Key words
cytotoxicity - melanoma - bioassay-guided fractionation - laccaridiones - fungal natural products - Montagnula sp. - structural elucidation
Introduction
Natural products are important sources of anticancer lead molecules [1]. More than 60% of the current
compounds with antineoplastic activity were originally isolated as natural products
or are natural product derivatives, and microbial metabolites are among the most
important of these chemotherapeutic agents [2]. Melanoma is a malignancy of pigment producing cells (melanocytes), which
are located primarily in the skin, but are also found in the ears, gastrointestinal
tract, eyes, oral and genital mucosa, and leptomeninges [3]. Malignant melanoma is the most aggressive
form of skin cancer and accounts for about 3% of all cases of malignant
tumors. Its incidence is increasing worldwide, and it is becoming resistant to
current therapeutic agents [4]. In a recent
report, 30 in vivo and in vitro natural active principles were
reviewed for their pharmacological effects on migration and/or metastasis of
melanoma cells, mapping the mechanisms of action for these underexploited
properties. They were described as acting mainly through their antagonistic effects
upon the TNF-α and EP2 receptors or the suppression of several
protein kinases involved in metastatic pathways such as RAS, PI3K, ERK, and FAK.
Some were able to reduce the level of mesenchymal biomarkers such as N-cadherin
and/or elevate the expression of other molecules such as E-cadherin [5].
Herein, we report the isolation and structural elucidation of the cytotoxic
polyketide laccaridione C (1) ([Fig.
1]) obtained from culture broths of the fungal strain CF-223743 after
fractionation of ethyl methyl ketone extracts based on melanoma targeted activity. A
phylogenetic placement of the strain CF-223743, based on its ITS/28S,
identified the fungus as a species of the genus Montagnula, an ascomycete of
the order Pleosporales ([Fig. 2]). The
related polyketides laccaridione A (2), laccaridione B (3) [6], and leptosphaerodione (4) [7] were also detected as minor components in
the extract after LC-DAD-HRMS-based dereplication [8].
Fig. 1 Structure of laccaridione C (1), the related
laccaridiones A (2) and B (3), and leptosphaerodione
(4).
Fig. 2 Consensus tree from Bayesian-phylogeny inferences based on
the 28S sequences of selected Montagnula strains and related genera
of Montagnulaceae. Clade probability values are indicated at the
branches. Massarinaceae strains were used as an outgroup.
Results and Discussion
Compound 1 was isolated from the ethyl methyl ketone extract of a culture of
Montagnula sp. (CF-223743) by melanoma activity-guided fractionation on
SP207SS resin followed by repeated semipreparative HPLC. The molecular formula of
1 was inferred to be C21H22O6 by
accurate mass measurements (ESI-TOF, see Supporting Information) (m/z
371.1489, [M+H]+, calcd. for
C21H23O6
+, 371.1479), which translates into 11 degrees of unsaturation.
The structure of 1 was established by 1D and 2D NMR spectroscopy ([Table 1] and Supporting Information).
The 1H NMR and HSQC spectra displayed signals corresponding to four
sp2 methines, two aliphatic methines, one of them oxygenated, one
aliphatic methylene, a methoxy group, and three aliphatic methyl groups with
singlet, doublet, and triplet multiplicities. Two hydroxy protons were also
observed, with one of them corresponding to a phenol involved in intramolecular
hydrogen bonding according to its sharp and remarkably deshielded singlet signal at
δ
H 12.4 ppm. Three of the four sp2 protons constituted isolated
spin systems. The presence of a key substructural moiety corresponding to a
1,3-dimethylpentenyl unit directly bonded to a quaternary oxygenated sp2
carbon was established by analysis of COSY and HMBC spectra (Supporting
Information). Searching for such a substructure in the Chapman &
Hall Dictionary of Natural Products Database (v25.1) revealed its presence in
laccaridiones A (2) and B (3) [6] and leptosphaerodione (4) [7]. The closely related molecular formulae, UV and NMR spectra reported
for these compounds unequivocally indicated that 1 belonged to the same
structural class. The UV (DAD) spectrum of 1
(Supporting Information) displayed the characteristic bands of the
ortho-benzoquinone ring system (λ
max 308 and 490 nm) responsible for the strong red coloration of
the compound. Further analysis of all key 1H-1H and
1H-13C long-range correlations observed in the COSY and
HMBC spectra of 1 ([Fig. 3]) allowed
for establishing its full structure, which turned out to be identical to that of
reported laccaridiones A (2) and B (3), but lacking the alkyl acetalic
substituent and thus being a hemiacetal. The E stereochemistry of the double bond in
the side chain was likewise established based on comparison of chemical shifts.
Since 1 contains a chiral center at position C-3’of the fixed
configuration, the two possible configurations at the hemiacetal carbon (two epimers
at C-11) render two possible diastereomers with almost identical chemical shifts.
The presence of a hemiacetal explains the splitting observed in the 1H
NMR spectrum for some signals of the lateral side chain. Two epimers at the chiral
center at position 3’ in an equimolar ratio were present in compound
1, as indicated by the intensity of the split NMR signals. This is not
surprising considering that formally the hemiacetal is the product of the
intramolecular nucleophilic addition of an enolic hydroxy group at position C-9 over
an aldehyde at position C-11. Since such an attack may take place over both faces of
the carbonyl with equal probability, two epimeric hemiacetals in an equimolar ratio
are formed. Thus, 1 was isolated as a mixture of diastereomers, and for this
reason, its optical rotation was not measured.
Fig. 3 Key COSY and HMBC correlations observed in the structure of
1.
Table 1 NMR data (500 MHz, DMSO-d
6, 24°C) for compound 1.
|
No.
|
1
|
|
|
δ
C (ppm)
|
δ
H, mult. (J in Hz)
|
|
1
|
179.3
|
|
|
2
|
175.9
|
|
|
3
|
151.7
|
|
|
4
|
113.3
|
6.67, s
|
|
5
|
135.6
|
|
|
6
|
118.2
|
6.78, s
|
|
7
|
141.1
|
|
|
8
|
99.6
|
6.21, s
|
|
9
|
156.4
|
|
|
10
|
-
|
|
|
11
|
87.2
|
6.49, d (6.1)
|
|
12
|
113.8
|
|
|
13
|
162.0
|
|
|
14
|
110.7
|
|
|
15
|
55.5
|
3.75, s
|
|
1’
|
127.7
|
|
|
2’
|
139.2
|
6.21, m
|
|
3’
|
34.1
|
2.49, m
|
|
4’
|
29.7
|
1.41, m; 1.31, m
|
|
5’
|
11.8
|
0.84, t (7.4)
|
|
6’
|
12.7
|
1.89, s
|
|
7’
|
20.2
|
0.99, d (6.6)
|
|
11-OH
|
|
7.43, d (6.1)
|
|
13-OH
|
|
12.41, s
|
The presence in the fungal extract of the related polyketides laccaridione A
(2), laccaridione B (3), and leptosphaerodione (4),
detected as trace components in the enriched fractions after LC-DAD-HRMS-based
dereplication, indicates their obvious biosynthetic relationship.
Compound 1 was tested against the melanoma (A2058) cell line used for its
bioassay-guided isolation, exhibiting an IC50 of
13.2±2.9 µM. It was also further tested against an extended
panel of six human cell lines, exhibiting potent activity after MTT assays against
the breast cancer MCF7 cell line with an IC50 value of
3.7±1.5 µM, neuroblastoma (SH-SY5Y) with an IC50
of 8.8±3.0 µM, liver carcinoma (Hep G2) with an
IC50 of 11.6±4.2 µM, pancreas carcinoma (MIA
PaCa-2) with an IC50 of 18.7±2.3 µM, lung
carcinoma (A549) with an IC50 of 29.0±0.7 µM, and
skin fibroblasts (CCD-25Sk, derived from normal skin of a patient that died from a
high-grade glioma) with an IC50 of 20.5±1.7 µM.
Doxorubicin gave IC50 values of 1.79±0.06 (A2058),
6.99±0.07 (MCF7), 0.69±0.07 (SH-SY5Y), 1.95±0.09 (Hep G2),
2.97±0.73 (MIA PaCa-2), 10.51±0.28 (A549), and
0.86±0.05 µM (CCD-25Sk) when tested as a positive control
under the same conditions. Methyl methanesulfonate (MMS) was used as a second
positive control at a concentration of 4 μM, which caused a
100% inhibition of all cell lines tested.
In conclusion, we report herein the isolation and cytotoxic properties of a new
bioactive polyketide, laccaridione C (1), from Montagnula sp., an
ascomycete from the Pleosporales order. The interest in the bioactivity of
laccaridione derivatives can be perceived by the previously published patents
concerning their use as antitumor, antifungal, antibacterial, and anti-HIV agents
[9]
[10]
[11]. A further literature review confirmed
these properties and indicates that these bioactive pigmented polyketides also have
potent protease inhibitory properties [6]
[12]
[13]
[14]
[15], which is most likely the main mechanism
of action of the bioactivity herein reported for 1.
Biosynthetically, compound 1 is the intermediate stage between the C-11
dehydroxylated form of leptosphaerodione (4) and the C-11 methoxylated
laccaridione A (2) or the C-11 ethoxylated laccaridione B (3).
Laccaridiones and related naphtoquinones are bioactive fungal natural products that
are interestingly reported from distant taxonomic groups and from
marine/terrestrial environments, e. g., leptosphaerodione was first
isolated from the marine-derived fungus Leptosphaeria oraemaris, an
ascomycete of the order Pleosporales [7], and
laccaridiones A and B were first isolated from the taxonomically distant terrestrial
basidiomycete Laccaria amethystea
[6]. Obionins A and B, two structurally
related molecules also having an ortho-benzoquinone ring system, were
isolated from the marine-derived fungus Leptosphaeria obiones of the
Pleosporales order [16] and from an
unidentified fungus also belonging to the Pleosporales order [15], respectively. New obionin derivatives were
also obtained from the Sooty Blotch fungus Microcyclospora malicola, an
ascomycete of the order Capnodiales [13].
Although rare, the presence of this natural product family has been reported in
terrestrial and marine fungal strains, Basidiomycetes and in two different orders of
Ascomycetes, which raises the possibility of horizontal gene transference between
these fungi. The strain CF-223743 Montagnula sp. used in this study was
isolated from the Grand Comoros Island in the Indic Ocean, raising the question if a
putative intra-kingdom horizontal gene transference occurred from a microbial marine
or terrestrial source. Phylogenetic placement suggests that this strain might be a
new species of the Montagnula genus ([Fig.
2]).
In summary, we report herein the isolation of laccaridione C (1) from the
fungus Montagnula sp., selected as an active compound against melanoma after
a microbial extract screening that increases the structural variety of this
interesting bioactive chemical class, and adds a new strain to the fungal taxonomic
group known to produce natural products from this polyketide heterocyclic
family.
Materials and Methods
General experimental procedures
IR spectra were measured with a JASCO FT/IR-4100 spectrometer equipped
with a PIKE MIRacle single reflection ATR accessory. NMR spectra were recorded
on a Bruker Avance III spectrometer (500 and 125 MHz for 1H
and 13C NMR, respectively) equipped with a 1.7 mm TCI
MicroCryoProbe using the signal of the residual solvent as the internal
reference (δ
H 2.50 and δ
C 39.5 ppm for DMSO-d
6). LC-UV-MS analysis was performed on an Agilent 1100 single
quadrupole LC-MS system using a Zorbax SB-C8 column (2.1 ×
30 mm, 5 μm, flow rate 0.3 mL/min,
40°C). HRESIMS spectra was acquired using a Bruker maXis QTOF mass
spectrometer coupled to the same HPLC system as described above [17]. Flash chromatography was performed
with a CombiFlash Teledyne ISCO Rf400x. Semipreparative HPLC was done using a
GILSON GX-281 322H2 coupled to a UV-VIS detector and an automatic fraction
collector. Methyl ethyl ketone used for extraction was of analytical grade. All
solvents employed for isolation were of HPLC grade.
Strain and fermentation
The producing fungus Montagnula sp. (CF-223743) was isolated from dung
collected from a forest in Zikaledjou (Grand Comoros Island). The biological
material was pretreated with hot water at 65°C (50 mL/g
of dung) to favor the growth of hyperthermophilic fungi. The mixture was
vortexed for 5 min and the supernatant was plated in Bandoni’s
sorbose yeast extract tetracycline agar medium [18]. Frozen stock cultures in 10% glycerol
(− 80°C) are maintained in the collection of
Fundación MEDINA.
DNA extraction, PCR amplification, and DNA sequencing were performed as
previously described [19]. The sequences
of the 28S rDNA region were compared with GenBank or the NITE Biological
Resource Center (http://www.nbrc.nite.go.jp/) databases
using BLAST. Species and genus groups were tested with Bayesian analysis
employing the Markov chain Monte Carlo approach using MrBayes 3.01
(http://mrbayes.sourceforge.net/) [20].
Ten mycelia agar plugs of the fungus were used to inoculate 50 mL of SMYA
seed medium (maltose 40 g/L, yeast extract
10 g/L, neopeptone 10 g/L, agar
4 g/L) that was fermented for 7 days. This seed culture was used
to inoculate (3% v/v) 1 L of YES medium (10 ×
100 mL in 500 mL Erlenmeyer) that contained Bacto yeast extract
(20 g/L; Difco),
MgSO4· 7H2O (0.5 g/L;
Merck), sucrose (150 g/L; Fisher), and 1 mL trace
elements (from a stock of ZnSO4·7H2O, PANREAC,
1 g/L and CuSO4·5H2O,
0.5 g/L; Merck). All cultivations were performed at 22°C
with 70% humidity, in agitation at 220 rpm for 14 days in Kuhner
incubators.
Extraction and isolation
A 1-L culture of the fungus in YES medium was extracted with 1 L of ethyl methyl
ketone (MEK). After adding the organic solvent, the mixture was subjected to
continuous shaking at 220 rpm for 1 h. The biomass was separated by
centrifugation, and the aqueous phase was decanted from the organic phase. The
organic phase was dried to generate an extract that was loaded on a column
packed with SP207SS reversed-phase resin (brominated styrenic polymer,
65 g, 32 × 100 mm) previously equilibrated with water.
The column was washed with water (1 L) and afterwards the sample was dissolved
in DMSO, loaded onto the column, and eluted at a flow rate of
8 mL/min using a gradient from 10–100% acetone
in water (for 30 min) with a final 100% acetone step
(15 min), collecting 19 fractions of 20 mL. Fractions were
concentrated to dryness on a centrifugal evaporator and activity against the
melanoma (A2058) cell line was found in fractions 10 and 11. Fraction 10,
selected for further purification, was dissolved in 700 μL of
DMSO and subjected to repeated injections
(100 μL/injection) in reversed-phase semipreparative
HPLC (Agilent Zorbax RX-C8, 9.4×250 mm, 5 µM,
3.6 mL/min, UV detection at 210 nm) with a double
isocratic solvent system of 40/60 acetonitrile/water for the
first 20 min, followed by a 45/60 acetonitrile/water
solvent system between minutes 20.5 and 46 to yield compound 1
(1.6 mg, retention time 43 min, 95.1% purity by HPLC-UV
at 210 nm) as responsible for the observed bioactivity.
(E)-1,10-Dihydroxy-7-methoxy-3-(4-methylhex-2-en-2-yl)-1H-benzo[g]isochromene-8,9-dione
(Laccaridione C) (1): Dark reddish amorphous solid; UV λ
max (nm): 242, 308, and 490 nm; IR (ATR) ν
max 2956, 2915, 2850, 1736, 1682, 1595, 1546 cm-1;
for 1H and 13C NMR data see [Table 1]. HRESIMS m/z
371.1489 [M + H]+ (calcd. for
C21H23O6
+, 371.1479).
Cytotoxicity MTT assays
MTT is a colorimetric assay for measuring the activity of cellular enzymes that
reduce the tetrazolium dye, MTT, to purple colored formazan crystals. This assay
measures mitochondrial metabolic activity via NAD(P)H-dependent cellular
oxidoreductase enzymes and may, under defined conditions, reflect the number of
viable cells present [21]. Six human tumor
cell lines, including A2058 (ATCC CRL-11147), MCF7 (ATCC HTB-22), SH-SY5Y (ATCC
CRL-2266), Hep G2 (ATCC HB-8065), MIA PaCa-2 (ATCC CRM-CRL-1420), and A549 (ATCC
CCL-185), and skin fibroblast cell line CCD-25Sk (ATCC CRL-1474) were used for
this study. Cells were seeded at a concentration of 1×104
cells/well in 100 µL culture medium and incubated at
37°C in 5% CO2. After 24 h, when the monolayer was
formed, the medium was replaced with a final volume of 195 μL
and 5 μL of extracts and controls (extract screening) or
199 μL and 1 μL of pure compounds and controls
(pure compound screening, a 10-point curve starting at a concentration of
50 μM with serial ½ dilutions). Methyl methanesulfonate
(MMS) (purity 99.8% by GC; Sigma-Aldrich) and DMSO were used as a
positive and negative controls, respectively. Doxorubicin (purity ≥ 98
% by HPLC; Sigma-Aldrich) was also tested using an 8-point curve with an
initial concentration of 50 μM and serial ½ dilutions.
After the extracts/compounds and controls were added, the plates were
incubated at 37°C in 5% CO2 for 72 h. After that
time, the MTT solution was prepared at 5 mg/mL in PBS 1×
and then diluted to 0.5 mg/mL in MEM without phenol red. The
sample solution in wells was tossed, the wells were washed lightly with PBS
1×, and 100 µL of MTT dye were added to each well. The
plates were then incubated for 3 h at 37°C in a 5%
CO2 atmosphere. The supernatant was removed and
100 µL of DMSO 100% were added. The plates were gently
shaken to solubilize the formed formazan crystals and the absorbance was
measured using a Wallac 1420 VICTOR microplate reader at a wavelength of
570 nm. The resulting data were analyzed with Genedata Screener 12.0.5
Standard Software.
Supporting information
NMR spectra of compound 1 (Figs. 1S– 5S), HRMS spectrum of 1 (Fig.
6S), UV spectrum of 1 (Fig. 7S), images of cultures of Montagnula
sp. in several solid media (Fig. 8S), and growth inhibition curves for
cytotoxic activity of compound 1 (Figs. 9S– 15S) are available as
Supporting Information (Fig. 16S)
