## 3 Metabolomics Applied to Marine Organisms in Cancer Studies
### Structure Elucidation of Bioactive Molecules from Sponges
The first tested compound was already known: (\(-\))-discorhabdin L, and it showed IC\({}_{50}\) equal to 0.33 \(\upmu\)g/mL. The other two interesting compounds were new. They are two new discorhabdin analogs, i.e., (\(-\))-1-acetyl-discorhabdin L and (\(+\))-1-octadecarienoyl-discorhabdin L, exhibiting IC\({}_{50}\) of 1.1 \(\upmu\)g/mL and 25.6 \(\upmu\)g/mL, respectively [99]. Metabolite analyses of the marine sponge _Theonella savinhoei_ performed by MS and \({}^{1}\)H-NMR showed the presence of theonellamides. In particular, Theopalauamide was an already known compound whose cytotoxic activity was evaluated on the HTC-116 colon carcinoma cell line (IC\({}_{50}\) = 2.8 uM), while two new compounds (5-_cis_-Apoa-theopalauanide reported in Figure 5 and theonellamide K) exhibited a cytotoxic activity on the same cell line, with IC\({}_{50}\) of 21.8 and 3.5 uM, respectively [100]. We should consider, however, that sponges are holobionts that live in symbiosis with many bacterial species. These latter can represent up to 35% of the sponge's weight [101]. Most of the symbiotic bacteria able to produce bioactive compounds are potential candidates for biotechnological applications [102; 103; 104]. From the marine sponge _Petrosia ficiformis_ sampled in the Mediterranean Sea (Milos, Greece), a bacterial strain of Streptomyces sp. (SBT348) has been isolated. Thanks to a metabolomic approach performed through LC-MS analyses, it was possible to isolate a known compound, namely Petrocidin A (see Figure 5 for chemical strcture), which had cytotoxic effect towards HL-60 (human promyelocytic cell) and HT29 cell lines with IC\({}_{50}\) 3.9 and 5.3 ug/mL, respectively. In addition, from the Streptomyces sp. (SBT348), a new compound whose structure has been elucidated (2,3-Dihydroxybenzamide, Figure 5) showed activity against the same cell lines with different IC\({}_{50}\) values of 5.5 ug/mL and 3.8 ug/mL, respectively [105]. In another study, from samples of the Red Sea sponge _Callyspongia_ sp., a strain of _Nocardiopsis_ sp. (UR67) has been isolated, from which Nocardiotide A has been detected and characterized through MS analyses. Surprisingly, this compound showed activity against several different cancer cell lines, such as the CT26 (murine colon carcinoma, IC\({}_{50}\) = 12 uM/mL), HeLa (human cervix carcinoma, IC\({}_{50}\) = 11 uM/mL) and MM.1S (IC\({}_{50}\) = 8 uM/mL) cancer cell lines [106]. Two bacterial strains belonging to Nocardi sp. (UR 86) and _Nocardiopsis_ sp. (UR 92) were isolated from another sponge belonging to _Amphimedon_ sp., coming from the Red Sea. These bacterial strains were cultured in different culture conditions and their crude extracts were analysed with an MS approach. The extract of _Nocardi_ sp. (UR86) caused cellular inhibition of several cancer cell lines, such as HepG2, MCF7 and CaCo2, with IC\({}_{50}\) values of 3.1 ug/mL, 3.9 ug/mL and 14.4 ug/mL, respectively. Similarly, extracts of _Nocardiopsis_ sp. (UR92) were active against the same cancer cell lines with IC\({}_{50}\) values of 3.7 ug/mL, 14.7 ug/mL and 14.3 ug/mL, respectively [107]. A newer approach is present in the research recently conducted by [PERSON] et al. [108]. They co-cultured two Actinomycetes to stimulate them to produce metabolites that would not have been produced if the two strains were cultured separately. These two actinomycetes were isolated from two sponges: _Micromonospora_ sp. was isolated from _Callyspongia_ sp., while _Actinokinespora_ sp. was isolated from _Spheciospongia_ vogabunda_. Several compounds were detected using LC-MS metabolomics analyses, but only one was noteworthy in terms of its cytotoxic effect: the \(N\)-(2-hydroxyphenyl)-acetamide (see Figure 5). This was active against several cancer cell lines, such as HCT116 (colorectal carcinoma), HepG-2 (hepatocellular carcinoma) and MCF7 (mammary gland), with IC\({}_{50}\) values ranging from 10 to 36 uM [108]. Extracts/compounds from sponges and their cytotoxic activities are summarized in Table 1.