Secondary metabolites represent an attractive and important source of natural products with a wide range of bioactivities and biotechnological applications. Organisms such as plants, algae, bacteria and fungi are able to produce bioactive compounds, many of which have been used as antitumor or antimicrobial agents, immunosuppressants, vaccine antigens or insecticides. Fungi especially are known to be one of the major producers of bioactive compounds. In fact, in 2012 the number of bioactive natural compounds characterized was 80,000–100,000, of which, 15,000 were of fungal origin [1
]. Some well-known examples of bioactive fungal compounds include penicillin, developed as an antibacterial [2
], lovastatin developed as a cholesterol-lowering medication [3
], cyclosporins developed as immunosuppressive agents [5
] and taxol, which has been proved to have an antitumor activity [6
]. Up to now, most of the characterized fungal natural products are from terrestrial habitats even though fungi also colonize fresh water and marine habitats. Aquatic fungi have been less well studied compared to terrestrial fungi, but they have generated an increasing level of interest in the last decade.
Fungi from marine habitats were first studied in the 1940s when Barghoorn and Linder (1944) demonstrated their occurrence [8
]. First exhaustive definition of marine fungi distinguished obligate and facultative organisms. In other words, marine fungi were divided between those “that grow and sporulate exclusively in a marine or estuarine habitat from those from freshwater or terrestrial milieus able to grow and possibly to sporulate in the marine environment” [9
]. In 2015, Jones et al. provided a review of the classification of marine fungi, including the accepted names of 1112 species of characterized fungi from marine habitats [10
]. Recently, there has been an increased level of interest in marine fungi, especially in defining their diversity and ecological role. Along with studies on marine filamentous fungi and yeasts, several studies have focused on the diversity of fungi in extreme environments such as the subseafloor [11
], hydrothermal fields [13
], or deep-sea hydrothermal vents [14
]. In many cases, their activity and function has also been described, using transcriptomics to better understand their ecological role in extreme ecosystems [16
]. Such studies have suggested that fungal communities from extreme ecosystems might represent an untapped reservoir of potential novel bioactive molecules, and, in recent years, secondary metabolites (also called specialized metabolites) obtained from fungi isolated from fresh water and marine habitats have gained considerable attention. According to a study by Rateb and Ebel (2011), marine fungal secondary metabolites have various biological and pharmaceuticals properties. In total, 690 novel structures of secondary metabolites have been identified from marine fungi between mid-2006 and 2010 [17
]. At this time, 6222 molecules have been referenced from Dikarya fungi in MarineLit database (http://pubs.rsc.org/marinlit/
). Evaluation of the biotechnological potential of marine-derived fungi has revealed that they produce mainly polyketides, terpenoids and peptides [17
]. In addition, whole genome sequence analysis suggests that many deep-sea fungi have the potential to produce bioactive compounds. Indeed, Rédou et al. (2015) provided evidence that almost all fungal isolates in their study (96% of the 200 filamentous fungi and yeasts) had at least one gene involved in a secondary metabolite biosynthesis pathway [19
], for example genes that encode enzymes involved in polyketides production (Polyketide Synthase, PKS), terpenoids (Terpene synthases, TPS) or non-ribosomal peptides (Non-Ribosomal Peptide Synthetase, NRPS). Along with this study, several studies have demonstrated antimicrobial activities of diverse marine fungi [20
], deep-subseafloor fungi [21
], and antimicrobial, antitumoral and antioxidant activities of sponge-associated marine fungi [22
Polyketides represent a major and highly diverse group of natural products [17
]. With a wide range of complex structures including macrolides, polyphenols, polyenes, and polyethers, polyketides comprise several groups of biologically important secondary metabolites such as flavonoids, phloroglucinols, resorcinols, stilbenes, pyrones, curcuminoids and chalcones [24
]. Polyketides have numerous functions and applications as pigments, antibiotics, immunosuppressants, antioxidants, antiparasitics, cholesterol-lowering, and antitumoral agents [26
]. Polyketide biosynthesis requires specific enzymes known as polyketide synthases (PKS). PKSs are a large group of enzymes, divided into three classes: type I, II or III [26
], where type I PKSs are large multi-domain enzymes able to function in either a modular or iterative manner, type II PKSs are dissociable multi-enzyme complexes functioning in an iterative manner and type III PKSs are homodimeric enzymes, mechanistically different from the two other subgroups of PKSs, and structurally simpler [25
]. Type III PKSs share common characteristics: they form dimers. Each monomer with a size of 40–45 kDa contains a conserved Cys-His-Asn catalytic triad within an active site cavity [25
]. Polyketides are produced by iterative decarboxylative condensations of malonyl-CoA and diverse acyl-CoA thioesters (as extender and starter units), and cyclization reactions [28
]. Despite their simplicity type III PKSs have an unusually wide range of substrates and play an important role in the biosynthesis of various bioactive polyketide compounds in different organisms [29
]. For example, chalcones are produced by land plants [30
]. Phlorotannins are unique to brown algae and phloroglucinols can be synthesized by diverse kind of organisms from prokaryotes (Pseudomonas
) to eukaryotes (brown algae) [31
]. Type III PKSs have also recently been discovered in fungi [36
]. The first characterized fungal type III PKS was isolated from Neurospora crassa
] and since then, several fungal type III PKSs have been reported. Among them, type III PKSs isolated from Aspergillus oryzae
] and another from Aspergillus niger
] have been characterized, as well as a type III PKS isolated from Sporotrichum laxum
which appears to be involved in the production of spirolaxine, a potential drug candidate with anti-Helicobacter pylori
]. A recent analysis reveals a total of 552 type III pks
genes from 1193 fungal genomes (JGI Mycocosm) [43
]. Only eleven type III PKSs have been biochemically characterized in fungal kingdom [43
]. Polyketides produced by fungal type III PKS are grouped into triketide pyrones, tetraketide pyrones and alkylesorcinols [25
]. All enzymes are isolated from terrestrial fungi. To date, no marine fungal enzymes have been described.
In this study we report the identification of a type III PKS from a marine yeast Naganishia uzbekistanensis
strain Mo29 (UBOCC-A-208024) (formerly named as Cryptococcus
]. Two enzyme forms, the whole protein PKSIII Mo29 (Std) and the reduced protein PKSIII Mo29 (Red) without the C-terminus extension were expressed as recombinant proteins in E. coli
and their biochemical enzyme activities were analyzed by LC/MS-MS. Our phylogenetic, biochemical and structural modelling of this new fungal type III PKS provide new insights into the biotechnological potential of deep-sea yeast.
Genes encoding type III Polyketides synthases are found in Dikarya such as Aspergillus niger
, Aspergillus oryzae
, Neurospora crassa
, Botrytis cinerea
or Sordaria macrospora
], and recent genomic studies by Navarro-Munoz and Collemare revealed that there are 522 genes encoding these enzymes among 1193 fungal genomes [43
]. However, currently, very few fungal pyrone synthase enzymes have been described biochemically. Indeed, there are only 11 PKSIII enzymes from fungi that have been functionally characterized and these all belong to fungal species of terrestrial origin. In this work, we have described the first biochemical characterization of PKSIII from marine fungi, a yeast, Naganishia uzbekistanensis
strain Mo29 (UBOCC-A-208024) (formerly named as Cryptococcus
Marine fungi were first described in the 1980s and 1990s [9
]. Thanks to numerous oceanographic cruises, we now know that it is possible to find many fungal species in the marine environment. Among these, the species found mainly pertained to Dikarya (Basidiomycota
). These species are found in different places in the marine environment such as the water column, associated with macro-organisms like algae, deep-sea sediments and hydrothermal vents [63
]. The N. uzbekistanensis
strain Mo29 (UBOCC-A-208024) was discovered during the MoMARDREAM-Naut oceanographic cruise (2007), on the exploration of the hydrothermal sources of the Rainbow site (-2300 m, Mid-Atlantic Ridge) [14
]. Genome sequencing of this fungal species led to identify a gene encoding a PKSIII [45
]. Phylogenetic analysis of the PKSIII protein sequence with enzymes from plants and bacteria, revealed that all fungal PKSIII enzymes (619 protein sequences) were grouped into a single branch. The PKSIII enzyme from N. uzbekistanensis
strain Mo29 strain (UBOCC-A-208024) was not included in the fungal group/cluster. Instead, it clustered with 3 other protein sequences identified in another strain of Naganishia
sp. and two other strains of Cryptococcus
]. This suggests that sequences of these three PKSIII proteins have followed an independent and early separation in the evolution compared to the other fungal sequences of PKSIII included in this study.
Interestingly, one of the features of these four enzymes is the presence of an additional 74 amino acids extension at the C-terminus. Previous studies in Sordaria macrospora
and Botrytis cinerea
have revealed that PKSIII may have a C-terminus extension [53
] but the contribution of this region to the enzymatic function in these organisms is not known. Here, by expressing forms of the PKSIII Mo29 protein with and without this C-terminus extension, we show that it is not required for the enzymatic activity. Both forms are functional and produce mainly pyrones and some alkylresorcinols.
Analysis of the predicted structure of PKSIII Mo29 identified three catalytic residues: cysteine at position 171 (171C), Histidine at position 352 (352H) and Arginine at position 385 (385N). The next step is to mutate one or the three catalytic residues. This experiment could prove that these residues are involved in the enzymatic activity.
Previous studies of PKSIII have shown that another essential structure for the PKSIII protein is the “tunnel” which allows the entry of different starters such as lauroyl-CoA or stearoyl-CoA. These starters will either be cyclized or branched to the cycle synthesized by malonyl-CoA in the tunnel structure. The majority of the amino acids involved in the tunnel in PKSIII Mo29 protein are identical or similar to those found in the structure of PKSIII from N. crassa
, from A. oryzae
and B. cinerea
and Figure S2
]. Based on the structure of the PKSIII of N. crassa
(3E1H) and the models of B. cinerea
and A. niger
, some amino acids explaining structural differences could be found. In position 144 of the PKSIII from N. uzbekistanensis
strain Mo29, there is a tyrosine (144Y) whereas in N. crassa
and the two other fungal proteins, there are two asparagines (PKSIIINc 125N and BcPKS 138N) and an alanine (AnPKS 140A) (amino acids not having the same chemical characteristics). Similarly, at position 212, there is a cysteine (212C), while in the three other fungal sequences, there is a non-polar amino acid (PKSIIINc 190V, BcPKS 203V and AnPKS 205L). In position 236, the PKSIII Mo29 has a serine (that is a polar amino acid) whereas in the three other fungi, there is a non-polar amino acid (PKSIIINc 206G, BcPKS 219G and AnPKS 221A). The other notable difference is in the amino acid that could bind to stearoyl-CoA. For the PKSIII Mo29, at position 355, there is a serine whereas in the other proteins, a nonpolar (309A), an aromatic (321A) or a positively charged amino acid (320R) are found, respectively, in PKSIIINc, BcPKS and AnPKS. These different amino acid changes in the structure of the PKSIII Mo29 protein could explain the fact that it does not synthetize short chain pyrones like some PKSIII proteins characterized biochemically in A. oryzae
(AnCsyA and AnCsyB) [39
We overexpressed the entire PKSIII Mo29 protein as well as a truncated form lacking the additional 74 aa at the C-terminus. These two proteins forms are dimeric and are active. Indeed, the 74 aa extension in C-terminus does not seem to be involved in the enzymatic activity as in B. cinerea
and in S. macrospora
]. The two proteins are able to produce compounds in the presence of malonyl-CoA but always in the presence of an acyl-CoA starter with a carbon chain longer than 6 units. PKSIII Mo29 does not incorporate small carbon chains (acetyl-CoA, malonyl-CoA (only) and hexanoyl-CoA). No molecules are synthesized in the presence of these three starters. The known fungal pyrone synthases catalyse reactions starting from the acyl-CoA chain and ending with aldol cyclization and/or lactonization [25
]. The fungal PKSIII that have been experimentally characterized are divided into two functional groups. The first group uses only long acyl-CoA chains with several malonyl-CoA. We find in this group enzymes from N. crassa
, A. niger
, B. cinerea
and S. macrospora
]. The second group consists of two enzymes found in A. oryzae
]. These two enzymes can use starter molecules with chains of different lengths to produce triketide, tetraketide pyrones and alkylresorcinols. These results show that the Mo29 enzyme belongs to the first group (like the enzymes of N. crassa
, A. niger
, B. cinerea
and S. macrospora
). PKSIII Mo29 produces triketide and tetraketide pyrones in the presence of starter such as octanoyl-CoA, decanoyl-CoA, lauroyl-CoA and palmytoyl-CoA (Table 2
). Another study allowed us to highlight that this protein could accommodate very long chain fatty acyl-CoA starter units produced by E. coli
. Indeed, molecules synthesized by proteins overexpressed by E. coli
are secreted into the culture medium. Analysis of molecules found in the culture medium show that the two types of proteins (Std and Red) are able to synthesis several molecules having 14 carbons (C14) to 28 carbons (C28). However, the majority of molecules possess 18 and 20 to 24 carbons. Among these molecules are pyrones and alkylresorcinols. The most synthesized molecules are C22
348.2663) and C20
320.2349) which are pyrones (triketide pyrones) and C23
346.2869) (alkylresorcinol) (Figure 5
The PKSIII Mo29 Std and PKSIII Mo29 Red synthesize the same molecules, pyrones and alkylresorcinols. However, the truncated form seems to produce less product than the entire recombinant protein. These results were observed in In vitro experiments and in E. coli culture medium. In the E. coli culture medium, all molecules are less produced by PKSIII Mo29 Red than PKSIII Mo29 Std. These results could be explained by the absence of the C-terminus 74 aa extension. The extension could be implicated in the dimer stability or substrates binding. In this study, we have tested only classical substrates and starters such as palmitoyl-CoA; however, we did not identify natural substrates in N. uzbekistanensis strain Mo29 strain (UBOCC-A-208024) yet. So, the extension could be playing a role in the entrance of other substrates than those used in vitro.
Several α-pyrones produced by marine and terrestrial fungi have shown cytotoxic activities against several cancer cell lines [58
]. The different molecules produced by PKSIII Mo29 In vitro have been brought into contact with different cancer cell lines, such as Caco-2 and THP1 (colon cancer cell lines and leukemic cell lines). Molecule produced by PKSIII Mo29 in the presence of malonyl-CoA and palmitoyl-CoA is a α-pyrone with a molecular formula of C20
321.2507). When the Caco-2 cells and the THP1 are exposed for 48 h in the presence of 0.012 g/L of this molecule, the cell viability decreases. Therefore, it would mean that this α-pyrone has a cytotoxic effect on these two tumors cell lines. The molecule produced by PKSIII Mo29 in the presence of malonyl-CoA and lauroyl-CoA In vitro is also a triketide pyrone with a molecular formula of C16
266.1884). THP1 cells were incubated for 48 h with 0.012 g/L of this molecule. Cell viability was reduced by 5%. These two molecules seem to have cytotoxic effects on cancer cell lines like Caco-2 and THP1.
4. Materials and Methods
4.1. Fungal Strains
The yeast strain studied in these experiments was a Naganishia uzbekistanensis
strain Mo29 (UBOCC-A-208024) isolated from hydrothermal vents of the Rainbow site (−2300 m, Mid-Atlantic Ridge) during the oceanographic cruise MoMARDREAM-Naut (2007) [14
]. Yeast was cultivated at 30 °C on culture medium composed of 2% malt extract, 0.3% yeast extract and 1.5% agar during 7 days.
4.2. Bacterial Strains
Escherichia coli SURE 2 Supercompetent Cells [e14(McrA-) Δ(mcrCB-hsdSMR-mrr) 171 endA1 gyrA96 thi-1 supE44 relA1 lac recB recJ sbsC umuC::Tn5 (Kanr) uvrC [F’ proAB lacIqZΔM15 Tn10 (Tetr)]] (Agilent Technologies, Santa Clara, CA, USA) was used as a host strain in order to verify the gene sequences. For protein expression, recombinant pQE-80L (Qiagen, Hilden, Deutschland) vectors were transformed into E. coli BL21-CodonPlus(DE3)-RIPL [E. coli B F– ompT hsdS(rB– mB–) dcm+ Tetr gal λ(DE3) endA Hte [argU proL Camr] [argU ileY leuW Strep/Specr]] (Agilent Technologies). Bacteria were cultivated at 37 °C and 18 °C on Luria-Bertani solid medium (0.1% peptone, 0.05% yeast extract, 0.1% sodium chloride, 0.1% agar) or Luria-Bertani liquid medium (0.1% peptone, 0.05% yeast extract, 0.1% sodium chloride).
4.3. Phylogenetic Analysis
The selected set of PKSIII was constituted of 755 aa sequences (Supplementary Material S1
). Fasta formatted sequences were inserted into the NGPhylogeny.fr pipeline “A la carte” (https://ngphylogeny.fr/
) and were analysed as followed. The protein sequences were first aligned using MAFFT then cleaned with the tool trimAl resulting in the selection of 271 informative positions over the 2957 aligned positions. Maximum likelihood phylogenetic analysis according to the best substitution model selection (LG +G + I) was carried out using the PhyML-SMS tool under the standard conditions. The bootstrap analysis of 100 replicates was used to provide estimates for the phylogenetic tree topology and it resulted in a newick file formatted with the program MEGA v10.1.1 to obtain the corresponding simplified tree figure.
4.4. Cloning of Polyketide Synthase and Sequence Validation
Total RNA was extracted from N. uzbekistanensis
strain Mo29 (UBOCC-A-208024) grown on culture medium agar using the RNeasy kit for Plant and Fungi (Qiagen, Hilden, Deutschland ) and cDNAs were synthesised using the GoScript™ Reverse Transcription System kit (Promega, Madison, WI, USA). Then, cDNA coding for type III pks
gene were amplified by PCR using specific primers. One amplification corresponds to the complete sequence of the gene encoding the entire protein (Standard PKSIII (Std)) whereas the other corresponds to the sequence truncated by the last 74 amino acids, encoding a reduced protein (truncated/reduced PKSIII (Red)). Amplification of the standard pksIII
transcript was performed with primers PKSIII_Mo29_Forward (5′-CATGCGAGCTCGGTACC
I restriction site is underline) and PKSIII_Mo29_StandardReverse (5′-TCAGCTAATTAAGCTT
III restriction site is underlined). PCR was performed using the Phusion®
HF DNA Polymerase kit (BioLabs, Ipswich, MA, USA), in 50 µL reaction volumes containing 1 × Phusion GC Buffer, 1 mM of dNTPs mix, 6% of DMSO, 2.5 µM of each primer, 1U of Phusion DNA polymerase and 50 ng of cDNA. The reduced pksIII
transcript was amplified with primers PKSIII_Mo29_Forward and PKSIII_Mo29_ReducedReverse (5′-TCAGCTAATTAAGCTT
III restriction site is underlined). PCR was performed using the Pfu DNA Polymerase kit (Promega), in 50 µL reaction volumes containing 1 × Pfu Buffer, 0.4 mM of dNTPs mix, 0.1 µM of each primer, 3U of Pfu DNA polymerase and 50 ng of cDNA. The PCRs were performed on PeqStar 2 × thermocycler (Peqlab). The amplification consisted of an initial denaturation step at 94 °C for 2 min, followed by 30 iterations of 30 s at 94 °C, 30 s at 56 °C, 2 min at 72 °C and a final extension step of 10 min at 72 °C. The amplified fragments fragments were extracted from agarose gels and purified using the NucleoSpin Gel PCR Clean-up kit (Macherey-Nagel, Hoerdt, France), then cloned into pQE-80L (Qiagen) using Kpn
I and Hind
III restriction sites and primers PKSIII_Mo29_Forward, PKSIII_Mo29_StandardReverse and PKSIII_Mo29_ReducedReverse. Ligated plasmids were transformed into E. coli
SURE 2 Supercompetent Cells (Agilent Technologies, Santa Clara, CA, USA) and transformants selected using LB solid medium supplemented with 100 μg/mL of ampicillin. Plasmids were extracted and verified by sequencing (Eurofins Genomics, Luxembourg). A multiple alignment was generated with the two obtained sequences and initial N. uzbekistanensis
strain Mo29 (UBOCC-A-208024) pksIII
gene sequence [45
], using on-line software MUSCLE (Multiple Sequence Comparison by Log-Expectation) and ClustalW2 program [64
4.5. Expression and Purification of Recombinant Proteins
For protein expression, E. coli BL21-CodonPlus-RIPL cells (Agilent Technologies, Santa Clara, CA, USA ) transformed with sequence-verified plasmids was grown at 37 °C under agitation in LB liquid medium containing 100 µg/mL of ampicillin until the OD600 nm reached a between 0.4 and 0.6. Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM and growth continued for 21 h at 20 °C with agitation. Cells were then collected by centrifugation at 9000 rpm for 15 min and pellets were resuspended in buffer A (50 mM Tris-HCl pH = 7.5, 500 mM NaCl and 50 mM imidazole) supplemented with lysozyme, protease inhibitor mixture and DNase before lysis by sonication. After centrifugation at 14,000 rpm at 4 °C for 30 min, supernatants were passed through 0.2 µm filter. Enzyme purification was performed on an Äkta Avant system at 20 °C (GE Healthcare, Chicago, IL, USA) with protein detection by UV at 280 nm. His-Tagged proteins were purified on immobilized Ni-nickel tetradentate absorbent (NTA) medium, using a HisTrap FF column (GE Healthcare). Proteins were eluted using an isocratic gradient from 0% to 100% of buffer B (50 mM Tris-HCl pH = 7.5, 500 mM NaCl, 500 mM imidazole). In total, 1–2 mL fractions were collected during the gradient. Fractions were concentrated by centrifugation at 4000 rpm and 4 °C for 20 min using 15 mL centricon filters. Proteins were then further purified on a Sephacryl-200 gel filtration column (GE Healthcare), equilibrated in buffer composed of 50 mM of Tris-HCl pH = 7.5 and 200 mM of NaCl. All protein samples were analysed for purity and integrity using 12% SDS-PAGE. Dynamic light scattering analysis was performed using a DynaPro-801 molecular-sizing instrument (Structural Biology platform; SB Roscoff, Roscoff, France) equipped with a microsampler (Protein Solutions, Wyatt Technology, CA, USA). A 50 µL sample was passed through a filtering assembly containing a 0.02 µm filter into a 12 µL chamber quartz cuvette. The data were analysed using the Dynamics 4.0 and DynaLS software (Wyatt Technology, CA, USA).
4.6. Enzyme Assays
Experiments were performed by individually testing six different starter acyl-CoAs at 200 μM (acetyl-CoA, hexanoyl-CoA, octanoyl-CoA, decanoyl-CoA, palmytoyl-CoA and lauroyl-CoA) in an assay mixture of 500 μL containing 20 μM malonyl-CoA, 50 μg of purified enzyme, 50 mM Tris HCl, pH 7.5, and 1 mM EDTA (final concentrations). Incubations were performed at room temperature for 2 h and stopped by adding 10 μL of 37% HCl. The products were then extracted with 1 mL of ethyl acetate with acetic acid (100 1).
Recombinant E. coli BL21-CodonPlus-RIPL cells were grown at 37 °C under agitation in Luria-Bertani liquid medium containing 100 µg/mL of ampicillin until the optical density at 600 nm reached a value between 0.4 and 0.6. Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM and growth continued for 21 h at 20 °C with agitation. For each construct (PKSIII Std and PKSIII Red), identical cultures were grown without addition of IPTG and used as control. 1 mL of cells was collected and centrifuged. Supernatants were collected and polyketide products were extracted twice with a solution of ethyl acetate and acetic acid (100:1). The organic phase was transferred to a glass vial and evaporated under a stream of nitrogen. Extracts were solubilized in acetonitrile RS Grade followed by a 0.22 µm filtration before LC-MS analyses.
The extracts were analysed on an Agilent 6530 Accurate-Mass Q-ToF LC/MS (Agilent Technologies, Santa Clara, CA, USA). Products were separated on a Zorbax Extend-C18 1.8 µm (2.1 × 50 mm) column (Agilent Technologies, Santa Clara, CA, USA) at a flow rate of 300 µL/min. Gradient elution was performed with water (containing 0.1% formic acid and 0.1 mM ammonium formate) and acetonitrile (containing 0.1% formic acid), from 15 to 100% acetonitrile in 25 min. Only negative Electrospray Ionization (ESI-) were used. Online LC-ESI-MS spectral analyses were performed using MassHunter Quantitative Analysis software and Mass Profiler. Identification of the products was performed by direct comparison with the authentic compounds using chemical formula or proposed from accurate m/z determination and fragmentation patterns. Spectral comparison was performed using MassHunter Mass Profiler software (Agilent Technologies, Santa Clara, CA, USA ).
4.8. Cell Culture Conditions
Caco-2 cells, derived from a human colorectal adenocarcinoma, were purchased from ECACC (number 88081201, Salisbury, UK). Cells were cultured at 37 °C and 5% (v/v) CO2 in DMEM supplemented with penicillin (100 UI/mL), streptomycin (100 μg/mL) containing 10% (v/v) heat-inactivated foetal bovine serum (FBS), 4.5 g/L glucose, 25 mM HEPES, 2% (v/v), 2 mM of L-glutamine and 1% (v/v) of non-essential amino acids (NEAA) (Invitrogen, Carlsbad, CA, USA) with weekly passage. Cells between passage 30 and 50 were seeded at a density of 80 × 103 cells/cm2 (Sigma–Aldrich, Saint-Louis, MO, USA). In these conditions, cells reached confluence in 3 days and differentiated completely in 21 days.
Human monocytic leukaemia cells (THP-1) were acquired from the European Collection of Cell Cultures (ECACC; number 88081201, Salisbury, UK). THP-1 suspensions were grown in RPMI-1640 medium supplemented with 10% heat-inactivated foetal bovine serum (FBS), 3652 mM L-glutamine, 10,000 Units/mL 1% penicillin 10,000 Units/mL and 10,000 µg/mL 1% streptomycin 10,000 µg/mL (Biochrom GmbH, Berlin, Germany) at 37 °C with 100% relative humidity (RH) in a 5% CO2 atmosphere. Cells were grown to a density between 0.2 and 1 × 106 cells/mL as recommended by ECACC. Culture medium was replaced every 3 days with fresh growth medium.
4.9. Lactate Dehydrogenase Assays
Caco-2 cells were grown until 21 days post-confluency (differentiated cells) in culture medium in the presence of different PKSIII Mo29 reaction products at concentrations specified. The viability of the Caco-2 monolayers was measured using the CytoTox 96 Non –Radioactive Cytotoxicity Assay (Promega, Madison, WI, USA). After 48 h, 50 µL of cell culture medium was collected from each well and incubated with the substrate solution for 30 min. Absorbance at 490 nm was measured by spectrophotometer. Experiments were repeated three times, six repetitions each. The relative LDH leakage (%) related to control wells containing cell culture medium with DMSO was calculated by [A]test/[A]control × 100. Where [A]test is the absorbance of the test sample and [A]control is the absorbance of the solvent control sample.
4.10. Evaluation of Cytotoxicity by Mitochondrial Activity
THP1 cells were plated in 96-well tissue culture plates at a density of 3 × 104 cells/well. The effects on the mitochondrial activity of THP-1 cells exposed to extracts as indicated in the text were studied using the MTS assay kit. Control cultures without extracts but with solvent (DMSO) were included. After 48 h of exposure (acute exposure), the cells were washed with PBS and 20 μL of a freshly prepared MTS/PMS solution was added to the wells. The cells were further incubated for 3 h. The amount of soluble formazan (MTS metabolite) was then quantified by reading the absorbance at 490 nm on a Multiskan FC plate reader (Thermo Scientific, Madison, WI, USA). Cell viability obtained for the negative control (cell cultures not exposed to molecules) was defined as 100% and affected fraction as 0%. Affected fraction mean percentages of three independent experiments ± standard error of the mean (SEM) were used for statistical analyses.
4.11. Statistical Analysis for Cell Viability
Statistical analyses were performed using Statgraphics Plus for Windows (version 1.4 StatPoint Technologies, Inc., Warrenton, VA, USA). After verifying the normal distribution of the data and the homogeneity of variances, one-way analyses of variance were used to detect significant differences among means. Least significant differences (LSD) tests were then applied to compare mean values obtained from different culture condition to negative control.