when tested against HepG2 cells. On the contrary, 1 and 2 showed lower activity against cancer cell lines with respect to the normal ones (Table 2).

Mechanism of Action
In order to elucidate the cell death metabolic pathway induced by mycalols, expression levels of selected genes by using a PCR array were analyzed in HepG2 cells treated in the presence of mycalols at 9 µM for 48 h. Gene transcription was considered affected by compounds if expression values were greater than two-fold difference with respect to the control (DMSO alone). Both differentially up-regulated and down-regulated genes are reported in Table 3. Gene expression analyses indicated that Unc-51-like kinase 1 (ULK1) and UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 5 (GALNT5) were the most down-regulated genes (−12.03 and −10.71-fold expression, respectively). ULK1 is involved in mammalian autophagic signaling [25] in hepatic cells and is known to be a regulator of different metabolic pathways/processes, such as mevalonate/cholesterol biosynthesis pathway [26] and lipotoxicity. Lipotoxicity is known to induce tissue damage and inflammation in metabolic disorders [27] and has been found to be related to ferroptosis [28,29], an iron-dependent programmed cell death triggered by the accumulation of lethal lipid species [30]. GALNT5 is involved in carcinogenesis and progression of choloangiocarcinoma, and activates the AKT/Erk (extracellular signalregulated kinases) pathway [31]. In addition, a decrease in its substrate concentration (UDP-N-acetylglucosamine) has been related to ferroptosis in hepatoma cells [32]. In the current study, the mitogen-activated protein kinase 8 (MAPK8; also known as JNK1), which has been directly related to ferroptosis as well [33], was up-regulated (3.71-fold regulation). On the other hand, gene expression analyses showed that the pro-apoptotic genes caspase 1 (CASP1), tumor necrosis factor receptor superfamily, member 11b (TNFRSF11; known to promote apoptosis in osteoclasts, https://www.uniprot.org/ uniprot/O00300) and apoptotic peptidase activating factor 1 (APAF) were down-regulated (−6.85, −5.57, and −2.03 fold regulation, respectively), while the anti-apoptotic genes nucleolar protein 3 (apoptosis repressor with CARD domain; NOL3) and B-cell lymphoma 2 A1 (BCL2A1) were the most up-regulated genes (7.40 and 5.14-fold regulation, respectively), suggesting that apoptosis was not involved in the mechanism of action of mycalol toxicity.
To confirm whether mycalols triggered ferroptosis, the levels of glutathione peroxidase 4 (GPX4) and nuclear receptor coactivator 4 (NCOA4) were evaluated in hepatocellular carcinoma at the protein level. Both GPX4 and NCOA4 have an important role in ferroptosis processes. GPX4 inhibition has been associated with the accumulation of lipid peroxides, which leads to ferroptosis [29]. On the contrary, NCOA4 overexpression has been associated with an improved sensitivity to ferroptosis [34]. In the current study, the HepG2 cell line was treated in the presence or in the absence of mycalols. As shown in Figure 4a,b, the level of GPX4 protein was strongly reduced in HepG2 treated in the presence of 9 µM mycalols with respect to cells treated in the presence of the DMSO alone (p < 0.05). On the contrary, NCOA4 protein level was increased after mycalols treatment (p < 0.01; Figure 4c,d) with respect to the treatment in the presence of the vehicle alone. The reduction in GPX4 and the increase in NCOA4 protein levels suggested that mycalols were able to induce ferroptosis in hepatocarcinoma cells.
Hs.442337 NM_176823 S100A7A S100 calcium binding protein A7A To confirm whether mycalols triggered ferroptosis, the levels of glutathione peroxidase 4 (GPX4) and nuclear receptor coactivator 4 (NCOA4) were evaluated in hepatocellular carcinoma at the protein level. Both GPX4 and NCOA4 have an important role in ferroptosis processes. GPX4 inhibition has been associated with the accumulation of lipid peroxides, which leads to ferroptosis [29]. On the contrary, NCOA4 overexpression has been associated with an improved sensitivity to ferroptosis [34]. In the current study, the HepG2 cell line was treated in the presence or in the absence of mycalols. As shown in Figure 4a and 4b, the level of GPX4 protein was strongly reduced in HepG2 treated in the presence of 9 μM mycalols with respect to cells treated in the presence of the DMSO alone (p < 0.05). On the contrary, NCOA4 protein level was increased after mycalols treatment (p < 0.01; Figure 4c,d) with respect to the treatment in the presence of the vehicle alone. The reduction in GPX4 and the increase in NCOA4 protein levels suggested that mycalols were able to induce ferroptosis in hepatocarcinoma cells. and NCOA4 (d) were normalized, respectively, with β-tubulin or glyceraldehyde 3-phosphate dehydrogenase (GAPDH), used as standard proteins, and are reported as relative to GPX4 and NCOA4 levels (* p < 0,05; ** p < 0.01).

Discussion
Sponges represent an important reservoir of bioactive marine natural compounds. Abdelaleem et al. [35] reported the number of isolated bioactive compounds in a taxon of the order Demospongiae between 2013 and 2019, showing that 195 new compounds and 189 known compounds were isolated. These compounds have been reported to have a Figure 4. Effects of the mycalol mixture on ferroptosis-related proteins glutathione peroxidase 4 (GPX4) and nuclear receptor coactivator 4 (NCOA4). Western blotting analyses with anti-GPX4 (a) or anti-NCOA4 (b) antibody of the extracts from HepG2 treated with the DMSO alone (control) or in the presence of mycalols. The intensity of the bands of GPX4 (c) and NCOA4 (d) were normalized, respectively, with β-tubulin or glyceraldehyde 3-phosphate dehydrogenase (GAPDH), used as standard proteins, and are reported as relative to GPX4 and NCOA4 levels (* p < 0.05; ** p < 0.01).

Discussion
Sponges represent an important reservoir of bioactive marine natural compounds. Abdelaleem et al. [35] reported the number of isolated bioactive compounds in a taxon of the order Demospongiae between 2013 and 2019, showing that 195 new compounds and 189 known compounds were isolated. These compounds have been reported to have a broad range of bioactivities, such as antimicrobial, cytotoxic, antioxidant, antiviral, anti-allergic, anti-parasitic, and anti-inflammatory [35].
Here, we report a new cytotoxic activity of mycalols against A549, HepG2, and melanoma cells (A2058), other than thyroid cancer cells. In addition to A549 and HepG2, suberitenones B also showed activity against melanoma cells. Suberitenones A was shown, for the first time, to be active against lung carcinoma, hepatocellular carcinoma, and melanoma (A549, HepG2, and A2058) cells. However, both suberitenones A and B also showed high toxicity against normal cells (MRC-5, lung fibroblasts). On the contrary, as previously reported on thyroid cells [20,21], cytoxicity of mycalols was weaker against normal cells than tumor cell lines. Particularly, mycalols showed the highest activity against hepatocarcinoma cells. It is worth noting that the members of this family of products show an in vitro activity that is dependent on the length and the substitution of the alkyl skeleton, with a good correlation with the lipophilicity of the products [21]. Reasonably, this suggests that the activity of one of these products may be significantly stronger than that we recorded with the mixture. In this study, we further investigated the cell death pathway triggered by the pool of mycalols in hepatocellular carcinoma cells. Expression levels of 84 genes, involved in different death pathways, were studied. Results showed that the differentially expressed genes could be associated with ferroptosis. In particular, ULK1, GALNT5 down-regulation and MAPK8 (also known as JNK1) upregulation have been previously related to ferroptosis processes [28,32,33,37]. Ferroptosis is a programmed cell death triggered by iron accumulation and iron-dependent lipid peroxidation. Ferroptosis has been associated to many diseases, such as cancer, kidney injury, and neurological diseases [38]. Thus, activating or blocking ferroptosis could be a strategy to treat various diseases [29,39]. GPX4 has a key role in ferroptosis and is involved in the inhibition of lipid peroxides. GPX4 converts glutathione (GSH) into its oxidized form (GSSG) and reduces lipid peroxides [29]. A marine terpenoid, named heteronemin, was recently isolated from Hippospongia sp., which induces apoptosis and ferroptosis in hepatocarcinoma cells, reducing GPX4 expression as well [40]. Ferroptosis has also been related to NCOA4 levels [41]. In fact, NCOA4 over-expression in cellular models increases sensitivity to ferroptosis [34]. In order to confirm that mycalols from Haliclona (Rhizoniera) dancoi triggered the ferroptosis pathway, we analyzed the protein levels of both GPX4 and NCOA4, in the hepatocarcinoma cell line. In cells treated in the presence of mycalols, GPX4 protein levels were significantly reduced, while NCOA4 levels increased, confirming our hypothesis.

Conclusions
The aim of this study was to screen different sponges collected in Antarctic waters, in the framework of the XXXIV Italian National Antarctic Research Program (PNRA) in November-December 2018, in order to find bioactive natural products. Four different species of sponges have been collected and for each species, both total extracts and their four SPE-fractions have been prepared and tested on different tumor cell lines. A mixture of mycalols as well as suberitenones A and B were found in the most active fractions. Mycalols showed the highest activity. Thus, we studied the mechanism of action of the mycalol mixture at the gene and protein level, and the results suggested the activation of the ferroptosis death pathway in the hepatocarcinoma cell line. Several cancer cells have been found to modulate lipid metabolism in order to reduce ferroptosis sensitivity [42], thus targeting ferroptosis and its mediators could become a promising strategy in cancer therapy [43].

Sampling
Three analyzed sponge specimens were previously identified by morphological and molecular approaches in our previous paper [7]. All samples belonged to the class Demospongiae and to the following two orders: Haplosclerida with two species, Hemigellius pilosus (Kirkpatrick, 1907) and Haliclona (Rhizoniera) dancoi (Topsent, 1901), and Poecilosclerida with one species, Mycale (Oxymycale) acerata (Kirkpatrick, 1907). The species of sample C7 (MNA code 13860) was identified as Hemimycale topsenti in the present work (Table 1) Samples were immediately washed with filtered and sterilized seawater to remove loosely attached bacteria and/or debris [7,44]. A small fragment of each sponge was stored in 70% ethanol for taxonomic identification, another fragment was preserved in RNAlater TM into sterile tubes and stored at −20 • C until use for DNA extraction, and the remaining sample was frozen and stored at −80 • C for chemical extraction procedures. In addition, sponge slides of spicules were deposited at the Italian National Antarctic Museum (MNA, Section of Genoa, Italy). The MNA voucher codes of each sample are reported in Table 1.

Morphological Analysis and Polymerase Chain Reaction (PCR) of 18S/28S rRNA and CO1 Markers
For the taxonomic identification, small fragments of each sponge were heat-dissolved in nitric acid, rinsed in water, and dehydrated in ethanol. Then, spicules were mounted on slides for microscopic analyses, following standard methods [45]. The skeletal architecture was examined under a light microscope and hand-cut sections of sponge portions were made as described in Hooper [46]. The taxonomic classification follows the updated nomenclature reported in the World Porifera Database (WPD) [47].

Chemical Extraction and SPE Fractionation
After lyophilization, the organic material was extracted with methanol ((Merk Life Science S.r.l., Milano, Italy) at room temperature and a small amount (about 50 mg) of raw extract (hereafter referred to as TE) was subjected to SPE on a GX-271 ASPEC Gilson apparatus by using CHROMABOND ® HRX cartridges (6 mL/500 mg, MACHEREY-NAGEL, Düeren, Germany) as reported by Cutignano et al. [22]. The active SPE-HRX fraction C (5.1 mg) of H. dancoi was purified by chromatography on a silica Pasteur pipette (SiO 2 ) eluted with a gradient of chloroform in methanol, to obtain 1.2 mg of mycalols mixture. 1 H NMR in pyr-d 5 ( Figure S9) and HR ESI + -MS were acquired to define the chemical composition ( Figure 2).

In Vitro Cell Viability Studies
To evaluate the in vitro effects of the extracts or fractions on the cell viability of HepG2, A2058, A549, and MRC5 cell lines were seeded in 96-well microtiter plates at a density of 1 × 10 4 cells/well and incubated at 37 • C to allow for cell adhesion in the plates. After 16 h, the medium was replaced with fresh medium containing increasing concentrations of extracts, fractions (0.01, 0.1, 1, 10, and 100 µg/mL) or purified compounds (0.05, 0.10, 0.19, 0.39, 0.78, 1.56, 3.12, 6.25, 12.5, 25, 50, 100 µM) dissolved in dimethyl sulfoxide (DMSO), for 72 h. The maximum concentration of DMSO used was 1% (v/v). Each concentration was tested at least in triplicate. After 72 h, the 3-(4,5-dimethyl-2-thizolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT; A2231,0001, AppliChem Panreac Tischkalender, GmbH, Darmstadt, Germany) assay was carried out. Briefly, the medium was replaced with a medium containing MTT at 0.5 mg/mL and the plates were incubated for 3 h at 37 • C. After incubation, the cells were treated with isopropyl alcohol (used as MTT solvent) for 30 min at room temperature. Absorbance was measured at OD = 570 nm by a microplate reader (Multiskan™ FC Microplate Photometer, Thermo Fisher Scientific, Waltham, MA, USA). Cell survival was expressed as a percentage of viable cells in the presence of the tested samples, with respect to untreated control cultures with only DMSO.

RNA Extraction and Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR)
HepG2 cells to be used for RNA extraction were seeded in 6 wells plates (at 500,000 per well) and kept 16 h for attachment. The seeded cells were then treated in the presence of mycalols at 9 µM (molarity was evaluated based on the average molecular weights of the mycalols into the mixture) for 48 h at 37 • C. Cells were washed by adding phosphatebuffered saline (PBS 1×).
Cells were lysed by adding 1 mL of Trisure Reagent (Meridian bioscience, Memphis, TN, USA). RNA was isolated as previously described [56]. RNA concentration, quality, and purity were assessed using an ND-1000 UV-Vis spectrophotometer (NanoDrop Technologies, Thermo Fisher Scientific, Waltham, MA, USA), monitoring the absorbance at 260 nm, and the 260/280 nm and 260/230 nm ratios (both ratios were about 2.0). RNA quality was evaluated by gel electrophoresis that showed intact RNA, with sharp ribosomal bands. About 500 ng of RNA was subjected to reverse transcription reaction using the RT2 first strand kit (cat.330401, Qiagen, Hilden, Germany) according to the manufacturer's instructions. The RT-qPCR analysis was performed in duplicate using the RT2 Profiler PCR Array kit (cat. PAHS-212ZE-4, Qiagen, Hilden, Germany) to analyze the expression of 84 genes involved in cell death signaling pathways. Plates were run on a ViiA7 (Applied Biosystems, Foster City, CA, USA, 384-well blocks). The PCR program consisted of a denaturation step at 95 • C for 20 s followed by 40 cycles at 95 • C for 15 s and 60 • C for 1 min. The cycle threshold (Ct)-values were analyzed with PCR array data analysis online software (GeneGlobe Data Analysis Center http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php accessed on May 2021, Qiagen, Hilden, Germany). Real-time data were expressed as the fold of expression, describing the changes in gene expression between cells treated in the presence of mycalols and cells treated in the presence of DMSO alone. Only expression values greater than a two-fold difference with respect to the controls were considered significant.

Protein Extraction and Western Blotting Analyses
The HepG2 cells to be used for protein extraction were seeded in 6-well plates (at 500,000 per well) and kept 16 h for attachment. The seeded cells were then treated in the presence of mycalols at 9 µM for 48 h at 37 • C. Cells were washed by adding phosphatebuffered saline (PBS 1×). Cells were lysed in RIPA Buffer (Cell signaling technology, Danvers, MA, USA) 1% Triton X-100, supplemented with protease inhibitors (#310A7779, AppliChem GmbH, Darmstadt, Germany). The lysates were centrifuged at 14,000 rpm for 30 min at 4 • C. The total amount of proteins in each lysate was measured using the Bradford assay. Before sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), each lysate was diluted in Laemmli sample buffer (#161-0747, Biorad, Hercules, CA, USA) containing β-mercapto-ethanol and then boiled for 5 min at 95 • C. For Western blotting analysis, proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (#1704156, Trans-blot Turbo transfer pack-Biorad) using the Trans blot turbo transfer system (Biorad, Hercules, CA, USA). After the transfer, PVDF membranes were incubated in a blocking solution (1× Tris-buffered saline, TBS, 5% BSA) for 1 h at 25 • C and then incubated with primary and secondary antibodies supplemented with 5% BSA. ECL (#170-5060, Biorad, Hercules, CA, USA) reactions were performed as per the manufacturer's instructions and immunoreactive bands were detected by chemiluminescence using ChemiDoc MP imaging system (Biorad, Hercules, CA, USA). The obtained immunoreactive bands were quantitated using Image Lab v6.0 (Biorad, Hercules, CA, USA). Different housekeeping proteins were used to normalize immunoreactive bands depending on the molecular weight of the detected proteins. B-tubulin and GAPDH were used to normalize GPX4 and NCOA4, respectively.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/md19080459/s1, Figure S1: MultiAlin output of CO1 PCR product indicated as C7_dgLCO1490/ dgHCO21 aligned to the BLAST highly similar sequence of the strain Hemimycale topsenti (ex Suberites topsenti; LN850246.1); Figure S2: Cell viability assay. The figure shows the effects on cell viability of fractions (B green bars; C blue bars; D yellow bars; E red bars) or total extracts (ex, black bars) of the samples (a) M. a., (b) H. d., (c) H.t. and (d) H. p. on A2058, A549, and HepG2 cell lines at increasing concentrations ( 10 and 100 ng/mL and 1, 10, and 100 µg/mL). Cell viability was normalized using cells with only DMSO as a control sample. Results are expressed as percent survival after 72 h exposure (n = 3; * for p < 0.05; ** for p < 0.01 and *** for p < 0.001, Student's t-test); Figure S3