Unsaturated genistein disaccharide glycoside as a novel agent affecting microtubules

Article abstract of DOI:10.1016/j.bmcl.2009.07.089

Genistein, due to its recognized chemopreventive and antitumor potential, is a molecule of interest as a lead compound in drug design. While multiple molecular targets for genistein have been identified, so far neither for this isoflavonoid nor for its na

Full text of DOI:10.1016/j.bmcl.2009.07.089

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4939–4943
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Unsaturated genistein disaccharide glycoside as a novel agent
affecting microtubules
a
a
a
a,
´
Aleksandra Rusin , Agnieszka Gogler , Magdalena Głowala-Kosinska , Daria Bochenek
,
Aleksandra Gruca a, Grzegorz Grynkiewicz b, Jadwiga Zawisza c, Wiesław Szeja c, Zdzisław Krawczyk a,c,
*
a
_
Department of Tumor Biology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Branch Gliwice, Wybrzeze AK 15, 44-100 Gliwice, Poland
b Pharmaceutical Research Institute, Rydygiera 8, 01-793 Warsaw, Poland
c Department of Organic, Bioorganic Chemistry and Biotechnology, Silesian Technical University, Krzywoustego 4, 44-100 Gliwice, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Genistein, due to its recognized chemopreventive and antitumor potential, is a molecule of interest as a
lead compound in drug design. While multiple molecular targets for genistein have been identified, so far
neither for this isoflavonoid nor for its natural or synthetic derivatives disruption of microtubules and
mitotic spindles has been reported. Here we describe such properties of the synthetic glycosidic deriva-
Received 7 May 2009
Revised 14 July 2009
Accepted 16 July 2009
Available online 22 July 2009
tive of genistein significantly more cytotoxic than genistein, 7-O-(2,3,4,6-tetra-O-acetyl-b-
D-galactopyr-
anosyl)-(1?4)-(6-O-acetyl-hex-2-ene- -erythro-pyranosyl)genistein, shortly named G21. We found
that G21 causes significant mitotic delay, frequent appearance of multipolar spindles, and alteration of
the interphase microtubule array.
a
-D
Keywords:
Genistein glycoside
Microtubule interfering drugs
Ó 2009 Published by Elsevier Ltd.
Epidemiological and experimental studies place genistein
among chemopreventive agents and a complementary drugs in a
treatment of cardiovascular diseases, postmenopausal syndromes,
and cancer.1,2 At lower concentrations, essentially not exceeding
affect proliferation of cancerous cells by mechanisms other than
inhibition of tyrosine kinases and chemical glycosylation seems a
viable option for such purpose.15 The genistein derivatives, glyco-
sides of 2,3-unsaturated sugars, were found to inhibit proliferation
of multiple cancer cell lines at a concentration significantly (5–10
times) lower than a parent compound, what makes them attractive
objects, both for pharmacological studies and as lead compounds
for further modifications. Of multiple novel derivatives, the most
20
lM, this isoflavonoid may act as a selective estrogen receptor
modulator (SERM), with higher affinity to ERb than ER
a, while at
higher doses genistein reveals antiproliferative activity. Although
multiple molecular targets have been identified or suggested, the
major mechanism by which genistein impedes growth of cancer-
ous cells are thought to be: the inhibition of the activity of tyrosine
promising obtained so far is: 7-O-(2,3,4,6-tetra-O-acetyl-b-
D-galac-
topyranosyl)-(1?4)-(6-O-acetyl-hex-2-ene-a-D-erythropyranosyl)
kinases, topoisomerase II, and transcription factor NF-
jB, as well as
genistein, shortly named G21.16,17
affecting activities which control cell cycle.3
In the present report we describe the results of our investiga-
tion which demonstrate, that the synthetic genistein glycoside,
G21 has the potential to affect microtubule dynamics, leading to
malformation of mitotic spindles. To our knowledge, the G21 is
the first derivative of genistein exhibiting features of a mitotic
spindle poison. We also describe the simplified method of regio-
and stereoselective synthesis of this compound.
These observations inspired several groups to synthesize vari-
ous derivatives of genistein with the intention to either improve
biochemical and pharmacokinetic characteristics of the parent
drug or to obtain compounds containing essential elements of
the parent substance but having novel properties and/or affecting
novel molecular targets.4–10 So far none of the novel derivatives
revealed better inhibitory action on tyrosine kinase activity than
genistein11,12 while some approaches targeting genistein to tyro-
sine kinases through conjugation of the isoflavone with EGF or spe-
cific antibodies gave promising results.13,14
This novel genistein derivative was earlier obtained in a multi-
step procedure based on palladium catalyzed exchange of unsatu-
rated anomeric carbonate esters,16,18 which resulted in the mixture
of 7-, 40- regio- and
a,b-stereoisomers and meticulous chromatog-
Much effort has been put into the design and synthesis of new
genistein derivatives with improved anticancer activity that could
raphy was required in order to isolate the desired compound. Here,
we describe a novel approach to synthesis of G21 which resulted in
high regio- and stereoselectivity of glycosylation of genistein agly-
con, in keeping with observation that usually the reactivity of the
phenolic hydroxyl group at 7-OH is higher than that of other posi-
tions in flavonoids.19–21 Both the anomeric leaving group and the
method of its activation have been changed. As a part of the
* Corresponding author. Tel.: +48 322789757; fax: +48 32 278 9840.
E-mail addresses: krawczyk@io.gliwice.pl, arusin@io.gliwice.pl (Z. Krawczyk).
Present address: ETH Zürich, Institut f. Biochemie, Schafmattstr. 18, 8093 Zürich,
Switzerland.
0960-894X/$ - see front matter Ó 2009 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2009.07.089

4940
A. Rusin et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4939–4943
AcO
AcO
O
O
AcO
AcO
O
S
N
i)
AcO
AcO
S
N
OAc
HS
O
O
S
O
OAc
OAc
OAc
OAc
2
1
3
AcO
O
O
AcO
AcO
HO
ii)
O
O
S
N
O
S
OAc
OH
OAc
OH
4
AcO
O
O
AcO
AcO
O
O
O
O
OAc
OAc
OH
OH
G21
Figure 1. Scheme of the stereoselective synthesis of G21. Reagents: (i) InCl3, CH2Cl2, yield 83%; (ii) Cu(OTf)2, CH2Cl2, yield 76%.
program to develop novel, versatile methods for the stereoselective
synthesis of glycosides derivatives of genistein we have become
interested in relatively novel class of glycosyl donors, substituted
thioimidoyl derivatives.22 A number of such moieties have already
been employed at the anomeric center in an attempt to achieve
better stereo control of the glycosylation23 and phosphorylation
process,24 although there was no precedence in their application
to glycal chemistry.
Admittedly, in case of glycals, unfavorable regioselectivity in
reaction of thiol group containing substrates could be expected
on the ground of hard and soft acids and bases theory25 but, since
benzotiazol derivatives were not examined before, we took a
chance of obtaining desired 1-thiolglycoside, even through isola-
tion form a mixture. Here we describe the synthesis of novel S-ben-
zothiazolyl derivatives of 2,3-unsaturated pyranoses and their
application for stereoselective and regioselective glycosylation of
genistein (Fig. 1). For this purpose lactal (1) was first reacted with
2-mercapto-benzothiazole (2) in the presence of a promoter in
methylene chloride at room temperature to afford benzothiazolyl
The advantages of this procedure in comparison with previously
reported one16,18 is the accessibility of the stable glycosylating
donor, and high regio- and stereoselectivity of its action under mild
activation conditions.27
The structure of obtained glycoside (G21) was confirmed by 1
H
NMR spectral data and by comparison with a known compound.
The physical and spectral properties of -glycoside were identical
as in literature data.16 The 1H NMR spectra of
-glycoside showed
a characteristic signal at d 5.72 ppm (ddd, 1H, J1,2 = 0.8 Hz, J1,3
a
a
=
ꢀ1.9 Hz, J1,4 = 0.7 Hz, H-1), d 5.92 ppm (dd, 1H, J3,4 = 2.76 Hz, H-3),
d 6.30 ppm (ddd, 1H, J2,3 = 10.28 Hz, J2,4 = ꢀ1.3 Hz, H-2). The 1H
NMR spectrum of b-glycoside gave H-1, 5.83 ppm (J1,2 = 1.65 Hz,
J1,3 = 1.25 Hz, J1,4 = 1.2 Hz, J1,5 = ꢀ0.4 Hz) H-2, 6.25 ppm (ddd, J2,3
=
10.24 Hz, J2,4 = ꢀ3.1 Hz), and H-3, 6.02 ppm (ddd, J3,4 = 1.5 Hz).
Relevant NMR spectra are available as Supplementary data.
Compound G21 was previously shown to inhibit proliferation of
many cancer cell lines, however, the mechanism of its action was
not studied in details.16,17 For biological evaluation of G21 activity
human prostate cancer cell line, DU 145 was chosen and grown un-
der standard conditions.28 These results, together with appropriate
control experiments, as well as additional data on cytotoxicity of
G21 against human colon carcinoma HCT 116 cells are available
as Supplementary data.
(2,3,4,6-tetra-O-acetyl-b-
D-galactopyranosyl)-(1?4)-6-O-acetyl-
hex-2-ene-1-thio- -erythropyranoside (3) in 83% yield. Having
a-D
obtained required intermediate thioglycoside we turned our atten-
tion to the evaluation of its glycosylating properties. It was hypoth-
esized that heavy metal salt-based promoter system would
complex sulfur and nitrogen atoms in benzothiazole aglycon,
improving the leaving group ability.26 Thus, thioglycoside was cou-
pled with genistein (4) in the presence of a thiophilic metal
promoter in dry methylene chloride. The highest regio- and stere-
oselectivities for the synthesis of a title compound were obtained
in the presence of AgOTf and Cu(OTf)2. When MeOTf was used
the methylation of genistein was observed. In reaction using NIS/
TMSOTf as a promoter the mixture of glycosides was obtained
In order to assign the mechanism of antiproliferative activity of
G21 we investigated the influence of genistein and G21 on the cell
cycle with flow cytometer.29
In subconfluent control cells we observed two main populations
of cells with different DNA content: 60% of cells were in G1 phase of a
cell cycle (2C DNA), and around 20% of cells were in G2/M phase of a
cycle (4C DNA). As shown inFigure 2. the treatment of cells with G21
resulted in a significant accumulation of cells in G2/M phase of a
cycle (4C DNA) as compared to cells treated with genistein.
In the next step we determined microscopically the fraction of
mitotic cells.30 Treatment of cells for 24 h with G21 led to the
increase of the mitotic index from 6%, observed in control, to
(a/b: 3/1) and products of reaction of genistein (not identified)
were formed. The scheme of the novel synthesis of G21 is shown
in Figure 1.

A. Rusin et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4939–4943
4941
Figure 2. Flow cytometry analysis of cell cycle and DNA ploidy of DU 145 cells treated with genistein and G21. The 2C DNA corresponds to cells at G1 phase of a cell cycle, and
the 4C DNA corresponds to cells at G2/M phase. Cells were collected after 24 h incubation with drugs at concentration indicated in the picture. Histograms are representative
for the treatment. Numerical data on the histograms represents mean number of cells with indicated DNA content standard deviation, calculated from three independent
experiments.
almost 30%. (Table 1). In contrast to significant increase of the
mitotic index values after G21 treatment, the mitotic index after
the dose of genistein which caused visible accumulation of 4C
DNA cells was lower than in untreated control. Whereas genistein
blocked cell cycle in G2 phase, G21 almost did not influence this
phase, blocking the cells in mitosis.
In the next step we decided to check if the mitotic block is
related to changes in mitotic spindle structure in immunofluores-
cently stained specimens.31 Analysis of mitotic spindle aberrations
after G21 treatment is presented in Figure 3.
assembly. However, the type of changes of interphase microtu-
bules array indicates whether a given chemical stabilizes or
destabilizes microtubules. The results of a study of interphase
microtubule array, performed on cells treated with G21, two
reference drugs which affect microtubule dynamics: vinblastine,
inhibiting microtubules assembly and paclitaxel—microtubules
assembly stimulating drug, and with genistein, which is known
not to interact with microtubules34 are presented in Figure 4.
In control cells most of mitotic spindles had two regular poles,
and genistein did not influence their structures even at 100
concentration (not shown).
lM
Multipolar or monopolar spindles occurred very rarely. On a
contrary, in cells treated with G21, aberrant, mainly multipolar
mitotic spindles were observed. The fraction of cells containing
aberrant mitotic spindle increased in a G21 dose-dependent man-
ner, and already at 5 lM most of mitosis were aberrant (Fig. 3B and
C). Multipolar spindles were also observed after treatment of cells
with equitoxic to G21 doses of known drugs altering microtubule
dynamics, such as vinblastine and paclitaxel (not shown), and as
described in literature.32,33 Since both, microtubule destabilizing
agents, that is, vinblastine, and assembly promoting ones, that is,
paclitaxel, may cause multipolar spindle formation,32,33 it is not
possible to assign if a drug inhibits or stimulates microtubules
Table 1
Mitotic index counted upon microscopic examination
Control
Genistein
100
2.2 0.7
G21
Figure 3. Types of mitotic spindles in cells treated with G21. (A) Influence of
growing concentration of G21 on the frequency of abnormal spindles. (B) Normal
bipolar spindle. (C) Multipolar spindle. (D) Monopolar spindle. In control cells
normal, bipolar spindles dominate. After treatment with G21 multipolarity is
50
6
l
M
l
M
5
l
M
10
lM
5.9 1.2
2.7
29.4 0.1
28.1 0.6
Means and standard deviations are calculated from three independent experiments.
frequently observed. Scale bar—20 lm.

4942
A. Rusin et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4939–4943
References and notes
1. Middleton, E.; Kandaswami, C.; Theoharides, T. C. Pharmacol. Rev. 2000, 52, 673.
2. Dixon, R. A.; Ferreira, D. Phytochemistry 2002, 60, 205.
3. Banerjee, S.; Li, Y.; Wang, Z.; Sarkar, F. H. Cancer Lett. 2008, 269, 226.
4. Somjen, D.; Amir-Zaltsman, Y.; Gayer, B.; Kulik, T.; Knoll, E.; Stern, N.; Lu, L. J.;
Toldo, L.; Kohen, F. J. Endocrinol. 2002, 173, 415.
5. Wang, S. F.; Jiang, Q.; Ye, Y. H.; Li, Y.; Tan, R. X. Bioorg. Med. Chem. 2005, 13,
4880.
6. Meng, Q. H.; Lewis, P.; Wahala, K.; Adlercreutz, H.; Tikkanen, M. J. Biochim.
Biophys. Acta 1999, 1438, 369.
7. Matsumoto, T.; Kobayashi, T.; Kikuchi, T.; Honda, T.; Kamata, K. J. Smooth
Muscle Res. 2005, 41, 23.
8. Wang, J.; Shang, F.; Jing, R.; Liu, L.; Wang, S.; Hou, J.; Huan, M.; Mei, Q. J.
Pharmacol. Sci. 2007, 104, 82.
9. Zhang, L. N.; Xiao, Z. P.; Ding, H.; Ge, H. M.; Xu, C.; Zhu, H. L.; Tan, R. X. Chem.
Biodivers. 2007, 4, 248.
10. Kohen, F.; Gayer, B.; Kulik, T.; Frydman, V.; Nevo, N.; Katzburg, S.; Limor, R.;
Sharon, O.; Stern, N.; Somjen, D. J. Med. Chem. 2007, 50, 6405.
11. Ogawara, H.; Akiyama, T.; Watanabe, S.; Ito, N.; Kobori, M.; Seoda, Y. J. Antibiot.
1989, 42, 340.
12. Booth, C.; Hargreaves, D. F.; Hadfield, J. A.; McGown, A. T.; Potten, C. S. Br. J.
Cancer 1999, 80, 1550.
13. Uckun, F. M.; Narla, R. K.; Zeren, T.; Yanishevski, Y.; Myers, D. E.; Waurzyniak,
B.; Ek, O.; Schneider, E.; Messinger, Y.; Chelstrom, L. M.; Gunther, R.; Evans, W.
Clin. Cancer Res. 1998, 4, 1125.
14. Messinger, Y.; Yanishevski, Y.; Ek, O.; Zeren, T.; Waurzyniak, B.; Gunther, R.;
Chelstrom, L.; Chandan-Langlie, M.; Schneider, E.; Myers, D. E.; Evans, W.;
Uckun, F. M. Clin. Cancer Res. 1998, 4, 165.
Figure 4. Immunofluorescent staining of b-tubulin in interphase DU 145 cells. Cells
were treated with a vehiculum (control) or indicated drugs for 24 h. (A) Control; (B)
100 lM genistein; (C) 5 lM G21; (D) 25 lM G21; (E) 1 nM vinblastine; (F) 2 nM
paclitaxel, (note that concentration in C, E, F are equitoxic and correspond to 50%
cytotoxicity). G21, vinblastine and paclitaxel cause different perturbations of
microtubule array described in details in the text. Genistein does not induce any
15. Grynkiewicz, G.; Szeja, W.; Boryski, J. Acta Pol. Pharm. 2008, 65, 655.
´
P.; Ramza, J.; Pucko, W.;
16. Polkowski, K.; Popiolkiewicz, J.; Krzeczynski,
Zegrocka-Stendel, O.; Boryski, J.; Skierski, J. S.; Mazurek, A. P.; Grynkiewicz,
G. Cancer Lett. 2004, 203, 59.
17. Popiolkiewicz, J.; Polkowski, K.; Skierski, J. S.; Mazurek, A. P. Cancer Lett.. 2005
2005, 229, 67.
changes in microtubule array. Scale bar—10 lm.
18. Achmatowicz, O.; Grynkiewicz, G.; Pucko, W.; Mazurek, A.; Polkowski, K.;
Boryski, J.; Szeja, W.; Szelejewski W.; Opolski, A.; Pastuch-Gawolek, G.;
Radzikowski, C.; Szechner, B.; Wietrzyk, J.; Skierski, J.; Wandzik, I.;
Krzeczyn´ ski, P. Polish Patent P-346955; PCT/PL02/000029, 2002.
19. Lewis, P. T.; Wahala, K.; Hoikkala, A.; Mutikainen, I.; Meng, Q. H.; Adlercreutz,
H.; Tikkanen, M. J. Tetrahedron 2000, 56, 7805.
20. Wang, S. F.; Jiang, Q.; Ye, Y. H.; Li, Y.; Tan, R. X. Bioorg. Med. Chem. 2005, 13,
4880.
21. Chern, J. H.; Shia, K. S.; Hsu, T. A.; Tai, C. L.; Lee, C. C. Bioorg. Med. Chem. Lett.
2004, 14, 2519.
22. ElAshry, H. S. H.; Awad, L. F.; Atta, A. I. Tetrahedron 2006, 62, 2943.
23. Szeja, W.; Grynkiewicz, G. In Handbook of Glycosylation; Demchenko, A., Ed.;
Wiley-VCH: Weinheim, 2008; pp 329–361.
24. Ferrieres, V.; Blanchard, S.; Fischer, D.; Plusquellec, D. Bioorg. Med. Chem. Lett.
2002, 12, 3515.
Genistein—as expected—caused no alteration of microtubule ar-
ray even at 100 M concentration. At the concentration of G21
ꢁIC50 (5 M) the microtubule network was markedly loosened at
l
l
the cell periphery (Fig. 4C) and microtubules acquired characteris-
tic, curly appearance, resembling the pattern described for vinblas-
tine used at ꢁIC50 concentration (Fig. 4E). This pattern was
essentially different from the one observed for cells treated with
paclitaxel (Fig. 4F), where microtubule network was noticeably
denser than in control cells or the ones treated with genistein,
G21 and vinblastine. It has to be noted that at high concentration
25. Priebe, W.; Zamojski, A. Tetrahedron 1980, 36, 287.
26. Demchenko, A. V.; Malysheva, N. N.; De Meo, C. Org. Lett. 2003, 5, 455.
27. Procedure for synthesis of benzothiazoyl glycosyl donor: A solution of the per-O-
acetyl-lactal (1 mM), 2-mercapto-benzothiazol (1 mM) in CH2Cl2 (2 mL) and
freshly activated molecular sieves (3 Å, 0.2 g) was stirred under argon for
10 min, InCl3 (0.15 mM) was added. The reaction mixture was then stirred for
60 min under reflux. Upon completion (TLC), the reaction mixture was diluted
with CH2Cl2 (10 mL), the solid was filtered-off and the residue was washed
with CH2Cl2 (5 mL). The combined filtrate was washed with 5% aq NaHCO3
(3 ꢂ 10 mL), dried (MgSO4) and concentrated in vacuo. The residue was
purified by column chromatography on silica gel (chloroform) to allow the
thioglycoside as colorless oil (600 mg, yield 83%). The structure was confirmed
by 1H NMR (CDCl3) d (ppm) 1.98 (s, 3H), 2.02 (s, 6H), 2.07 (s, 3H), 2,18 (s,3H),
3.90–4.34 (m, 7H), 4.62 (d, 1H, J = 8 Hz), 5.05 (dd, 1H, J1 = 10.2 Hz, J2 = 3.4 Hz),
5.24 (dd, 1H, J2 = 7.6 Hz), 5.42 (d, 1H, J = 2.8 Hz), 5.72 (d, 1H, J = 2.6 Hz), 5.94
(ddd, 1H, J1 = 10.2 Hz, J2 = J3 = 2.5 Hz), 6.31(ddd, 1H, J2 = J3 = 0.8 Hz), 6.54 (d,
1H, J = 2.2 Hz), 6.62 (d,1H, J = 2.2 Hz), 6.95 (dd, 2H, J1 = 6.6 Hz, J2 = 2.2 Hz), 7.53
(dd, 2H), 8.01 (d, 1H), 8.19 (d, 1H). Procedure for synthesis of G21: A mixture of
the glycosyl donor (Fig. 1, Formula 3) (0.1 mM) and genistein (Fig. 1, Formula
4) (0.15 mM) in CH2Cl2 (2 mL) was sonificated for 30 min, freshly activated
molecular sieves (3 Å, 0.2 g), Cu(OTf)2 (0.15 mM) was added and reaction
mixture was stirred under argon for 1 h at room temp. Upon completion, the
reaction mixture was diluted with CH2Cl2 (10 mL), the solid was filtered-off
and the residue was washed with CH2Cl2 (10 mL). The combined filtrate was
washed with aq NaHCO3 and water (3 ꢂ 10 mL), dried (MgSO4) and
concentrated in vacuo. The residue was purified by column chromatography
on silica gel (chloroform) to allow the genistein glycoside as amorphous solid
(630 mg, yield 76%).
of G21 (25 lM) microtubules disappeared almost completely
(Fig. 4D). The ability of G21 to influence the structure of microtu-
bule network, and to induce mitotic spindle aberration was not
limited to DU 145 cells, and comparable effects were also observed
in other cell lines under study (not shown).
In summary, we have found a new method for the regio- and
stereoselective synthesis of the glycosyl genistein derivative,
G21. Extensive biological studies have shown that this compound
acts as a microtubule destabilizing agent. This ability has not been
reported previously for any isoflavonoid derivative.
Acknowledgments
We are grateful to Dr Tadeusz Bieg for his help with NMR exper-
iments and to Krystyna Klyszcz for technical assistance. This work
was supported by grant of Foundation for Supporting of Polish
Pharmacy and Medicine (06/FB/2004) and partially by grant of
Ministry of Science and Higher Education (PBZ-MNiI-1/1/2005).
Supplementary data
28. Cell culture conditions: Cells were grown at 37 °C, in humidified atmosphere
enriched with CO2 (5%) in RPMI 1640 medium (Sigma–Aldrich, Germany)
supplemented with non-inactivated fetal bovine serum (FBS) (10%; GIBCO
Effect of G21, lactal, genistein and mixture of these compounds
on the proliferation of human prostate cancer cells DU 145 and
human colon carcinoma cells HCT 116. NMR spectra of G21. Sup-
plementary data associated with this article can be found, in the
online version, at doi:10.1016/j.bmcl.2009.07.089.
Invitrogen, UK) and gentamicin sulfate (40 l
g mLꢀ1; Sigma–Aldrich, Germany).
29. Flow cytometry: Floating cells were collected and added to adherent cells,
harvested by trypsinization. Cells were washed with PBS and then fixed in ice-
cold ethanol (70%) for 30 min, treated with RNase (100 l
g mLꢀ1) and stained

A. Rusin et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4939–4943
g mLꢀ1). The DNA content was analyzed
using Becton Dickinson FACSCanto cytometer (BD Company, USA).
30. Mitotic index determination: Cells were grown in 3 cm culture dishes and
collected after 24 h treatment. Floating cells were pooled with adherent cells
after trypsinisation, washed with PBS, cytospinned, fixed with
4943
with propidium iodide (PI) (100
l
treatment with genistein, G21, vinblastine or paclitaxel. Microtubules were
immunostained as described by Huang, Y. T.; Huang, D. M.; Guh, J. H.; Chen, I.
L.; Tzeng C. C.; Teng, C. M. J. Biol. Chem. 2005, 280, 2771. Cells were examined
under an Nikon ECLIPSE E800 microscope using an oil immersion objective
100ꢂ. The type of mitotic spindle (bipolar = normal, multipolar or monopolar)
was counted in 100 of mitotic cells per treatment. Experiments were repeated
three times.
paraformaldehyde (4%) in PBS for 60 min, stained with DAPI (3 lM; Sigma–
Aldrich, Germany), and mounted in DAKOÒ Fluorescent Mounting Medium
(Dako, USA). Slides were examined under Nikon ECLIPSE E800 microscope using
40ꢂ objective. The mitotic cells were counted per 500 cells of the specimen.
31. Microtubule array analysis: Cells growing on 8-well chamber slides (Lab-Tek
PermanoxÒ slides, Nalgen Nunc International, Rochester, NY, USA) after
32. Hornik, J. E.; Bader, J. R.; Tribble, E. K.; Trimble, K.; Breunig, J. S.; Halpin, E. S.;
Vaughan, K. T.; Hinchcliffe, E. H. Cell Motil. Cytoskeleton 2008, 65, 595.
33. Jordan, M. A.; Thrower, D.; Wilson, L. J. Cell Sci. 1992, 102, 401.
34. Metzler, M.; Pheiffer, E. Health Perspect. 1995, 103, 21.