Marine-Derived Compounds Targeting Topoisomerase II in Cancer Cells: A Review

Cancer affects more than 19 million people and is the second leading cause of death in the world. One of the principal strategies used in cancer therapy is the inhibition of topoisomerase II, involved in the survival of cells. Side effects and adverse reactions limit the use of topoisomerase II inhibitors; hence, research is focused on discovering novel compounds that can inhibit topoisomerase II and have a safer toxicological profile. Marine organisms are a source of secondary metabolites with different pharmacological properties including anticancer activity. The objective of this review is to present and discuss the pharmacological potential of marine-derived compounds whose antitumor activity is mediated by topoisomerase II inhibition. Several compounds derived from sponges, fungi, bacteria, ascidians, and other marine sources have been demonstrated to inhibit topoisomerase II. However, some studies only report docking interactions, whereas others do not fully explain the mechanisms of topoisomerase II inhibition. Further in vitro and in vivo studies are needed, as well as a careful toxicological profile evaluation with a focus on cancer cell selectivity.


Introduction
Cancer is the second leading cause of death in the world after cardiovascular diseases, affecting an estimated 19 million people and causing approximately 10 million deaths in 2020 [1].
Chemotherapy represents the main anticancer therapeutic approach. Nowadays, the principal clinically employed anticancer drugs are natural products, or their structural analogs [2][3][4][5][6]. However, several factors limit their effectiveness: (i) their efficacy is inversely proportional to disease progression; (ii) occurrence of chemoresistance; (iii) severe toxicity caused by lack of selectivity against cancer cells [7,8]. For this reason, the discovery of anticancer agents characterized by an improved pharmaco-toxicological profile remains a major aim of pharmacological research.
One of the principal targets of drugs used in chemotherapy to stop the aberrant proliferation of cancer cells is topoisomerase (topo) II [9].
Topo is a class of nuclear enzymes essential for cell survival. They regulate the topology of DNA and are involved in replication, transcription, proliferation, and chromosome segregation during the cell cycle. Vertebrates express two different isoforms of topo II-α and β-and although they possess 70% sequence homology and show similar enzyme activity, they are expressed and regulated differently [10].  (1); flexing of the G-segment in the presence of metals ions (2); formation of the cleavage complex (3); closing the gate to constrain the T-segment to pass through the G-segment (4); ligation of the G-segment (5); release of the T-segment (6); release of the G-segment (7); enzyme ready for a new catalytic cycle (8).
Thus, the inhibition of topo activity allows the blocking of the cell cycle and then conduces to cell death [11]. Topo II-mediated DNA breakage is a critical step for cell survival and must be finely regulated to avoid a possible fragmentation of the entire genome [9]. In a healthy cell, there is fine control of the formation of cleavage complexes, which are short-lived and reversible. Topo II inhibitors are compounds capable of modulating the formation of cleavable complexes and altering this equilibrium. Thus, the inhibition of topo activity allows the blocking of the cell cycle and then conduces to cell death [11]. Topo II-mediated DNA breakage is a critical step for cell survival and must be finely regulated to avoid a possible fragmentation of the entire genome [9]. In a healthy cell, there is fine control of the formation of cleavage complexes, which are short-lived and reversible. Topo II inhibitors are compounds capable of modulating the formation of cleavable complexes and altering this equilibrium.
There are two different mechanisms described for topo II inhibition: (i) poisoning or (ii) catalytic inhibition. Poisoning is the main mechanism and acts on the stabilization of the cleavable complex, leading to maintaining the permanent breakage of DNA. Indeed, when the levels of cleavable complexes become high, they cannot be repaired by topo II, thus becoming irreversible DNA lesions that activate different signaling pathways and result in cell death by apoptosis [12]. On the other hand, catalytic inhibition implies that the inhibitor prevents the formation of the cleavage complex. If the amount of cleavage Neo was highly cytotoxic in several tumor cell lines [25,26]. In addition, neo was equally cytotoxic in wild-type A2780 ovarian cancer cells and in multidrug-resistant (MDR)-expressing A2780AD cell line (Table 1). Of note, taxol, DOXO, and amsacrine (m-AMSA) had a 15-, 33-, and 8-fold lower cytotoxicity than neo [25]. In vivo, the administration of neo (12.5-50 mg/kg for 19 days) to Balb/c nu/nu mice bearing HCT-116 and KB xenograft reduced tumor growth (Table 1) and displayed the same efficacy as ETO [25]. DT was cytotoxic on different tumor cell lines. Additionally, DT had a selective cytotoxic effect on tumor cells, since the cell viability of rat alveolar macrophage NR8383 cells was more than 80% after exposure to the highest tested concentration of the compound [35]. In the same study, DT (0.01-10 μg/mL) was found to inhibit topo IIα using a cell-free DNA cleavage assay with an enzyme-mediated negatively supercoiled pHOT1 plasmid DNA. In the presence of topo IIα, DT at low concentrations (0.01, 0.1, and 1 μg/mL) caused DNA relaxation, and at high concentrations (2.5, 5, and 10 μg/mL) blocked DNA relaxation. This means that DT interferes with the topo IIα catalytic cycle [35]. However, the compound did not generate linear DNA [35], which is associated with the stabilization of topo II-DNA cleavage complex typical of topo II poisons [37].
The link between the inhibition of topo IIα and the apoptotic activity of DT is controversial. DT increased the apoptotic fraction of K562 cells at concentrations of 2.5, 5.0, and 10 μg/mL. Moreover, the compound at 0.5 and 1.0 μg/mL activated caspase-3 (Casp-3) and cleaved poly (ADP-ribose) polymerase (PARP), while at 5 μg/mL it decreased Casp-3 activity and PARP cleavage. DT also induced the phosphorylation of various DNA damage-related proteins, including H2A histone family member X (H2A.X), ataxia telangiectasia mutated (ATM), breast cancer gene (BRCA), and ataxia-telangiectasia rad3-related (ATR) in the same concentration-dependent manner. Additionally, while 2.5 μg/mL of DT increased intracellular reactive oxygen species (ROS) levels in a timedependent manner (0-60 min), at 5 μg/mL, ROS levels rose up to 30 min and then gradually decreased time-dependently [35]. This could possibly explain the lower activation of Casp-3 and the lower phosphorylation of DNA damage-related proteins in cells treated with DT 5 μg/mL. At the same time, the pre-treatment of cells with the ROS DT was cytotoxic on different tumor cell lines. Additionally, DT had a selective cytotoxic effect on tumor cells, since the cell viability of rat alveolar macrophage NR8383 cells was more than 80% after exposure to the highest tested concentration of the compound [35]. In the same study, DT (0.01-10 µg/mL) was found to inhibit topo IIα using a cell-free DNA cleavage assay with an enzyme-mediated negatively supercoiled pHOT1 plasmid DNA. In the presence of topo IIα, DT at low concentrations (0.01, 0.1, and 1 µg/mL) caused DNA relaxation, and at high concentrations (2.5, 5, and 10 µg/mL) blocked DNA relaxation. This means that DT interferes with the topo IIα catalytic cycle [35]. However, the compound did not generate linear DNA [35], which is associated with the stabilization of topo II-DNA cleavage complex typical of topo II poisons [37].
The link between the inhibition of topo IIα and the apoptotic activity of DT is controversial. DT increased the apoptotic fraction of K562 cells at concentrations of 2.5, 5.0, and 10 µg/mL. Moreover, the compound at 0.5 and 1.0 µg/mL activated caspase-3 (Casp-3) and cleaved poly (ADP-ribose) polymerase (PARP), while at 5 µg/mL it decreased Casp-3 activity and PARP cleavage. DT also induced the phosphorylation of various DNA damagerelated proteins, including H2A histone family member X (H2A.X), ataxia telangiectasia mutated (ATM), breast cancer gene (BRCA), and ataxia-telangiectasia rad3-related (ATR) in the same concentration-dependent manner. Additionally, while 2.5 µg/mL of DT increased intracellular reactive oxygen species (ROS) levels in a time-dependent manner (0-60 min), at 5 µg/mL, ROS levels rose up to 30 min and then gradually decreased time-dependently [35]. This could possibly explain the lower activation of Casp-3 and the lower phosphorylation of DNA damage-related proteins in cells treated with DT 5 µg/mL. At the same time, the pre-treatment of cells with the ROS scavenger N-acetyl cysteine (NAC) inhibited the apoptotic activity and the protein expression of phosphorylated H2A.X (γ-H2A.X) induced by DT at 5 µg/mL [35]. This result points out that, although inhibition of topo IIα is associated with the activation of DNA damage-related proteins, overproduction of ROS also contributes to increase DNA damage and seems to be the major pro-apoptotic trigger. ROS-induced apoptosis by DT has been found to involve the IKK (IκB kinases)/NFκB (nuclear factor kappa B) and PI3K (phosphatidylinositol 3-kinase)/Akt signaling pathways, as demonstrated by the reduced expression of IKK/NFκB-related proteins and the increased phosphorylation of Akt [35]. Given that the continuous activation of IKK/NF-κB pathway promotes tumorigenesis [38], its inhibition by DT could be considered an additional mechanism of its antitumor effect. However, Akt activation is associated with tumor aggressiveness and drug resistance [39]. Hence, further investigation should be carried out to clearly understand the effects of DT resulting from the activation of Akt.
Regarding apl-1, Shih and colleagues explored its antitumor activity on leukemic and prostatic cancer cell lines, focusing also on its ability to inhibit topo II. Apl-1 was highly cytotoxic (Table 1) and induced apoptosis through the dysregulation of the oxidative balance, as demonstrated by the excess of ROS and NOX (active nicotinamide adenine dinucleotide phosphate oxidase) production [36]. In addition, apl-1 reduced the activity of the PI3K/Akt/mTOR (mammalian target of rapamycin) pathway, a mechanism associated with an antitumor activity [40]. Moreover, apl-1 inhibited the relaxation of supercoiled DNA, showing an IC 50 (concentration that inhibited the 50% of DNA relaxation) value of 1.37 µM ( Table 1). As DT, apl-1 did not generate linear DNA [36], meaning that it could not stabilize the DNA cleavage complex. A further study determined that apl-1, despite increasing phosphorylation of H2A.X, did not produce DNA single strand breaks (SSBs) and DSBs, and did not increase the number of nuclear γ-H2A.X foci [41]. All these findings show that apl-1, in contrast to its oxidized derivative, acts as a topo IIα catalytic inhibitor, without inducing DNA damage.
Apl-1 inhibited the protein expression of heat shock protein 90 (Hsp90) in PC-3 and Du145 prostate cancer cells, making it a dual target inhibitor [36]. Hsp90 chaperon ensures the stability, integrity, shape, and function of critical oncogenic proteins (also called Hsp90 client proteins), which play critical roles in signal transduction, cell proliferation and survival, cell-cycle progression and apoptosis, as well as invasion, tumor angiogenesis, and metastasis [42]. Other marine topo II inhibitors, in addition to apl-1, possess this dual inhibitory activity of topo II and Hsp90, as discussed in the next sections. This is probably due to the similar ATPase domain structures of topo II and Hsp90 [43]. Other studies found that apl-1 inhibited the Wnt/β-catenin pathway through the proteasomal degradation of βcatenin [44] and the epidermal growth factor (EGF)-dependent proliferation of breast cancer cells (MCF-7 and ZR-75-1), probably by blocking the phosphorylation of EGF receptor [45]. Moving toward the later stages of the carcinogenic process, apl-1 showed antimetastatic and antiangiogenic effects: in PC-3 and Du145 cells, it inhibited cell migration and colony formation, and suppressed the EMT process induced by the transforming growth factor-β1 (TGF-β1) [36].
Overall, apl-1 exerted a marked antitumor activity in different tumor cell models and modulated multiple targets. Despite this, conflicting results are reported regarding its selective activity toward cancer cells. In normal rat macrophage cells (NR8383) and normal human skin cells (CCD966SK), the IC 50 , calculated for its cytotoxic effects, was almost 4− and 17−fold higher, respectively, than the average IC 50 calculated for tumor cells (0.39 µM) [36]. However, apl-1 induced apoptosis and blocked cell-cycle progression indiscriminately in leukemia (THP-1 and NOMO-1) cells and in bovine aortic endothelial cells [41]. Thus, the toxicological profile of apl-1 needs more in-depth studies.

Makaluvamines
Another type of alkaloids produced by sponges are pyrroloiminoquinones, which include makaluvamines and batzellines.
Makaluvamines ( Figure 4) were isolated from sponges mainly belonging to the Zyzza genus. In the 1990s, these compounds were the subject of intensive studies to evaluate their antitumor activity. All makaluvamines (A-V) exhibited a marked cytotoxic activity. [46][47][48]. In addition, makaluvamine A and C reduced the tumor mass of human ovarian carcinoma OVCAR3-xenograft in Balb/c nu/nu athymic mice (Table 1) in vivo [49].
Another type of alkaloids produced by sponges are pyrroloiminoquinones, which include makaluvamines and batzellines.
Makaluvamines ( Figure 4) were isolated from sponges mainly belonging to the Zyzza genus. In the 1990s, these compounds were the subject of intensive studies to evaluate their antitumor activity. All makaluvamines (A-V) exhibited a marked cytotoxic activity. [46][47][48]. In addition, makaluvamine A and C reduced the tumor mass of human ovarian carcinoma OVCAR3-xenograft in Balb/c nu/nu athymic mice (Table 1) in vivo [49]. Regarding the ability of makaluvamines to inhibit topo II, the results are somewhat ambiguous: makaluvamine G did not inhibit topoisomerase II; for the other makaluvamines, there are conflicting data on whether they act as topo II catalytic inhibitors or poisons. Makaluvamine N inhibited more than 90% of the relaxation of supercoiled pBR322 DNA at 5.0 μg/mL [46,49], while makaluvamines A-F modulated topo II-mediated decatenation of kinetoplast DNA (kDNA) differently [49,50]. Overall, makaluvamine B was inactive, while makaluvamine A and F were the most effective, exhibiting IC90 (concentration that inhibits 90% of kDNA decatenation) values of 41 μM and 25 μM, respectively [49]. Later, Matsumoto et al. demonstrated that different Regarding the ability of makaluvamines to inhibit topo II, the results are somewhat ambiguous: makaluvamine G did not inhibit topoisomerase II; for the other makaluvamines, there are conflicting data on whether they act as topo II catalytic inhibitors or poisons. Makaluvamine N inhibited more than 90% of the relaxation of supercoiled pBR322 DNA at 5.0 µg/mL [46,49], while makaluvamines A-F modulated topo II-mediated decatenation of kinetoplast DNA (kDNA) differently [49,50]. Overall, makaluvamine B was inactive, while makaluvamine A and F were the most effective, exhibiting IC 90 (concentration that inhibits 90% of kDNA decatenation) values of 41 µM and 25 µM, respectively [49]. Later, Matsumoto et al. demonstrated that different makaluvamines promoted the formation of cleavable complex. Makaluvamine C, D, and E (33-466 µM) cleaved radiolabeled pUC 19 DNA in the presence of human topo II in a concentration-dependent manner, although they showed fewer and weaker cleavage sites than ETO and mitoxantrone. In addition, when also testing other makaluvamines at 91 mM using a cell-free cleavage assay with radiolabeled rf M13 mp 19 plasmid DNA, they found that makaluvamine I and H were the most efficient in inducing topo II-mediated cleavage of plasmid DNA, showing a 61% and 33% of cleavage, respectively, compared to the 100% of ETO, at the same tested concentration (Table 1). In both assays, makaluvamine D and E exhibited a comparable behavior, i.e., a weak and marked formation of cleavable complex, respectively, whereas makaluvamine C was more efficient in cleaving plasmid DNA than radiolabeled pUC 19 DNA [51]. Overall, this latter study points out that makaluvamines may act as topo II poisons. In support of this hypothesis, there are various data. Firstly, makaluvamine A intercalated into DNA and induced DNA DSBs in the neutral filter elution assay, which measures the formation of protein-linked DNA DSBs, compatible with the generation of DNA cleavable complex. The effect was comparable to that of the known DNA intercalating topo II poison m-AMSA [49]. Similar findings were reported for makaluvamine C [50]. Secondly, the most active makaluvamines (A and F) were much more cytotoxic in CHO xrs-6 cells compared to CHO BR1 cells (DSBs repair-competent): they exhibited a hypersensitive factor (HF, i.e., the ratio of IC 50 on xrs-6 to that on BR1 cells) equal to 9 (for makaluvamine A) and 6 (for makaluvamine F), and thus equal to or higher than that of m-AMSA (HF = 6) [49]. Similarly, makaluvamine I showed a 5-fold lower IC 50 in xrs-6 cells (0.4 µM) compared to AA8 DNA repair-competent cells (2 µM) [51]. This evidence shows a typical behavior of DNA intercalating topo II poisons. Overall, it is very likely that some makaluvamines have the formation of cleavable complexes as their predominant mechanism and thus act as a poison. However, the lack of extensive studies does not allow to clearly identify the mechanism of topo II inhibition of the different compounds. In addition, further experiments on their activity on in vitro or in vivo models are needed to identify their potential use as anticancer agents.
Recently, different makaluvamine analogs as well as a hybrid derived from makaluvamine A and ellipticine have been found to inhibit the catalytic activity of topo II and block DNA relaxation [52,53]. However, the hybrid derivative was equally cytotoxic on both prostate cancer cells and normal fibroblasts, thus demonstrating a non-selective activity toward tumor cells [53].

Batzellines
Batzellines are a group of alkaloids isolated from the marine sponge Batzella sp. (Figure 5), structurally linked to other marine substances such as makaluvamines and discorhabdins.
Mar. Drugs 2022, 20, x FOR PEER REVIEW 9 of 52 Among them, isobatzelline A, isobatzelline C, isobatzelline D, and secobatzelline A were highly cytotoxic on a panel of pancreatic cancer cell lines (Table 1). Surprisingly, cytotoxic activity was found to be inversely proportional to the inhibition of topo IImediated DNA decatenation [54]. Isobatzelline E and batzelline B, which are not among the most cytotoxic, inhibited 95% and the 63%, respectively, of DNA decatenation at 25 μg/mL; at the same concentration, isobatzellines A, C, and D, which are the most cytotoxic, inhibited 36%, 27%, and 26% of topo II-mediated DNA decatenation, respectively. These latter significantly intercalated into DNA, while the most potent topo II inhibitor isobatzelline E was the less potent DNA-intercalating compound [54]. This different Among them, isobatzelline A, isobatzelline C, isobatzelline D, and secobatzelline A were highly cytotoxic on a panel of pancreatic cancer cell lines (Table 1). Surprisingly, cytotoxic activity was found to be inversely proportional to the inhibition of topo IImediated DNA decatenation [54]. Isobatzelline E and batzelline B, which are not among the most cytotoxic, inhibited 95% and the 63%, respectively, of DNA decatenation at 25 µg/mL; at the same concentration, isobatzellines A, C, and D, which are the most cytotoxic, inhibited 36%, 27%, and 26% of topo II-mediated DNA decatenation, respectively. These latter significantly intercalated into DNA, while the most potent topo II inhibitor isobatzelline E was the less potent DNA-intercalating compound [54]. This different behavior seems to influence the mechanism by which batzellines interfere with cell-cycle progression in a different way. In fact, only the most potent topo II inhibitor isobatzelline E blocked cells in the G2 phase of the cell cycle, whereas all the others, characterized by a less pronounced inhibitory activity on topo II and a greater ability to intercalate into DNA, blocked cell-cycle progression in the S phase [54]. Overall, these results indicate that batzellines cytotoxicity relies upon both topo II inhibition and DNA-intercalation, and that the more batzellines intercalate into the DNA, the greater the cytotoxicity of the specific compound [54]. Bearing in mind the close similarity with makaluvamines and, especially, the marked ability of isobatzellins A, C, D to intercalate with DNA, more in-depth studies should be carried out to assess whether batzellines induce DNA damage and act as topo II poisons by promoting the formation of DNA cleavable complex.

Hippospongic Acid A
Hippospongic acid A (HA-A) is a triterpene isolated from the marine sponge Hippospongia sp.
Both the natural enantiomer (R)-HA-A ( Figure 6a) and the racemate (±)-HA-A (Figure 6b), which consists of the natural stereoisomer [(R)-HA-A] and the unnatural one [(S)-HA-A], dosedependently inhibited both human and yeast topo II relaxation activity, showing an IC 50 value of 15 µM. Inhibition of topo I has also been observed, although with a higher IC 50 value (25 µM), together with the inhibition of DNA polymerases within 2-fold higher IC 50 values [55]. (R)-HA-A and (±)-HA-A at 10 µM blocked cell-cycle progression in both G1 and G2/M phases, and induced apoptosis in NUGC-3 human gastric cancer cells. The G1-phase arrest was probably due to the inhibition of DNA polymerases, while the G2/M-phase block was mainly due to the inhibition of topoisomerases [55]. Based on these results, it seems likely that several mechanisms, namely inhibition of topo I, topo II, and DNA polymerases, are involved in the compound's antitumor activity rather than the exclusive inhibition of topo II.
Mar. Drugs 2022, 20, x FOR PEER REVIEW 10 of 52 fold higher IC50 values [55]. (R)-HA-A and (±)-HA-A at 10 μM blocked cell-cycle progression in both G1 and G2/M phases, and induced apoptosis in NUGC-3 human gastric cancer cells. The G1-phase arrest was probably due to the inhibition of DNA polymerases, while the G2/M-phase block was mainly due to the inhibition of topoisomerases [55]. Based on these results, it seems likely that several mechanisms, namely inhibition of topo I, topo II, and DNA polymerases, are involved in the compound's antitumor activity rather than the exclusive inhibition of topo II.

10-Acetylirciformonin B
10-Acetylirciformonin B (10AB) (Figure 7) is a furanoterpenoid derivative isolated with other terpenoid-derived metabolites from the marine sponge Ircinia sp. [56]. Among all the isolated compounds, 10AB was the most cytotoxic (Table 1). Interestingly, it seems to exert a selective cytotoxic effect for cancer cells: in HL-60 cells, 10AB at 6.0 μM induced 80% apoptosis; in rat alveolar NR8383macrophages, it suppressed cell viability by 18.3% [57]. A previous study reported that in HL-60 cells 10AB induced Casp-dependent apoptosis and promoted the formation of DNA DSBs, accompanied by the phosphorylation of H2A.X and checkpoint kinase 2 (Chk2), two markers of nuclear DNA damage [58]. A more recent study showed that 10AB-induced DNA damage may be related to its ability to inhibit topo IIα catalytic activity: 10AB (1.5, 3.0, 6.0, and 12.0 μM) inhibited DNA relaxation without producing linear DNA (like the topo IIα poison ETO), and at 3 μM decreased the protein expression of topo IIα in HL-60 cells. All these findings indicate that 10AB could act as a DNA damaging agent and compromise the topo IIα catalytic cycle, leading to apoptotic cell death [57]. In this regard, in HL-60 cells 10AB (1.5, 3.0, and 6.0 μM) disrupted MMP (mitochondrial membrane potential) and reduced the protein expression of anti-apoptotic proteins (Bcl-2 and Bcl-X) as well as of other proteins involved in the apoptotic process, as X-linked inhibitor of apoptosis protein (XIAP) and survivin. 10AB also generated ROS, activated the mitogenactivated protein kinases (MAPK)/extracellular signal-regulated kinase (ERK) pathway, and inhibited the PI3K/PTEN/Akt/mTOR signaling pathway [57]. Akt transcriptionally regulates the expression of hexokinase II (HK-II) [59]. HKs are enzymes that catalyze the Among all the isolated compounds, 10AB was the most cytotoxic (Table 1). Interestingly, it seems to exert a selective cytotoxic effect for cancer cells: in HL-60 cells, 10AB at 6.0 µM induced 80% apoptosis; in rat alveolar NR8383macrophages, it suppressed cell viability by 18.3% [57]. A previous study reported that in HL-60 cells 10AB induced Caspdependent apoptosis and promoted the formation of DNA DSBs, accompanied by the phosphorylation of H2A.X and checkpoint kinase 2 (Chk2), two markers of nuclear DNA damage [58]. A more recent study showed that 10AB-induced DNA damage may be related to its ability to inhibit topo IIα catalytic activity: 10AB (1.5, 3.0, 6.0, and 12.0 µM) inhibited DNA relaxation without producing linear DNA (like the topo IIα poison ETO), and at 3 µM decreased the protein expression of topo IIα in HL-60 cells. All these findings indicate that 10AB could act as a DNA damaging agent and compromise the topo IIα catalytic cycle, leading to apoptotic cell death [57]. In this regard, in HL-60 cells 10AB (1.5, 3.0, and 6.0 µM) disrupted MMP (mitochondrial membrane potential) and reduced the protein expression of anti-apoptotic proteins (Bcl-2 and Bcl-X) as well as of other proteins involved in the apoptotic process, as X-linked inhibitor of apoptosis protein (XIAP) and survivin. 10AB also generated ROS, activated the mitogen-activated protein kinases (MAPK)/extracellular signal-regulated kinase (ERK) pathway, and inhibited the PI3K/PTEN/Akt/mTOR signaling pathway [57]. Akt transcriptionally regulates the expression of hexokinase II (HK-II) [59]. HKs are enzymes that catalyze the phosphorylation of glucose, i.e., the first step of glycolysis, and are upregulated in many tumors characterized by a high glycolytic activity. Moreover, HK-II has a pro-survival activity and protects mitochondria against mitochondrial apoptotic cell death by interfering with anti-and pro-apoptotic proteins and decreasing ROS generation [59]. Thus, downregulation of HK allows the shift of cancer cells' metabolism to oxidative phosphorylation and increases ROS levels, which leads to cell death. The demonstrated ability of 10AB to downregulate p-Akt protein expression may lead to the downregulation of HK-II. This means that 10AB-induced apoptosis seems to be mediated by topo IIα inhibition and oxidative stress, as well as the perturbation of metabolic and cell survival pathways.

Manoalide-Like Sesterterpenoids
In 1994, Kobayashi et al. isolated four sesterterpenes from the sponge Hyrtios erecta [60]. Among them, manoalide 25-acetals ( Figure 8) inhibited the DNA-unknotting activity of calf thymus topo II, showing an IC 50 value of about 25 µM. In addition, it exhibited antitumor activity on CDF 1 mice inoculated whit P388 leukemia cells, with a T/C% score (the ratio between the tumor volume in the treated group and in the untreated control group) of 150% at 1 mg/kg (Table 1) [60].

Manoalide-Like Sesterterpenoids
In 1994, Kobayashi et al. isolated four sesterterpenes from the sponge Hyrtios erecta [60]. Among them, manoalide 25-acetals ( Figure 8) inhibited the DNA-unknotting activity of calf thymus topo II, showing an IC50 value of about 25 μM. In addition, it exhibited antitumor activity on CDF1 mice inoculated whit P388 leukemia cells, with a T/C% score (the ratio between the tumor volume in the treated group and in the untreated control group) of 150% at 1 mg/kg (Table 1) [60].  All the derivates were tested on multiple leukemia cell lines ( Table 1). The compounds L2, L4, M7, and M9, bearing a 24R, 25S configuration, were the most effective, thus assuming that the cytotoxic activity was configuration-dependent [61]. The administration of M7 to immunodeficient athymic mice (1 μg/kg every day for 33 days) reduced the tumor growth of Molt-4 xenograft by about 66%, without affecting body weight [61].
M7 has been shown to act as a catalytic inhibitor of topo IIα. Moreover, it inhibited DNA relaxation with an IC50 value of 1.18 μM and promoted the formation of supercoiled DNA products in the presence of topo IIα [61]. Compared to manoalide 25-acetals, the inhibitory activity of M7 toward topo II was greatly higher, although purified topo II from two different organisms were used: human for M7 [61] and calf thymus for manoalide 25acetals [60]. The topo IIα catalytic inhibitor activity was associated with DNA damage, as demonstrated by its ability to promote the phosphorylation of ATM, Chk2, and H2A.X and to induce DNA DSBs at 0.75 μM in Molt-4 cells. M7-induced DNA damage has been found to activate apoptotic cell death, as indicated by and the activation of Casp-3, -8, and All the derivates were tested on multiple leukemia cell lines ( Table 1). The compounds L2, L4, M7, and M9, bearing a 24R, 25S configuration, were the most effective, thus assuming that the cytotoxic activity was configuration-dependent [61]. The administration of M7 to immunodeficient athymic mice (1 µg/kg every day for 33 days) reduced the tumor growth of Molt-4 xenograft by about 66%, without affecting body weight [61].
M7 has been shown to act as a catalytic inhibitor of topo IIα. Moreover, it inhibited DNA relaxation with an IC 50 value of 1.18 µM and promoted the formation of supercoiled DNA products in the presence of topo IIα [61]. Compared to manoalide 25-acetals, the inhibitory activity of M7 toward topo II was greatly higher, although purified topo II from two different organisms were used: human for M7 [61] and calf thymus for manoalide 25-acetals [60]. The topo IIα catalytic inhibitor activity was associated with DNA damage, as demonstrated by its ability to promote the phosphorylation of ATM, Chk2, and H2A.X and to induce DNA DSBs at 0.75 µM in Molt-4 cells. M7-induced DNA damage has been found to activate apoptotic cell death, as indicated by and the activation of Casp-3, -8, and -9, the disruption of MMP, and the cleavage of PARP [61].

Heteronemin
Another marine sesterterpenoid-type product, heteronemin ( Figure 10), was separated from the Hippospongia sp. sponge [62]. Heteronemin was able to induce apoptosis as well as inhibit the proliferation different cancer cell lines [63,64]. Interestingly, in hepatocellular carcinoma HA22T HA59T cells, heteronemin induced both apoptosis and ferroptosis [65], a non-apopt programmed cell death mechanism characterized by the iron-dependent accumulation lipid ROS [66]. Due to the well-known occurrence of multi-drug resistance caused by deregulation of apoptosis [67], the evidence that heteronemin is a ferroptosis induce very interesting.
Deepening the molecular mechanisms involved in heteronemin's cytotoxicity prostate cancer cells, Lee et al. found that it induced both autophagy and apoptosis [ Autophagy promotes either cell survival or cell death in a context-and cell-depend manner [68]. Autophagy induced by heteronemin seems to possess a cytoprotective ef rather than a pro-apoptotic one [62]. Indeed, heteronemin (1.28 and 2.56 μM) activa LC3-B II (LC3-phosphatidylethanolamine conjugate), a marker of autophagy, but at 5 μM, when apoptosis was markedly induced, autophagy was blocked. Moreover, the p treatment with two autophagy inhibitors (3-methyladenine and chloroquine) raised percentage of LNCaP apoptotic cells [62]. Similarly, in A498 renal carcinoma cells, inhibition of autophagy increased the pro-apoptotic activity of heteronemin [69].
The marine sesterterpene completely inhibited DNA relaxation in the cell-free D cleavage assay and reduced topo IIα protein expression in LNCaP cells, which resulted the block of the total catalytic activity of the enzyme. Heteronemin did not produce lin DNA, thus assuming its inability to stabilize DNA-topo II cleavable complex [62].
Mechanisms other than the inhibition of topo II are possibly involved in antitumor activity of heteronemin.
Heteronemin suppressed the expression of Hsp90 and that of its client proteins, t being able to modulate the expression of oncogenic proteins and transcription fact involved in tumorigenesis [62]. Moreover, it blocked NF-κB activation via proteaso inhibition in K562 cells [70] and the activation of ERK1/2 and STAT3 in breast cancer c [63,64]. In LnCaP cells, heteronemin (1.28-5.12 μM) disrupted MMP, foster Heteronemin was able to induce apoptosis as well as inhibit the proliferation of different cancer cell lines [63,64]. Interestingly, in hepatocellular carcinoma HA22T and HA59T cells, heteronemin induced both apoptosis and ferroptosis [65], a non-apoptotic programmed cell death mechanism characterized by the iron-dependent accumulation of lipid ROS [66]. Due to the well-known occurrence of multi-drug resistance caused by the deregulation of apoptosis [67], the evidence that heteronemin is a ferroptosis inducer is very interesting.
Deepening the molecular mechanisms involved in heteronemin's cytotoxicity in prostate cancer cells, Lee et al. found that it induced both autophagy and apoptosis [62]. Autophagy promotes either cell survival or cell death in a context-and cell-dependent manner [68]. Autophagy induced by heteronemin seems to possess a cytoprotective effect rather than a pro-apoptotic one [62]. Indeed, heteronemin (1.28 and 2.56 µM) activated LC3-B II (LC3-phosphatidylethanolamine conjugate), a marker of autophagy, but at 5.12 µM, when apoptosis was markedly induced, autophagy was blocked. Moreover, the pre-treatment with two autophagy inhibitors (3-methyladenine and chloroquine) raised the percentage of LNCaP apoptotic cells [62]. Similarly, in A498 renal carcinoma cells, the inhibition of autophagy increased the pro-apoptotic activity of heteronemin [69].
The marine sesterterpene completely inhibited DNA relaxation in the cell-free DNA cleavage assay and reduced topo IIα protein expression in LNCaP cells, which resulted in the block of the total catalytic activity of the enzyme. Heteronemin did not produce linear DNA, thus assuming its inability to stabilize DNA-topo II cleavable complex [62].
Mechanisms other than the inhibition of topo II are possibly involved in the antitumor activity of heteronemin.
Heteronemin suppressed the expression of Hsp90 and that of its client proteins, thus being able to modulate the expression of oncogenic proteins and transcription factors involved in tumorigenesis [62]. Moreover, it blocked NF-κB activation via proteasome inhibition in K562 cells [70] and the activation of ERK1/2 and STAT3 in breast cancer cells [63,64]. In LnCaP cells, heteronemin (1.28-5.12 µM) disrupted MMP, fostering mitochondrial dysfunction. Due to the overproduction of ROS and Ca 2+ release, heteronemin promoted oxidative and endoplasmic reticulum (ER) stress, therefore triggering the unfolded protein response (UPR) signaling network to re-establish ER homeostasis [62]. Oxidative and ER stress results from the activation of protein tyrosine phosphatases (PTPs) [62]. PTPs modulate the levels of cellular protein tyrosine phosphorylation and control cell growth, differentiation, survival, and death. PTPs exert both tumor-suppressive and oncogenic functions in a context-dependent manner [71]. Pre-treatment of LnCaP with a PTP inhibitor reduced heteronemin-induced ROS generation and ER stress, thus demonstrating that in this experimental setting, PTPs exhibits a tumor-suppressive mechanism and participates in the antitumor activity of heteronemin [62].
Oxidative stress was also involved in the heteronemin-induced anticancer effects in Molt-4 cells. In this cell line, it enhanced γ-H2A.X protein expression, probably due to apoptosis rather than DNA damage occurrence. Indeed, although γ-H2A.X is the most sensitive biomarker of DNA damage, its measure by ELISA and/or immunoblotting allows to evaluate the total H2A.X protein levels in a sample, but apoptotic cells with pan-nuclear H2A.X expression cannot be differentiated from surviving cells, which may alter H2A.X quantification. In contrast, the fluorescent microscopic quantification of foci is the most sensitive approach and can distinguish between pan-nuclear staining and foci formation [72]. The increased γ-H2A.X protein expression induced by heteronemin in Molt-4 cells was demonstrated by using Western Blot, as for all the other sponge-derived topo II inhibitors, and, unlike other studies, the expression of other DNA damage-related proteins was not evaluated. Thus, it is not clear whether heteronemin induces DNA damage in this experimental model.
In vivo, heteronemin inhibited the growth of Molt-4 and LnCaP xenograft in Balb/c nude mice and in immunodeficient athymic mice, respectively, treated with 0.31 µg/g (three times a week for 24 days) and 1 mg/kg (every day for 29 days) of heteronemin [62,73].
However, considering the marked antitumor activity of SS1, a possible in vivo study of this compound should be considered as well.

SS1, SS2
, and TPL were cytotoxic on many tumor cell lines [74] (Table 1). All th compounds inhibited DNA relaxation, reaching almost 100% inhibition at the high tested concentration (20 μg/mL). There was no information regarding the production linear DNA [74]. Topo II inhibition was associated with DNA damage: SS1 (0.0625μg/mL) increased the protein expression of γ-H2A.X and, at 0.0625 μg/mL; it also indu DNA DSBs in Molt-4 cells [74]. Although SS2 enhanced γ-H2A.X protein expression, difficult to associate this event exclusively with DNA damage since neither other mar of DNA damage nor the formation of DSBs have been evaluated. SS1, like heterone [62], promoted ROS generation and ER stress and induced mitochondrial apoptosis [ In addition, SS1 shared with heteronemin the ability to inhibit Hsp90 protein express
Mar. Drugs 2022, 20, x FOR PEER REVIEW 15 o and that of its client proteins [74]. Although Lai and colleagues investigated SS1 m deeply than TPL, the latter was also tested in a Molt-4 cells xenograft animal mo showing that its daily administration (1.14 μg/g) for 33 days inhibited almost 50% xenograft tumor growth in male immunodeficient athymic mice [74]. Authors justif their choice to only test TPL in vivo by the small amount they were able to isolate for other two compounds. However, considering the marked antitumor activity of SS possible in vivo study of this compound should be considered as well.
Both compounds strongly inhibited either the topo II-catalyzed DNA relaxation a the protein expression of topo IIα in Molt-4 [75,76] and K562 cells [76]. For D relaxation, xestoquinone showed an IC50 value of 0.094 μM [76], and halenaquin showed an IC50 about 5.5-fold lower (0.017 μM) [75]. These results indicate that they as potent catalytic inhibitors of topo II. However, they did not form DNA-topo II cleav complex, since no linear DNA was observed in the cell-free DNA relaxation assay [75, Additionally, molecular docking studies reported that xestoquinone was capable binding topo II with a docking score of −26.9, although a similar or even a lower va Halenaquinone and xestoquinone exhibited a comparable cytotoxic activity [75,76]. In vivo, the administration of halenaquinone (1 µg/g for 30 days) and xestoquinone (1 µg/g for 50 days) suppressed the growth of Molt-4 xenograft in immunodeficient athymic mice, without affecting body weight (Table 1) [75,76].
Both compounds strongly inhibited either the topo II-catalyzed DNA relaxation and the protein expression of topo IIα in Molt-4 [75,76] and K562 cells [76]. For DNA relaxation, xestoquinone showed an IC 50 value of 0.094 µM [76], and halenaquinone showed an IC 50 about 5.5-fold lower (0.017 µM) [75]. These results indicate that they act as potent catalytic inhibitors of topo II. However, they did not form DNA-topo II cleavage complex, since no linear DNA was observed in the cell-free DNA relaxation assay [75,76]. Additionally, molecular docking studies reported that xestoquinone was capable of binding topo II with a docking score of −26.9, although a similar or even a lower value was observed for topo I (−24.0) and Hsp90 (−15.5) [76]. These results demonstrate that the compound can bind to multiple targets. Xestoquinone (7.84 µM) treatment of Molt-4 cells markedly increased the expression of multiple DNA damage markers (p-Chk1, p-Chk2, and γ-H2A.X), pointing out that its topo II catalytic activity inhibition induced DNA damage [76]. No markers of DNA damage were evaluated for the congener halenaquinone. Nonetheless, given the close similarities in the antitumor mechanisms of both compounds, it cannot be excluded that congener halenaquinone was a topo II catalytic inhibitor. In fact, both compounds have been shown to inhibit the activity of histone deacetylase (HDAC) in vitro [75,76] and in a Molt-4 xenograft mouse in vivo model [76]. This is not so surprising, as several studies report that topo II and HDAC mutually modulate their activity [43]. In addition to this, ROS overproduction [75,76], induction of ER stress, and binding to protein Hsp90 [76] recorded for both compounds led to apoptosis. Notably, the two polycyclic quinone-type metabolites promoted both apoptotic pathways as the disruption of MMP, decrease in anti-apoptotic proteins (Bcl-2, Bcl-X, Bid), increase in pro-apoptotic ones (Bax, Bak) (all markers of intrinsic apoptosis), and activation of Casp-8 and -9 (markers of extrinsic apoptosis) were observed in Molt-4 and K562 cells [75,76].
Alongside halenaquinone and xestoquinone, other polycyclic quinone-type metabolites were isolated from the sponge Xestospongia sp. [77]. All studied compounds inhibited topo II (Table 1). Among those, adociaquinone B ( Figure 13) was the most potent with an IC 90 (the concentration inducing the 90% of inhibition) < 11 µM and 78 µM for DNA decatenation and relaxation, respectively. In contrast to xestoquinone and halenaquinone, adociaquinone B was a non-intercalating DNA topo II poison. In fact, it strongly promoted the formation of the enzyme-DNA cleavable complex to the same extent as mitoxantrone, a known topo II poison [78]. However, in contrast to mitoxantrone, adociaquinone B did not intercalate into DNA since it was not able to displace ethidium bromide from calf thymus DNA [77]. Secoadociaquinone A and B, two other Xestospongia sp. metabolites, inhibited topo II activity in the cell-free DNA decatenation assay without exhibiting cytotoxicity since they were unable to permeate cell membranes. Thus, it is not sufficient to test the inhibitory activity of topo II only on cell-free systems, as very often the physicochemical properties of the tested compounds prevent their entry into cells and consequently a possible interaction with intracellular targets, such as topo II [77].
Mar. Drugs 2022, 20, x FOR PEER REVIEW 16 o Alongside halenaquinone and xestoquinone, other polycyclic quinone-t metabolites were isolated from the sponge Xestospongia sp. [77]. All studied compou inhibited topo II (Table 1). Among those, adociaquinone B ( Figure 13) was the most po with an IC90 (the concentration inducing the 90% of inhibition) < 11 μM and 78 μM DNA decatenation and relaxation, respectively. In contrast to xestoquinone halenaquinone, adociaquinone B was a non-intercalating DNA topo II poison. In fac strongly promoted the formation of the enzyme-DNA cleavable complex to the sa extent as mitoxantrone, a known topo II poison [78]. However, in contrast mitoxantrone, adociaquinone B did not intercalate into DNA since it was not abl displace ethidium bromide from calf thymus DNA [77]. Secoadociaquinone A and B, other Xestospongia sp. metabolites, inhibited topo II activity in the cell-free D decatenation assay without exhibiting cytotoxicity since they were unable to permeate membranes. Thus, it is not sufficient to test the inhibitory activity of topo II only on c free systems, as very often the physicochemical properties of the tested compou prevent their entry into cells and consequently a possible interaction with intracell targets, such as topo II [77].

Leptosin F
Leptosin F (LEP, Figure 14) is an indole derivative containing sulphur that is derived from the fungus Leptoshaeria sp., which grows on the marine alga Sargassum tortile [82].

Leptosin F
Leptosin F (LEP, Figure 14) is an indole derivative containing sulphur that is derived from the fungus Leptoshaeria sp., which grows on the marine alga Sargassum tortile [82]. Yanagihara and colleagues demonstrated that LEP potently inhibited the growth of RPMI-8402 T cell acute lymphoblastic leukemia cells-more powerfully than ETO and with an IC50 value in the nM range-and induced apoptosis [82]. A pro-apoptotic effect has also been reported for LEP in normal human embryo kidney cells (293 cell line), where it activated Casp-3 at doses as low as 1 to 10 μM [82]. These results could indicate that LEP does not act selectively against cancer cells, but rather on all rapidly proliferating cells.
The in vitro kDNA decatenation assay revealed its ability to inhibit topo II [82]. Gel electrophoresis of the kDNA after decatenation assay showed that LEP did not act as a catalytic inhibitor of topo II, as the authors instead stated. Further studies would be necessary to define the exact mechanism of interaction between LEP and the enzyme. Moreover, since the compound concentration required to exert cytotoxic activity on RPMI-8402 cells was extremely lower (nM range) than that required to inhibit topo II (µM range), the cytotoxicity of LEP at the cellular level might involve other pathways in addition to the inhibition of topo II.

Pericosine A
Pericosine A (PA, Figure 15) is a metabolite produced by a strain of Periconia byssoides OUPS-N133, a marine fungus originally separated from the sea hare Aplysia kurodai [83]. Some studies reported the ability of PA to induce growth inhibition on different cancer cell lines [83,84] (Table 2). Furthermore, in mice inoculated with P388 leukemic cells, PA increased the median survival days compared to vehicle (13.0 versus 10.7 days) Yanagihara and colleagues demonstrated that LEP potently inhibited the growth of RPMI-8402 T cell acute lymphoblastic leukemia cells-more powerfully than ETO and with an IC 50 value in the nM range-and induced apoptosis [82]. A pro-apoptotic effect has also been reported for LEP in normal human embryo kidney cells (293 cell line), where it activated Casp-3 at doses as low as 1 to 10 µM [82]. These results could indicate that LEP does not act selectively against cancer cells, but rather on all rapidly proliferating cells.
The in vitro kDNA decatenation assay revealed its ability to inhibit topo II [82]. Gel electrophoresis of the kDNA after decatenation assay showed that LEP did not act as a catalytic inhibitor of topo II, as the authors instead stated. Further studies would be necessary to define the exact mechanism of interaction between LEP and the enzyme. Moreover, since the compound concentration required to exert cytotoxic activity on RPMI-8402 cells was extremely lower (nM range) than that required to inhibit topo II (µM range), the cytotoxicity of LEP at the cellular level might involve other pathways in addition to the inhibition of topo II.

Pericosine A
Pericosine A (PA, Figure 15) is a metabolite produced by a strain of Periconia byssoides OUPS-N133, a marine fungus originally separated from the sea hare Aplysia kurodai [83].

Leptosin F
Leptosin F (LEP, Figure 14) is an indole derivative containing sulphur that is derived from the fungus Leptoshaeria sp., which grows on the marine alga Sargassum tortile [82]. Yanagihara and colleagues demonstrated that LEP potently inhibited the growth of RPMI-8402 T cell acute lymphoblastic leukemia cells-more powerfully than ETO and with an IC50 value in the nM range-and induced apoptosis [82]. A pro-apoptotic effect has also been reported for LEP in normal human embryo kidney cells (293 cell line), where it activated Casp-3 at doses as low as 1 to 10 μM [82]. These results could indicate that LEP does not act selectively against cancer cells, but rather on all rapidly proliferating cells.
The in vitro kDNA decatenation assay revealed its ability to inhibit topo II [82]. Gel electrophoresis of the kDNA after decatenation assay showed that LEP did not act as a catalytic inhibitor of topo II, as the authors instead stated. Further studies would be necessary to define the exact mechanism of interaction between LEP and the enzyme. Moreover, since the compound concentration required to exert cytotoxic activity on RPMI-8402 cells was extremely lower (nM range) than that required to inhibit topo II (µM range), the cytotoxicity of LEP at the cellular level might involve other pathways in addition to the inhibition of topo II.

Pericosine A
Pericosine A (PA, Figure 15) is a metabolite produced by a strain of Periconia byssoides OUPS-N133, a marine fungus originally separated from the sea hare Aplysia kurodai [83]. Some studies reported the ability of PA to induce growth inhibition on different cancer cell lines [83,84] (Table 2). Furthermore, in mice inoculated with P388 leukemic cells, PA increased the median survival days compared to vehicle (13.0 versus 10.7 days) Some studies reported the ability of PA to induce growth inhibition on different cancer cell lines [83,84] (Table 2). Furthermore, in mice inoculated with P388 leukemic cells, PA increased the median survival days compared to vehicle (13.0 versus 10.7 days) ( Table 2). In the same study, the authors reported that PA at 100-300 mM inhibited topo II and at 449 µM inhibited the epidermal growth factor receptor (EGFR) by 40−70%. Since PA seems to exert its inhibitory effects on topo II at very high concentrations, it is unlikely that this mechanism of action was responsible for its in vitro and in vivo antitumor effects. The inhibition of EGFR, a protein kinase known to promote cell proliferation and counteract apoptosis [85], could be a more plausible mechanism [83]. The lack of important information on its antitumor activity in vitro and in vivo does not permit a clear characterization of the anticancer activity of PA. Therefore, further experiments should be conducted to fully understand the potential usefulness of PA in the oncological area.

Marinactinone B
Marinactinone B (MB, Figure 16) is a γ-pyrone derivate isolated from the bacterial strain Marinactinospora thermotolerans SCSIO 00606, found in the sediments of the northern South China Sea [86].
Mar. Drugs 2022, 20, x FOR PEER REVIEW 30 of 52 (Table 2). In the same study, the authors reported that PA at 100-300 mM inhibited topo II and at 449 μM inhibited the epidermal growth factor receptor (EGFR) by 40−70%. Since PA seems to exert its inhibitory effects on topo II at very high concentrations, it is unlikely that this mechanism of action was responsible for its in vitro and in vivo antitumor effects. The inhibition of EGFR, a protein kinase known to promote cell proliferation and counteract apoptosis [85], could be a more plausible mechanism [83]. The lack of important information on its antitumor activity in vitro and in vivo does not permit a clear characterization of the anticancer activity of PA. Therefore, further experiments should be conducted to fully understand the potential usefulness of PA in the oncological area.

Marinactinone B
Marinactinone B (MB, Figure 16) is a γ-pyrone derivate isolated from the bacterial strain Marinactinospora thermotolerans SCSIO 00606, found in the sediments of the northern South China Sea [86]. MB was evaluated for its anticancer activity against breast (MCF-7), pancreatic (SW1990), hepatic (HepG2 and SMCC-7721), lung (NCI-H460), and cervical (HeLa) cancer cell lines. It exhibited cytotoxicity at medium-elevated concentration values only against SW1990 (99 μM) and SMCC-7721 (45 μM) cell lines. It was also a very weak inhibitor of topo II with an IC50 value of 607 μM [86]. With such a high IC50 value, MB is not a promising compound per se. However, given its interaction with topo II, MB could constitute the basis for the development of analogues with antitumor activity.

Aspergiolide A
Aspergiolide A (ASP, Figure 17) is an anthracycline [87] isolated from Aspergillus glaucus, which was obtained from the marine sediment around mangrove roots harvested in the Chinese province of Fujian [88].  MB was evaluated for its anticancer activity against breast (MCF-7), pancreatic (SW1990), hepatic (HepG2 and SMCC-7721), lung (NCI-H460), and cervical (HeLa) cancer cell lines. It exhibited cytotoxicity at medium-elevated concentration values only against SW1990 (99 µM) and SMCC-7721 (45 µM) cell lines. It was also a very weak inhibitor of topo II with an IC 50 value of 607 µM [86]. With such a high IC 50 value, MB is not a promising compound per se. However, given its interaction with topo II, MB could constitute the basis for the development of analogues with antitumor activity.

Aspergiolide A
Aspergiolide A (ASP, Figure 17) is an anthracycline [87] isolated from Aspergillus glaucus, which was obtained from the marine sediment around mangrove roots harvested in the Chinese province of Fujian [88].
Mar. Drugs 2022, 20, x FOR PEER REVIEW 30 of 52 (Table 2). In the same study, the authors reported that PA at 100-300 mM inhibited topo II and at 449 μM inhibited the epidermal growth factor receptor (EGFR) by 40−70%. Since PA seems to exert its inhibitory effects on topo II at very high concentrations, it is unlikely that this mechanism of action was responsible for its in vitro and in vivo antitumor effects. The inhibition of EGFR, a protein kinase known to promote cell proliferation and counteract apoptosis [85], could be a more plausible mechanism [83]. The lack of important information on its antitumor activity in vitro and in vivo does not permit a clear characterization of the anticancer activity of PA. Therefore, further experiments should be conducted to fully understand the potential usefulness of PA in the oncological area.

Marinactinone B
Marinactinone B (MB, Figure 16) is a γ-pyrone derivate isolated from the bacterial strain Marinactinospora thermotolerans SCSIO 00606, found in the sediments of the northern South China Sea [86]. MB was evaluated for its anticancer activity against breast (MCF-7), pancreatic (SW1990), hepatic (HepG2 and SMCC-7721), lung (NCI-H460), and cervical (HeLa) cancer cell lines. It exhibited cytotoxicity at medium-elevated concentration values only against SW1990 (99 μM) and SMCC-7721 (45 μM) cell lines. It was also a very weak inhibitor of topo II with an IC50 value of 607 μM [86]. With such a high IC50 value, MB is not a promising compound per se. However, given its interaction with topo II, MB could constitute the basis for the development of analogues with antitumor activity.

Aspergiolide A
Aspergiolide A (ASP, Figure 17) is an anthracycline [87] isolated from Aspergillus glaucus, which was obtained from the marine sediment around mangrove roots harvested in the Chinese province of Fujian [88].  ASP was cytotoxic on different human and murine cancer cell lines (Table 2) [88].

Wang et al. have delved into the antitumor efficacy of ASP in vitro and in vivo.
The compound induced Casp-dependent apoptosis as early as 12 h after treatment [87]. In addition, ASP increased γ-H2A.X protein expression. Considering its anthracyclinic structure, it has been hypothesized that the inhibition of topo II could be involved in its apoptotic activty. The kDNA decatenation assay demonstrated that ASP inhibited the enzyme in a fashion comparable to DOXO. The results of in vivo experiments in H22 hepatoma-bearing mice and on BEL-7402 cancer xenografts (Table 2) corroborated the in vitro findings. ASP reduced tumor volume dose-dependently in H22 mice and showed comparable activity to that of DOXO (2 mg/kg). In BEL-7402 xenografts, ASP showed significantly milder activity than DOXO. Interestingly, in both in vivo models, ASP altered mice body weight considerably less than DOXO, suggesting less toxicity than the benchmark anthracycline [87]. The study also investigated the pharmacokinetic profile of ASP, which has been shown to distribute throughout the body in a perfusion-and bloodflow-dependent manner, and was able to concentrate in tumor tissues. Additionally, ASP penetrated the blood brain barrier. No clinical signs of toxicity or organs morphological changes were found in mice treated with the maximal tolerable dose of ASP (more than 400 mg/kg) [87], which is considerably higher than the dose necessary to produce the antitumor effects. The genotoxic potential of ASP was also evaluated via the in vivo bone marrow erythrocyte micronucleus assay. The number of micronuclei produced following treatment with ASP was comparable to the negative control, suggesting that ASP was not genotoxic [87].
Anthracyclines are proven to cause significant cardiotoxicity and electrocardiogram abnormalities including long QT syndrome, a potentially lethal condition induced by several drugs [89]. Long QT syndrome has been found to be caused by the blockade of hERG (human ether-a-go-go-related gene), a gene codifying the pore-forming subunit of the potassium channels, which are relevant for cardiac repolarization [90]. Thus, Li et al. investigated the in vitro inhibitory rates of ASP on the hERG current. The resulting values indicated that ASP was unable to inhibit the hERG channel, and hence it is unlikely to produce cardiotoxicity through this mechanism [87].
On the whole, the studies reported above identify ASP as an attractive candidate in the oncological area. However, further studies will be necessary to clarify whether the effects of the compound can be attributed to topo II inhibition.

Jadomycin DS
Jadomycin DS (JAD, Figure 18) is a polyketide produced by the bacterium Streptomyces venezuelae ISP5230 under stress conditions [91].  [87]. In addition, ASP increased γ-H2A.X protein expression. Considering its anthracyclinic structure, it has been hypothesized that the inhibition of topo II could be involved in its apoptotic activty. The kDNA decatenation assay demonstrated that ASP inhibited the enzyme in a fashion comparable to DOXO. The results of in vivo experiments in H22 hepatoma-bearing mice and on BEL-7402 cancer xenografts (Table 2) corroborated the in vitro findings. ASP reduced tumor volume dose-dependently in H22 mice and showed comparable activity to that of DOXO (2 mg/kg). In BEL-7402 xenografts, ASP showed significantly milder activity than DOXO. Interestingly, in both in vivo models, ASP altered mice body weight considerably less than DOXO, suggesting less toxicity than the benchmark anthracycline [87]. The study also investigated the pharmacokinetic profile of ASP, which has been shown to distribute throughout the body in a perfusion-and bloodflow-dependent manner, and was able to concentrate in tumor tissues. Additionally, ASP penetrated the blood brain barrier. No clinical signs of toxicity or organs morphological changes were found in mice treated with the maximal tolerable dose of ASP (more than 400 mg/kg) [87], which is considerably higher than the dose necessary to produce the antitumor effects. The genotoxic potential of ASP was also evaluated via the in vivo bone marrow erythrocyte micronucleus assay. The number of micronuclei produced following treatment with ASP was comparable to the negative control, suggesting that ASP was not genotoxic [87].
Anthracyclines are proven to cause significant cardiotoxicity and electrocardiogram abnormalities including long QT syndrome, a potentially lethal condition induced by several drugs [89]. Long QT syndrome has been found to be caused by the blockade of hERG (human ether-a-go-go-related gene), a gene codifying the pore-forming subunit of the potassium channels, which are relevant for cardiac repolarization [90]. Thus, Li et al. investigated the in vitro inhibitory rates of ASP on the hERG current. The resulting values indicated that ASP was unable to inhibit the hERG channel, and hence it is unlikely to produce cardiotoxicity through this mechanism [87]. On the whole, the studies reported above identify ASP as an attractive candidate in the oncological area. However, further studies will be necessary to clarify whether the effects of the compound can be attributed to topo II inhibition.

Jadomycin DS
Jadomycin DS (JAD, Figure 18) is a polyketide produced by the bacterium Streptomyces venezuelae ISP5230 under stress conditions [91].  JAD shares three common features with ETO and DOXO: (i) a lactone ring, (ii) a quinone moiety, and (iii) a copper-mediated DNA cleavage activity. To estimate the molecular interactions of JAD, binding studies were conducted using a nuclear magnetic resonance spectroscopy (NMR) method that allows the identification of molecules capable of binding a ligand-protein with binding affinity (K D ) in the µM−mM range [92,93]. JAD bound topo IIβ. However, the overall K D for JAD-topo IIβ complex was equal to 9.4 mM, suggesting that the bond formed between JAD and topo IIβ is weak [91]. The high binding constant between the compound and topo IIβ does not depict JAD as an attractive anti-cancer drug. Moreover, JAD interacted unselectively with several unrelated enzymes including serum albumin [91], making it difficult to determine its actual mode of action and severely compromise its hypothetic in vivo application.

3.6.
2RA was cytotoxic [94], blocked the cell cycle in the G2/M phase, and triggered Caspdependent apoptosis in HepG2 cells. To determine whether 2RA was able to interact with human topo IIα, a molecular docking study was performed, demonstrating that 2RA was able to bind to the active receptor pocket with a binding energy of −7.84 kJ/mol [94]. In addition, an increased formation of hydrogen bonds in the protein-ligand complex was recorded compared to the protein, indicating that the protein-ligand complex had a higher binding affinity and stability than the protein [94]. However, in vitro studies should be conducted to demonstrate that 2RA is a topo II α inhibitor.

Streptomyces sp. VITJS4 Ethyl Acetate Crude Extract
Streptomyces sp. VITJS4 bacterial strain was isolated from the marine environment in Tamil Nadu, India [95]. VITJS4 ethyl acetate crude extract exerted cytotoxic effects against HepG2 and HeLa cancer cells with identical IC50 values of 50 μg/mL and induction of apoptosis. Hence, this would suggest a cell line-independent mechanism of action [95]. Gas chromatography-mass spectrum analysis (GC-MS) identified a phthalate derivative, namely 1, 2-benzenedicarboxylic acid, mono-(2-ethylhexyl) ester, as the major bioactive metabolite among the 52 bioactive compounds of the ethyl acetate extract, which is probably responsible for the activity observed on the two human cancer cell lines. Molecular docking analysis was conducted to assess the interaction between the compound and topo IIα. What emerged is the formation of bonds at the active pocket of protein with a binding energy of −5.87 kJ/mol [95].

Sulochrin
Sulochrin ( Figure 20) is a benzophenone derivative isolated from Aspergillus falconensis after cultivating it on a solid rice medium containing 3.5% of (NH4)2SO4 [96]. 2RA was cytotoxic [94], blocked the cell cycle in the G2/M phase, and triggered Caspdependent apoptosis in HepG2 cells. To determine whether 2RA was able to interact with human topo IIα, a molecular docking study was performed, demonstrating that 2RA was able to bind to the active receptor pocket with a binding energy of −7.84 kJ/mol [94]. In addition, an increased formation of hydrogen bonds in the protein-ligand complex was recorded compared to the protein, indicating that the protein-ligand complex had a higher binding affinity and stability than the protein [94]. However, in vitro studies should be conducted to demonstrate that 2RA is a topo II α inhibitor.

Streptomyces sp. VITJS4 Ethyl Acetate Crude Extract
Streptomyces sp. VITJS4 bacterial strain was isolated from the marine environment in Tamil Nadu, India [95]. VITJS4 ethyl acetate crude extract exerted cytotoxic effects against HepG2 and HeLa cancer cells with identical IC 50 values of 50 µg/mL and induction of apoptosis. Hence, this would suggest a cell line-independent mechanism of action [95]. Gas chromatography-mass spectrum analysis (GC-MS) identified a phthalate derivative, namely 1, 2-benzenedicarboxylic acid, mono-(2-ethylhexyl) ester, as the major bioactive metabolite among the 52 bioactive compounds of the ethyl acetate extract, which is probably responsible for the activity observed on the two human cancer cell lines. Molecular docking analysis was conducted to assess the interaction between the compound and topo IIα. What emerged is the formation of bonds at the active pocket of protein with a binding energy of −5.87 kJ/mol [95].

Sulochrin
Sulochrin ( Figure 20) is a benzophenone derivative isolated from Aspergillus falconensis after cultivating it on a solid rice medium containing 3.5% of (NH 4 ) 2 SO 4 [96]. Sulochrin was cytotoxic on L5178Y murine lymphoma cell line with an IC50 value of 5.1 μM [96]. The compound was not cytotoxic on MDA-MB-231 human breast cancer cells; however, at a concentration of 70 μM, it dramatically reduced cell migration [96]. Molecular docking studies indicated the interaction of sulochrin with topo II. With a free binding energy of −12.11 kcal/mol, the compound showed a robust stability through the formation of several stable bonds within the active sites, comparable to that exerted by DOXO (−16.28 kcal/mol). Molecular docking studies also demonstrated the capacity of the compound to even bind within the active sites of two further enzymes: the cyclindependent kinase 2 (CDK2) involved in cell-cycle progression, and the matrix metalloproteinase 13 (MMP-13) involved in the EMT process, with moderate free binding energies [96].

3-hydroxyholyrine A
3-hydroxyholyrine A (3HA, Figure 21) is an indolocarbazole produced by the marine-derived bacterium Streptomyces strain OUCMDZ-3118 in the presence of 5hydroxy-L-tryptophan [97]. 3HA exerted cytotoxic effects on many tumor cell lines (Table 2) and reduced the expression of the antiapoptotic protein survivin more potently than ETO in MKN45 cells [97]. In supercoiled plasmid DNA relaxation assay, 3HA potently inhibited the activity of topo IIα enzyme at 1.0, 5.0, and 10.0 μM. Of note, 3HA exhibited an inhibitory activity at concentrations lower than ETO (50 μM). The inhibition of topo IIα resulted in DNA damage, as demonstrated by the concentration-dependent increase in the expression of γ-H2A.X. Sulochrin was cytotoxic on L5178Y murine lymphoma cell line with an IC 50 value of 5.1 µM [96]. The compound was not cytotoxic on MDA-MB-231 human breast cancer cells; however, at a concentration of 70 µM, it dramatically reduced cell migration [96]. Molecular docking studies indicated the interaction of sulochrin with topo II. With a free binding energy of −12.11 kcal/mol, the compound showed a robust stability through the formation of several stable bonds within the active sites, comparable to that exerted by DOXO (−16.28 kcal/mol). Molecular docking studies also demonstrated the capacity of the compound to even bind within the active sites of two further enzymes: the cyclin-dependent kinase 2 (CDK2) involved in cell-cycle progression, and the matrix metalloproteinase 13 (MMP-13) involved in the EMT process, with moderate free binding energies [96].

3-Hydroxyholyrine A
3-hydroxyholyrine A (3HA, Figure 21) is an indolocarbazole produced by the marinederived bacterium Streptomyces strain OUCMDZ-3118 in the presence of 5-hydroxy-Ltryptophan [97]. Sulochrin was cytotoxic on L5178Y murine lymphoma cell line with an IC50 value of 5.1 μM [96]. The compound was not cytotoxic on MDA-MB-231 human breast cancer cells; however, at a concentration of 70 μM, it dramatically reduced cell migration [96]. Molecular docking studies indicated the interaction of sulochrin with topo II. With a free binding energy of −12.11 kcal/mol, the compound showed a robust stability through the formation of several stable bonds within the active sites, comparable to that exerted by DOXO (−16.28 kcal/mol). Molecular docking studies also demonstrated the capacity of the compound to even bind within the active sites of two further enzymes: the cyclindependent kinase 2 (CDK2) involved in cell-cycle progression, and the matrix metalloproteinase 13 (MMP-13) involved in the EMT process, with moderate free binding energies [96].

3-hydroxyholyrine A
3-hydroxyholyrine A (3HA, Figure 21) is an indolocarbazole produced by the marine-derived bacterium Streptomyces strain OUCMDZ-3118 in the presence of 5hydroxy-L-tryptophan [97]. 3HA exerted cytotoxic effects on many tumor cell lines ( Table 2) and reduced the expression of the antiapoptotic protein survivin more potently than ETO in MKN45 cells [97]. In supercoiled plasmid DNA relaxation assay, 3HA potently inhibited the activity of topo IIα enzyme at 1.0, 5.0, and 10.0 μM. Of note, 3HA exhibited an inhibitory activity at concentrations lower than ETO (50 μM). The inhibition of topo IIα resulted in DNA damage, as demonstrated by the concentration-dependent increase in the expression of γ-H2A.X. 3HA exerted cytotoxic effects on many tumor cell lines ( Table 2) and reduced the expression of the antiapoptotic protein survivin more potently than ETO in MKN45 cells [97]. In supercoiled plasmid DNA relaxation assay, 3HA potently inhibited the activity of topo IIα enzyme at 1.0, 5.0, and 10.0 µM. Of note, 3HA exhibited an inhibitory activity at concentrations lower than ETO (50 µM). The inhibition of topo IIα resulted in DNA damage, as demonstrated by the concentration-dependent increase in the expression of γ-H2A.X.

Wakayin
Wakayin ( Figure 22) is a pyrroloiminoquinone alkaloid isolated from an ascidian, commonly called sea squirt, belonging to the species Clavelina [99]. In early studies evaluating its activity, wakayin induced cytotoxic effects on the human colon HCT-116 cancer cell line with an IC50 value of 0.5 μg/mL. On the same cell line, it inhibited topo II enzyme at a concentration of 250 μM [99]. Moreover, wakayin exhibited a higher cytotoxicity on DSBs repair-deficient CHO xrs-6 cells than on DSBs repair-proficient CHO BR1 cells. Their IC50 ratio was indeed 9.8, higher than that of ETO corresponding to 7.0. Those results clearly indicate DSB induction as a mechanism involved in the cytotoxicity of wakayin [100]. Taking into account this evidence and the planar quinonic structure of wakayin, it was hypothesized and then demonstrated that wakayin inhibited the decatenation of kDNA in a concentration-dependent manner in the range of 40 to 133 μg/mL [100]. However, the difference between the concentration inhibiting the purified enzyme (40-133 μg/mL) and the concentration exerting the cytotoxic effects (0.5 μg/mL) suggests that other mechanisms, not just topo II inhibition, could contribute to wakayin-induced DNA damage.

Ascididemin
Ascididemin (ASC, Figure 23) is a pyridoacridine alkaloid isolated from the mediterranean ascidian Cystodytes dellechiajei collected near the Balearic Islands [101] as well as from Okinawan ascidian Didemnum sp., from Kerama Islands [102]. It has been reported that ASC was 10-fold more cytotoxic in CHO xrs-6 (DSBs repair deficient) than in CHO BR1 (DSBs repair proficient) cells, while exhibiting identical toxicity in CHO-BR1 (SSB repair-proficient) and CHO-EM9 (SSB repair-deficient) cells, raising the hypothesis that DSBs were involved in its in vitro anticancer activity [103]. Moreover, ASC was cytotoxic on human leukemia, colon, and breast cancer cell lines In early studies evaluating its activity, wakayin induced cytotoxic effects on the human colon HCT-116 cancer cell line with an IC 50 value of 0.5 µg/mL. On the same cell line, it inhibited topo II enzyme at a concentration of 250 µM [99]. Moreover, wakayin exhibited a higher cytotoxicity on DSBs repair-deficient CHO xrs-6 cells than on DSBs repair-proficient CHO BR1 cells. Their IC 50 ratio was indeed 9.8, higher than that of ETO corresponding to 7.0. Those results clearly indicate DSB induction as a mechanism involved in the cytotoxicity of wakayin [100]. Taking into account this evidence and the planar quinonic structure of wakayin, it was hypothesized and then demonstrated that wakayin inhibited the decatenation of kDNA in a concentration-dependent manner in the range of 40 to 133 µg/mL [100]. However, the difference between the concentration inhibiting the purified enzyme (40-133 µg/mL) and the concentration exerting the cytotoxic effects (0.5 µg/mL) suggests that other mechanisms, not just topo II inhibition, could contribute to wakayin-induced DNA damage.

Wakayin
Wakayin ( Figure 22) is a pyrroloiminoquinone alkaloid isolated from an ascidian, commonly called sea squirt, belonging to the species Clavelina [99]. In early studies evaluating its activity, wakayin induced cytotoxic effects on the human colon HCT-116 cancer cell line with an IC50 value of 0.5 μg/mL. On the same cell line, it inhibited topo II enzyme at a concentration of 250 μM [99]. Moreover, wakayin exhibited a higher cytotoxicity on DSBs repair-deficient CHO xrs-6 cells than on DSBs repair-proficient CHO BR1 cells. Their IC50 ratio was indeed 9.8, higher than that of ETO corresponding to 7.0. Those results clearly indicate DSB induction as a mechanism involved in the cytotoxicity of wakayin [100]. Taking into account this evidence and the planar quinonic structure of wakayin, it was hypothesized and then demonstrated that wakayin inhibited the decatenation of kDNA in a concentration-dependent manner in the range of 40 to 133 μg/mL [100]. However, the difference between the concentration inhibiting the purified enzyme (40-133 μg/mL) and the concentration exerting the cytotoxic effects (0.5 μg/mL) suggests that other mechanisms, not just topo II inhibition, could contribute to wakayin-induced DNA damage.

Ascididemin
Ascididemin (ASC, Figure 23) is a pyridoacridine alkaloid isolated from the mediterranean ascidian Cystodytes dellechiajei collected near the Balearic Islands [101] as well as from Okinawan ascidian Didemnum sp., from Kerama Islands [102]. It has been reported that ASC was 10-fold more cytotoxic in CHO xrs-6 (DSBs repair deficient) than in CHO BR1 (DSBs repair proficient) cells, while exhibiting identical toxicity in CHO-BR1 (SSB repair-proficient) and CHO-EM9 (SSB repair-deficient) cells, raising the hypothesis that DSBs were involved in its in vitro anticancer activity [103]. Moreover, ASC was cytotoxic on human leukemia, colon, and breast cancer cell lines It has been reported that ASC was 10-fold more cytotoxic in CHO xrs-6 (DSBs repair deficient) than in CHO BR1 (DSBs repair proficient) cells, while exhibiting identical toxicity in CHO-BR1 (SSB repair-proficient) and CHO-EM9 (SSB repair-deficient) cells, raising the hypothesis that DSBs were involved in its in vitro anticancer activity [103]. Moreover, ASC was cytotoxic on human leukemia, colon, and breast cancer cell lines [102]. Cytotoxicity elicited by ASC (Table 3) was related to the induction of Casp-dependent apoptosis, even at the lowest concentrations [102,104]. Meanwhile, it inhibited the growth of the non-malignant African green monkey kidney cell line BSC-1, revealing a lack of selectivity against cancer cells [103].
ASC was shown to inhibit topo II activity at a concentration equal to 30 µM [105]. Nearly 10 years later, Dassonneville and colleagues evaluated its interaction with topo II and demonstrated that this compound can (i) inhibit DNA ligation after it has been cleaved by topo II, and (ii) stimulate DNA cleavage with most cleavage sites having a C on the side of the cleaved bond [104]. Based on these results, ASC could be defined as a site-specific topo II poison for the purified enzyme, although its activity appeared to be inferior compared to the positive control ETO [104]. However, the capability of ASC to function as a topo II poison was not demonstrated in cellular assays. Indeed, comparing the cytotoxic activity of ASC on human leukemia cells sensitive (HL-60) or resistant (HL-60/MX2) to mitoxantrone, ASC was cytotoxic with similar IC 50 values (0.48 µM for HL-60 and 0.65 µM for HL-60/MX2) [104]. Matsumoto and coworkers performed a cell-free assay to clarify the mechanism of action of ASC. The results proved that ASC was able to cleave the DNA in a concentration-and time-dependent manner, even in the absence of topo II. Moreover, experimental results demonstrated (i) the generation of ROS, (ii) that antioxidants treatment protected against DNA cleavage, and (iii) that cells deficient in ROS-induced damage repair system were more susceptible to ASC. On the whole, those results suggest that ROS production is involved in the cytotoxicity of ASC [106]. The production of ROS could be due to the direct reduction of ASC iminoquinone heterocyclic ring to semiquinone, with production of H 2 O 2 [106]. Considering the potential of ASC to intercalate in DNA, it is probable that ROS production occurs in proximity of the nucleic acid, thereby producing DNA damage [106].
Umemura and coworkers evaluated different GA3P formulations bearing high (>80%) and low (<20%) lactic acid percentage (GA3Pl+ and GA3Pl−, respectively) [108]. Both preparations of GA3P inhibited kDNA decatenation with similar IC 50 values (0.048 µg/mL for GA3P+ and 0.052 µg/mL for GA3P−), proving that GA3P was a topo II inhibitor and that lactic acid percentage had no impact on topo II inhibition [108]. Gel electrophoresis of pT2GN plasmid DNA revealed that GA3P+ did not induce the accumulation of cleavable complexes and acted as a catalytic inhibitor. Furthermore, the analysis of plasmid DNA showed that GA3P+, when simultaneously added to teniposide, inhibited the stabilization of teniposide-induced cleavable complexes [108].
In a large panel of cells, the polysaccharide slightly inhibited cell proliferation with GI 50 values ranging from 0.67 to 11 µg/mL [108]. However, no further cellular assays were undertaken to elucidate the cytotoxic activity or the possible death mechanism exerted by the compound. Despite evidence showing that GA3P+ was a topo II catalytic inhibitor, its chemical profile and high molecular weight can hamper its entry into the nucleus and its interaction with DNA or topo II. Certainly, further studies will be required to clarify the mechanism of action of GA3P against cancer cells.

Echinoside A
Echinoside A (ECH, Figure 24) is a saponin isolated from the sea cucumber Holothuria nobilis (Selenka), an echinoderm retrieved from the sea ground of the Dongshan Island (P. R. China) [109]. ECH exerted a broad-spectrum anticancer activity against a panel of 26 human and murine cancer cell lines with very similar IC50 ranging from 1.0 to 6.0 μM [109]. Fluorescent TUNEL staining of ECH-treated HL-60 cells and DNA fragmentation indicated that the observed cytotoxicity resulted from Casp-dependent apoptosis. The potent effects observed in cancer cells were confirmed by in vivo experiments on animal cancer models (Table 3).
An extensive and comprehensive set of in vitro experiments with topo IIα enzyme was conducted to investigate its topo II inhibitor activity. The results indicate that ECH effectively reduced the pBR322 plasmid DNA relaxation and suppressed kDNA decatenation [109]. An assay with top IIα extracted from HL-60 cells proved that ECH 0.5 μM induced the formation of stable cleavage complexes, which is a common mechanism for topo II poisons, along with intercalation in DNA. However, two different experiments (Table 3) reported that ECH was a non-intercalative agent, even at high concentrations [109]. The activity of ECH toward topo IIα-DNA binding was evaluated using a fluorescence anisotropy assay, which revealed that ECH inhibited the binding between the enzyme and DNA. Molecular docking studies clarified that ECH, through its sugar moiety, established strong hydrogen bonds with the DNA binding site of topo IIα, working as a catalytic inhibitor that competes with DNA for the substrate [109]. Further studies explored the effects of ECH on the cleavage/religation equilibrium using a cell-free assay. ECH produced an increase in DNA cleavage and enhanced DSBs formation, without significant effects on religation [109]. The ability of ECH to promote DNA cleavage without affecting DNA ligation makes it similar to topo II poisons such as ellipticin, genistein, and quinolones [110,111], which act with the same mechanism. However, ECH has been found to possess the peculiar characteristics of i) blocking the ECH exerted a broad-spectrum anticancer activity against a panel of 26 human and murine cancer cell lines with very similar IC 50 ranging from 1.0 to 6.0 µM [109]. Fluorescent TUNEL staining of ECH-treated HL-60 cells and DNA fragmentation indicated that the observed cytotoxicity resulted from Casp-dependent apoptosis. The potent effects observed in cancer cells were confirmed by in vivo experiments on animal cancer models (Table 3).
An extensive and comprehensive set of in vitro experiments with topo IIα enzyme was conducted to investigate its topo II inhibitor activity. The results indicate that ECH effectively reduced the pBR322 plasmid DNA relaxation and suppressed kDNA decatenation [109]. An assay with top IIα extracted from HL-60 cells proved that ECH 0.5 µM induced the formation of stable cleavage complexes, which is a common mechanism for topo II poisons, along with intercalation in DNA. However, two different experiments (Table 3) reported that ECH was a non-intercalative agent, even at high concentrations [109]. The activity of ECH toward topo IIα-DNA binding was evaluated using a fluorescence anisotropy assay, which revealed that ECH inhibited the binding between the enzyme and DNA. Molecular docking studies clarified that ECH, through its sugar moiety, established strong hydrogen bonds with the DNA binding site of topo IIα, working as a catalytic inhibitor that competes with DNA for the substrate [109].
Further studies explored the effects of ECH on the cleavage/religation equilibrium using a cell-free assay. ECH produced an increase in DNA cleavage and enhanced DSBs formation, without significant effects on religation [109]. The ability of ECH to promote DNA cleavage without affecting DNA ligation makes it similar to topo II poisons such as ellipticin, genistein, and quinolones [110,111], which act with the same mechanism. However, ECH has been found to possess the peculiar characteristics of (i) blocking the noncovalent binding of topo IIα to DNA by competing with DNA for the DNA-binding domain of the enzyme, and (ii) hindering topo IIα-mediated pre-strand passage cleavage/religation equilibrium. Taken together, the studies presented above suggest that ECH is a potent non-intercalative topo II inhibitor with a peculiar mechanism of action. It acts as a topoisomerase poison (stabilization of cleavable complexes and induction of DSBs) and a catalytic inhibitor (inhibition on the topo II-DNA binding, interference with the pre-strand passage cleavage/religation equilibrium). Due to these characteristics, it constitutes a promising starting point for the development of anticancer drugs based on topo II inhibition

Eusynstyelamide B
Eusynstyelamide B (EUB, Figure 25) is a bis-indole alkaloid extracted from the marine ascidian Didemnum candidum found in the Great Barrier Reef [112].
Mar. Drugs 2022, 20, x FOR PEER REVIEW 40 of 52 noncovalent binding of topo IIα to DNA by competing with DNA for the DNA-binding domain of the enzyme, and ii) hindering topo IIα-mediated pre-strand passage cleavage/religation equilibrium. Taken together, the studies presented above suggest that ECH is a potent non-intercalative topo II inhibitor with a peculiar mechanism of action. It acts as a topoisomerase poison (stabilization of cleavable complexes and induction of DSBs) and a catalytic inhibitor (inhibition on the topo II-DNA binding, interference with the pre-strand passage cleavage/religation equilibrium). Due to these characteristics, it constitutes a promising starting point for the development of anticancer drugs based on topo II inhibition

Eusynstyelamide B
Eusynstyelamide B (EUB, Figure 25) is a bis-indole alkaloid extracted from the marine ascidian Didemnum candidum found in the Great Barrier Reef [112]. EUB was able to induce cytotoxicity in breast MDA-MB-231 and LNCaP prostate cancer cells [112,113]. Table 3 reports the differences in gene and protein expression between MDA-MB-231 and LNCaP cell lines, emphasizing the cell line-specific mechanisms of EUB. The COMET assay and the quantitative evaluation of γ-H2A.X foci supported the production of DNA damage via DSBs in both cell lines.
With the aim to investigate whether the observed DNA damage derived from the direct interaction of EUB with DNA, a displacement assay and a DNA melting temperature analysis were performed. Both demonstrated that EUB did not directly interact with DNA but instead acted as a topo II poison [113]. EUB was also highly cytotoxic in two non-transformed cell lines (NFF primary human neonatal foreskin fibroblasts and RWPE-1 epithelial prostate cell line), with IC50 values even lower than that reported on tumor cell lines. NFF and RWPE-1 cells are highly proliferating and express high levels of topo IIα [114]. This means that the effects of EUB were not specific for cancer cells. Further in vitro and in vivo studies have to be performed to assess the safety profile of EUB. EUB was able to induce cytotoxicity in breast MDA-MB-231 and LNCaP prostate cancer cells [112,113]. Table 3 reports the differences in gene and protein expression between MDA-MB-231 and LNCaP cell lines, emphasizing the cell line-specific mechanisms of EUB. The COMET assay and the quantitative evaluation of γ-H2A.X foci supported the production of DNA damage via DSBs in both cell lines.
With the aim to investigate whether the observed DNA damage derived from the direct interaction of EUB with DNA, a displacement assay and a DNA melting temperature analysis were performed. Both demonstrated that EUB did not directly interact with DNA but instead acted as a topo II poison [113]. EUB was also highly cytotoxic in two' nontransformed cell lines (NFF primary human neonatal foreskin fibroblasts and RWPE-1 epithelial prostate cell line), with IC 50 values even lower than that reported on tumor cell lines. NFF and RWPE-1 cells are highly proliferating and express high levels of topo IIα [114]. This means that the effects of EUB were not specific for cancer cells. Further in vitro and in vivo studies have to be performed to assess the safety profile of EUB.

Conclusions
Of the compounds discussed in this review, only a few acts as topo II poisons (adociaquinone B and EUB) and as catalytic inhibitors (neo and apl-1). Several others exhibit topo II inhibitory activity but, due to the paucity of experimental evidence, their mode of inhibition has not been elucidated, making it difficult to establish their mechanism of action.
Although topo II inhibitors, particularly topo II poisons, are successfully used as anticancer agents, the occurrence of drug resistance and severe side effects, such as cardiotoxicity and the development of secondary malignancies, limit their use [43]. An approach to overcome these limitations could be the use of dual inhibitors. Multiple marine-derived compounds described in this review such as 25-acetals manoalide, xestoquinone, HA-A, and M7, inhibit both topo I and topo II [55,60,61,76], while for others, topo II inhibitory activity is accompanied by the inhibition of Hsp90 [36,62,74] or HDAC [75,76]. The resulting advantages are manifold. Simultaneous inhibition of topo I and topo II could reduce the possible onset of resistance. The same advantage can be achieved by inhibiting topo II and Hsp90 [43]. Concerning topo II and HDAC inhibition, HDAC inhibition-mediated histone hyperacetylation increases chromatin decondensation and DNA accessibility. These effects may promote topo II binding and enhance topo II inhibiting activity [43]. Among the marine compounds presented in this review, heteronemin is the most interesting. Indeed, its cytotoxic activity was highly multimechanistic, with inhibition of the catalytic activities of both topo I and topo II and inhibition of Hsp90, associated with oxidative and ER stress. However, the dual inhibitors are often compounds with a high molecular weight [119], which could limit their druggability and their safety profile as well as indicate that their pharmacokinetics should be thoroughly explored Another issue to consider is the ability of topo II inhibitors to cause DNA lesions that, if not repaired or not cytotoxic, could lead to chromosome aberrations and secondary malignancies such as leukemias [120]. Although topo II catalytic inhibitors are usually associated with no or limited direct DNA damage [121], some marine-derived topo II catalytic inhibitors presented in this review induce DNA DSBs and/or increase the protein expression of DNA damage-related proteins. Thus, it would be of great relevance to clarify whether their genotoxicity results from their topo II catalytic inhibition or involves different mechanisms. A further concern related to the toxicological profile is the lack of selectivity toward cancer cells exhibited by some marine compounds, which prompts more extensive studies on non-transformed cells to assess the safety of such molecules.
Lastly, some marine compounds exhibited a strong binding affinity for topo II, demonstrated through molecular docking studies. Among those, the most interesting are neo, ECH, and sulochrin, which are characterized by a binding energy of -61.8, -39.21, and -12.11 kcal/mol, respectively. However, in some cases, this interaction has not been confirmed by cellular assays, making it difficult to know whether topo II binding leads to the actual inhibition of the enzyme activity. Thus, at least DNA decatenation and/or relaxation assays are necessary to confirm their topo II inhibitory activity. These cell-free assays certainly provide early indications of the effective inhibition of topo II. However, they may not be sufficient because, as shown for secoadociaquinone A and B and GA3P [77,108], their inhibitory activity on the purified enzyme does not necessarily lead to the inhibition of topo II at the cellular level.
In conclusion, in this review, we reported current studies on marine-derived compounds targeting topo II, highlighted their pharmacological potential, and discussed their toxicological issues.