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Marine Fungi: A Source of Potential Anticancer Compounds

Metabolites from marine fungi have hogged the limelight in drug discovery because of their promise as therapeutic agents. A number of metabolites related to marine fungi have been discovered from various sources which are known to possess a range of activities as antibacterial, antiviral and anticancer agents. Although, over a thousand marine fungi based metabolites have already been reported, none of them have reached the market yet which could partly be related to non-comprehensive screening approaches and lack of sustained lead optimization. The origin of these marine fungal metabolites is varied as their habitats have been reported from various sources such as sponge, algae, mangrove derived fungi, and fungi from bottom sediments. The importance of these natural compounds is based on their cytotoxicity and related activities that emanate from the diversity in their chemical structures and functional groups present on them. This review covers the majority of anticancer compounds isolated from marine fungi during 2012–2016 against specific cancer cell lines.


INTRODUCTION
Marine fungi are important source of secondary metabolites useful for the drug discovery purposes. Even though marine fungi are less explored in comparison to their terrestrial counterparts, a number of useful hits have been obtained from the drug discovery perspective adding to their importance in the natural product discovery (Molinski et al., 2009;Butler et al., 2014), which have yielded a wide range of chemically diverse agents with antibacterial, antiviral and anticancer properties in animal systems. Starting with the celebrated example of cephalosporins, marine fungi have provided unique chemical skeletons that could be used to develop drugs of clinical importance (Bhadury et al., 2006;Saleem et al., 2007;Javed et al., 2011;Sithranga and Kathiresan, 2011). Fungi, in general, have been generous source of drugs as evidenced by the isolation of many drugs in use such as paclitaxel, camptothecin, vincristine, torreyanic acid and cytarabine to name a few. In this light, marine are important not just from the perspective of new drugs but also as a source of new scaffolds that can be modified further to obtain the desired action. Despite significant progress in the drug discovery that has provided treatment for some major ailments, minor infections, and epidemics; new drugs are required to combat global resistance to drugs for existing diseases and new infections that have been reported in recent times (such as SARS, dengue and Zika viruses). In addition to drug resistance in diseases such as tuberculosis and malaria, cancer & HIV-AIDS (Passaes and Sáez-Cirión, 2014) have been biological targets with limited success toward therapeutics development.
In addition to terrestrial sources, oceans have been a huge reservoir of a variety of biologically active compounds, which have often been the resulting metabolite of marine life (König et al., 2006;Chen G. et al., 2014;Agrawal et al., 2016;Deshmukh et al., 2017). Though, why marine fungi produce such complex and diverse set of metabolites in not fairly understood, it is largely assumed that they play key roles in chemical defense and communication. The biosynthesis of these metabolites in dependent on ecological, physical and biological factors and, therefore, small changes in these conditions can generate entirely new set of metabolites (Pejin and Maja, 2017). The contribution of marine based therapeutics can be gauged from the fact that during 1981-2002, more than half of the FDA approved drugs had originated from marine life. Most of the marine based drugs have come from invertebrates (sponges, tunicates, mollusks, and bryozoans); two-thirds of which, belong to the class of non-ribosomal peptides. Some of these are already in the market ( Polymixin B, pristinamycin, gramicidin, vancomycin, bleomycin, actinomycin D) as antibiotic and anti-cancer agents while several others are in clinical trials (Manoalide, discodermolide) (Singh et al., 2008). In this regard, there have not been many reports of drugs from marine fungi that are used clinically which can be partly attributed to lack of systematic and comprehensive approaches as well as lack of optimization which has precluded a large number of potential hits from becoming actual drugs. Therefore, metabolites marine fungi constitute a group of underrepresented resource for discovering novel therapeutics (Imhoff, 2016).
Several classes of chemically distinct metabolites from marine fungi have been reported in recent years which have a wide range of activities against different targets (Wu et al., , 2016. From marine fungi alone, over thousand metabolites have been reported to have potential to be developed as drugs (Gomes et al., 2015), with several as anticancer compounds (please also see a detailed review, Bugni and Ireland, 2004 for historical inputs, taxonomy, ecological roles, distribution and chemistry as well biological activities of marine fungi), none of these have reached the market till now. However, for majority of these findings, complete taxonomy studies, biological targets and modes of interaction have not been identified yet. Due to these limitations, in this review, we cover anticancer compounds reported from marine fungi obtained from different sources such as deep-sea sediments, algae, sponge, mangrove endophytic and other marine fungi, discovered during 2012-2016 with a focus on summarizing the important findings and highlighting the lead compounds. Wherever explored, the biological targets and efficacies have been discussed as well. Novel anticancer compounds reported from marine fungi are given in Supplementary  Table 1. They are arranged on the basis of sources the fungi isolated.

METABOLITES ISOLATED FROM DEEP-SEA SEDIMENT FUNGI
Deep-sea fungi inhabit at depths of thousand meters or below the surface (Swathi et al., 2013) where the sea environments are extreme; which are typically characterized by the absence of sunlight irradiation, predominantly low temperature, high hydrostatic pressure, and oligotrophy. Many reports indicate abundance and diversity of fungi in these environments (Hua et al., 2011;Mahé et al., 2013). Here, we present an account of metabolites reported from the deep-sea fungi during 2012-2016 that have displayed anticancer activities in various cell lines.
Linear peptides, simplicilliumtides A, E, G, and H (1-4; Figure 1) were isolated from a culture broth of the deep-seaderived fungal strain Simplicillium obclavatum EIODSF 020e collected in the East Indian Ocean. Simplicilliumtides A and G showed weak cytotoxicity toward human leukemia HL-60 cell line with IC 50 values of 64.7 and 100 µM, and simplicilliumtides E and H showed weak cytotoxicity toward K562 cell line with IC 50 values of 39.4 and 73.5 µM (Liang et al., 2016). Using a combination of fermentation and subsequent chromatographic separation, acaromycin A (5) and (+)-cryptosporin (6; Figure 1) were isolated from the deep-sea derived fungus Acaromyces ingoldii FS121 which was obtained from the South Sea in China. Using a combinaion of one and two-dimensional NMR as well as mass spectroscopic techniques, the chemical structures were elucidated and the absolute configuration was further determined by circular dichroism (CD) experiments. Compounds (5) and (6) exhibited considerable growth inhibition against tumor cell lines MCF-7, NCI-H460, SF-268, and HepG-2 with IC 50 values <10 µM. The inhibitory effect of compound (5) against MCF-7 cell line was comparable to cytoxicity of cisplatin which was used as a positive control (Gao et al., 2016a). A new tetranorlabdane diterpenoid, asperolide E (7; Figure 1) was isolated from the deep sea sediment-derived fungus Aspergillus wentii SD-310. The cytotoxicity of compound (7) was evaluated against HeLa, MCF-7, and NCI-H446 cell lines which showed IC 50 values of 10.0, 11.0, and 16.0 µM respectively .
Asperethers A-E (8-12; Figure 1), five new 20-norisopimarane diterpenoids having a 14,16-cyclic ether unit and a unique 6/6/6/5 tetracyclic skeleton, were discovered from the culture extract of Aspergillus wentii SD-310 from the deep-sea sediment sample. The chemical structure of these compounds was determined by mass spectrometry and NMR techniques ( 1 H NMR, COSY, HSQC, HMBC) and the absolute configurations were supported by NOESY, X-ray crystallography, CD and computational methods. Compounds (8-12) displayed cytotoxic activities against A549 cell line with the IC 50 values of 20, 16, 19, 17, and 20 µM, respectively, which were moderately higher than the positive control Adriamycin .

COMPOUNDS FROM MANGROVE ENDOPHYTIC FUNGI
Using one strain many compounds (OSMAC) approach, spirobrocazines C (69) and brocazine G (70) were obtained from mangrove-derived fungus Penicillium brocae MA-231 (Figure 3). Their chemical structures and absolute stereochemical configurations were determined by spectroscopic analysis, computational calculations and X-ray diffraction. Spirobrocazine C (69) showed moderate activity against A2780 cells (IC 50 59 µM) while compound (70) showed strong activity against A2780 and A2780 CisR cell with the IC 50 values of 664 and 661 nM respectively, which were much better than that of the positive control cisplatin, which gave IC 50 values of 1.67 and 12.63 µM respectively . 2,4-Dihydroxy-6nonylbenzoate (71; Figure 3) was isolated from a mangrove endophytic fungus, Lasiodiplodia sp. 318, which was collected from Excoecaria agallocha in Mangrove National Nature Reserve in Gaoqiao, Zhanjiang city, Guangdong Province, China. Its structure was established by spectroscopic techniques (one and two-dimensional NMR, HR-ESI-MS), and electronic CD experiment. Compound (71) exhibited cytotoxicity against MMQ and GH3 cell lines with the IC 50 values of 5.2 and 13.0 µM respectively (Huang et al., 2017). Endophytic fungus, Lasiodiplodia theobromae ZJ-HQ1 was isolated from a healthy leaf of the marine mangrove A. ilicifolius, which was collected from Zhanjiang Mangrove Nature Reserve in Guangdong Province, China. This fungus gave two new chlorinated preussomerins, chloropreussomerins A and B (72, 73) together with spreussomerin K (74), preussomerin H (75), preussomerin G (76), and preussomerin F (77) as their metabolites (Figure 3). Their chemical structures were elucidated by a combination of spectroscopic techniques. The absolute configurations of (72) and (73) were determined by single-crystal X-ray diffraction techniques. Compounds (72) and (73) were the first chlorinated compounds in the preussomerins family, which showed potent in vitro cytotoxicity against A549 and MCF-7 human cancer cell lines with IC 50 values ranging from 5.9 to 8.9 µM. Compounds (74-77) exhibited significant bioactivity against A549, HepG2, and MCF-7 human cancer cell lines with the IC 50 values of 2.5-9.4 µM . 7-O-methylnigrosporolide (78), pestalotioprolides D-F (79-81) were isolated from mangrove derived endophytic fungus Pestalotiopsis microspore (Figure 3), which was obtained from fresh healthy fruits of Drepanocarpus lunatus (Fabaceae) collected from Douala, Cameroon. Co-culture of P. microspora with Streptomyces lividans resulted in roughly ten-fold enhancement in the production accumulation of compounds (80) and (81) compared to axenic fungal control. Their chemical structures were determined by the analysis of NMR and mass spectroscopy data. The cytotoxicity of these compounds (78-81) was significant against murine lymphoma cell line L5178Y, which showed IC 50 values of 0.7, 5.6, 3.4, and 3.9 µM respectively. Compound (80) was potent against the human ovarian cancer cell line A2780 with an IC 50 value of 1.2 µM . Campyridones D (82) and ilicicolin H (83) were isolated from Campylocarpon sp. HDN13-307 (Figure 3), which was obtained from the root of mangrove plant Sonneratia caseolaris. Their chemical structures and absolute configurations were determined on the basis of spectroscopic analysis and electronic CD results. Campyridone D (82) and ilicicolin H (83) were cytotoxic against HeLa cell with the IC 50 values of 8.8 and 4.7 µM respectively .
Endophytic fungus Pestalotiopsis clavispora isolated from the mangrove plant Rhizophora harrisonii was the source of a new polyketide derivative pestalpolyol I (96; Figure 3). The chemical structure of the new compound was determined using one and two-dimensional NMR spectroscopy, as well as by high-resolution mass spectrometry. Compound (96) displayed cytotoxicity against the mouse lymphoma cell line L5178Y activity with an IC 50 value of 4.10 µM (Perez et al., 2016). Four highly oxygenated chromones, rhytidchromone A, B, C, and E (97-100) were isolated from the culture broth of a mangrovederived endophytic fungus, Rhytidhysteron rufulum, which was obtained from Thai Bruguiera gymnorrhiza (Figure 3). Their structures were determined by analysis of 1D and 2D NMR spectroscopic data. The structure of rhytidchromone A (97) was further confirmed by single-crystal X-ray diffraction analysis. Compounds (97-100) displayed cytotoxicity against Kato-3 cell lines with the IC 50 values ranging from 16.0 to 23.3 µM, while rhytidchromones A and C were active against MCF-7 cells with the IC 50 values of 19.3 and 17.7 µM respectively (Chokpaiboon et al., 2016).
Another compound ethyl-2,4-dihydroxy-6-(8 ′hydroxynonyl)-benzoate (101; Figure 3) was isolated from a mangrove endophytic fungus, Lasiodiplodia sp. 318# and its complete chemical structure was elucidated by spectroscopic techniques. The compound (101) was cytotoxic against several cell lines with the IC 50 values of 10.1 µM (MDA-MB-435), 12.5 µM (HepG2), 11.9 µM (HCT-116), 13.31 µM (A549), and 39.74 µM (THP1) respectively . Mangrove derived endophytic fungus Fusarium sp. (No. DZ27) in the South China Sea was the source of beauvericin (102), a cyclic peptide (Figure 3), and its chemical structure was deduced by spectroscopic methods and also using the reference data from the literature. Beauvericin (102) was potent in the growth inhibition of KB and KBv200 cells with the IC 50 values of 5.76 and 5.34 µM. Further analysis of beauvericin (102) activity was done, which showed that it induced apoptosis through the decrease of reactive oxygen species generation, loss of mitochondrial membrane potential, release of cytochrome C, activation of Caspase-9 and -3, and cleavage of PARP and did not regulate Bcl-2 or Bax expression (Tao et al., 2015).
Mangrove associated endophytic fungus Penicillium sp.FJ-1 of Avicennia marina, which was collected in Fujian, China was the source of two new metabolites; compounds (103) and (104) as shown in Figure 3. Their chemical structures were determined using NMR and mass spectroscopy. The antiproliferative activity of compound (103) was weak against Tca8113 and MG-63 cells with the IC 50 values of 26 and 35 µM respectively. The positive control, taxol, gave the IC 50 values of 46 and 10 nM with Tca8113 and MG-63 cell lines respectively. The IC 50 value of compound (104) on Tca8113 and normal liver cell line WRL-68 was 10 and 58 µM respectively. Compound (104) also showed anti-tumor effect on MG-63 cells with an IC 50 value of 55 nM. Compound (104) was also tested against nude mice, which showed significant inhibition of tumor growth of human osteosarcoma (Zheng et al., 2014). A known diterpenoid 3,4-seco-sonderianol (105; Figure 3) was isolated from endophytic fungus J3 of Ceriops tagal collected in the mangrove reserve of Dong Zhai Gang, Hainan province, China. Its structure was elucidated using spectroscopic methods including 1D and 2D NMR (HMQC, 1 H-1 H COSY and HMBC). Compound (105) exhibited activities against K562, SGC-7901, and BEL-7402 cell lines with the IC 50 values of 9.2, 15.7, and 25.4 µg/mL respectively. Paclitaxel was used as the positive control, which displayed the IC 50 values of 5.1 µg/mL against K562, 1.6 µg/mL against SGC-7901 and 6.3 µg/mL against BEL-7402 cel lines respectively (Zeng et al., 2015). Waol A (106), pestalotiopene A (107), cytosporone E (108) were obtained from the endophytic fungus Acremonium strictum, isolated from the mangrove tree Rhizophora apiculata (Figure 3). The chemical structures of the isolated compounds were elucidated on the basis of comprehensive NMR and mass spectrometry analysis. Compounds (106-108) showed moderate cytotoxic activity against human cisplatin-sensitive (IC 50 values 27.1, 76.2, and 8.3 µM respectively) and resistant A2780 cell lines (IC 50 values 12.6, 30.1, and 19.0 µM respectively) (Hammerschmidt et al., 2014).
Mangrove endophytic fungus Dothiorella sp., which was obtained from the bark of the mangrove tree Aegiceras corniculatum at the estuary of Jiulong River, Fujian Province of China, was the source of two new polyketides, named dothiorelones F (109) and G (110) as shown in Figure 3. Their chemical structures were determined on the basis of NMR data and mass spectrometry. Dothiorelones F (109) and G (110) showed significant cytotoxicity against Raji cancer cell line with an IC 50 value of 2 µg/mL (Du and Su, 2014). The mangrove endophytic fungus Aspergillus terreus (No. GX7-3B), which was obtained from a branch of Bruguiera gymnoihiza (Linn.) growing on the coastal salt marsh of the South China Sea was the source of compounds (111, 112) and beauvericin (102) as shown in Figure 3. Their chemical structures were determined by the analysis of the spectroscopic data. The cytotoxicity of compounds (111) and (102) ranged from moderate to strong against MCF-7, A549, HeLa, and KB cell lines with the IC 50 values of 4.98 and 2.02 (MCF-7), 1.95 and 0.82 (A549), 0.68 and 1.14 (HeLa) and 1.50 and 1.10 µM (KB) respectively. The inhibitory activity of compound (112) was weak against these tumor cell lines (Deng C. M. et al., 2013). Endophytic fungus Aspergillus niger MA-132 was isolated from mangrove plant Avicennia marina, which was the source of two sterol derivatives nigerasterols A and B (113, 114) as shown in Figure 3. The chemical structures and absolute configurations of these compounds were determined using spectroscopic methods. Modified version of Mosher's method was used to confirm the absolute configuration of compound (113). Nigerasterols A and B (113, 114), which represent the first 5,9-epidioxy-sterol compounds of marine origin were evaluated for cytotoxicity. Nigerasterol B (114) displayed potent activity against the tumor cell line HL60 with an IC 50 value of 1.50 µM, while nigerasterol A (113) displayed stronger activity with an IC 50 value of 0.30 µM. Both compounds (113) and (114) displayed potent activities against A549 cell line with the IC 50 values of 1.82 and 5.41 µM respectively .
A new isobenzofuranone, 4-(methoxymethyl)-7-methoxy-6methyl-1(3H)-isobenzofuranone (115; Figure 3) was isolated from the mangrove endophytic fungus Penicillium sp. ZH58, which was obtained from the South China Sea coast. Its chemical structure was determined by the analysis of spectroscopic data. Compound (115) exhibited cytotoxicity against KB and KB V200 cells with the IC 50 values of 6 and 10 µg/mL, respectively . A new xanthone derivative (116; Figure 3) was isolated from the culture of mangrove endophytic fungus, Phomopsis sp. (ZH76). Its chemical structure was determined on the basis of spectroscopic data. Compound (116) inhibited the growth of HEp-2 and HepG2 cells with the IC 50 values of 9 and 16 µM respectively . Mangrove fungus Aspergillus terreus (No. GX7-3B) led to the production of two metabolites: compound (117) and compound (118) as shown in Figure 3. The chemcial structures of these compounds were determined on the basis of spectroscopic data. Compound (117) showed inhibitory activity toward MCF-7 and HL-60 cancer cell lines with the IC 50 values of 4.4 and 3.4 µM, respectively. The cytotoxicity of compound (118) was promising against HL-60 cell line with an IC 50 value of 0.6 µM . Mangrove endophytic fungus, Penicillium sp. ZH16 was obtained from the South China Sea, which produced furanocoumarin derivative (119) as shown in Figure 3. Its chemical structure was determined by the analysis of NMR and mass spectroscopic data. Compound (119) was cytotoxic against KB and KB V 200 cells with the IC 50 values 5 and 10 µg/mL respectively . Endophytic fungus Bionectria ochroleuca, which was obtained from the inner leaf tissues of the plant Sonneratia caseolaris in Hainan island (China) produced pullularin A (120), pullularin C (121), verticillin D (122) and pullularins E and F (123, 124) as shown in Figure 3. Their chemical structures were established using NMR spectroscopy and high-resolution mass spectrometry. Compounds (120-124) were cytotoxic against the mouse lymphoma cells (L5178Y) with the EC 50 values between 0.1 and 6.7 µg/mL (Ebrahim et al., 2012).
Meroterpenes (125-127) were isolated from the marine fungus Penicillium sp. 303 cultured from sea water samples obtained from Zhanjiang Mangrove National Nature Reserve in Guangdong Province, China (Figure 3). The isolated compounds are structurally related to the miniolutelide class of meroterpenoids and were identified as derivatives of miniolutelide B. Compounds  Figure 3. Its chemical structure was determined by spectroscopic methods and it was found to be cytotoxic against HEp-2 and HepG2 cells with the IC 50 values of 8 and 9 µg/mL (Yang et al., 2013c). The endophytic fungus Nigrospora sp. MA75 was obtained from the marine semimangrove plant Pongamia pinnata that led to the production of a new quinone derivative (130; Figure 3) which was isolated from Nigrospora sp. MA75, an. The chemical structure of compound (130) was elucidated by detailed spectroscopic analysis and absolute configuration determination. Compound (130) showed potent inhibition growth of MCF-7, SW1990, and SMMC7721 tumor cell lines with the IC 50 values of 4, 5, and 7 µg/mL respectively (Shang et al., 2012b). Anthracene derivative (131) was isolated from mangrove endophytic fungus No.5094 which was collected in the South China Sea as shown in Figure 3. The compound was identified on the basis of spectral analysis. Compound (131) showed strong inhibitory activity with KB and KBv200 cell lines having the LD 50 values of 5.5 and 10.2 µM respectively (Yang et al., 2013a).
The marine-derived fungus Aspergillus sp., which was obtained from the sponge Xestospongia testudinaria, was collected from the South China Sea that gave two phenolic bisabolane sesquiterpenoid dimers, disydonols A and C (156, 157) as shown in Figure 5. Their chemical structures were determined on the basis of spectroscopic analysis. Compound (156) exhibited in vitro moderate cytotoxicity toward HepG-2 and Caski human tumor cell lines with the IC 50 values of 9.31 and 12.40 µg/mL respectively. Compound (157) also displayed selectivity against HepG-2 and Caski human tumor cell lines with the IC 50 values of 2.91 and 10.20 µg/mL respectively . A new polyacetylene, xestospongiamide (158) was obtained from the Red Sea sponge, Xestospongia sp. which was collected from deep waters of Sharm Obhur, Jeddah, Saudi Arabia (Figure 5). Compound (158) showed antitumor effect against both Ehrlich ascites carcinoma and lymphocytic leukemia (LD 50 5.0 µM each) (Ayyad et al., 2015).
A marine-derived fungus of the genus Stachylidium was isolated from the sponge Callyspongia cf. C. flammea. Chemical investigation of the bioactive fungal extract led to the isolation of the novel phthalimidine derivatives marilines A1 and A2 (159, 160) whose chemical structures are shown in Figure 5. The absolute configurations of the enantiomeric compounds (159) and (160) were assigned using a combination of experimental circular dichroism (CD) investigation and quantum chemical CD calculations. The skeleton of marilines is unusual and its biosynthesis was suggested to require uncommon biochemical reactions in fungal secondary metabolism. Both enantiomers, marilines A1 (159) and A2 (160) inhibited human leukocyte elastase (HLE) with an IC 50 value of 0.86 µM (Almeida et al., 2012).

COMPOUNDS FROM OTHER MARINE DERIVED FUNGUS
Aspergillus versicolor Y31-2, which was obtained from seawater samples in the Indian Ocean, gave a quinolinone derivative (161) as shown in Figure 6. Compound (161) was cytotoxic against MCF-7 and SMMC-7721 cell lines with the IC 50 values of 16.6 and 18.2 µmol/L . Fermented products of marine fungus Penicillium sclerotiorum M-22 which was isolated from a rotten leaf sample collected on the west coast of Haikou, Hainan province, China gave two azaphilonidal derivatives penicilazaphilones B (162) and C (163) as shown in Figure 6. Cytotoxicity studies revealed that penicilazaphilones B (162) and C (163) were selective against melanoma cells B-16 and human gastric cancer cells SGC-7901 with the IC 50 values of 0.29, 0.44 and 0.06, 0.72 µM respectively. The control experiments with normal mammary epithelial cells M10 at the same concentration did not show significant toxicity (Zhou et al., 2016). A furan derivative (164) was isolated from marine-derived fungus Penicillium chrysogenum HGQ6 which was obtained from Lianyungang sea mud sample (Figure 6). The compound (164) was active against BGC823 cell line with an IC 50 value of 0.19 mg/mL, which was lower than that of adriamycin with an IC 50 value of 0.06 mg/mL (Guo et al., 2016). A mutant from diethyl sulfate (DES) mutagenesis of a marine-derived fungus Penicillium purpurogenum G59 produced epiremisporine B (165), epiremisporine B1 (166) and isoconiochaetone C (167) as shown in Figure 6. Epiremisporine B (165) exhibited cytotoxicity against K562, HL-60, with the IC 50 values of 69.0 and 62.9 µg/mL. Similarly epiremisporine B1 (166) exhibited cytotoxicity against K562, HL-60 cell lines with the IC 50 values of 53.1 and 54.7 µg/mL respectively while the percent inhibition rate for isoconiochaetone C (167) were 20.4 and 26.0 at 100 µg/ mL against K562 and HL-60 cell lines respectively . Penicitrinine A (168) a novel alkaloid with a unique spiro skeleton was isolated from a marine-derived fungus Penicillium citrinum (Figure 6). Penicitrinine A (168) showed toxicity against A-375, SPC-A1, and HGC-27 cancer cell lines with IC 50 values of 20.1, 28.6 and 29.4 µM respectively. Morphological evaluation, apoptosis rate analysis, Western blot and real-time quantitative PCR (RT-qPCR) results showed that penicitrinine A could significantly induce A-375 cell apoptosis by decreasing the expression of Bcl-2 and increasing the expression of Bax. Additionally, anti-metastatic effects of penicitrinine A in A-375 cells by wound healing assay, trans-well assay, Western blot and RT-qPCR were also investigated. These results showed penicitrinine A significantly suppressed metastatic activity of A-375 cells by regulating the expression of MMP-9 and its specific inhibitor TIMP-1 (Liu Q. Y. et al., 2015).
Aspergillus sp. was found in the gut of a marine isopod Ligia oceanica, which was collected in the seaside of Dinghai in Zhoushan, Zhejiang Province of China, that produced aspochalasin V (169; Figure 6). Apochalasin V (169) showed moderate activity against PC3 and HCT116 cell line with the IC 50 values of 30.4 and 39.2 µM respectively (Liu et al., 2014). Fungus Aspergillus terreus SCSGAF0162 was obtained from the tissue of gorgonian Echinogorgia aurantiaca collected in Sanya, Hainan Province, China which produced a cytotoxic and antiviral cyclic tetrapeptide asperterrestide A (170) as shown in Figure 6. Compound (170) was cytotoxic to human carcinoma U937 and MOLT4 cell lines with the IC 50 values of 6.4 and 6.2 µM respectively . Aculeatusquinones B and D (171,172) were produced from marine-derived fungus Aspergillus aculeatus (Figure 6). The chemical structures of these compounds were determined by spectroscopic methods. Compounds (171) and (172) were cytotoxic to HL-60, K562, and A-549 cell lines with the IC 50 values in the range of 5.4-6.1 µM (Chen et al., 2013).
Diorcinol D (173) and diorcinol E (174) (Figure 6) were produced from the marine-derived fungus Aspergillus versicolor. Their chemical structures were determined using spectroscopic analysis. Compound (173) was moderately cytotoxic against HeLa and K562 cell lines with the IC 50 values of 31.5 and 48.9 µM respectively while compound (174) showed cytotoxicity against only HeLa cell line with the IC 50 value 36.5 µM (Gao et al., 2013b). A new pyridinone, chaunolidone A (175; Figure 6) was isolated from marine-derived fungus Chaunopycnis sp. (CMB-MF028) which was obtained from the inner tissue of a pulmonate false limpet Siphonaria sp. that was collected from rock surfaces in the intertidal zone of Moora Park, Shorncliffe, Queensland, Australia. Chaunolidone A (175) was found to be a selective and potent inhibitor of human non-small cell lung carcinoma cell NCI-H460 with the IC 50 value 0.09 µM (Shang et al., 2015).
Marine-derived fungus Penicillium oxalicum SCSGAF 0023, which was isolated from the South China Sea gorgonian Muricella flexuosa, produced oxalicumone A (179; (Figure 6). The compound (179) was cytotoxic against A375 and SW-620 cell lines with IC 50 values of 11.7 and 22.6 µM . Compound (180; Figure 6) was isolated from the fungal strain Aspergillus sydowii SCSIO 00305 which was collected from a healthy tissue of Verrucella umbraculum. The compound (180) showed significant cytotoxicity against A375 cell lines with the IC 50 value of 5.7 µM (He et al., 2012). A cytotoxic compound AGI-B4 (181; Figure 6) was obtained from the culture of a marine-derived fungus Neosartorya fischeri strain 1008F1. The chemical structure of the isolated compound was elucidated on the basis of spectroscopic data. Compound (181) showed toxicity aginst human gastric cancer cell line SGC-7901 with an IC 50 value of 0.29 µM and against hepatic cancer cells BEL-7404 with an IC 50 value of 0.31 µM (Tan et al., 2012). Fungus Chondrostereum sp. which was collected from soft coral Sarcophyton tortuosum in Hainan Sanya National Coral Reef Reserve, China produced chondrosterin J (182; Figure 6). The chemical structure of the compound was determined using NMR, mass spectrometry and single crystal X-ray diffraction techniques. The compound (182) was cytotoxic against human nasopharyngeal cancer cell lines CNE-1 and 2 with the inhibitory concentration (IC 50 ) values of 1.32 and 0.56 µM respectively . Fungus Ascotricha sp. ZJ-M-5 was obtained from a mud sample in Fenghua, China which produced compound (183) and (+)-6-Odemethylpestalotiopsin C (184; Figure 6). Compounds (183) and (184) were cytotoxic against HL-60 and K562 with the IC 50 values 6.9 and 12.3 µM respectively .
The trichodermamides are modified dipeptides isolated from a wide variety of fungi, including Trichoderma virens. Previous studies have reported that trichodermamide B initiated cytotoxicity in HCT-116 colorectal cancer cells. In the present study trichodermamide B (188; Figure 6) showed an IC 50 value of 3.1 µM in HeLa cell line. Compound (189) caused S-phase accumulation and cell death in HeLa cells, suggesting response to DNA double strand breaks (Jans et al., 2017). Chromosulfine (189; Figure 6), a novel cyclopentachromone sulfide, was isolated from a neomycin-resistant mutant of the marine-derived fungus, Penicillium purpurogenum G59. Its structure, including stereochemistry, was determined using spectroscopic methods using NMR, electronic CD (ECD) analysis and Mosher's method. The compound (189) showed toxicity against K562, HL-60, BGC-823, HeLa, and MCF-7 cell lines with IC 50 values of 60. 8, 16.7, 73.8, 75.4, and 59.2 µM (Yi et al., 2016). Neohydroxyaspergillic (190) and neoaspergillic acid (191) (Figure 6) were isolated from the marine-derived fungus (strain CF07002) of the genus Aspergillus. Their structures were determined by the interpretation of NMR spectroscopic data which were corroborated by subsequent synthesis. Compound (191) exhibited toxicity against Jurkat, K562, U937, and Raji cell lines with the IC 50 values of 31.6, 50.1, 42.6, and 54.9 µM respectively. Compound (190) was poorly active against Jurkat cell lines with an IC 50 value of 60.2 µM (Cardoso-Martinez et al., 2015). Aspergillus glaucus was obtained from the marine sediment in Fujian province of the People's Republic of China which gave a novel anthraquionone derivative aspergiolide A (192). The active components of this fungus were isolated which resulted in the identification of a novel naphtho[1,2,3de]chromene-2,7-dione skeleton. Compound (192) acts by topoisomerase II inhibition similar to adriamycin activity. Further experiments with BEL-7402 cells showed that (192) reduced cancer growth via a caspase dependent pathway . Marine-derived fungus, Aspergillus fumigatus was isolated from marine green algae in Seosaeng-myeon, Ulsan in the Republic of Korea which produced isosclerone (193) as shown in Figure 6. It showed cytotoxicity toward MCF-7 human breast cancer cells with the IC 50 value 63 µM after 24 h incubation. Further experiments showed that compound (193) inhibited the protein and gene expressions of MMP-2,-9 in MCF-7 human breast cancer cells by altering MAPK signaling pathway . Marine gorgonianassociated fungus Penicillium oxalicum SCSGAF 0023 produced oxalicumone E (194) and oxalicumone A (195; Figure 6). The chemical structures of these compounds were determined by spectroscopic analysis. Compounds (194) and (195) exhibited cytotoxicity against eight cell lines (H1975, U937, K562, BGC823, MOLT-4, MCF-7, HL60, and Huh-7) with the IC 50 values of < 10 µM respectively (Bao et al., 2014). Deoxybostrycin (196) is an anthraquinone compound which was obtained from the marine mangrove fungus Nigrospora sp. No. 1403 as shown in Figure 6. The in vitro cytotoxicity of deoxybostrycin against MDA-MB-435, HepG2, and HCT-116 cancer cell lines were determined with the IC 50 values of 3.1, 29.9, and 5.6 µM respectively . Three new alkaloids auranomides A and B (197, 198) and auranomide C (201) were isolated from the marine-derived fungus Penicillium aurantiogriseum (Figure 6). The chemical structures of compounds (197-199) were elucidated by using spectroscopic methods such as IR, high-resolution mass spectroscopy and two-dimensional NMR spectroscopy. Auranomides A-C (197-199) exhibited moderate cytotoxic activity against K562, ACHN, HEPG2, and A549 cell lines. Auranomide B (199) displayed the best activity among them with an IC 50 value of 0.097 µmol/mL against HEPG2 cells .

AN OVERVIEW OF CYTOTOXICITY RESULTS
As discussed before in different sections in this review article, a total of 199 compounds isolated from marine fungi have shown considerable promise as cytotoxic agents with potential to be developed as anticancer agents in recent years. About half of these compounds have been known to be isoloated from terrestrial or other natural sources previously but they have been reported to be isolated from the marine soruces for the first time. The Supplementary Table 1 outlines the novelty of these compounds with known previous anticancer acitivities, if any. A number of compounds reported in this review article have shown considerable anticancer activity comparable to positive controls (which are currently used anticancer drugs). Many of these metabolites have displayed inhibitory concentrations in the low micromolar range which obviously mark their potential to be developed as anticancer drugs. However, there is a definite need to improve these inhibitory concentrations since lower dosage would help in eliminating undesired side effects. The exploration in this direction has to be a two pronged approach: one, where the actual cellular targets that lead to cytotoxic effects need to be identified while the other, needs to focus on identifying the structural moieties that are responsible for cytotoxicity. The latter effort would lead to structure-activity based drug design programs to alter chemical functionalities in order to achieve higher efficacy.
Additionally, since several of these metabolites possess structural features (such as compounds 84, 88, 116, 119, 129,  131, 192, and 196) that would enable binding to DNA and RNA, cancer targets that involve nucleic acid recognition should be probed. For example, chromosomal DNA ends in humans, which are rich in guanines, have been shown to form a unique four stranded structures called G-quadruplexes. Both in vitro and in vivo studies have shown the formation of these noncanonical structures which use assembly of four guanines (called G-tetrads), hydrogen bonded in a Hoogsteen fashion (Ranjan et al., 2010). After every cell division, the telomeric DNA gets shortened by certain bases and this process continues until reaching a threshold (called Hayflick limit) where senescence is initiated in a normal cell cycle. However, in the majority of cancer cases, a ribonucleoprotein called telomerase gets activated. The telomerase contains an RNA unit complementary to human telomeric repeat sequence TTAGGG. Binding of this RNA unit of telomerase initiates reverse transcription process to regenerate the curtailed telomere (Camarena et al., 2007;Fakhoury et al., 2007). Many research efforts have, therefore, targeted inhibiting/disrupting telomerase interaction with the telomeric DNA as a means to develop new therapies for cancer treatment (Mergny and Hélène, 1998). One of the ways in which this inhibition can be achieved, is by folding the telomeric ends as stable G-quadruplex structures since telomerase recognizes only the linear form of the telomeric DNA. As a result, small molecules that target these G-quadruplex structures have been tested to see if they could function as inhibitors of telomerase interaction. A number of small molecule inhibitors have been reported that bind to G-quadruplexes and enhance their stability (Xue et al., 2011;. Such stabilizations are known to disfavor telomerase binding and thereby stopping the telomere regeneration. An important feature of G-quadruplex stabilization by small molecule is making stabilizing interactions with the G-tetrads by means of π-bonding. Some other molecules have shown interactions exclusively with the grooves whereas few have shown interactions both with the tetrads and the grooves. Several of the molecules reported in this review have features that would enable binding both with the G-tetrads and the grooves (for example compound 116). Furthermore, topoisomerases are enzymes that remove supercoiling in DNA during the replication process and repairs strand breaks (Tse-Dinh, 2009;Pommier, 2013). Human DNA topoisomerase has been an attractive cancer target and anticancer drug camptothecin is known to elicit its effect by forming a ternary complex between the enzyme and the DNA. Some of the molecules reported in this review (e.g., compound 194) have already shown topoisomerase inhibitory function. Stalling topoisomerase functions by its stabilization with small molecules is another target for anticancer therapy. Since several of these molecules possess structural features that would enable binding to nucleic acids, a screen that targets all forms of nucleic acid structures should be done. This would not only identify the lead compounds for cancer therapy but would also result in identifying the compound that could be of potential use in antibacterial and antiviral therapy.
In addition to this, there are many new discoveries that could have protein targets within the cancer cells. In fact, the majority of FDA approved anticancer drugs target proteins such as cyclin dependent kinases and histone deacytylase. Could these proteins targets be the potential mechanism by which these metabolites induce cytotoxicity? For some of the compounds (64, 65, 168), Bcl-2 downregulation was established as one factor that led to the apoptosis. Do these metabolites function by upregulation of tumor suppressor proteins such as p53 and Bax? The curiosities can be answered only when all protein targets are screened for binding; at least for the ones whose cellular functioning is fairly understood. This would usher a new beginning in the development of natural product based small molecules and possibly identify structural motifs that target specific regions in the protein binding pockets. Such leads can then be used to launch structure activity relationship programs to improve the potency of these leads whose inhibitory concentrations are mostly in the micromolar range. Overall, the discoveries presented in this review highlights many structural classes including some new skeletons that these metabolites produce, which have potential to be developed as clinically useful anticancer drugs. However, in the absence of more detailed studies that focuses on deciphering the cellular events that lead to cell death, their true potential as an anticancer compound might remain to be under-appreciated.

CONCLUSIONS AND PERSPECTIVES
Marine life has been the source of several clinically useful drugs. The findings covered in this review highlight the discoveries of many new natural small molecules, some of them with novel skeletons, that have anticancer activity against a variety of cancer cell lines. The anticancer activity of these compounds is varied with inhibitory concentrations ranging from low to high micromolar concentrations. Some of these metabolites have inhibitory concentrations comparable or better than some of the currently used anticancer drugs. Clearly, these leads have not been explored in detail to determine the actual cellular targets that result in the cytotoxicity and that has been an area whose complete exploration may result in a paradigm shift in the current drug discovery efforts. However, other parallel efforts are needed to facilitate and accentuate marine based drug discovery. One such need is setting-up national and international centers for culture collection since many of the new metabolites reported here have been collected from harsh and hostile environments where the human reach is not easily achievable. This would also help not just in retaining these precious cultures but also in allowing wider reach of these metabolites to specialized groups. A major impediment in marine based drug discovery and, in general, natural product based drug discovery has been the lack of centers that foster programs at the interface of chemistry and biology. Clearly, such specialized centers that have expertise both in chemistry and biology could help in realizing true properties of these metabolites. Morever, lack of complete taxonomy details of the new species and bureaucratic difficulties in the implementation of Nagoya protocol hinder smooth access of knowledge and resources. Therefore, international agreements that clearly address these problems and seek solutions to it, could also greatly help in the smooth exchange of resources. Another important improvement in the area would be developing sustainable biochemical production processes of the screening hits as demonstrated in the case of anticancer compounds Scopularide A and B (Yu et al., 2008;Kramer et al., 2014). Additionally, efforts should also be initiated to look beyond anticancer properties of these molecules.

AUTHOR CONTRIBUTIONS
SD, VP, and NR reviewed the contents critically. VP and NR drew chemical structures and assisted in the preparation of Supplementary Table 1. SD and NR wrote the review.