J Appl Phycol DOI 10.1007/s10811-015-0657-7
Impact of temperature on the biosynthesis of cytotoxically active carbamidocyclophanes A–E in Nostoc sp. CAVN10 Michael Preisitsch 1 & Ha Thi Ngoc Bui 1,2 & Christian Bäcker 1 & Sabine Mundt 1
Received: 6 February 2015 / Revised and accepted: 28 June 2015 # Springer Science+Business Media Dordrecht 2015
Abstract The effects of different temperatures on biomass production and carbamidocyclophane biosynthesis of the cyanobacterium Nostoc sp. CAVN10 were investigated under batch cultivation conditions over 30 days. Cyanobacterial growth correlated with increasing temperatures from 18 to 33 °C but revealed different specific growth rates within the cultivation period. The accumulation of carbamidocyclophanes A–E was investigated at different growth stages, and their levels were quantified by HPLC-UV analysis. The highest dry weight content of 1.5 % for carbamidocyclophane A, 1.0 % for carbamidocyclophane B, and 1.1 % for carbamidocyclophane C was found around the 15th day at 28 °C. At 33 °C, however, yields of these compounds decreased significantly, but the content of carbamidocyclophanes D and E continuously increased to 0.4 % on the 25th day. In general, carbamidocyclophanes showed cytotoxic activity against LN18 glioblastoma cells and 5637 human urinary bladder carcinoma cells with half maximal inhibitory concentration (IC 50 ) values of 2.1–3.1 μM and 0.8–2.1 μM. Only carbamidocyclophane D exhibited a less potent cytotoxicity against 5637 cells with an IC50 value of 10.1 μM.
Keywords Cyanobacteria . Nostoc . Cultivation . Temperature . [7.7]Paracyclophanes . Carbamidocyclophanes . Cytotoxicity
* Michael Preisitsch
[email protected] 1
Department of Pharmaceutical Biology, Institute of Pharmacy, Ernst-Moritz-Arndt-University, Friedrich-Ludwig-Jahn Straße 17, 17489 Greifswald, Germany
2
Public Health Center Laboratory, Hanoi School of Public Health, 138 Ginag Võ, Hanoi, Vietnam
Introduction With regard to raising the number of potential drug candidates for the developing pharmaceutical industry, ongoing screening and isolation attempts have been initiated to explore new bioactive compounds and to identify potent lead structures from different natural sources, such as terrestrial plants, fungi, and microorganisms, or marine organisms including microalgae and macroalgae, aquatic plants, fungi, actinomycetes, and sponges (König et al. 2006; Harvey 2008; Gerwick and Moore 2012; Newman and Cragg 2012; Blunt et al. 2013; Tarman et al. 2013; Evidente et al. 2014). Consequently, in the field of natural product drug discovery, cyanobacteria are considered as promising producers of novel and biologically active secondary metabolites (Dixit and Suseela 2013; Singh et al. 2011; Nunnery et al. 2010). In fact, more and more is recognized that previously isolated compounds from macroorganisms are often originally biosynthesized by the associated microbes, for example, cyanobacteria and heterotrophic eubacteria (König et al. 2006; Gerwick and Moore 2012). According to Burja et al. (2001), marine cyanobacterial secondary metabolites consist either of cyclic and linear lipopeptides, macrolides, amides, derivatives of fatty acids and amino acids or of combined biosynthetic origin revealing pronounced anticancer, antimicrobial, antifungal, antiviral, enzyme inhibiting, antifeedant, and even immunosuppressive activities, but mostly associated with a generalized cytotoxicity. Especially from mixed polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS) pathways and NRPSPKS assembly, respectively, derived ketopeptides and peptoketides, for example, the cytotoxic curacin A and the neurotoxic jamaicamide A, are structurally diverse and bioactive compounds. Frequently accompanied structural modifications including not only N-methylations, halogenations, and
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glycosylations but also the incorporation of α/β-hydroxyl acids or unusual amino acids as well as oxidations increase the structural and thus bioactive capacities (Burja et al. 2001; Jones et al. 2009; Tan 2010; Tidgewell et al. 2010). According to the phycological classification system of cyanobacteria (Anagnostidis and Komárek 1988; Komárek and Anagnostidis 1989), the filamentous orders Oscillatoriales and Nostocales tend to be a rich source of interesting natural products. Especially, marine tropical strains belonging to the genera Moorea (formerly often incorrectly classified as Lyngbya due to morphological resemblance) (Engene et al. 2012) and Oscillatoria are predominantly reported for their rich bioactive secondary metabolite spectrum (Gerwick et al. 2008; Liu and Rein 2010; Tidgewell et al. 2010). Nevertheless, as reviewed by Boopathi and Ki (2014), many marine and freshwater cyanobacteria of the order Nostocales, for example, strains of Anabaena, Anabaenopsis, Aphanizomenon, Cylindrospermopsis, Nodularia, Nostoc, Raphidiopsis, Scytonema, and Sphaerospermopsis, produce a variety of strong acting natural biotoxins such as the hepatotoxic microcystins (MCYSTs) and nodularins (NODs) as well as the hepatotoxic alkaloid cylindrospermopsin (CYN) or the neurotoxic alkaloids anatoxins (ANTXs) and saxitoxins (SXTs). In contrast to those, one of the most prominent classes of cyanobacterial anticancer agents, the cryptophycins, is originally isolated from various terrestrial Nostoc strains (Schwartz et al. 1990; Trimurtulu et al. 1994). These tubulindepolymerizing depsipeptides are also derived from a mixed PKS-NRPS pathway (Magarvey et al. 2006). Especially, cryptophycin 1, initially considered as a potent fungicide, and its synthetic derivative cryptophycin 52 are active against drug-sensitive as well as drug-resistant tumor cell lines in the low picomolar range (Smith et al. 1994; Al-awar and Shih 2012). The latter had entered phase II of different clinical trials (Edelman et al. 2003; D’Agostino et al. 2006). Due to limited efficacy, neurotoxic side effects, and high production costs, however, Eli Lilly stopped further development (Niedermeyer and Brönstrup 2012; Al-awar and Shih 2012). In contrast to the cryptophycins, efforts regarding dolastatin 10, a potent antimitotic peptide biosynthesized from Symploca and Moorea species (Harrigan and Goetz 2002; Luesch et al. 2002), were considerably more successful. The linking of the synthetic, highly cytotoxic, but antineoplastic active, monomethylauristatin E (MMAE) to monoclonal antibodies opened up new scopes of action for natural products with pronounced cytotoxicity. Thus, the invention of these antibody-drug conjugates (ADCs) led to the first commercially available drug derived from a cyanobacterial metabolite by Takeda in cooperation with Seattle Genetics in 2011, named brentuximab vedotin (Adcetris®) (Niedermeyer and Brönstrup 2012). To enhance the understanding of the regulatory complexity and its alteration by environmental factors, cyanotoxin
biosyntheses have been investigated by numerous studies both on the molecular and transcriptional level, respectively (for detailed overview, see in particular Neilan et al. 2013; Boopathi and Ki 2014), as well as on the metabolic compound level. Up to now, cultivation studies regarding the influence of environmental factors on the secondary metabolite content levels have been investigated on a variety of cyanobacterial strains. Especially, cyanotoxins have been in the focus of research, in particular already mentioned MCYSTs (Van der Westhuizen and Eloff 1985; Codd and Poon 1988; Sivonen 1990; Rapala et al. 1997; Rapala and Sivonen 1998; Kurmayer 2011; Monchamp et al. 2014), NOD (Blackburn et al. 1996; Lehtimäki et al. 1997; Repka et al. 2001; Jonasson et al. 2008), CYN (Saker and Neilan 2001; Bácsi et al. 2006; Dyble et al. 2006; Bar-Yosef et al. 2010; Cirés et al. 2011; Mohamed and Al-Shehri 2013; Burford et al. 2014), ANTXs (Rapala et al. 1993; Rapala and Sivonen 1998; Kearns and Hunter 2000; Gagnon and Pick 2012), and the SXTs (Dias et al. 2002; Castro et al. 2004; Carneiro et al. 2009). Besides mainly investigated parameters such as light, nitrogen, phosphorous, salinity, and pH, the temperature seems to have a significant impact on both the biosynthesis of natural products and the growth of the investigated cyanobacteria (Lehtimäki et al. 1997; Rapala et al. 1997; Rapala and Sivonen 1998; Dias et al. 2002; Pomati et al. 2004; Cirés et al. 2011; Kurmayer 2011) and moreover for eukaryotic algae (Bouterfas et al. 2002) or marine diatoms (Berges et al. 2002). Unfortunately, due to non-harmonized cultivation procedures, the absence of uniform specific growth controls, the use of different quantification methods, or different biovolume surrogates, studies of environmental stimuli are difficult to compare with each other (Meissner et al. 2013; Neilan et al. 2013). Furthermore, for some environmental factors, conflicting results were reported when investigating their impact on different cyanobacterial strains (Meissner et al. 2013; Neilan et al. 2013; Boopathi and Ki 2014). Until now, cytotoxic [7.7]paracyclophanes have not been investigated on any correlation of cultivation parameters to biosynthetic productivity rate. The paracyclophane family possesses a unique scaffold consisting primarily of two benzene cores that are connected with each other in their para-positions by two aliphatic chains (Fig. 1). Due to the ability to form inclusion complexes by utilization of intramolecular cavities, larger synthetic cyclophanes are already applied in host-guest chemistry (Smith et al. 2001). Until now, naturally occurring compounds, originated by fatty acid recruitment and subsequent polyketide pathway (Bobzin and Moore 1993; Nakamura et al. 2012), have been isolated less frequently from cyanobacterial Cylindrospermum and Nostoc species. The compounds can be divided into four subgroups, namely, the cylindrocyclophanes, carbamidocyclophanes, nostocyclophanes, and merocyclophanes. They differ in a diverse substitution pattern of functional groups attached to the core structure (Chen et al.
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1991; Moore et al. 1992; Bui et al. 2007; Kang et al. 2012). In deviation from other paracyclophanes, the carbamidocyclophanes are mainly characterized by the presence of one or two carbamate groups within the molecule. Furthermore, congeners differ in a varying degree of chlorine atoms in the butyl side chains. In a previous report, our working group described this subtype of cyclophanes for the first time by utilizing a bioassay-guided approach to isolate carbamidocyclophanes A–E from the biomass of the Vietnamese terrestrial cyanobacterium Nostoc sp. CAVN10 (Bui et al. 2007). Recently, further carbamoylated derivatives have been discovered in two more phylogenetically closely related Nostoc sp. strains (Luo et al. 2014; Preisitsch et al. 2015). The carbamidocyclophanes exhibit cytotoxicity against various human cancer cell lines as well as anti-MRSA and anti-Mycobacterium tuberculosis activity in the low micromolar range (Bui et al. 2007; Luo et al. 2014; Preisitsch et al. 2015). Here, we describe the influence of temperature on the growth of Nostoc sp. CAVN10 as well as the impact of this environmental factor on the biosynthesis of carbamidocyclophanes A–E. With the aim of revealing optimized temperature-related culture conditions to enhance the compound yields, we monitored their respective contents simultaneously in obtained biomasses via HPLC-UV analysis. Furthermore, we evaluated the cytotoxic potential of carbamidocyclophanes A–E against 5637 and LN18 cancer cells, and we completed the evaluation of these compounds against MCF7 and FL cells.
To investigate the influence of temperature on the production of carbamidocyclophanes in different growth stages, 100-mL aliquots of pooled stock cultures were transferred into 1.8-L Fernbach flasks and BG-11 was added up to 500 mL. Flasks were incubated at respective study conditions for 5 days before 10 mL (corresponding to 10±1 mg biomass) of Nostoc sp. culture was used as inoculum for the test cultivation. The cyanobacterium was cultured in 500-mL Erlenmeyer flasks containing 300 mL of BG-11. The flasks were gently shaken (25 rpm) at 24± 1, 28±1, and 33±1 °C in an orbital shaking incubator (OSFT-LS/R-32, TEQ, Germany) and exposed to an illumination of 30 μmol photons m−2 s−1 (fluorescent lamp TL-D 30 W/830, Philips, the Netherlands) utilizing a light/ dark rhythm of 12/12 h. For the determination of cyanobacterial growth over 30 days, at least three independent cultivation experiments were performed with two technical replicates per treatment. To avoid content fluctuations based on any response to illumination, the biomass was harvested in 5-day steps always at the same time within the cycle by centrifugation (Rotanta 96 R, Hettich Zentrifugen, Germany; 3300g, 15 min at 10 °C), washed with distilled water, and dried by lyophilization. Growth of Nostoc sp. CAVN10 was determined by measuring the dry weights of every 5-day harvest and is represented by growth curves. The related growth rates were estimated by calculating the specific growth rate (μg) based on following equation (Wood et al. 2005):
Material and methods The terrestrial cyanobacterium Nostoc sp. CAVN10 is integrated in the culture collection of the Institute of Pharmacy, University of Greifswald (Bui et al. 2007). For maintenance of a laboratory culture, 5 mL of a 3-week-old cyanobacterial stock culture was used to inoculate 100-mL Erlenmeyer flasks containing 50 mL BG-11 medium (Waterbury and Stanier 1981) but added with micronutrient solution according to Kuhl and Lorenzen (1964). Stock cultures were continuously illuminated by cool-white fluorescent lighting (fluorescent lamp Lumilux 36 W/840, Osram, Germany) of 8 μmol photons m−2 s−1 at 20±1 °C.
Fig. 1 Structural overview of carbamidocyclophanes A–E
μg ¼
InN t InN 0 Δt
where Δt is the length of the time interval (tt −t0) and N0 and Nt are the corresponding biomasses. For isolation of carbamidocyclophanes A–E (Fig. 1), Nostoc sp. CAVN10 was cultured in a 40-L glass fermenter in 35 L of BG-11 medium with an inoculum of three 20-day cultivated Fernbach flasks according to Mundt et al. (2001). The pH of the culture was adjusted to 8.5 using CO2 supplementation. The fermenter was continuously illuminated by
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cool-white fluorescent lighting (fluorescent lamp Lumilux 36 W/840, Osram, Germany) of 20 μmol photons m−2 s−1 at 20±1 °C. After 4 weeks, the biomass was collected by continuous flow centrifugation (Stratos D37520, Heraeus Instruments, Germany; 4000g, 20 °C). The biomass was lyophilized and stored at −20 °C until use. The yield of freeze-dried biomass was 0.6 g L−1.
Isolation of carbamidocyclophane reference standards The entire extraction, separation, and isolation procedure to provide carbamidocyclophanes A–E as external reference substances was performed in accordance to Bui et al. (2007). To ensure sufficient supply of carbamidocyclophanes A–E for cytotoxicity assays, compounds were also isolated from the cyclophane-producing cyanobacterium Nostoc sp. CAVN2 as previously described (Preisitsch et al. 2015).
Processing and quantification of temperature-related cultivation samples Corresponding biomass samples cultivated under equal conditions were pooled and homogenized. Aliquots of 30 mg were suspended in 50-mL centrifuge tubes with 30 mL of MeOH, homogenized for 10 min by ultrasonication (ultrasonication bath Transsonic 460, Elma, Germany), and subsequently extracted at 750 rpm for 16.5 h on a magnetic stirrer (Variomag, H+P Labortechnik, Germany) utilizing 10×6 mm stirring bars (VWR, Germany). Samples were centrifuged (Rotanta 96 R, Hettich Zentrifugen, Germany; 3300g, 10 min at 4 °C), and the supernatant was filtered (filter paper 595 ½ Ø 185 mm, 12–25 μm, Schleicher and Schuell, Germany). The extraction procedure was repeated twice with 20 mL MeOH for 1 h. The supernatants were combined and reduced to dryness by rotary evaporation. The crude extract was dissolved in 2 mL of 80 % MeOH, filtered (syringe filter PTFE Ø 13 mm, 0.2 μm, VWR, Germany), and subjected to HPLC-DAD quantification consisting of previously listed components (Bui et al. 2007). Separation was achieved by utilizing a Synergi Polar RP HPLC column (4 μm, 80 Å, 250×4.6 mm, Phenomenex, USA) and a binary gradient (flow rate 1.0 mL min−1) of MeOH in deionized water from 10 to 60 % in 5 min, followed by 60 to 85 % in 25 min, and 85 to 100 % in 3 min. Calculation of the carbamidocyclophane A–E contents was performed at wavelength 226 nm with Gemynix software (version 1.91, Flowspek, Switzerland) by using six calibration levels of carbamidocyclophanes A–E as external references in a concentration range from 0.03 to
1.00 mg mL−1 each. The specific carbamidocyclophane production rates (μCARB) were calculated based on gravimetric concentration data according to Orr and Jones (1998).
Cytotoxicity assays The cytotoxicity evaluation of carbamidocyclophanes A–E was investigated against the human urinary bladder carcinoma cell line 5637 and against human glioblastoma cells (LN18) by utilizing 3-amino-7-dimethylamino-2methylphenazine hydrochloride and the neutral red uptake (NRU) assay as previously described (Bäcker et al. 2014). Additionally, all five carbamidocyclophanes were tested for cytotoxicity against the breast adenocarcinoma cell line MCF7 and human amniotic epithelial fibroblast-like (FL) cells by using the crystal violet staining (CVS) assay according to Bracht et al. (2006) and Bui et al. (2007). Tests were performed at least for three times with four to six technical replicates of compound concentrations, originated by serial dilution, between 31.25 and 0.24 μg mL−1. Half maximal inhibitory concentration (IC50) values were obtained from dose-response curves. Etoposide (Alexis Biochemicals, USA) was carried as positive control (IC50 values of 0.6 μM for 5637 and LN18 cells, IC50 value of 1.0 μM for MCF7 cells).
Statistical analyses Except for the quantification results of 1–5, for the μg-μCARB analysis, and for the characterization of correlation factors, values are expressed as mean±standard error of the mean (SEM) and as mean+SEM, respectively, of at least three independent experiments. Data of each experimental variable were tested for Gaussian distribution utilizing the Kolmogorov-Smirnov test and the D’Agostino-Pearson omnibus normality test. Testing for equivalence, based on consideration of the 90 % confidence intervals, was performed by utilizing the MS ACOMED Excel tool (version 2, Acomed, Germany). Student’s unpaired t test with Welch’s correction (t test), one-way analysis of variance (ANOVA), and two-way ANOVA each followed by Tukey’s multiple comparison test (Tukey test) were used to analyze statistical differences related to growth, cyclophane contents, and cytotoxicity analysis. Non-parametric data were performed utilizing one-way ANOVA followed by Dunn’s multiple comparisons test (Dunn’s test). The Mann-Whitney U test (U test) was used in case of at least one non-parametric data set for specific comparison. A value of P 0.05, one-way ANOVA with Tukey test; P > 0.05, t test). In addition, the temperature reduction from 28 to 18 °C also showed a significant up to 1.8fold decline of the growth rate (P < 0.05, U test). However, a closer look to the data revealed that CAVN10 responded differently to temperature during the log phase. Therefore, we determined an early (5th–
Fig. 2 Growth curves of Nostoc sp. CAVN10 cultivated at different temperatures. Values shown are expressed as mean ± SEM (n ≥ 3). Statistically significant differences of dry weights (DW) on the 15th day, DW(18 °C) versus DW(33 °C) (P < 0.05); on the 20th day, DW(18 °C) versus DW(24 °C) (P
