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Stepanova et al.
20 mmol) were added to a suspension of compound 2f (5.72 g,
10 mmol) in butanol (100 mL). The mixture was stirred for 48 h
with access to air. The charcoal was filtered off and the filtrate
was twice washed with water and concentrated. The residue was
triturated with ether, filtered off, and washed with ether. The
yield of compound 1f was 5.32 g (80%), red amorphous powder.
Its physicochemical and spectroscopic characteristics are idenꢀ
tical with those of an authentic sample.6
Tris(1ꢀpentylꢀ1Hꢀindolꢀ3ꢀyl)methylium chloride. A solution
of FeCl3 (4.86 g, 30 mmol) in water (50 mL) was added to
a solution of compound 2f (5.72 g, 10 mmol) in THF (200 mL).
The mixture was stirred for 24 h and concentrated in vacuo. The
residue was diluted with water and the resulting precipitate was
filtered off and dried. The yield of tris(1ꢀpentylꢀ1Hꢀindolꢀ3ꢀ
yl)methylium chloride was 4.43 g (75%), red amorphous powꢀ
der. Its physicochemical and spectroscopic characteristics are
identical with those of an authentic sample.6
To study the internucleosomal DNA degradation, the cells incuꢀ
bated with compound 1f (c = 1 μmol L–1) for 24 h were precipiꢀ
ated at 1000×g, the supernatant was centrifuged for 15 min at
12 000×g, the precipitates were combined and lysed in a buffer
(pH 7.4) containing 0.02 M TrisꢀHCl, 0.35 M NaCl, 0.5% NPꢀ40,
0.002 M MgCl2, and 0.001 M dithiothreitol. DNA was extracted
with phenol—chloroform (1 : 1) and precipiated with ethanol in
the presence of 0.3 M sodium acetate at –20 °C. The precipitate
was treated with RNAse A at 65 °C for 20 min and subjected to
electrophoresis in a 1.5% agarose gel. Experiments were carried
out at least three times.
Immunocytochemical determination of NFkB. Jurkat cells
(6•104 cells mL–1) were incubated for 48 h with the test comꢀ
pounds in nontoxic concentrations, washed twice with a cold
physiological buffer, and centrifuged onto the slide surfaces. Cells
were fixed with ethanol and acetone and made permeable in
a buffer containing 1% Triton Xꢀ100 for 5 min. Bovine serum
albumin (final concentration 5%) was added. Primary antibodies
for the active subunit NFkB p65 (MAB3026, Chemicon) were
applied and kept for 1 h. Antibodies conjugated with Alexa Fluor
594 (A11005, Invitrogen) were used as secondary antibodies. The
Tris(1ꢀpentylꢀ1Hꢀindolꢀ3ꢀyl)methylium acetate. A 10% soluꢀ
tion of NaOH (30 mL) was added to a suspension of compound
1f (6.65 g, 10 mmol) in ether (40 mL). The mixture was stirred
for 30 min, whereupon the organic layer was separated and conꢀ
centrated. The yield of tris(1ꢀpentylꢀ1Hꢀindolꢀ3ꢀyl)methanol
was 5.75 g (98%), light yellow amorphous solid.
preparations were stained up with Hoechstꢀ33258 (2 μg mL–1
)
and analyzed on a Nikon 80i fluorescence microscope (400x magꢀ
Acetic acid (1.2 mL, 20 mmol) was added to a solution of
tris(1ꢀpentylꢀ1Hꢀindolꢀ3ꢀyl)methanol (5.87 g, 10 mmol) in THF.
The mixture was stirred for 20 min and evaporated to dryness
in vacuo. The residue was triturated with ether and the resulting
precipitate was filtered off and washed with ether. The yield of
tris(1ꢀpentylꢀ1Hꢀindolꢀ3ꢀyl)methylium acetate was 5.6 g (90%),
red powder. Its physicochemical and spectroscopic characterisꢀ
tics are identical with those of an authentic sample.6
The cytotoxic activity of the compounds obtained was tested
in a concentration range from 0.1 to 50 μmol L–1. The percentꢀ
age of viable cells was determined after a 48ꢀh incubation. The
average inhibiting doses required for the compounds to block the
viability of 50% of cells (IC50) are given in Table 1.
nification).
Estimation of the antibacterial and antifungal activities. The
minimum inhibitory concentrations (MIC) for Gramꢀpositive
and Gramꢀnegative bacteria were determined by the microdiluꢀ
tion method in a cationꢀadjusted Müller—Hinton medium (Becꢀ
ton Dickinson and Company, Cockeysville (MD), USA) as deꢀ
scribed earlier.15 The activity of the test compounds against varꢀ
ious cultures of yeast and mycelial fungi was estimated using the
previously described16 micromethod by twofold serial dilutions
in the nutrient medium RPMI 1640 (liquid, with Lꢀglutamine,
without sodium bicarbonate).
The data obtained were statistically processed using a tꢀtest
procedure.
Cell lines. Cells were cultured in RPMIꢀ1640 with addition
of 10% fetal calf serum, 2•10–3
M Lꢀglutamine, penicillin
This work was financially supported by the Federal
Agency for Science and Innovations of the Russian Federꢀ
ation (State Contract No. 02.512.12.2035), the Council
on Grants at the President of the Russian Federation
(State Support Program for Leading Scientific Schools of
the Russian Federation, Grant NShꢀ5290ꢀ2010.4), and
the Russian Foundation for Basic Research (Project
No. 10ꢀ03ꢀ00210ꢀa).
(100 IU mL–1), and streptomycin (100 μg mL–1). The Mel Kor
cell line was obtained from a disseminated skin melanoma samꢀ
ple at the Laboratory for Experimental Diagnostics and Biotherꢀ
apy of Tumors of the N. N. Blokhin Russian Oncological Scienꢀ
tific Center (Russian Academy of Medical Sciences).14 The subꢀ
line HCT116p53KO was obtained at the laboratory headed by
B. Fogelstein (Johns Hopkins University) and provided by B. P.
Kopnin. Other cell lines were taken from the American Type
Culture Collection. The chemicals were purchased from Sigꢀ
ma—Aldrich, unless otherwise specified.
References
Estimation of cell death. Cells were stained with a conjugate
of Annexin V with FITC using an Annexin V Apoptosis Detecꢀ
tion kit (BD Pharmingen). Jurkat cells (6•104 cells mL–1) were
incubated together with the test compounds for 48 h, washed
with a cold physiological buffer, and transferred to an Annexin
Vꢀbinding buffer. The conjugate (5 μL) and propidium iodide
(5 μL) were added and the mixture was incubated for 15 min.
Cell fluorescence was determined on a flow cytofluorimeter at
wavelengths of 525 (green channel) and 675 nm (red channel).
At least 10 000 cells were analyzed. After the incubation with
a test compound was completed, part of cells were lysed in
a buffer containing 0.1% sodium citrate, the 0.3% detergent NPꢀ40,
RNAse A (100 μg mL–1), and propidium iodide (50 μg mL–1).
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