Mangrove-Associated Fungi: A Novel Source of Potential Anticancer Compounds

Cancer is the second leading cause of death worldwide, and the number of cases is increasing alarmingly every year. Current research focuses on the development of novel chemotherapeutic drugs derived from natural as well as synthetic sources. The abundance and diversity in natural resources offer tremendous potential for the discovery of novel molecules with unique mechanisms for cancer therapy. Mangrove-derived fungi are rich source of novel metabolites, comprising novel structure classes with diverse biological activities. Across the globe, coastal areas are primarily dominated by mangrove forests, which offer an intensely complex environment and species that mostly remain unexplored. In recent years, many structurally diverse compounds with unique skeletons have been identified from mangrove fungi and evaluated for their antiproliferative properties. These compounds may serve as lead molecules for the development of new anticancer drugs. Mangrove endophytes can be modulated using epigenetic means or culture optimization methods to improve the yield or to produce various similar analogs. The present review provides an insight into the bioactive metabolites from mangrove endophytes reported during the period from 2012 to 2018 (up to April, 2018) along with their cytotoxic properties, focusing on their chemical structures and mode of action, as indicated in the literature.


Introduction
Mangroves are salt-tolerant forest ecosystems, representing a lively ecosystem with an amalgam of land-dwelling and marine habitats with high biodiversity and socio-economic importance [1]. Marine fungi are among the most prominent species existing in mangrove forests, and support nutrient replenishment [2]. As per reports on marine fungi, in this ecological niche mangrove fungi make up the second largest group [3]. These fungi may occur as saprophytes, symbiotically or as a parasites in the mangrove ecosystem. In addition, these fungi belong to both the lower class, such as oomycetes and thraustochytrids, and the upper class, such as ascomycetes and basidiomycetes. Fungal secondary metabolites are structurally quite diverse, and their functions mostly depend on self-defense against other microorganisms [4]. Most often mangrove fungi flourish in challenging habitats, making them a rich source of bioactive metabolites. Endophytes are one of the various groups of mangrove fungi that have resulted in the identification of a large number of new bioactive metabolites of nutraceutical and pharmaceutical importance. These include antibiotic, anticancer, antidiabetic, antioxidant, antiviral, anti-inflammatory and immunosuppressive drugs, along with other pharmaceutical agents [5].
Cancer affects different organs, and is identified by the unchecked proliferation of abnormal cells that invade other healthy tissue. The treatment is primarily confined to chemotherapy. Besides being expensive, chemotherapy is known to cause severe side effects, making treatment problematic. The non-effectiveness of many existing drugs along with multi-drug resistance further aggravates the problem, making cancer treatment difficult. For medicinal chemists, the primary goal remains the discovery and identification of chemotherapeutic agents derived from natural products. Secondary metabolites have opened new avenues for the development of novel therapeutic agents [6,7]. Endophytic fungi, which are a less-explored area of the microbial community, have a tremendous potential to produce new metabolites that can be used for pharmaceutical applications. Since the initial report of the identification of paclitaxel, derived from an endophyte associated with Northwest Pacific yew by Stierle et al. [8], scientists have identified many other crucial anticancer molecules from fungal endophytes [6]. Many researchers were attracted to marine mangrove fungi because of their diversity, which may lead to the discovery of several novel natural products. With the remarkable advancements in spectroscopic techniques, separation methods and microplate-based sensitive in vitro assays, the natural product exploration of mangrove fungi has attracted special attention regarding novel and unexplored chemical scaffolds [9]. Of the various existing groups of mangrove fungi, endophytes have been identified as producers of new bioactive metabolites with pharmaceutical and nutraceutical importance.
Most of the endophytes have the potential to produce novel bioactive metabolites, which will undoubtedly boost novel drug discovery. However, higher similarity among microbes leads to the frequent identification of the same compound in the endophytes. During axenic cultivation, a specific portion of the biosynthetic genes are expressed while growing in vitro, and various genes stay masked or silent and do not express in laboratory conditions. For this reason, the routine method of fermentation yields metabolites without chemical diversity. Co-cultivation could help to overcome this problem and is preferred, with two or more microbes allowed to grow together. This approach offers a better competitive environment, allowing the increased production of constitutive as well as cryptic compounds that are not traced out in axenic cultures [10]. Several co-cultivation strategies such as different combinations of fungi, the co-cultivation of fungi with bacteria and the co-cultivation of different bacteria have been reported for the enhancement of the chemical diversity of marine-derived microorganisms [10].
The development of methodologies to induce the expression of biosynthetic transcription as well as the suppression of these genes plays a vital role in the search for new secondary metabolites. The regulation of the enzymes that control metabolite production can be achieved by changing epigenetic mechanisms such as DNA methylation and histone modifications (acetylation and phosphorylation) by using epigenetic modifiers [11]. As an example of the importance of epigenetic modulation in producing unknown natural products, when Aspergillus niger is cultivated over a two-week period in vermiculite-based semi-solid medium treated with suberoylanilide hydoxamic acid (SAHA), it leads to the isolation of a new fungal metabolite nygerone [12]. Hence the epigenetic approach can be a game changer in the production/enhancement of secondary metabolites.
The present review provides a comprehensive overview of the bioactive metabolites identified from mangrove endophytes during the period from 2012 to 2018 (up to April, 2018) including eighty novel compounds of the total 181 reported. The total number of compounds as well as novel compounds isolated from mangrove fungi during this period is presented in Figure 1. The origin, chemical structure of the biological targets and efficacies of these compounds are also discussed where available. The anticancer properties of many of these compounds are presented in Table 1. They are arranged based on the broader category of the taxonomic class of the cytotoxic compounds producing fungi. An attempt has also been made to review recent developments such as co-cultivation and epigenetic modifications in endophytic fungi to enhance the secondary metabolite production.

Compounds Produced by Coelomycetes
Pestalotiopsis is the most noteworthy coelomycetous fungi and the species of Pestalotiopsis are known to produce the diverse array of novel compounds. Strobel and Long [13] described Pestalotiopsis as the "E. coli of the temperate and tropical rainforest systems". The species Pestalotiopsis is widely recognized to be a prolific producer of a diverse array of metabolites that include alkaloids, chromones, coumarins, isocoumarin derivatives, lactones, peptides, phenols, phenolic acids, quinones, semiquinones, xanthones, terpenoids and xanthone derivatives along with an array of antimicrobial, antifungal, antitumor, antiviral, antineoplastic, and antioxidant compounds [14,15]. Some of the cytotoxic compounds reported from this genus such as demethylincisterol A3 (1), ergosta-5,7,22-trien-3-ol (2), stigmastan-3-one (3), stigmast-4-en-3-one (4), stigmast4-en-6-ol-3-one (5), and flufuran (6) (Figure 2), were discovered from Pestalotiopsis spp., associated with Chinese mangrove Rhizophora mucronata. Compounds 2-6 showed cytotoxicity against human cancer cell lines HeLa, A549, and HepG, with IC 50 values in the range of 11.44-102.11 µM. Compound 1 had the most potential, with IC 50 values reaching the nM activity level from 0.17 to 14.16 nM. Flow cytometric investigation demonstrated that compound 1 inhibited the cell cycle at the G0/G1 phase in a dose-dependent manner with a significant induction of apoptosis on the three tested cell lines. The involvement of the mitochondria in compound-1-induced apoptosis was demonstrated using MMP [16].
The compounds 7-O-methylnigrosporolide (7), pestalotioprolides D-F (8, 9, 10) ( Figure 2), were extracted from the Pestalotiopsis microspora, endophytic fungus obtained from the fruits of Drepanocarpus lunatus collected from Douala, Cameroon. An approximately ten-fold increase in the yield of compounds 9 and 10 compared to axenic fungal control was observed when P. microspora was co-cultured with Streptomyces lividans. Compounds 7-10 exhibited cytotoxicity against the L5178Y cell line with IC 50 values of 0.7, 5.6, 3.4, and 3.9 µM, respectively, and compound 9 also showed potent activity against the A2780 cell line displaying an IC 50 value of 1.2 µM [17].
Another study by Hemphill et al. reported a new compound pestalpolyol I (11) (Figure 2) of the polyketide group from Pestalotiopsis clavispora, the endophytic fungus obtained from petioles of the Rhizophora harrisonii, growing in Port Harcourt (Nigeria). Compound 11 showed cytotoxicity against the L5178Y cell line with an IC 50 value of 4.10 µM [18].
A new aromatic amine, pestalamine A (12) (Figure 2), was isolated from P. vaccinia from a branch of Kandelia candel, a viviparous mangrove species widely distributed in coastal and estuarine areas of southern China. The structure of pestalamine A 12 was determined by spectroscopic methods, especially 2D NMR analyses. Compound 12 showed moderate cytotoxicity against MCF-7, HeLa, and HepG2 human cancer cell lines with IC 50 values of 40.3, 22.0, and 32.8 µM, respectively [19].
Phomopsis sp. HNY29-2B, an endophyte isolated from the branch of Acanthus llicifolius collected from the South China Sea in Hainan province, China, is reported as a source of the known phomoxanthones, including dicerandrol A (22) (Figure 2

Compounds Produced by Ascomycetes
A new diketopiperazine derivative, saroclazine B (34) (Figure 3), was isolated from the mangrove-derived fungus Sarocladium kiliense HDN11-84 isolated from the rhizosphere soil of the mangrove plant Thespesia populnea, collected in Guangxi Province, China. Compound 34 showed cytotoxicity against HeLa cell lines with an IC 50 value of 4.2 µM [30].
Benzofluoranthene metabolites and daldinone I (35) (Figure 3) were extracted from Annulohypoxylon sp., an endophytic fungus associated with Rhizophora racemose, collected in Cameroon. Compound 35 exhibited average to potent cytotoxicity with IC 50 values of 14.1 and 6.6 µM, against Jurkat J16 and Ramos cell lines, respectively. It was reported that compound 35 induces apoptotic cell death caused by the induction of intrinsic apoptosis [31].
A new anthraquinone rubrumol (36) (Figure 3) with poly-hydroxyl groups was isolated from a halo-tolerant endophytic fungus Eurotium rubrum, isolated from the salt-tolerant wild plant Suaeda salsa L. collected from the "BoHai" seaside, China. The biological effect of compound 36 on Topo I to relax supercoiled pBR322 DNA was investigated in the cleavable complex assay. The results indicated that compound 36 displayed biological activity compared to the positive control camptothecin. The relaxation activity of rubrumol (36) was stronger than that of camptothecin at the concentration of 100 µM. The band backward shifting and trailing of rubrumol (36) was observed at 100, 50, 10, 5 and 1 µM. Compound 36 also exhibited cytotoxic activities against A549, MDA-MB-231, PANC-1 and HepG2 human cancer cell lines, by MTT method. The inhibition rate for compound 36 against these four cancer cell line was less than 60% at 100 µg/mL, which implied that it displayed no significant cytotoxic activity [32].    Two new chlorinated preussomerins, chloropreussomerins A (50), and B (51), and a known preussomerin analog preussomerin K (52), preussomerin H (53), preussomerin G (54), preussomerin F (55), preussomerin D (56) (Figure 4), were obtained from the endophytic fungus Lasiodiplodia theobromae ZJ-HQ1 isolated from Excoecaria agallocha collected from Guangdong Province, China. Compounds 50-51 and 56 were found to be active against the A549 and MCF-7 cell lines with IC 50 values ranging from 5.9-8.9 µM, and compounds 52-55 showed cytotoxicity against A549, HepG2, and MCF-7 human cancer cell lines with IC 50 values of 2.5-9.4 µM [37].
Stemphylium globuliferum, an endophytic fungus associated with the Egyptian mangrove plant Avicennia marina, was the source of dihydroaltersolanol C (63), altersolanols A, B, N (64, 65, 66), and alterporriol E (67) (Figure 4) [40]. Compounds 63, 64, 65, and 67 showed cytotoxicity with IC 50 values of 3.4, 2.5, 3.7 and 6.9 µM, respectively, towards L5178Y cells [41]. Compound 66 also showed good activity, with IC 50 values in the low micro-molar range towards L5178Y cells [42]. Mishra et al. [43] reported that compound 64 exhibited cytotoxicity against 34 human cancer cell lines in vitro, with mean IC 50 (IC 70 ) values of 0.005 µg/mL (0.024 µg/mL). It has also been reported that compound 64 is a kinase inhibitor and induces cell death by apoptosis through the caspase-dependent pathway, and that kinase inhibition might be the mechanism for the cytotoxic activity [44]. The pro-apoptotic and anti-invasive activity of compound 64 that occurred through the inhibition of the NF-κB transcriptional activity may be responsible for its antitumor potential [45].
A marine anthraquinone derivative SZ-685C (73) ( Figure 5) has been isolated from the mangrove endophytic fungus Halorosellinia sp. (No. 1403), which was found in the South China Sea. The IC 50s of SZ-685C in nonfunctioning pituitary adenoma (NFPA), MMQ, and RPC cells were 18.76, 14.51, and 56.09 µM, respectively. Hoechst 33342 dye/propidium iodide (PI) double staining and fluorescein isothiocyanate-conjugated Annexin V/PI (Annexin V-FITC/PI) apoptosis assays detected an enhanced the rate of apoptosis in cells treated with SZ-685C. Enhanced expression levels of caspase 3 and phosphate and tensin homologs were determined by Western blotting. The protein expression levels of Akt were decreased when the primary human NFPA cells were treated with SZ-685C. It has been observed that SZ-685C (73) induces the apoptosis of human NFPA cells through the inhibition of the Akt pathway in vitro. These findings suggest that SZ-685C may be a potentially promising Akt inhibitor and anti-cancer agent for the treatment of NFPA [47].
cells. It has been suggested that SZ-685C induces MMQ cell apoptosis in a miR-200c-dependent manner [48].
Two new polyketides, named dothiorelons F (74) and G (75) ( Figure 5), were isolated from Dothiorella sp., an endophytic fungus associated with the bark of the mangrove tree Aegiceras corniculatum at the estuary of Jiulong River, Fujian Province, China. Compounds 74 and 75 showed significant cytotoxicity against the Raji cancer cell line, with an IC50 value of 2 µg/mL [49]. SZ-685C (73), was previously reported to inhibit the proliferation of certain tumor cells. SZ-685C inhibited MMQ cell growth in a dose-dependent manner but showed little toxicity toward rat pituitary cells. The IC 50 of SZ-685C in MMQ cells and RPCs were 13.2 and 49.1 µM, respectively. Increasing numbers of apoptotic cells were observed in response to escalating concentrations of SZ-685C, and the expression level of prolactin was inhibited. Nevertheless, the level of prolactin mRNA was unchanged. Additionally, miR-200c was upregulated in MMQ cells compared with RPCs, and downregulation of miR-200c was observed in SZ-685C-treated MMQ cells. Furthermore, the overexpression of miR-200c weakened the effect of the SZ-685C-induced apoptosis of MMQ cells. It has been suggested that SZ-685C induces MMQ cell apoptosis in a miR-200c-dependent manner [48].
Two new polyketides, named dothiorelons F (74) and G (75) ( Figure 5), were isolated from Dothiorella sp., an endophytic fungus associated with the bark of the mangrove tree Aegiceras corniculatum at the estuary of Jiulong River, Fujian Province, China. Compounds 74 and 75 showed significant cytotoxicity against the Raji cancer cell line, with an IC 50 value of 2 µg/mL [49].

Compounds Produced by Hyphomycetes
A new compound Penibenzophenone B (108) (Figure 7), was obtained from the endophytic fungus Penicillium citrinum HL-5126 isolated from the mangrove Bruguiera sexangula var. rhynchopetala collected in the South China Sea. The new compound 108 displayed cytotoxic activity against human A549 cell lines with an IC 50 value of 15.7 µg/mL [60].
Five new derivatives of macrolide antibiotic Brefeldin A (109), along with Brefeldin A 7-O-acetate (110) (Figure 7), were produced by an endophytic fungus, Penicillium sp., which was isolated from the healthy root of Panax notoginseng. Compounds 109-110 exhibited cytotoxic activity against the 293, HepG2, Huh7 and KB cell line with an ID 50 values from 0.024 to 0.62 µM. Further, studies of the cellular mechanism of compounds 109-110 showed that they arrested HepG2 cells at the S phase [61].
A new chaetoglobosin, penochalasin K (111) (Figure 7), was extracted from the mangrove endophytic fungus Penicillium chrysogenum V11. Its structure was elucidated by 1D, 2D NMR spectroscopic analysis and high resolution mass spectroscopic data. Using the one strain many compounds (OSMAC) approach, new diketopiperazines, spirobrocazine C (118) and brocazine G (119) (Figure 7) were characterized from Penicillium brocae MA-231, an endophytic fungus associated with Avicennia marina collected at Hainan Island, China. Compound 119 exhibited potent cytotoxic activity against the A2780 and A2780 CisR cell lines, with   Zhang et al. [76] reported the production of a new depsidone, botryorhodine H (146) (Figure 8), by co-culturing mangrove endophytic fungus Trichoderma sp. 307 and Acinetobacter johnsonii B2. Compound 146 exhibited good cytotoxic activity against the MMQ and GH3 cell lines with IC 50 values of 3.09 and 3.64 µM, respectively.
A known cyclic peptide, beauvericin (104) (Figure 6), was obtained from Fusarium sp. (No. DZ27) an endophytic fungus residing inside the bark of Kandelia candel from Dongzhai mangrove, Hainan, China, in the South China Sea. Compound (104) showed cytotoxic activity against the KB and KBv200 cell lines with IC 50 values of 5.76 and 5.34 µM, respectively. It induces apoptosis through the mitochondrial pathway, including the decrease of relative oxygen species generation, the loss of mitochondrial membrane potential, the release of cytochrome c, the activation of Caspase-9 and -3, and the cleavage of PARP. Additionally, the regulation of Bcl-2 or Bax was not involved in the apoptosis induced by beauvericin in KB and KBv200 cells [79].
An inhibitor of histone deacetylase, Apicidin (149) (Figure 8), was isolated from Fusarium sp., an endophytic fungus associated with the leaf of mangrove Kandelia candel planted at Dongzhai Harbor on Hainan Island, China. Apicidin showed good cytotoxic activity against GLC-82 cells with the IC 50 value of 6.94 ± 0.27 µM. Apicidin suppressed proliferation and invasion, and induced apoptosis via the mitochondrial pathway in GLC-82 cells, including the loss of ∆Ψm, the release of cytochrome c from mitochondria, the activation of caspase-9 and -3, and the cleavage of poly-ADP-ribose polymerase [80]. Apicidin 149, was previously isolated from the mangrove endophytic fungus ZZF42 from the South China Sea and exhibited selective in vitro cytotoxicity towards KB and KBv200 with IC 50 values of less than 0.78 µg/mL [81].
Mangrove endophytic fungus No.5094, which was collected in the South China Sea was the source of anthracene derivative (159) (Figure 9). Compound 159 exhibited potent activity towards the KB and KBv200 cell lines with LD 50 values of 5.5 and 10.2 µM, respectively [87].
Marinamide (160) and methyl marinamide (161) (Figure 9) were obtained by co-cultures of two marine-derived mangrove endophytic fungi (strains Nos. 1924 and 3893) from the South China Sea coast. Their structures were elucidated using comprehensive spectra methods. Compound 160 was found to be cytotoxic with IC 50

Methods Used for the Activation of Silent Biosynthetic Genes
Recent studies in the marine-based microorganisms have shown that these microorganisms are a rich source for novel bioactive compounds. Salinosporamide A (marizomib), a microbial compound isolated from marine Salinispora bacteria with proteasome inhibitory activity is expected to be a future anti-cancer drug, and is presently under clinical trials [95]. However, the reoccurrence of the same compound as discovered in terrestrial sources, in marine microorganisms often leads to serious issues. Advances in molecular biology have enhanced our understanding regarding how to exploit the genetic potential of bacteria and fungi to produce newer chemical entities apart from those that are currently known, which have yet to be explored [96,97]. It has been reported that under laboratory conditions, biosynthetic genes are not expressed as such, as only limited bioactive compounds are produced by these microbes. To overcome these limitations, different strategies have been proposed, including culturing promising strains in varying culture media and under a variety of culture conditions [98], mixing cultures of two or more microbe variants and epigenetic modifications that treat microbes with epigenetic modifiers such as histone deacetylase inhibitors or DNA methyl transferase to initiate the transcription of silent genes [99,100] to enhance the variation and diversity of the produced metabolites.

The Co-Culture Strategy
Microbes in natural ecosystem conditions always harbor and flourish in co-existence with a variety of microbes. Antagonism and competition for limited resources often lead to high competition among species, and microbes adopt various defense strategies, which favor the production of important bioactive secondary metabolites [101]. The co-culturing of two or more different microbes at the laboratory scale might mimic the ecological setting and induce the cascade of genes responsible for biosynthesis that are normally are masked under optimum culture parameters. Co-cultivation of two Aspergillus species derived from mangroves produced the new alkaloid aspergicin and the previously recognized compounds neoaspergillic acid and ergosterol, with antibacterial activity [102]. Li et al. [103] co-cultured two mangrove epiphytes and identified a novel xanthone derivative compound that showed antifungal activity. Two new alkaloids, marinamide, and marinamide methylether, were reported from mangrove-derived endophytic fungi with a cytotoxic effect when grown in mixed fermentation [88]. Pestalone, a chlorinated prenylsecoanthraquinone, was produced by the marine-derived fungus Pestalotia sp. when grown in the presence of the marine-derived bacterium Thalassopia sp., which belongs to the Gram-negative group. [104]. When Libertella sp., a marine-based fungi, were cultured in the presence of the bacteria Thalassopia sp., it resulted in the production of diterpenoid libertellenones of fungal origin [105]. In another set of studies, when the bacterium Sphingomonas sp. was grown in the presence of Aspergillus fumigatus, a novel compound glionitrin A, a diketopiperazine disulfide, was identified and appeared to show strong cytotoxicity against HCT-116, A549, AGS and DU145 cells [106]. These studies suggest that co-cultivation has tremendous potential to generate novel chemical entities from microbes when cultured under laboratory conditions.

Epigenetic Modification
The addition of epigenetic modifiers to fungi would allow us to induce cryptic fungal gene clusters. This technique can be applied to any fungal strain and does not require strain-dependent genetic manipulation. Williams et al. [107] reported that epigenetic modifiers could be rationally employed to access silent natural product pathways. Histone deacetylase (HDAC) or DNA methyltransferase (DMAT) are often used as epigenetic agents to change the transcription rate of some genes [108]. Henrikson et al. [12] reported the identification of nygerone A from A. niger when grown with suberoylanilide hydoxamic acid (SAHA). Wang et al. [109] reported induced metabolite generation in Penicillium citreonigrum when grown in the presence of methyl transferase inhibitor, 5-azacytidine (5-AZA). When Hypoxylon sp., an endophytic fungi, was treated with the epigenetic modifiers SAHA and AZA it enhanced the production of volatile organic compounds (VOCs) [110].
The marine endophytic fungus Leucostoma persoonii from Rhizophora mangle enhanced the production of cytosporones B, C, E and R in HDAC inhibited fermentation [111]. These studies provide evidence that the use of epigenetic modifiers modulate secondary metabolite production, resulting in different gene expressions.

Conclusions
Mangrove fungi are a ubiquitous source of novel bioactive metabolites with the potential to display anticancer properties. It is interesting to observe the chemical diversity in these metabolites, which include simple glycoside (27) and peptide molecules (pullularins E, 89; F, 90 and apicidin, 149) as well as complex stereospecific structures such as cytochalasin H (18), phomopsichalasin G (19), aniquinazolines A-D (98-101) and penitrem A, B and F (178-180). Chemical diversity plays an important role in the drug discovery pipeline, as this provides structurally diverse scaffolds that display similar activity via different modes and/or mechanisms of action. This phenomenon is also observed in mangrove fungal metabolites, as they show potent anticancer activity via different mechanisms of action such as apoptotic cell death (SZ-685C, 73; beauvericin, 104), the inhibition of kinase proteins involved in signal transduction pathways (Mycoepoxydiene, 32; Altersolanol A, 64; and the inhibition of topoisomerase I (36). Although many metabolites demonstrated moderate cytotoxic activities against cancer cell lines, only a few displayed superior activity than the standard anticancer drugs (98-101, 119, 124). It can be suggested that the rational derivatization of metabolites may provide molecules with better activity against a wide range of cancer cell lines. In addition, the identified metabolites with broad-spectrum anticancer activity need to be investigated to establish their mechanisms of action and to develop as novel anticancer therapeutics.
Author Contributions: The manuscript was critically evaluated by S.K.D., V.P., M.S.R. and M.K.G. The chemical structures were drawn by V.P. and M.K.G. and they assisted in the preparation of Table 1. The manuscript has been read and approved by all named authors.
Funding: This research received no external funding.