New C14-surfactin methyl ester from the marine bacterium Bacillus pumilus KMM 456

Article abstract of DOI:10.1007/s11172-010-0369-8

New C₁₄-surfactin methyl ester and eight earlier described surfactins were isolated from the marine bacterium Bacillus pumilus associated with zoantharia Palythoa sp. The structure of the compounds isolated was established based on the data from amino acid analysis, one- and two-dimensional NMR spectroscopy, and tandem nanoelectrospray mass spectrometry. The absolute configurations of amino acids were determined by Marfey's method. Cytotoxic activity of a number of surfactins with respect to the tumor cells HCT-116 and MDA-MB-231 was studied.

Full text of DOI:10.1007/s11172-010-0369-8

Russian Chemical Bulletin, International Edition, Vol. 59, No. 11, pp. 2137—2142, November, 2010
2137
New C14ꢀsurfactin methyl ester
from the marine bacterium Bacillus pumilus KMM 456
O. I. Zhuravleva,a Sh. Sh. Afiyatullov,b S. P. Ermakova,b O. I. Nedashkovskaya,b
P. S. Dmitrenok,b V. A. Denisenko,b and T. A. Kuznetsovab†
aFarꢀEastern State University,
8 ul. Sukhanova, 690000 Vladivostok, Russian Federation
bThe Pacific Institute of Bioorganic Chemistry, FarꢀEastern Branch of the Russian Academy of Sciences,
159 prosp. 100 let Vladivostoku, 690022 Vladivostok, Russian Federation.
Fax: +7 (423) 231 4050. Eꢀmail: afiyat@piboc.dvo.ru
New C14ꢀsurfactin methyl ester and eight earlier described surfactins were isolated from the
marine bacterium Bacillus pumilus associated with zoantharia Palythoa sp. The structure of the
compounds isolated was established based on the data from amino acid analysis, oneꢀ and
twoꢀdimensional NMR spectroscopy, and tandem nanoelectrospray mass spectrometry. The
absolute configurations of amino acids were determined by Marfey's method. Cytotoxic activity
of a number of surfactins with respect to the tumor cells HCTꢀ116 and MDAꢀMBꢀ231 was
studied.
Key words: marine isolate of bacterium Bacillus pumilus, surfactins, tandem nanoelectroꢀ
spray mass spectrometry.
Bacteria of the family Bacillus produce a wide range of
biologically active lipopeptides, which are highly promisꢀ
ing for the use in medicine. Surfactins, cyclic depsipepꢀ
tides, containing a βꢀhydroxy acid and several amino acꢀ
ids, are of special interest among them. Compounds
of this large class are mixtures of isoforms differing
from each other in both the amino acid composition of
the peptide core and the length (C12—C15) and branching
(isoꢀ, anteisoꢀ, nꢀ) of their hydrophobic fattyꢀacid moiety.
The history of surfactins began from the report1 on
isolation of new lipopeptide with exceptional surfactant
properties from the culture medium of the bacterium
Bacillus subtillus. Then, a large body of works were devotꢀ
ed to the study of the structures and biological action of
this class of compounds. It was shown that they possess
hemolytic,2 antiviral,3,4 and antitumor5,6 activity, exhibit
hypocholesterinemic effect,7 and are strong biosurfacꢀ
tants.1,8 High biological activity of surfactins opens wide
prospects for their use in medicine and biotechnology,
while the presence of a large amount of isoforms allows
one to study dependence of the biological activity of these
compounds on their structures.
phase chromatography) were isolated by chromatography
from the ethyl acetate extract of bacterial cells.
The structures of compounds isolated were established
based on the data from amino acid analysis, oneꢀ and
twoꢀdimensional NMR spectroscopy, tandem nanoelecꢀ
trospray mass spectrometry, as well as on the comparison
of their spectra with those reported earlier. The absolute
configurations of amino acids were determined by Marfey´s
method. One of the depsipeptides, compound 7, was isoꢀ
lated from bacteria of the family Bacillus for the first time,
the rest are identical to the those described earlier.
The ESIꢀMS of compound 7 contains peaks of the
cationized molecules at m/z 1058 [M + Na]+ and 1080
[M + 2 Na – H]+ (Fig. 1). The molecular formula of 7 was
Irel•105 (rel. units)
4
1080.68793
3
2
Continuing search for the producents of biologically
active compounds among marine bacteria isolates, we
found that the strain Bacillus pumilus KMM 456 syntheꢀ
sizes compounds with high hemolytic activity. The indiꢀ
vidual peptides 1—9 (in the order of elution in reversedꢀ
1058.70543
[M + Na]+
1
685.45651
0
300 400 500 600 700 800 900 1000 1100 m/z
Fig. 1. The ESI mass spectrum of surfactin 7.
†
Deceased.
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 11, pp. 2083—2088, November, 2010.
1066ꢀ5285/10/5911ꢀ2137 © 2010 Springer Science+Business Media, Inc.

2138
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 11, November, 2010
Zhuravleva et al.
determined as C53H93N7O13 based on the analysis of
the ESIꢀTOF high resolution mass spectrum (found:
m/z 1058.6719 [M + Na]+, calculated 1058.6729).
Amino acid analysis of hydrolyzate of compound 7
indicates the presence of four amino acids: Asp, Glu, Val,
and Leu in the ratio 1 : 1 : 1 : 4.
the formation of two series of fragment ions:9
707→594→481→382→267
(Leu—Leu—Val—Asp)
(Fig. 3, b) and 1058→927→814→671→572 (Fig. 3, c).
The difference in 18 mass units between the fragment ions
832 (Leu—Leu) and 814 (Leu—Leu—OH2) (Fig. 3, a and
c) indicates that leucine is the Cꢀterminal amino acid,
while the ester bond in the lipopeptide is formed between
the carbonyl group of leucine and the hydroxy group of the
aliphatic moiety. These data allowed us to establish the
amino acid sequence in compound 7: N—Leu—Leu—
Val—Asp—Leu—Leu—C. The ion fragmentation unamꢀ
biguously shows that aspartic acid is not methylated and,
therefore, compound 7 contains methyl ester of glutꢀ
amic acid.
The spectrum contains an ion with m/z 392.27 correꢀ
sponding to the residue of the fatty acid and GluOMe (see
Fig. 3, a). The difference between this ion and GluOMe
is 249 molecular mass units, which corresponds to
the residue of a βꢀhydroxy acid with 14 carbon atoms
(C13H26ONaCO–). The 13C NMR spectrum of compound
7 (DMSOꢀd6) exhibits characteristic signals at δ 13.9 and
31.3, which indicate the linear structure of the acid.10
The absolute configuration of amino acids in comꢀ
pound 7 was determined by a modified Marfey's method
using their LꢀFDAAꢀderivatives11 (Table 1).
1
The H NMR spectrum of compound 7 exhibits sigꢀ
nals for seven amide protons in the region δ 7.57—8.40,
seven αꢀmethine protons at δ 4.04—4.54, and an intensive
signal for the methylene groups of the fattyꢀacid chain at
δ 1.24. A singlet signal for the methyl protons at δ 3.58
indicates the presence of the —COOMe group in the molꢀ
ecule, while the resonance at δ 5.07 evidences the presꢀ
ence of the —CHOC(O)— group. The 13C NMR specꢀ
trum of compound 7 contains ten signals for the carbonyl
carbon atoms, seven signals for Cα, and a signal for the
methoxy group at δ 51.3. The correlations observed in the
COSYꢀ45 spectrum indicate the presence of the isolated
spin system —CH2—CH(O)—CH2—CH2— in the strucꢀ
ture of 7.
The amino acid sequence, position of the methoxy
group, and the length of the fattyꢀacid chain in 7
were established based on the tandem mass spectrometric
data for the ion with m/z 1058.69 [M + Na]+ (Fig. 2).
A series of fragment ions obtained by simple cleavꢀ
age of the lipopeptide in the ESIꢀMS/MS
(1058→945→832→717→618→505→392) determines the
Cꢀterminal sequence of amino acids Leu—Leu—Asp—
Val—Leu—Leu (Fig. 3, a). Cleavage of the peptide by the
mechanism of double hydrogen transfer leads to
The ESIꢀMS spectra of compounds 1 and 3—5 exhibit
peaks of cationized molecules at m/z 1030, 1044, 1044,
and 1058 ([M + Na]+) which correspond to the molecular
masses 1007 (1), 1021 (3), 1021 (4), and 1035 (5), respecꢀ
tively. Amino acid analysis of hydrolyzates of these comꢀ
Irel•104 (rel. units)
2.0
1.5
1.0
0.5
0
300
400
500
600
700
800
900
1000
m/z
Fig. 2. The tandem nanoelectrospray mass spectrum (ESIꢀMS) of surfactin 7.

New C14ꢀsurfactin methyl ester
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 11, November, 2010 2139
Table 1. Stereochemistry of amino acids of surfactin 7
Amino
acid
tRa/min (configuration)
I
II
Glu
Asp
Val
5.17 (L), 6.25 (D)
8.74 (L), 9.55 (D)
20.30 (L), 25.27 (D)
23.71 (L), 28.04 (D)
5.19 (L)
8.65 (L)
20.23 (L)
23.70 (L)b, 28.02 (D)b
Leu
a Retention times of derivatives of the individual amino acids (I)
and derivatives of the amino acides in peptide 7 (II) are given.
b The ratio L : D = 1 : 1.
data for [Leu7]ꢀsurfactin,12 while the fragmentation in
the ESIꢀMS/MS on simple cleavage and cleavage with
the double hydrogen transfer corresponds to the literature
data.9 The length and type of the fattyꢀacid chains were
pounds indicates the presence of four amino acid residues:
Asp, Glu, Val, and Leu (1 : 1 : 1 : 4). The 1H and 13C NMR
spectra of 1 and 3—5 (in C5D5N) agree with the spectral
Fig. 3. Fragmentation of the ion with m/z 1058 on simple cleavage (a) and cleavage with the double hydrogen transfer (b, c).

2140
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 11, November, 2010
Zhuravleva et al.
determined based on the molecular mass values and chemꢀ
ical shifts for the terminal methyl groups. As a result, the
structures of depsipeptides 1, 3, 4, and 5 were established
as [Leu7]ꢀsurfactins with the fattyꢀacid chains of C13, C14,
C14, and C15 in length and anteisoꢀ, isoꢀ, nꢀ, and anteisoꢀ
structure, respectively.
shows the presence of five amino acid residues: Asp, Glu,
Val, Leu, and Ile (1 : 1 : 1 : 3 : 1). Fragmentation of the
ion with m/z 1036 [M + H]+ in ESIꢀMS/MS corresponds
to the fragmentation of [Leu7]ꢀsurfactin with the C15ꢀisoꢀ
fattyꢀacid chain.14 The 13C NMR spectrum of compound
8 (in C5D5N) exhibits signal for the Cα atom at δ 58.1
and signal for the γꢀmethyl group at δ 16.2, which are
characteristic of the isoleucine residue.12 The signal
for the methyl group at δ 11.5 indicates the anteisoꢀstrucꢀ
ture of the fatty acid. Thus, compound 8 was idenꢀ
tified as [Ile7]ꢀsurfactin with the C15ꢀanteisoꢀfattyꢀ
acid chain.
The ESIꢀMS spectra of compound 9 exhibit peak of
the cationized molecule at m/z 1072 [M + Na]+. Amino
acid analysis shows the presence of four amino acid resiꢀ
dues: Asp, Glu, Val, Leu (1 : 1 : 1 : 4). The 13C NMR specꢀ
trum of compound 9 (in C5D5N) shows a signal at δ 51.4,
while the 1H NMR spectrum exhibits a threeꢀproton sinꢀ
glet, which indicates the presence of a methoxy group in
the molecule. The ESIꢀMS/MSꢀfragmentation of the ion
with m/z 1072 on the simple cleavage and cleavage with
the double hydrogen transfer corresponds to the fragmenꢀ
tation of the C15ꢀsurfactin methyl ester, recently15 isolatꢀ
ed from the strain Bacillus subtilis. The signal for the meꢀ
thyl group at δ 11.5 indicates the anteisoꢀstructure of
the fatty acid. Thus, peptide 9 was identified as the surfacꢀ
tin [Leu7]ꢀOꢀmethyl ester with the C15ꢀanteisoꢀfattyꢀ
acid chain.
In the ESIꢀMS of compound 2, there are peaks of the
protonated and cationized molecules at m/z 1008 [M + H]+
and 1030 [M + Na]+. Amino acid analysis of compound 2
shows the presence of the same four amino acid residues,
viz., Asp, Glu, Val, and Leu (1 : 1 : 1 : 4). The series
of fragment ions 1030→917→804→689→590→477,
707→594→481→382→267, 786→643→544→431 obꢀ
tained in the ESIꢀMS/MS allowed us to establish the seꢀ
quence N—Glu—Leu—Leu—Val—Asp—Leu—Leu—C in
peptide 2. They differ by 14 mass units from the correꢀ
sponding series of peaks for C14ꢀsurfactin.9 The 13C NMR
spectrum exhibits signals at δ 14.0 and 31.3 indicating the
linear structure of the fatty acid.10 Thus, peptide 2 was
identified as [Leu7]ꢀsurfactin with the C13ꢀ(n)ꢀfattyꢀacid
chain. Earlier,13 this surfactin was isolated from the culꢀ
ture medium of Bacillus natto.
In the ESIꢀMS spectra of compound 6, there are peaks
of the protonated and cationized molecules at m/z 1022
[M + H]+ and 1044 [M + Na]+. Amino acid analysis shows
the presence of four amino acid residues: Asp, Glu, Val,
and Leu (1 : 1 : 2 : 3). The series of fragment ions obtained
on the cleavage of the ion with m/z 1022 [M + H]+ in
ESIꢀMS/MS (1022→923→810→695→596→483→370)
evidences the sequence C—Val—Leu—Asp—Val—Leu—
Leu—Glu—N in compound 6. The 13C NMR spectrum of
compound 6 (in C5D5N) exhibits two additional (as comꢀ
pared to the spectrum of peptide 5) signals for Cγ atoms of
valine at δ 19.3 and 19.6 and a signal for Cα at δ 59.1 The
signal for the methyl group at δ 11.5 indicates the anteisoꢀ
structure of the fatty acid. Thus, compound 6 was idenꢀ
tified as [Val7]ꢀsurfactin with the C15ꢀanteisoꢀfattyꢀ
acid chain.
The structure of all surfactins obtained are given in
Table 2.
Determination of cytotoxic activity
of the isolated compounds
Cytotoxic activity of surfactins 4—6 and 8 in the conꢀ
centration 100 μmol L–1 was studied with respect to two
lines of tumor cells: the human intestine cancer HCTꢀ116
(ATCC # CCLꢀ247) and the human breast cancer
MDAꢀMBꢀ231 (ATCC # HTBꢀ26) using MTSꢀreagent.
Surfactins 4—6 exhibit weak effect on the HCTꢀ116 cells
growth after 72 h. At the same time, surfactin 8, differing
The ESIꢀMS spectra of compound 8 contain peaks of
the protonated and cationized molecules at m/z 1036
[M + H]+ and 1058 [M + Na]+. Amino acid analysis
Table 2. Structures of surfactins from the bacterium B. pumilus KMM 456
Surfactin
βꢀHydroxy acid
Amino acid sequence
[M + H]+
1
2
3
4
5
6
7
8
9
anteisoꢀC13
nꢀC13
isoꢀC14
N—Glu—Leu—Leu—Val—Asp—Leu—Leu—C
N—Glu—Leu—Leu—Val—Asp—Leu—Leu—C
N—Glu—Leu—Leu—Val—Asp—Leu—Leu—C
N—Glu—Leu—Leu—Val—Asp—Leu—Leu—C
N—Glu—Leu—Leu—Val—Asp—Leu—Leu—C
N—Glu—Leu—Leu—Val—Asp—Leu—Val—C
N—Me—O—Glu—Leu—Leu—Val—Asp—Leu—Leu—C
N—Glu—Leu—Leu—Val—Asp—Leu—Ile—C
N—Me—O—Glu—Leu—Leu—Val—Asp—Leu—Leu—C
1008
1008
1022
1022
1036
1022
1036
1036
1050
nꢀC14
anteisoꢀC15
anteisoꢀC15
nꢀC14
anteisoꢀC15
anteisoꢀC15

New C14ꢀsurfactin methyl ester
Cell survivability (%)
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 11, November, 2010 2141
Experimental
a
Optical rotation was measured on a Perkin—Elmer 343 poꢀ
100
80
1
larimeter (Germany). H and 13C NMR spectra were recorded
on a Bruker DRXꢀ700 spectrometer (700 and 175.6 MHz, reꢀ
spectively) using Me4Si as an internal standard. Mass spectra
ESIꢀTOF were obtained on an Agilent 6510 LC QꢀTOF specꢀ
trometer (USA). The voltage on the capillary, nozzle, skimmer
was 3500, 215, and 65 V, respectively, the impact energy was
35 V. Column chromatography was performed on silica gel L
(40/100 μm, Chemapol, Czechoslovakia), HPLC was performed
on an Agilent 1100 Series chromatograph (USA) using a Supelꢀ
co Discovery C18 column (25 cm × 4.6 mm, 5 μm). Amino acid
analysis was performed on a Biochromꢀ30 analyzer (England)
using a Vetropac 8 μm resin (200 × 4.6 mm column) and the
reaction with a ninhydrin reagent at 135 °C.
Cultivation of bacterium B. pumilus (KMM 456). The bacteꢀ
rium was isolated from the zoantharia Palythoa sp. gathered in
the Vietnam waters (KuꢀLaoꢀRe lake) at the depth of 2 m. Ferꢀ
mentation was performed on a shaker at 25 °C for 24 h in the
medium of the following composition (C/g L–1): K2HPO4, 0.07;
NH4Cl, 1.0; yeast extract, 5.0; tripton, 5.0; sodium citrate, 0.01;
glucose, 5.0; 1 M Trisꢀbuffer (pH 7.5), 20.0; artificial sea
water, 200.0.
Isolation of compounds 1—9. The culture media (1.8 L) were
centrifuged for 30 min at 5000 g. The cells obtained were susꢀ
pended in DI water and destroyed by sonication. The suspension
of the destroyed cells were thrice extracted with ethyl acetate.
The combined extract was dried with Na2SO4, filtered, concenꢀ
trated to dryness, and the residue (440 mg) was subjected to
chromatography on a column with silica gel in the gradient sysꢀ
tem hexane—ethyl acetate (5 : 1, 3 : 1, 1 : 1, 1 : 4). The fraction
eluted with the system 1 : 4 (215 mg) was subjected to HPLC on
a Supelco Discovery C18 column (25 cm × 4.6 mm, 5 μm) in the
system acetonitrile—0.01% aq. CF3COOH (80 : 20) to obtain
individual surfactins 1 (8 mg), 2 (2 mg), 3 (8 mg), 4 (10 mg), 5
(12 mg), 6 (3 mg), 7 (2 mg), 8 (2 mg), and 9 (2 mg) with the
retention times 11.6, 12.5, 14.5, 15.7, 18.4, 20.2, 21.8, 24.0, and
25.4 min, respectively.
6
8
4
60
5
40
20
0
24
48
t/h
Cell survivability (%)
b
4
100
80
60
40
20
0
8
6
5
24
48
t/h
Fig. 4. Effect of surfactins 4—6 and 8 on the survival of cells
MDAꢀMBꢀ231 (a) and HSTꢀ116 (b).
from 5 and 6 by the replacement of the Leu7 and Val7
residues by Ile7, inhibits growth of these cells by 80%.
Surfactins 5, 6, and 8 shows weak effect with respect to
the MDAꢀMBꢀ231 cells. Surfactin 4, after 72 h showed
inhibition of growth of these cells by 49% (Fig. 4).
The activity of surfactins 4—8 in the concentraꢀ
tion 100 μmol L–1 with respect to formation and growth
of the cells colonies MDAꢀMBꢀ231 was studied also usꢀ
ing the soft agar method (the cell neoplastic transformaꢀ
tions). It was shown that surfactins 4, 5, 6, 7, and 8 inhibit
growth of colonies of these cells by 20, 79, 57, 41,
and 69%, respectively, as compared to the untreated
cells (Fig. 5).
C14ꢀSurfactin methyl ester (7). Molecular formula C53H93N7O13,
white powder, [α]20D –32.5 (c 0.5, MeOH).
ESIꢀHRMS, found: m/z 1058.6719 [M + Na]+, calculated
1058.6729.
Total acid hydrolysis of surfactins. A depsipeptide (0.4 mg) in
a glass tube was dissolved in HCl (5.6 M, 0.8 mL), evacuated,
and heated for 24 h at 105 °C. The cooled reaction mixture was
diluted with biDI water, concentrated in vacuo, and analyzed on
the amino acid analyzer.
Colonies/8000 cells
The absolute configurations of amino acids were determined
1000
800
600
400
200
by Marfey´s method.11
Culturing of cancer cells. The human intestine cancer cells
HCTꢀ116 (ATCC # CCLꢀ247) and human breast cancer cells
MDAꢀMBꢀ231 (ATCC # HTBꢀ26) were cultured in the McCoy
5a medium and DMEM, respectively, 10% FBS with addition of
penicillin (100 units L–1) and streptomycin (100 μg L–1) in
an MCOꢀ18AIC incubator (Sanyo, Japan) at 37 °C, the content
of CO2 was 5%.
Determination of cytotoxicity of surfactins. The cells HCTꢀ116
and MDAꢀMBꢀ231 (1.5•105 mL–1) were dispersed in 96ꢀwell
plates and cultured in 200 μL of 10% aq. McCoy 5a and DMEM,
respectively in a CO2ꢀincubator at 37 °C for 24 h. Then the cells
Contr
4
7
5
6
8
Fig. 5. Effect of surfactins 4—8 on the formation and growth of
colonies of cells MDAꢀMBꢀ231; Contr is control.

2142
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 11, November, 2010
Zhuravleva et al.
were treated with surfactins (100 μmol L–1) in the fresh medium
and incubated for 24 h. After incubation, 5ꢀ(3ꢀcarboxymethꢀ
oxyphenyl)ꢀ3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2ꢀ(4ꢀsulfophenyl)ꢀ2Hꢀ
tetrazolium (15 μL) was added into each well and incubatꢀ
ed for 4 h (37 °C, 5% CO2). Optical density was measured on
a BioꢀTek Instruments spectrophotometer (USA) at two waveꢀ
lengths (490 and 630 nm) and the ratio A490/630 was determined
(see Ref. 16).
2. N. G. Prokof´eva, N. I. Kalinovskya, P. A. Luk´yanov, E. B.
Shensova, T. A. Kuznetsova, Toxicon, 1999, 37, 801.
3. D. Vollenbroich, M. Ozel, J. Vater, R. M. Kamp, G. Pauli,
Biologicals, 1997, 25, 289.
4. M. Kracht, H. Rokos, M. Ozel, M. Kowall, G. Pauli, J. Vater,
Biochem. Pharmacol., 1999, 55, 975.
5. Y. Kameda, S. Oira, K. Matsui, S. Kanamoto, T. Hase, Chem.
Pharm. Bull., 1974, 22, 938.
Neoplastic transformation of cells (the soft agar method). The
action of surfactins on the formation and growth of colonies of
human breast cancer cells MDAꢀMBꢀ231 were determined using
6. S.ꢀY. Kim, J. Y. Kim, S.ꢀH. Kim, H. J. Bae, H. Yi, S. H.
Yoon, B. S. Koo, M. Kwon, J. Y. Cho, C.ꢀE. Lee, S. Hong,
FEBS Lett., 2007, 581, 865.
7. Y. Imai, H. Sugino, T. Fujita, A. Kakinuma, J. Takeda Res.
Lab., 1971, 30, 728.
8. D. G. Cooper, C. R. McDonald, S. J. B. Duff, N. Kosaric,
Appl. Environ. Microbiol., 1981, 42, 4308.
9. ShiꢀZong Yang, DongꢀZhi Wei, BoꢀZhong Mu, J. Biochem.
Biophys. Methods, 2006, 68, 69.
10. JinꢀShan Tang, Hao Gao, Kui Hong, Yang Yu, MiaoꢀMiao
Jiang, HaiꢀPeng Lin, WenꢀCai Ye, XinꢀSheng Yao, Magn.
Reson. Chem., 2007, 45, 792.
11. K. Fujii, T. Shimoya, Y. Ikai, H. Oka, K. Harada, Tetraheꢀ
dron Lett., 1998, 39, 2579.
method of soft agar. The cells MDAꢀMBꢀ231 (2.4•104 mL–1
)
were treated with surfactins (100 μmol L–1) in 1 mL of 0.33% aq.
BMEꢀagar containing 10% FBS over 3.5 mL of 0.5% aq. BMEꢀ
agar containing 10% FBS and 100 μM of surfactin. The cells
were cultivated for 21 days at 37 °C, 5% CO2. Colonies of human
intestine cancer cells were evaluated using a Motic AE 20 reꢀ
versed microscope (China) and the MoticImagePlus program.
Statistical processing of the data were performed using the
Student tꢀcriterion under conditions of a given confidence level
equal to 95% (the SigmaPlot 2000 program, version 6 (SPSS
Inc., USA)).
12. H. Itokawa, T. Miyashita, H. Morita, K. Takeya, T. Hirano,
M. Homma, K. Oka, Chem. Pharm. Bull., 1994, 42, 604.
13. Sh. Kanamoto, S. Nagai, K. Ohki, Y. Yasuda, Yakugaku
Zasshi, 1995, 115, 756.
14. K. Oka, T. Hirano, M. Homma, H. Ishii, K. Murakami,
S. Mogami, A. Motizuki, H. Morita, K. Takeya, H. Itokawa,
Chem. Pharm. Bull., 1993, 41, 1000.
This work was financially supported by the Russian
Foundation for Basic Research (Project Nos 09ꢀ04ꢀ00388
and 08ꢀ04ꢀ00289), the Presidium of the Russian Academy
of Sciences (Program "Molecular and Cell Biology"), and
the Federal Agency for Science and Innovations of the
Russian Federation (State Contract No. 02.518.11.7169).
15. X.ꢀY. Liu, Sh.ꢀZh. Yang, BoꢀZh. Mu, Process Biochem.,
2009, 44, 1144.
References
16. N. H. Colburn, E. J. Wendel, G. Abrusso, Proc. Natl. Acad.
Sci. USA, 1981, 78, 6912.
1. K. Arima, A. Kakinuma, G. Tamura, Biochem. Biophys. Res.
Commun., 1968, 31, 488.
Received June 30, 2010