Keywords

12.1 Introduction

Marine microorganisms are widely recognized as rich sources of novel natural products [1, 2]. In recent years, numerous novel bioactive compounds discovered from marine actinobacteria have been reported [3–6]. They are responsible for the production of about half of the discovered bioactive secondary metabolites notably antibiotics, antitumor agents, and immunosuppressive agents [7–10].

Marine actinobacteria also constitute an important and potential source of novel bioactive compounds. They produce different types of antibiotics, because the environmental conditions of the ocean differ greatly from terrestrial conditions [11]. Novel actinobacteria with biopharmaceutical potential have been increasingly isolated from marine habitats [12–15]. A new major marine Streptomyces genus have recently been described and shown to produce biological activities, including antibiotics [16, 17]. The antibiotics are entirely new and unique when compared to those from the terrestrial ones.

Around 23,000 bioactive secondary metabolites produced by microorganisms have been reported and over 10,000 of these compounds are produced by actinobacteria, representing 45 % of all bioactive microbial metabolites discovered [18]. Among actinobacteria, around 7600 compounds are produced by Streptomyces ­species [18]. Many of these secondary metabolites are potent antibiotics, which has made streptomycetes the primary antibiotic-producing organisms exploited by the pharmaceutical industry [18]. Members of this group are producers, in addition, of clinically useful drugs such as anthracyclines, peptides, aureolic acids, enediynes, antimetabolites, carzinophilin, mitomycins and others [19]. However, the search for novel drugs is still a priority goal for cancer therapy, due to the rapid development of resistance to multiple chemotherapeutic drugs. In addition, the high toxicity usually associated with cancer chemotherapy drugs and their undesirable side effects increase the demand for novel anticancer drugs active against untreatable tumors, with fewer side effects and/or with greater therapeutic efficiency [20]. This book chapter discusses the current status of novel anticancer drugs from marine actinobacteria

12.2 Actinobacteria from Marine Environment

Actinobacteria are Gram-positive, often filamentous, bacteria known for their unsurpassed capacity for the production of secondary metabolites with diverse biological activities. Extensive screening of terrestrial actinobacteria, started in the early 1950s, has yielded many important drug leads, later developed into antimicrobial (amphotericin B, erythromycin, vancomycin), anticancer (daunorubicin, bleomycin, mitomycin) and immunosuppressive (rapamycin) drugs. Despite this apparent success, most of the actinomycete-based screening programs at big pharmaceutical companies have been abandoned in the recent years due to several reasons. One of the reasons was high costs of the internal screening programs, combined with the low number of new drug leads and relatively low profit on such drugs as new anti-infectives [21]. Another reason has been frequent re-discovery of the same compounds, mostly due to the redundancy of the samples, as well as strain isolation and screening technologies [22].

In the recent years, actinobacteria isolated from the marine environment (­sediments, sponges, tunicates , neuston, etc.) have attracted considerable attention [23]. True marine actinobacteria are usually considerably more difficult to culture compared to their terrestrial relatives, most likely due to the special growth requirements. However, development of both sampling and cultivation techniques allowed isolation of representatives of several true marine actinomycete genera producing novel compounds with interesting biological activities [12].

12.3 Anticancer Activity

Cancer still remains one of the most serious human health problems and breast cancer is the second most universal cause of cancer deaths in women . ­Therapeutic methods for cancer treatment are surgery, radiotherapy, immunotherapy and chemotherapy and these techniques are individually useful in particular situations and when combined, they offer a more efficient treatment for tumor. Many of the antitumor compounds from marine drugs are derived from marine actinobacteria and these metabolites play an important role in identification of pharmaceutical compounds. Currently, it appears that there have been only a few studies focusing on finding bioactive compounds derived from marine actinobacteria to be used as anticancer agents, as well as agents against infectious organisms .

Pure active compounds extracted from the marine actinobacterium, Salinispora tropica have shown inhibitory effects in many malignant cell types [24]. In particular, Salinosporamide A (1) (Fig. 12.1) is a novel rare bicyclic beta-lactone gamma-lactam isolated from an obligate marine actinobacterium, Salinispora tropica [25, 26]. Salinosporamide A is an orally active proteasome inhibitor that induces apoptosis in multiple myeloma cells with mechanisms distinct from the commercial proteasome inhibitor anticancer drug Bortezomib [27]. It is being developed by Nereus Pharmaceuticals, Inc. (as NPI-0052) and was scheduled to enter clinical studies for treatment of cancer in humans in 2006. NPI-0052 is currently being evaluated in multiple phase I trials for solid tumors, lymphoma and multiple myeloma (http://www.nereuspharm.com/NPI-0052.shtml). NPI-0052 represents the first clinical candidate for the treatment of cancer produced by saline fermentation of an obligate marine actinobacterium [28] .

Fig. 12.1
figure 1

Chemical structures of salinosporamide A, caprolactones, actinofuranone A, actinofuranone B, resistoflavine, usabamycins and piperazimycins

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Prudhomme et al. [24] tested Salinosporamide A for its utility as an anticancer and antimalarial drug. It was shown to have inhibitory activity against parasite development in vitro (Plasmodium falciparum) and in vivo (P. yoelii). The exact mode by which salinosporamide A inhibits Plasmodium erythrocytic development is unknown; however, it is likely due to the inhibition of the proteasome complex. It is interesting to note that chloroquine resistant strains are still sensitive to Salinosporamide A. Targeting the proteasome system has a huge therapeutic implication as it can restrain growth and survival of most cell types [24]. These attributes, taken with the fact that it is already in phase I clinical trials as an antitumor agent , make it an excellent candidate for alternative therapies, such as antibacterial, anti-parasitic, antifungal or antiviral treatments .

Caprolactones (2) (Fig. 12.1) are new antibiotics isolated from Streptomyces sp. showing moderate phytotoxicity and promising activity against cancer cells with concomitant low general cytotoxicity [29]. Two new polyketides, actinofuranones A (3) and B (4) (Fig. 12.1), were isolated from the culture extract of a marine-derived Streptomyces strain, designated as CNQ766. It showed weak in ­vitro ­cytotoxicity against mouse splenocyte T-cells and macrophages with IC50 values of 20 μg/mL and were inactive against human colon carcinoma HCT-116 cells [30]. Resisto­flavine (5) (Fig. 12.1) is a cytotoxic compound, isolated from S. chibaensis AUBN1/7. It showed cytotoxic activity against human gastric adenocarcinoma HMO2 and ­hepatic carcinoma HePG2 cell lines [31]. Usabamycins (6) (Fig. 12.1) are new anthramycin-type analogues isolated from Streptomyces sp. NPS853. Usabamycins show weak inhibition of HeLa cell growth and selective inhibition of serotonin (5-hydroxytrypamine) 5-HT2B uptake [32] .

Piperazimycins (7) (Fig. 12.1) are cyclic hexadepsipeptides isolated from the fermentation broth of a Streptomyces sp. strain CNQ-593, isolated from marine sediments at a depth of approximately 20 m near the island of Guam. Cytotoxic activities of piperazimycins were initially evaluated in vitro against the human colon carcinoma HCT-116 cell line. All compounds exhibited significant cytotoxicity with an average GI50 of 76 ng/mL for each. Piperazimycin A also showed potent biological activity when evaluated against the NCI’s cancer cell line panel, with mean GI50, TGI and LC50 values for all the cell lines of 100 nM, 300 nM and 2 μM, respectively. Overall, piperazimycin A exhibited a nearly 3-fold more potent activity against solid tumors (average LC50 of 13.9 μM) than against the leukemia cell lines tested (average LC50 of 31.4 μM). It was most active against the melanoma (average LC50 of 0.3 μM), central nervous system (average LC50 of 0.4 μM) and prostate cell lines (average LC50 of 0.6 μM) cancers [33].

Neomarinones (8) (Fig. 12.2) are sesquiterpenoid naphthoquinones with a mixed polyketide-terpenoid origin [34] . Neomarinone, isomarinone, hydroxydebromomarinone and methoxydebromomarinone were produced by the actinobacterial isolate CNH-099 obtained from sediments at 1 m depth in Batiquitos Lagoon, North of San Diego, California. These compounds showed moderate in vitro cytotoxicity, (IC50 of 8 μg/mL) against human colon carcinoma HCT-116 cells. In addition, neomarinone generated a mean IC50 value of 10 μM in the NCI’s 60 cancer cell line panel [35, 36]. Nonactin (9) (Fig. 12.2), a cyclic polyether also known as ­macrotetrolide, has been isolated from cultures of Streptomyces sp. KORDI-3238, isolated from deep-sea sediments collected at Ayu Trough in the Western Pacific Ocean [37]. ­Biosynthesis of gene cluster of nonactin has previously been isolated and characterized from Streptomyces griseus DSM40695 [38], revealing that it is synthesized by a non-iteratively acting type II PKS that involves five ketosynthases and lacks the acyl carrier protein. Nonactin exhibited significant cytotoxicity against the multidrug-resistant human erythroleukemia cell line K-562 [39].

Fig. 12.2
figure 2

Chemical structures of neomarinones, nonactin and lucentamycins

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Lucentamycins (10) (Fig. 12.2), 3-methyl-4-ethylideneproline-containing peptides, are produced by Nocardiopsis lucentensis strain CNR-712, isolated from the sediments of a shallow saline pond from the island of Little San Salvador, in the Bahamas. Lucentamycins A and B showed significant in vitro cytotoxicity against human colon carcinoma HCT-116 cell line with IC50 values of 0.20 and 11 μM, respectively. However, lucentamycins C and D were not cytotoxic in the same assay, suggesting that the presence of an aromatic ring is essential for the biological activity of this class of compounds [40] .

Mansouramycin C (11) (Fig. 12.3) is an isoquinolinequinones antibiotic isolated from Streptomyces sp. Cytotoxicity profiling of the mansouramycins in a panel of up to 36 tumor cell lines indicated significant cytotoxicity of several derivatives, with pronounced selectivity for non-small cell lung cancer, breast cancer , melanoma and prostate cancer cells [41]. Four new polyketides, salinipyrones A (12) (Fig. 12.3) and B (13) (Fig. 12.3), and pacificanones A (14) and B (15) (Fig. 12.3) have been isolated from cultures of the obligate marine actinobacteria Salinispora pacifica CNS-237, found in the sediments collected from the Palau island, Western Pacific Ocean. Biological activity of these compounds is currently being examined in diverse bioassays. In the initial screening, salinipyrones and the pacificanones displayed no significant activity in a cancer cytotoxicity assay using HCT-116 ­human colon cancer cells. In an isolated mouse splenocyte model of allergic inflammation, salinipyrone A displayed moderate inhibition of interleukin-5 production by 50 % at 10 μg/mL without measurable human cell cytotoxicity [42].

Fig. 12.3
figure 3

Chemical structures of mansouramycin C, salinipyrones A, salinipyrones B, pacificanones A, pacificanones B, pyridinium and arenamides A

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Pyridinium (16) (Fig. 12.3) is a salt antibiotic isolated from Amycolatopsis alba. The compound showed potent cytotoxic activity against cancer cell lines of cervix (HeLa) , breast (MCF-7) and brain (U87MG) in vitro and also exhibited antibacterial activity against Gram-positive and Gram-negative bacteria [43]. This new α-pyrone containing secondary metabolite was detected by HPLC–DAD analysis in a culture filtrate extract of Streptomyces sp. NTK 227, a strain isolated from the Atlantic Ocean sediments and found to be a member of the Streptomyces ­albidoflavus 16S rRNA gene clade .

Three new cyclohexadepsipeptides, arenamides A-C (17) (Fig. 12.3), were isolated from the fermentation broth of a marine actinobacterial strain identified as Salinipora arenicola CNT-088 which was obtained from the marine sediments at a depth of 20 m off the Great Astrolab Reef, in the Kandavu Island chain, Fiji. Arenamides A and B exhibited weak in vitro cytotoxicity against human colon carcinoma HCT-116 with IC50 values of 13.2 and 19.2 μg/mL, respectively [44]. In addition, arenamides have been associated to chemoprevention of carcinogenesis by suppression of NFκB activation. NFκB regulates the expression of a number of genes, the products of which are involved in tumorigenesis [45, 46]. Effect of arenamides on NFκB activity was studied with stably transfected 293/NFκB-Luc human embryonic kidney cells, induced by treatment with tumor necrosis factor (TNF). Arenamides A and B blocked TNF-induced activation in a dose- and time dependent manner with IC50 values of 3.7 and 1.7 μM, respectively [44] .

Albidopyrone (18) (Fig. 12.4) showed moderate inhibitory activity against protein-tyrosine phosphatase B [47]. Two new cytotoxic antibiotics, piericidins C7 and C8 (19) (Fig. 12.4), were isolated from a marine Streptomyces sp. [48]. ­Biological activity of piericidins was examined using rat glial cells transformed with the adenovirus E1A gene (RG-E1A-7), Neuro-2a mouse neuroblastoma cells, C6 rat glioma cells and 3Y1 rat normal fibroblast. Adenovirus E1A gene product inactivated the retinoblastoma tumor suppressor protein that plays an important role in cell-cycle and apoptosis control in mammalian cells and is inactivated during the development of a wide variety of cancers [49]. Piericidins C7 and C8 showed selective cytotoxicity against RG-E1A-7 cells (IC50 of 1.5 nM and 0.45 nM, respectively), and inhibited the growth of Neuro-2a cells (IC50 of 0.83 nM and 0.21 nM, respectively) without cytotoxic cell death. On the other hand, C6 rat glioma cells and 3Y1 rat normal fibroblast were not affected by piericidins [50].

Fig. 12.4
figure 4

Chemical structures of albidopyrone, piericidins, chinikomycin A, glyciapyrroles A, aureoverticillactam and marinomycins

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Chinikomycins (20) (Fig. 12.4) are two novel antitumor antibiotics isolated from Streptomyces sp. They exhibited antitumor activity against different human cancer cell lines, but were inactive in antiviral, antimicrobial and phytotoxicity test [51]. ­Glyciapyrroles A (21) (Fig. 12.4) is a new pyrrolosesquiterpenes antibiotic isolated from Streptomyces sp. (NPS008187). Glyciapyrroles A possesses potent antitumor activity against the pair tumor cell lines at concentration up to 1 mM [17] . Aureoverticillactam (22) (Fig. 12.4) is a 22-membered macrocyclic lactam produced by Streptomyces aureoverticillatus NPS001583 isolated from marine sediments. ­Aureoverticillactam was found to possess moderate growth inhibitory activity against human colorectal adenocarcnioma HT-29, Jurkat leukemia and mouse melanoma B16F10 cell lines [4]. Marinomycins (23) (Fig. 12.4) are new antitumor antibiotics isolated from Marinispora sp. Marinomycins show significant antimicrobial activities against drug resistant bacterial pathogens and demonstrate impressive and selective cancer cell cytotoxicities against six of the eight melanoma cell lines in the National Cancer Institute’s 60 cell line panel. The discovery of these new compounds from a new, chemically rich genus further documents that marine actinobacteria are a significant resource for drug discovery [52].

A new cytotoxic substance named mechercharmycin A (24) (Fig. 12.5) was isolated from marine-derived Thermoactinomyces sp. YM3-251. Mechercharmycin A exhibited relatively strong antitumor activity, whereas mechercharmycin B exhibited no such activity [53]. A higher number of type I polyketide derived compounds with antitumor activity have been isolated from marine actinobacteria. Once such compound is arenicolides (25) (Fig. 12.5), 26-membered polyunsaturated macrolactones, produced by the obligate marine actinobacteria S. arenicola strain CNR-005, isolated from the marine sediments, at a depth of 20 m from the coastal around the island of Guam. In particular, arenicolide A was found to exhibit moderate ­cytotoxicity toward the human colon adenocarcinoma cell line HCT-116 with an IC50 of 30 μg/mL [54]. Chalcomycin (26) (Fig. 12.5), a 16-membered macrolide, is produced by Streptomyces sp. M491 isolated from the Qingdao coast (China). Chalcomycin has been found to inhibit protein synthesis in HeLa human cervix carcinoma cell line [55, 56] .

Fig. 12.5
figure 5

Chemical structures of mechercharmycin A, arenicolides, chalcomycinand manumycin A

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Manumycins constitute a class of compounds with antibiotic, cytotoxic, and other biological activities. It has been reported that manumycin A (27) (Fig. 12.5) and its analogues inhibit Ras farnesyl transferase and the growth of Ki-ras-activated murine fibrosarcoma in mice [57]. The side chains in manumycins appear to be a typical polyketide-derived moiety, differing with respect to their combinations of starter and elongation units. The central cyclohexene ring may be derived from the polyketide as in the case of manumycins or from some modified amino acid like 3-amino-5-hydroxybenzoic acid. Manumycin A and chinikomycins A and B (the quinone form of chinikomycin A) were isolated from Streptomyces sp. M045 derived from the sediments of Jiaozhou Bay in China [51] .

Aureolic acid (28) (Fig. 12.6) (Chromomycin B, A2 and A3) are a new antitumor antibiotics isolated from Streptomyces sp. WBF16. These compounds showed strong cytotoxicity against SGC7901, HepG2, A549, HCT116 and COC1 and ­HUVEC [58]. Daryamides are new antitumor-antibiotics isolated from marine-derived Streptomyces strain CNQ-085. Daryamides A (29) (Fig. 12.6), B (30) and C (31) (Fig. 12.6) were subjected to cytotoxicity evaluation against the human colon carcinoma cell line HCT-116. Daryamide A exhibited significantly more potent cancer cell cytotoxicity, with an IC50 of 3.15 μg/mL, than daryamides B and C and very weak antifungal activity against Candida albicans [59].

Fig. 12.6
figure 6

Chemical structure of aureolic acid, daryamides A, daryamides B and daryamides C

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Diazepinomicin (32) (Fig. 12.7) is an unique farnesylated dibenzodiazepinone produced by a Micromonospora strain [3]. It possesses antibacterial, anti-inflammatory and antitumor activity. It has a broad spectrum of in vitro cytotoxicity and has demonstrated in vivo activity against glioma, breast and prostate cancer in mouse models. Chlorinated dihydroquinones (33) (Fig. 12.7) are novel antibiotics produced by a new marine Streptomyces sp [15]. The compounds formally possess new carbon skeletons, but are related to several previously reported metabolites of the napyradiomycin class. Structures of the new molecules possess significant antibacterial and cancer cell cytotoxicities. Caboxamycin (34) (Fig. 12.7) is a new benzoxazole antibiotic and was detected by HPLC-diode array screening in extracts of Streptomyces sp. NTK 937, another strain which was isolated from the sediments collected from the Canary ­Basin. The compound, caboxamycin was named after the first letters of the collection site from where the organism was isolated and from letters drawn from its chemical structure. Caboxamycin showed inhibitory activity against both Gram-positive bacteria and against the tumor cell lines gastric adenocarcinoma (AGS), hepatocellular carcinoma (Hep G2) and breast carcinoma cells (MCF7). The antibiotic also showed an inhibitory activity against the enzyme phosphodiesterase [60].

Fig 12.7
figure 7

Chemical structures of diazepinomicin, chlorinated dihydroquinones, caboxamycin, chandrananimycin A and N-(2-hydroxyphenyl)-2-phenazinamine (NHP)

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Chandrananimycin A (35) (Fig. 12.7) is a novel antibiotic isolated from Actinomadura sp. Chandrananimycin A possesses potent antifungal activity against ­Mucor miehei. It also exhibits antialgal activity against the microalgae Chlorella vulgaris and C. sorokiniana and antibacterial activity against Staphylococcus ­aureus and ­Bacillus subtilis, along with anticancer activity [61]. N-(2-hydroxyphenyl)-2-phenazinamine (NHP) (36) (Fig. 12.7) is a new antibiotic isolated from ­Nocardia ­dassonvillei. The new compound showed significant antifungal activity against Candida albicans, with a MIC of 64 µg/mL and high cancer cell cytotoxicity against HepG2, A549, HCT-116 and COC1 cells [62] .

12.4 Conclusions

Actinobacteria and, in particular the genus Streptomyces, have been well known during the last seventy years as prolific producers of novel bioactive compounds , anticancer drugs included. With the increasing development of oceanographic studies leading to the isolation of new actinobacteria from marine sources, new prolific genera in the production of useful compounds have been found, such as ­Salinispora. However, the Ocean, without any doubt, is keeping a myriad of new actinobacteria providing novel structural diversity to be discovered and used. In addition, the continuous effort for unravel the biosynthesis of the already known compounds and the isolation and characterization of their biosynthesis gene clusters will lead to the development of new anticancer compounds, hopefully with improved therapeutic properties, by using combinatorial biosynthesis approaches.