Synthesis and investigation of the anticancer effects of estrone-16-oxime ethers in vitro

Article abstract of DOI:10.1016/j.steroids.2012.10.009

An expanding body of evidence indicates the possible role of estrane derivatives as useful anticancer agents. The aim of this study was to describe the cytotoxic effects of 63 newly synthetized estrone-16-oxime ethers on human cancer cell lines (cervix carcinoma HeLa, breast carcinoma MCF7 and skin epidermoid carcinoma A431), studied by means of the MTT assay. Four of the most promising compounds were selected for participation in additional experiments in order to characterize the mechanism of action, including cell cycle analysis, morphological study and the 5-bromo-2′-deoxyuridine incorporation assay. The cancer selectivity was tested on a noncancerous fibroblast cell line (MRC-5). Since apoptosis and cell cycle disturbance were observed, caspase-3 activities were further assayed for the two most effective agents. These estrone-16-oxime analogs activated caspase-3 and changed the mRNA level expression of endogenous factors regulating the G1-S phase transition (retinoblastoma protein, CDK4 and p16). The repression of retinoblastoma protein was reinforced at a protein level too. These experimental data lead to the conclusion that estrone-16-oxime ethers may be regarded as potential starting structures for the design of novel anticancer agents.

Full text of DOI:10.1016/j.steroids.2012.10.009

Steroids 78 (2013) 69–78
Contents lists available at SciVerse ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis and investigation of the anticancer effects of estrone-16-oxime
ethers in vitro
Ágnes Berényi a, Renáta Minorics a, Zoltán Iványi b, Imre Ocsovszki c, Eszter Ducza a, Hubert Thole b,
b
b
b
b
b
b,
⇑
}
Josef Messinger , János Wölfling , Gergo Mótyán , Erzsébet Mernyák , Éva Frank , Gyula Schneider ,
István Zupkó a,
⇑
a Department of Pharmacodynamics and Biopharmacy, University of Szeged, Eötvös u. 6, H-6720 Szeged, Hungary
b Department of Organic Chemistry, University of Szeged, Dóm tér 8, H-6720 Szeged, Hungary
c Department of Biochemistry, University of Szeged, Dóm tér 9, H-6720 Szeged, Hungary
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 June 2012
Received in revised form 28 September 2012
Accepted 10 October 2012
Available online 2 November 2012
An expanding body of evidence indicates the possible role of estrane derivatives as useful anticancer
agents. The aim of this study was to describe the cytotoxic effects of 63 newly synthetized estrone-16-
oxime ethers on human cancer cell lines (cervix carcinoma HeLa, breast carcinoma MCF7 and skin epider-
moid carcinoma A431), studied by means of the MTT assay. Four of the most promising compounds were
selected for participation in additional experiments in order to characterize the mechanism of action,
including cell cycle analysis, morphological study and the 5-bromo-20-deoxyuridine incorporation assay.
The cancer selectivity was tested on a noncancerous fibroblast cell line (MRC-5). Since apoptosis and cell
cycle disturbance were observed, caspase-3 activities were further assayed for the two most effective
agents. These estrone-16-oxime analogs activated caspase-3 and changed the mRNA level expression
of endogenous factors regulating the G1–S phase transition (retinoblastoma protein, CDK4 and p16).
The repression of retinoblastoma protein was reinforced at a protein level too. These experimental data
lead to the conclusion that estrone-16-oxime ethers may be regarded as potential starting structures for
the design of novel anticancer agents.
Keywords:
Estrone-16-oxime ethers
Cell viability
Apoptosis
Cell cycle blockade
Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction
nism of the growth-inhibitory action of the compound in several
types of malignant cells [8]. Similarly, steroidal glycoalkaloids,
Endogenous steroidal compounds exert an outstandingly broad
spectrum of physiological functions in spite of the conserved struc-
ture, indicating that minor chemical changes may lead to mole-
cules with substantial property differences. The steroidal
skeleton is therefore the backbone for the design and synthesis
of original drug candidates with a wide range of pharmacological
activities. Many plant steroids are reported to possess inotropic,
antihypercholesterolemic, anti-inflammatory and antioxidant
properties [1–5]. The anticancer capacity is one of the most inten-
sively investigated characters of natural steroid products and syn-
thetic analogs. Diosgenin, one of the most widely studied saponins,
exerts antiproliferative activities against several human cancer cell
lines, such as osteosarcoma 1547, HER2-overexpressing breast
cancer, melanoma M4Beu, leukemia K562, HEL, and colon HT-29
[6–11]. Apoptosis induction and up-regulation of tumor suppressor
p53 have been reported to be crucial components of the mecha-
including a-tomatine and solanine, displayed an appreciable inhi-
bition of human colon (HT-29) and liver (HepG2) cancer cell prolif-
eration [12]. These capacities were greater than those of the
aglycones tomatidine and solanidine, respectively, indicating that
the steroidal backbone is not an exclusive factor determining the
overall cytostatic activities of these natural products.
Besides those of the active components isolated from plants, the
outstanding anticancer and cardioprotective properties of endoge-
nous estrogen hormone metabolites have been described [13]. The
final steps in the oxidative conversion of estradiol involve hydrox-
ylation of rings A and D of the estrone skeleton. 2-Methoxyestradi-
ol, an intensively investigated metabolite synthesized by catechol-
O-methyltransferase, exerts a pronounced anticancer effect on a
broad variety of human malignant cell lines of reproductive origin
[14–16].
Introduction of an oxime function into an appropriate skeleton
is a reasonable approach to the preparation of potent cytotoxic
agents. A set of oxime-containing flavone and isoflavone deriva-
tives have been synthetized and tested against three human adher-
ent cell lines, and the effective agents were further evaluated
⇑
Corresponding authors. Tel.: +36 62 546839 (I. Zupkó).
E-mail addresses: Schneider@chem.u-szeged.hu (G. Schneider), zupko@pharm.
u-szeged.hu (I. Zupkó).
0039-128X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.steroids.2012.10.009

70
Á. Berényi et al. / Steroids 78 (2013) 69–78
against the full panel of 60 human tumor cell lines derived from
nine cancer types. Flavon-6-yl oximes proved to exhibit the most
pronounced cytostatic effects, with the lowest mean GI50 value
as low as 0.08 lM. The apoptosis induction of the most effective
compound was evidenced by flow-cytometry.
tion was generally complete within 30–45 min. O-Benzoylation of
oximes 3a–e and 4a–d with benzyl bromide or substituted benzyl
bromides yielded O-benzyl oximes 7f,g, 9e–g, 10e,f, 11c–e and
12a–c. The reactions at room temperature needed 6–12 h, depend-
ing on the electron-donating or electron-withdrawing character of
the benzyl bromide substituent. 3-Sulfamoyloxy-16-alkoximinoe-
strone derivatives (6a,b) were prepared by the reaction of sulfa-
moyl chloride and 16-alkoximinoestrone (5b,c) in N,N-
dimethylformamide. O-Acyl oximes 7h,i, 8d,e, 9h,i, and 10g,h were
prepared with acetic anhydride or propionic anhydride in pyridine.
The crude products were purified by flash chromatography.
The antiproliferative activity of a 5-nitrofuran-2-yl-vinyl quino-
line derivative was increased when its oxo function was substi-
tuted with
a H-bond-donating oxime group. The derivative
containing the H-bond-accepting methyloxime was less favorable
against cancer cells of reproductive origin [17]. The (Z) and (E) iso-
mers of cholest-4-en-6-one oxime also exhibited moderate, but
cancer-selective growth-inhibitory properties [18].
The aim of the present study was the synthesis of a set of es-
trone oximes and a two-step investigation of their direct action
against the viability of human adherent cancer cell lines HeLa (cer-
vix adenocarcinoma), MCF7 (breast adenocarcinoma) and A431
(skin epidermoid carcinoma). Compounds exhibiting noteworthy
IC50 values were subjected to further experiments targeting the
cancer selectivity and the mechanism of action, including cell cycle
analysis, fluorescent microscopy, and determination of the cas-
pase-3 activity and the expression of crucial cell cycle-regulating
factors.
2.3. Synthesis of 16-oximinoestrone derivatives (3a–e and 4a–d;
general method)
Estrone (1a) and its 3-substituted derivatives (1b–e) in the nat-
ural series (13b-methyl) and their 13a-methyl analogs (2a–d)
(10.0 mmol) were stirred under nitrogen in 40 ml of freshly dis-
tilled tert-butanol for 10 min. Potassium tert-butoxide (55 mmol,
6.0 g) was added to the suspension, and stirring was continued
for 30 min, when dissolution was complete. n-Butyl nitrite
(20 mmol, 2.8 ml) was added from a syringe, and the solution
turned deep-red and then orange within a few minutes. A second
portion of n-butyl nitrite (20 mmol, 2.8 ml) was added after 6 h,
and stirring was continued for an additional 12 h. The reaction
mixture was next quenched in 100 g of ice and acidified with gla-
cial acetic acid. The precipitate that formed was filtered off,
washed until neutral with water, and dried over P2O5. The crude
products 3a–d and 4a–d were subjected to chromatographic sepa-
ration on a silica gel column with tert-butyl methyl ether/light
petroleum (20:90). 3e was chromatographed with ethyl acetate/
dichloromethane (10:90).
2. Experimental
2.1. General
Melting points (mp) were determined on a Kofler block and are
uncorrected. Specific rotations were measured in CHCl3 (c 1) at
20 sC with a POLAMAT-A (Zeiss-Jena) polarimeter and are given
in units of 10ꢀ1 cm2 gꢀ1. Elementary analysis data were deter-
mined with a Perkin-Elmer CHN analyzer model 2400. The reac-
tions were monitored by TLC on Kieselgel-G (Merck Si 245 F)
layers (0.25 mm thick); solvent systems (ss): (A) tert-butyl methyl
ether/dichloromethane (5:95, v/v), (B) tert-butyl methyl ether/
dichloromethane (10:90, v/v), (C) acetone/toluene/n-hexane
(35:30:30, v/v). The spots were detected by spraying with 5% phos-
phomolybdic acid in 50% aqueous phosphoric acid. The Rf values
were determined for the spots observed by illumination at 245
2.4. Synthesis of 16-alkoximinoestrone derivatives (5a–d, 7a–e, 8a–c,
9a–d, 10a–d and 11a,b; general method)
The 16-oximinoestrone derivative (3a–d and 4a–d)
(1.00 mmol) and 700 mg (3.00 mmol) of Ag2O were suspended in
anhydrous diethyl ether (30 ml) and the suspension was stirred
at room temperature for 30 min. Alkyl iodide (25 mmol) was then
added dropwise to the reaction mixture, which was stirred until
completion of the reaction (monitored by TLC). The excess Ag2O
and silver salt were filtered off from the suspension and washed
with diethyl ether (50 ml), and the organic phase was evaporated.
The crude product was purified by flash chromatography. 5a–d
were chromatographed with tert-butyl methyl ether/light petro-
leum (20:80), and 7a–e, 8a–c, 9a–d, 10a–d and 11a,b with dichlo-
romethane/petroleum ether (30:70) or dichloromethane.
and 365 nm. Flash chromatography: silica gel 60, 40–63 lM. All
solvents were distilled prior to use. NMR spectra were recorded
on a Bruker DRX500 instrument at 500 (1H NMR) or 125 MHz
(
13C NMR). Chemical shifts are reported in ppm (d scale), and cou-
pling constants (J) in Hz. For the determination of multiplicities,
the J-MOD pulse sequence was used.
2.2. Materials and methods
The search for steroids with novel biological effects led to the
synthesis of 16-oximinoestrone derivatives. The starting materials
of the syntheses were 3-hydroxyestra-1,3,5(10)-trien-17-one (1a),
3-methoxyestra-1,3,5(10)-trien-17-one (1b), 3-benzyloxyestra-1,3,
5(10)-trien-17-one (1c), 3-p-methoxybenzyloxyestra-1,3,5(10)-tri-
en-17-one (1d), 3-sulfamoyloxyestra-1,3,5(10)-trien-17-one (1e),
2.5. Synthesis of 16-benzyloximinoestrone derivatives (7f,g, 9f,g, 10e,f,
11d,e and 12a–c; general method)
and their 13a-methyl epimers (2a–d). The different steroidal oxi-
mes were synthetized as outlined in Fig. 1.
The 16-oximinoestrone derivative (3a–d and 4a–d)
(1.00 mmol) and 700 mg (3.00 mmol) of Ag2O were suspended in
anhydrous diethyl ether (50 ml) and the suspension was stirred
at room temperature for 30 min. Benzyl bromide or substituted
benzyl bromide (4.00 mmol) was then added to the reaction mix-
ture, which was stirred until completion of the reaction (monitored
by TLC). The excess Ag2O and silver salt were next filtered off and
washed with diethyl ether (50 ml), and the organic phase was
evaporated. The residual crude product was chromatographed
with dichloromethane/light petroleum (1:1) or dichloromethane.
Introduction of the oxime group at position 16 was achieved
after the deprotonation of 1a–e and 2a–d through the action of
potassium tert-butoxide and reaction with an excess of isoamyl ni-
trite, affording oximes 3a–e and 4a–d in yields of 85–90%.
O-Alkylation of 3a–e and 4a–d with a large excess of alkyl io-
dide in the presence of 4 equivalents of Ag2O in diethyl ether affor-
ded the required O-alkyl oximes 5a–d, 7a–e, 9a–d and 11a,b in the
natural (13b-methyl) series. The same methods were used in the
13-epimer (13a-methyl) series, yielding 8a–c and 10a–d. The reac-

Á. Berényi et al. / Steroids 78 (2013) 69–78
71
Fig. 1. Chemical structures of the synthetized and investigated estrone-16-oxime ethers. Reagents: (i) isoamyl nitrite, KOtBu, tBuOH; (ii) BnBr or substituted BnBr, Ag2O, Et2O.
2.6. Synthesis of 3-sulfamoyloxy-16-alkoxyiminoestrone derivatives
graphed on silica gel with tert-butyl methyl ether/light
(6a,b)
petroleum (20:80).
The 16-alkoxyiminoestrone derivative (5b,5c) (1.00 mmol) was
dissolved in dimethylformamide (25 ml) and sulfamoyl chloride
(10 mmol) was added in small portions during cooling in ice. The
reaction mixture was stirred at 0 sC for 6 h, and was then poured
into ice-water. The precipitate that formed was filtered off and dis-
solved in dichloromethane, and the solution was chromato-
2.7. Synthesis of 16-acetoximinoestrone derivatives (7h, 8d, 9h and
10g) and 16-propionyloxyestrone derivatives (7i, 8e, 9i and 10h)
The 16-oximinoestrone compound (3b,c and 4b,c) (1 mmol)
was dissolved in pyridine (5 ml), and acetic anhydride (5 ml) or
propionic anhydride (5 ml) was added during cooling in ice. The

72
Á. Berényi et al. / Steroids 78 (2013) 69–78
reaction mixture was allowed to stand at room temperature for
24 h, and was then poured into ice-water. The precipitate that
formed was filtered off, washed, dried and recrystallized from ace-
tone/light petroleum to yield 7h, 8d, 9h and 10g, or 7i, 8e, 9i and
10h.
for 1 h at 37 °C and were then photographed by means of a Nikon
Eclipse microscope equipped with an epifluorescence attachment
containing the appropriate optical blocks and a QCapture CCD
camera. The staining allowed the identification of live, early-apop-
totic, late-apoptotic and necrotic cells. Hoechst 33258 permeates
all the cells and makes the nuclei appear blue. Apoptosis was re-
vealed by nuclear changes such as chromatin condensation and nu-
clear fragmentation. The necrotic and the late-apoptotic cells were
identified as cells with propidium iodide uptake, which indicates
loss of membrane integrity, the cell nuclei being stained red [21].
2.8. Cell culturing
Human cancer cell lines (HeLa, MCF7 and A431, isolated from
cervix and breast cancers and skin epidermoid carcinoma, respec-
tively) and noncancerous MRC-5 human lung fibroblasts were
maintained in minimal essential medium supplemented with
10% fetal bovine serum (FBS), 1% non-essential amino acids and
an antibiotic–antimycotic mixture (AAM). A2780 cells (isolated
from ovarian cancer) were maintained in RPMI medium supple-
2.12. 5-Bromo-20-deoxyuridine (BrdU) incorporation assay
BrdU incorporation into the cellular DNA was determined with
the BrdU Labeling and Detection Kit I (Roche Diagnostic, Mann-
heim, Germany) on HeLa cells (3000/well) treated with the test
compound for 24 h. The incorporation of BrdU in place of thymi-
dine was monitored as a parameter for DNA synthesis. In accor-
dance with the manufacturer’s instructions, cells were labeled
with BrdU for 1 h, followed by fixation. The cellular DNA was par-
tially digested by nuclease treatment and labeled with mouse
monoclonal antibody. Finally, the fluorescein-conjugated anti-
mouse antibody was added and the wells were examined by fluo-
rescent microscopy with the use of an appropriate optic block (ex:
465–495 nm, em: 515–555 nm, dichromatic mirror: 505 nm). At
least 400 cells were counted from four parallel wells for the
expression of the BrdU-positive cells.
mented with 10% FBS, 1% AAM and 1% L-glutamine. All cell lines
were purchased from the European Collection of Cell Cultures
(Salisbury, UK). For pharmacological investigations, 10 mM stock
solutions of the tested compounds were prepared with dimethyl
sulfoxide (DMSO). The highest DMSO concentration of the medium
(0.3%) did not have any substantial effect on the determined cellu-
lar functions. All the chemicals, if otherwise not specified, were
purchased from Sigma–Aldrich Ltd. (Budapest, Hungary).
2.9. Antiproliferative activity measured by MTT assay
The effects on the viability of malignant cells were determined
in vitro on the four human cancer cell lines: HeLa, MCF7, A431 and
A2780. The cells were grown in a humidified atmosphere of 5% CO2
at 37 °C. Cells were seeded onto 96-well plates at a density of
5000 cells/well and allowed to stand overnight, after which the
medium containing the tested compound was added. After a 72 h
incubation period, viability was determined by the addition of
2.13. Caspase-3 assay
The activity of caspase-3 from treated cells was determined in
triplicate by means of a commercially available colorimetric kit
in accordance with the instructions of the supplier (Sigma–Aldrich,
Budapest, Hungary). Briefly, HeLa cells (16 million per condition)
were exposed to the test item for 48 h and then scraped, counted
20 lL of MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoli-
um bromide]) solution (5 mg/mL). The precipitated formazan crys-
tals were solubilized in DMSO and the absorbance was read at
545 nm with an ELISA reader. Two independent experiments were
performed with five parallel wells; cisplatin, an agent administered
clinically in some gynecological malignancies, was used as positive
control. Sigmoidal dose–response curves were fitted to the mea-
sured points, and the IC50 values were calculated by means of
GraphPad Prism 4.0 (GraphPad Software; San Diego, CA, USA) [19].
and resuspended in lysis buffer (10 lL for 1 million of cells). The
caspase-3 activity was measured by the addition of substrate
(Ac-DEVD-pNA) and the amount of product (pNA) was measured
at 405 nm after incubation for 17 h. Results on treated cells are gi-
ven as fold-increase by direct comparison with the untreated con-
trol results.
2.14. Reverse transcription-polymerase chain reaction (RT-PCR)
studies
2.10. Analysis of cell cycle by flow cytometry
Flow cytometric analysis was performed in order to character-
ize the cellular DNA content of treated HeLa cells. After treatment
for 24 or 48 h, the cells (200,000/condition) were trypsinized (Gib-
co BRL, Paisley, U.K.), washed with phosphate-buffered saline (PBS)
and fixed in 1.0 mL of cold 70% ethanol for 30 min on ice. After two
washing steps in cold PBS, the DNA was stained with propidium io-
The effects of the tested compounds on the mRNA expression
pattern of the regulator factors retinoblastoma protein (Rb), cy-
clin-dependent kinases 2 (CDK2), 4 (CDK4) and 6 (CDK6), and
p16, p21, p27 and p53, which play crucial roles in the transition
from the G1 to the S phase, were determined by RT-PCR in HeLa
cells. After a 24 h incubation period, the media containing the var-
ious test compounds were discarded and the total RNA was iso-
lated from the cells (5 ꢁ 105) by using the TRIzol Reagent in
accordance with the instructions of the manufacturer (Molecular
Research Center, Cincinnati, OH, USA) [22]. The pellet was resus-
dide (10 lg/mL) in the presence of RNA-ase (50 lg/mL). The sam-
ples were then analyzed by FACStar (Becton–Dickinson;
Mountain View, CA, USA). In each analysis, 20,000 events were re-
corded, and the percentages of the cells in the different cell-cycle
phases (subG1, G1, S and G2/M) were calculated by using win-
MDI2.9. The subG1 fractions were regarded as the apoptotic cell
population [20].
pended in 100
RNA concentrations of the samples were determined from their
absorbances at 260 nm. The RNA (0.5 g) was mixed with DNase-
and RNase-free distilled water and 20 M oligo(dT) (Invitrogen,
Carlsbad, CA, USA) in a final reaction volume of 10 L, which was
lL of DNase- and RNase-free distilled water. The
l
l
2.11. Hoechst 33258–propidium iodide double staining
l
incubated at 70 °C for 5 min. After the mixture had been cooled
to 4 °C, 20 U of RNase inhibitor, 20 U of MMLV reverse transcrip-
Near-confluent HeLa cells were seeded into a 96-well plate
(5000 cells/well). After incubation for 24 h with the test com-
pound, Hoechst 33258 and propidium iodide were added to the
tase (both from Promega, Madison, WI, USA), 200
50 mM Tris–HCl, pH 8.3, 75 mM KCl and 5 mM MgCl2 in a final
reaction volume of 10 L were added. The mixture was incubated
at 37 °C for 60 min. The PCR was carried out with 5 L of cDNA,
lM dNTP in
culture medium to give final concentrations of 5 and 2
l
g/mL,
l
respectively. The cells were incubated with the staining mixture
l

Á. Berényi et al. / Steroids 78 (2013) 69–78
73
Table 1
25
antisense primer of Rb, CDK2, CDK4, CDK6, p16, p21, p27 or p53
and 16 L of DNase- and RNase-free distilled water. The primer se-
lL of ReadyMix Taq PCR reaction mix, 2 lL of 20 pM sense and
Calculated antiproliferative IC50 values of the tested estrone-16-oxime ethers.
Compounds IC50 values (l
M)a
l
quences used to amplify Rb, CDK2, CDK4, p16, p21 and p27 were
described by Gao et al. [23], that for p53 by Matsuhashi et al.
[24] and that for CDK6 by Huang et al. [25]. Human glyceraldehyde
3-phosphate dehydrogenase primers were used as internal control
in all samples (Supplementary Table 1). The PCR was performed
with an ESCO SWIFT MAXI thermal cycler (Esco Technologies, Phil-
adelphia, PA, USA) and the products were separated on 2% agarose
gels, stained with ethidium bromide and photographed under a UV
transilluminator. Semiquantitative analysis was performed by den-
sitometric scanning of the gel with Kodak Image Station 2000R
(Eastman Kodak, Rochester, NY, USA).
HeLa
cells
MCF7
cells
A431
cells
A2780
cells
MRC-5
cells
3a
3e
10h
11a
4.41
4.04
3.52
5.63
5.66
>30
>30
4.13
>30
7.99
>30
>30
>30
13.25
8.81
18.28
11.96
4.61
25.05
0.86
>30
>30
>30
6.94
4.13
Cisplatin
a
Mean value from two independent determinations with five parallel wells,
standard deviation less than 15%.
hydroxy group was etherified with benzyl or p-methoxybenzyl
alcohol (9a–i, 10a–j, 11a–e, 12a–c). Among these more effective
compounds, 13b-methyl generally seemed to be preferred, though
with some exceptions (9i–10h, 9h–10g). In the light of these re-
sults, four oximes were selected for further in vitro investigations
(3a, 3e, 10h and 11a). The viability assays were repeated with a
2.15. Western blotting studies
HeLa cells were harvested in 60-mm dishes at a density of
2 ꢁ 105 cells/mL and treated with compounds 3a and 3e. Whole-
cell extracts were prepared by washing the cells with PBS and sus-
pending them in lysis buffer (50 mM Tris, 5 mM EDTA, 150 mM
NaCl, 1% NP-40, 0.5% deoxycholic acid, 1 mM sodium orthovana-
set of dilutions of these agents (0.1–30 lM) and extended to ovar-
ian cancer cell line A2780, and IC50 values were calculated (Table 1,
Supplementary Fig. 1). All of these estrone oximes affected the pro-
liferation of HeLa cells comparably to the reference agent cisplatin,
while MCF7, A413 and A2780 cells were less sensitive. The viability
of noncancerous fibroblast cell line MRC-5 was affected only by
11a, with a higher calculated IC50 value than that of cisplatin.
Agents with an unsubstituted oxime function (3a and 3e) could
be regarded as selective for HeLa cells, with some modest action
against ovarian cancer (A2780) cell line, while 10h and 11a dis-
played a broader spectrum of activities.
date, 100 lg/mL PMSF and protease inhibitors) [26]. 50 lg of pro-
tein per well was subjected to electrophoresis on 4–12% NuPAGE
Bis–Tris Gel in XCell SureLock Mini-Cell Units (Invitrogen, Carls-
bad, CA, USA). Proteins were transferred from gels to nitrocellulose
membranes, using the iBlot Gel Transfer System (Invitrogen,
Carlsbad, CA, USA). Antibody binding was detected with the West-
ernBreeze Chromogenic Western blot immunodetection kit (Invit-
rogen, Carlsbad, CA, USA). The blots were incubated on a shaker
with Rb, phosphorylated Rb (pRb) and b-actin polyclonal antibody
(Santa Cruz Biotechnology, Santa Cruz, CA, USA) 1:200 in the block-
ing buffer.
3.2. Morphological studies and cell cycle distribution
2.16. Statistical analysis
HeLa cells were incubated with compounds 3a and 3e at 3, 10
Statistical analysis was carried out by analysis of variance (AN-
OVA), followed by the Dunnett post-tests. RT-PCR data were ana-
lyzed by ANOVA, followed by the Neuman–Keuls post-tests. All
statistical analyses of the data were performed with GraphPad
Prism 4.0 (GraphPad Software; San Diego, CA, USA).
and 30 lM and with 10h and 11a at 10 and 30 lM for 24 h. The
presence of apoptosis or necrosis was determined according to
the cell morphology and membrane integrity. Separate photos
were recorded, in which Hoechst 33258 and propidium iodide fluo-
rescence served as morphological markers (Fig. 2). Concentration-
dependent increases in nuclear condensation and fragmentation
and in membrane permeability were generally observed. The most
markedly perturbed membrane integrity was seen in the case of
3e, while treatment with 10h resulted in pronounced nuclear con-
3. Results
3.1. Effects on viability of cancer cells (MTT assays)
densation with poor staining, even at 30
cell death.
lM, indicating apoptotic
The results of MTT assays with the 63 tested estrone analogs on
three human adherent cancer cell lines are presented in Supple-
mentary Table 2. Although a lower level of formazan production
can be attributed to a decreased metabolic activity of the treated
cells, the MTT assay is generally considered to be a suitable method
for the determination of cell viability. Estrone (1a) exhibited pro-
nounced action on HeLa cells, limited action on MCF7 cells and
no relevant action on the A431 cell line. None of the estrone ethers
Treatments with these selected agents resulted in significant
changes in the cell cycle distribution of HeLa cells (Fig. 3). A 24 h
incubation with unsubstituted oximes (3a and 3e) caused a pro-
nounced decrease in the synthetic (S) phase and an increase in
the G1 phase. At the highest concentration applied (30 lM), an in-
crease in the subdiploid (subG1) population was detected.
Although 11a caused a decrease in the G1 phase, the actions of
agents with substituted oximes were less obvious. After a longer
incubation (48 h), 3a and 3e resulted in a concentration-dependent
increase in the subG1 cells, but the S population was reduced. This
induction in subdiploid ratio was characteristic for 10h too,
whereas the action of 11a was limited to a slight S phase decrease
(1b–d) proved more potent and the 13a epimers (2a–d) were also
less effective. Introduction of an oxime function at position 16 of
the estrane skeleton resulted in a general increase in cytostatic ac-
tion. 3a and 3e were highly effective, especially against HeLa cells.
Estrone analogs with a substituted oxime function (5a–d) were
completely ineffective and their 3-sulfamoyloxy relatives (6a–c)
likewise exhibited only limited actions. Estrone methyl ethers with
a substituted oxime group at position 16 (7a–i and 8a–e) were
similarly minimally active, without any marked effect depending
on the configuration of the methyl function at position 13. Sub-
stantially more marked effects were detected when the aromatic
at 10 lM.
3.3. BrdU incorporation
The amount of synthetic thymidine analog BrdU incorporated
was used to detect the overall activity of DNA synthesis. HeLa cells

74
Á. Berényi et al. / Steroids 78 (2013) 69–78
Fig. 2. Fluorescence microscopy images of Hoechst 33258–propidium iodide double staining. Two separate pictures from the same field were taken for the two markers. HeLa
cells were treated with the vehicle (control), or with 3a, 3e, 10h or 11a at the given concentrations. The blue fluorescence (left panels) indicates Hoechst 33258, and the red
coloration (right panels) is a result of cellular propidium iodide accumulation. The bar in the Hoechst 33258 control picture denotes 100 lM.
Fig. 3. Effects of compounds 3a, 3e, 10h and 11a on the HeLa cell cycle distribution after incubation for 24 (panel A) or 48 h (panel B). ꢂ, ꢂꢂ and ꢂꢂꢂ indicate p < 0.05, p < 0.01
and p < 0.001, respectively, as compared with the control cells.
were treated with two or three concentrations of the investigated
compounds (3, 10 and 30 M) for 24 h. The incorporation of BrdU
3.4. Caspase-3 activity
l
into the DNA was substantially and statistically significantly inhib-
ited by all of the selected agents (Fig. 4). Compound 3e was the
most potent in inhibiting BrdU incorporation and, similarly to 3a,
exhibited a clear concentration dependence; their actions were
comparable to that of cisplatin.
The results detailed above led to two of the investigated com-
pounds (3a and 3e) being selected for additional experiments,
including determinations of the activity of caspase-3. The activity
of this apoptosis-executing key enzyme was increased substan-
tially and statistically significantly by 3a and 3e (Fig. 5). While

Á. Berényi et al. / Steroids 78 (2013) 69–78
75
two tested steroids. This ratio in the control cells was 1.53, which
was decreased to 0.59 and 0.46 by 3a, and to 1.06 and 0.80 by 3e, in
concentrations of 3 and 10 lM, respectively. The expressions of
this regulating factor at mRNA and protein levels were in good
agreement.
4. Discussion
A substantial amount of evidence has become available con-
cerning the antiproliferative properties of natural steroidal com-
pounds and synthetic analogs. Digitalis glycosides have long been
used in the treatment of congestive heart failure, and convincing
observational data bear witness to their anticancer action
[27,28]. The early reports have been reinforced and the mechanism
of this action has been postulated, leading to the generally ac-
cepted belief that the indications of cardiac glycosides may be ex-
tended to some cancers in the near future [29]. Diosgenin and
many related steroidal alkaloids characteristic of the Solanum
and Dioscorea genera exhibit potent anticancer effects, and may
suggest starting structures for novel synthetic antiproliferative
drugs [12,30].
Fig. 4. Incorporation of 5-bromo-20-deoxyuridine into HeLa cells after incubation
for 24 h. ꢂ, ꢂꢂ and ꢂꢂꢂ indicate p < 0.05, p < 0.01 and p < 0.001, respectively, as
compared with the control cells.
Cytotoxic steroids containing an oxime function at position 6
have been isolated from marine sponges and promising synthetic
analogs were recently designed which exhibit activities similar to
those of the present agents [31]. (Z)- and (E)-cholest-4-en-7-one
oximes and their lactam derivatives were reported to exert anti-
proliferative action against HeLa and chronic myelogenous leuke-
mia (K-562) cells, and the apoptosis-inducing capacity was
additionally evidenced by means of morphological and biochemi-
cal approaches [18]. Huang et al. have reported the synthesis and
screening of a set of ring A-modified cholestanes bearing an oxime
function at position 6. Some of these compounds exhibited consid-
erable activities against HeLa, human liver (SMMC 7404) and hu-
man gastric carcinoma (MGC 7901) cells [32]. These oximes are
stated to exhibit anticancer properties similar to or lower than
those of the currently investigated 16-oximes.
Fig. 5. Induction of caspase-3 activity after incubation with compounds 3a and 3e
for 48 h. The activity of untreated cells was taken as one unit. ꢂꢂꢂ indicates p < 0.001
as compared with the control cells.
3e resulted in a clear concentration–effect relationship, 3a caused
slightly lower activity at 30 lM than at 10 lM.
Estrogens may be responsible for carrying a pharmacophore
moiety to estrogen receptor-expressing cells, and therefore deter-
mining the molecular targeting of the agent [33,34]. We earlier re-
ported on some estrane-based antiproliferative compounds with
presumably hormone-independent actions [35,36]. The most
intensively investigated estrane is an endogenous estradiol metab-
olite, 2-methoxyestradiol, which does not exhibit hormonal activ-
ity, but which seems to be highly effective against a broad range of
cancer cell lines in vitro, and some limited but promising in vivo
data are also available. [13].
The aim of the present study was the design, synthesis and
pharmacological investigation of novel estrone-16-oximes. We
have found no previous report describing anticancer effects of
compounds from this class. The screening of the 63 newly synthe-
tized compounds against three human cancer cell lines pointed to
some structure–activity relationships. As a general rule, though
with a few exceptions, the b orientation of the 13-methyl group
3.5. RT-PCR studies
The expressions of four cell cycle-regulating factors (CDK4,
CDK6, p16 and Rb) that play key roles in the early G1–S transition
were additionally determined by means of a semiquantitative RT-
PCR technique (Fig. 6A). On the basis of the results of the cell cycle
analyses and the BrdU incorporation assays, two compounds (3a
and 3e) were included at two concentrations (3 and 10 lM), with
exposure for 24 h. The expression of tumor suppressor gene p16
was substantially and statistically significantly increased at the
mRNA level under all tested conditions. Treatment with these se-
lected agents resulted in a concentration-dependent repression of
CDK4, but not of CDK6. Retinoblastoma protein was significantly
repressed by 3a and 3e at the above concentrations. 3e seemed
more potent than 3a in this respect. Further assayed factors in-
volved in the regulation of the cell cycle (CDK2, p21, p53 and
p27) did not exhibit statistically significant differences as com-
pared with the control values (Fig. 6C).
is preferred. 3-Benzyl substitution typically favors the
a position
of the 13-methyl function. An unsubstituted oxime is generally
preferred over an alkyl-substituted one, but an aromatic group
(e.g. benzyl) may be considered. The hydroxy group on ring A
may be unsubstituted, sulfamoyloxylated or substituted with an
aromatic group (benzyl or p-methoxybenzyl). From the data relat-
ing to the anticancer efficacy, four compounds were selected for
further investigations. Two of them (3a and 3e) may be regarded
as HeLa-selective, with limited action on A2780 cells; the mole-
cules containing substituted oxime groups (10h and 11a) exerted
substantial action on MCF7 and A431 cells too. Tumor selectivity
is one of the most critical challenges in the development of a novel
3.6. Western blotting
Western blot analysis was performed to determine the expres-
sion of Rb and postsynthetically phosphorylated Rb at a protein le-
vel. Treatment with 3a and 3e at 3 and 10 lM reduced the
expression of both forms of Rb relative to the untreated cells
(Fig. 6B). The relative expression of pRb, expressed as the density
ratio Rb/pRb, was concentration-dependently decreased by the

76
Á. Berényi et al. / Steroids 78 (2013) 69–78
Fig. 6. Expression of Rb, CDK4, CDK6 and p16 at the mRNA level after incubation with compounds 3a, and 3e for 24 h. ꢂ, ꢂꢂ and ꢂꢂꢂ indicate p < 0.05, p < 0.01 and p < 0.001,
respectively, as compared with the control condition (panel A). Expressions of Rb and phosphorylated Rb at the protein level after incubation with compounds 3a and 3e for
24 h (panel B). Expression of CDK2, p21, p53 and p27 at the mRNA level after incubation with compounds 3a and 3e for 24 h (panel C).
anticancer agent. Although the MTT assay on intact human fibro-
blasts cannot be viewed as a thorough toxicological evaluation, it
is undoubtedly advantageous that three of the four selected com-
pounds did not exert substantial action on MRC-5 cells.
A set of additional in vitro experiments was devoted to an
experimental approach to the possible mechanism of action of
these agents. Most of the currently available drugs used in antican-
cer treatment have the capacity to induce programmed cell death

Á. Berényi et al. / Steroids 78 (2013) 69–78
77
either by fortifying apoptotic signaling or by inhibiting antiapopto-
tic signaling [37]. Treatment-dependent morphological changes
were recorded and qualitatively evaluated by means of Hoechst
33258–propidium iodide fluorescent staining after incubation for
24 h. Typical apoptotic markers, such as cellular shrinkage, nuclear
condensation and increased membrane permeability, were de-
tected, especially at the highest concentrations of 3a and 3e. Corre-
spondingly, flow cytometric cell cycle analysis was performed for a
quantitative determination of the DNA content of HeLa cells as a
function of the treatment. After a short exposure, 3a and 3e re-
sulted in a marked suppression of the synthetic phase, and the sub-
seems to be more beneficial than that of the reference agent cis-
platin. Accordingly, the estrone oxime skeleton is suggested as an
appropriate scaffold for the design and development of novel
antiproliferatve agents.
Acknowledgments
This publication is supported by the European Union and co-
funded by the European Social Fund. Project number: TÁMOP-
4.2.2/B-10/1-2010-0012. The authors thank the Hungarian Scien-
tific Research Fund (OTKA K 101659) for financial support.
diploid population increased only at high concentration (30 lM).
The same compounds elicited more pronounced cell cycle pertur-
bation, including the accumulation of subG1 cells, after a longer
incubation. It is therefore concluded that a period of 24 h is suffi-
cient for the development of the morphological hallmarks of apop-
tosis, but not for the complete activation of the self-decomposing
enzymatic procedure, which may explain why the appearance of
cells with subdiploid DNA requires 48 h. Besides apoptosis induc-
tion, a substantial contribution of necrosis cannot be excluded,
especially at higher concentration.
Compounds 3a and 3e significantly increased the caspase-3
activity, confirming the induction of programmed cell death.
Although caspase-independent cell death can manifest apoptotic
morphology, and crucial caspases, including caspase-3, may be in-
volved in non-lethal intracellular signaling, the assessment of exe-
cutioner caspase activity remains an important part of apoptosis
detection [38].
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.steroids.2012.
10.009.
References
[1] Ahlem CN, Page TM, Auci DL, Kennedy MR, Mangano K, Nicoletti F, et al. Novel
components of the human metabolome: the identification, characterization
and anti-inflammatory activity of two 5-androstene tetrols. Steroids
2011;76:145–55.
[2] Aperia A. New roles for an old enzyme: Na, K-ATPase emerges as an interesting
drug target. J Intern Med 2007;261:44–52.
[3] Minorics R, Szekeres T, Krupitza G, Saiko P, Giessrigl B, Wölfling J, et al.
Antiproliferative effects of some novel synthetic solanidine analogs on HL-60
human leukemia cells in vitro. Steroids 2011;76:156–62.
[4] Prokai-Tatrai K, Perjesi P, Rivera-Portalatin NM, Simpkins JW, Prokai L.
Mechanistic investigations on the antioxidant action of a neuroprotective
estrogen derivative. Steroids 2008;73:280–8.
Since 3a and 3e exerted a substantial inhibition of DNA synthe-
sis, comparable to that of the reference agent cisplatin, a further set
of experiments was devoted to the determination of cycle-regulat-
ing factors at the mRNA level by means of a RT-PCR technique.
The transition from the G1 to the S phase is tightly regulated by
the expression of Rb, CDK4 and CDK6, and p16 factors, the latter
regarded as crucially important [39]. Entry into the S phase, and
hence cell proliferation, is inhibited as long as Rb remains unphos-
phorylated by a complex containing cyclin D, CDK4 and CDK6.
Phosphorylated Rb dissociates from a heterodimeric complex of
E2F, allowing the transcription of S-phase specific genes [40]. The
importance of this cyclin D–CDK4–CDK6–p16–Rb–E2F pathway is
indicated by the fact that it has been found to be altered in virtu-
ally all human tumors [41]. An innovative agent intervening in this
pathway may therefore be considered especially advantageous.
In view of our results, it seems conceivable that treatments with
the two best compounds lead to the up-regulation of p16 (also re-
ferred to as CDK4 inhibitor). The hypofunction of this protein has
been associated with several malignancies and its expression cor-
relates with the chemotherapy response in patients with solid tu-
mors [42]. Since Rb is typically regulated postsynthetically by
phosphorylation, its overall activity cannot be fully characterized
by determining its expression at the mRNA level [40]. Our RT-
PCR data were therefore supplemented with Western blot analy-
ses, which indicated that the two most effective compounds have
the capacity to decrease the expressions of Rb and pRb at a protein
levels, and also the proportion of pRb. Since the expression of sig-
nificant members of a pathway parallel to that detailed above,
including CDK2, p21, p27 and p53, did not exhibit any appreciable
treatment-dependent differences, the signal mechanism via pro-
teins p16 and CDK4 governing the phosphorylation of Rb may be
suggested as a predominant mechanism of action of the tested es-
trone analogs.
[5] Rocha M, Banuls C, Bellod L, Jover A, Victor VM, Hernandez-Mijares A. A review
on the role of phytosterols: new insights into cardiovascular risk. Curr Pharm
Des 2011;17:4061–75.
[6] Chiang CT, Way TD, Tsai SJ, Lin JK. Diosgenin, a naturally occurring steroid,
suppresses fatty acid synthase expression in HER2-overexpressing breast
cancer cells through modulating Akt, mTOR and JNK phosphorylation. FEBS
Lett 2007;581:5735–42.
[7] Corbiere C, Battu S, Liagre B, Cardot PJ, Beneytout JL. SdFFF monitoring of
cellular apoptosis induction by diosgenin and different inducers in the human
1547 osteosarcoma cell line. J Chromatogr B Analyt Technol Biomed Life Sci
2004;808:255–62.
[8] Corbiere C, Liagre B, Terro F, Beneytout JL. Induction of antiproliferative effect
by diosgenin through activation of p53, release of apoptosis-inducing factor
(AIF) and modulation of caspase-3 activity in different human cancer cells. Cell
Res 2004;14:188–96.
[9] Leger DY, Liagre B, Corbiere C, Cook-Moreau J, Beneytout JL. Diosgenin induces
cell cycle arrest and apoptosis in HEL cells with increase in intracellular
calcium level, activation of cPLA2 and COX-2 overexpression. Int J Oncol
2004;25:555–62.
[10] Liu MJ, Yue PY, Wang Z, Wong RN. Methyl protodioscin induces G2/M arrest
and apoptosis in K562 cells with the hyperpolarization of mitochondria.
Cancer Lett 2005;224:229–41.
[11] Raju J, Patlolla JM, Swamy MV, Rao CV. Diosgenin,
a steroid saponin of
Trigonella foenum graecum (Fenugreek), inhibits azoxymethane-induced
aberrant crypt foci formation in F344 rats and induces apoptosis in HT-29
human colon cancer cells. Cancer Epidemiol Biomarkers Prev 2004;13:1392–8.
[12] Lee KR, Kozukue N, Han JS, Park JH, Chang EY, Baek EJ, et al. Glycoalkaloids and
metabolites inhibit the growth of human colon (HT29) and liver (HepG2)
cancer cells. J Agric Food Chem 2004;52:2832–9.
[13] Mueck AO, Seeger H. 2-Methoxyestradiol – Biology and mechanism of action.
Steroids 2010;75:625–31.
[14] Dahut WL, Lakhani NJ, Gulley JL, Arlen PM, Kohn EC, Kotz H, et al. Phase I
clinical trial of oral 2-methoxyestradiol, an antiangiogenic and apoptotic
agent, in patients with solid tumors. Cancer Biol Ther 2006;5:22–7.
[15] Li L, Bu S, Backstrom T, Landstrom M, Ulmsten U, Fu X. Induction of apoptosis
and G2/M arrest by 2-methoxyestradiol in human cervical cancer HeLaS3 cells.
Anticancer Res 2004;24:873–80.
[16] Sweeney C, Liu G, Yiannoutsos C, Kolesar J, Horvath D, Staab MJ, et al. A phase II
multicenter, randomized, double-blind, safety trial assessing the
pharmacokinetics,
pharmacodynamics,
and
efficacy
of
oral
2-
methoxyestradiol capsules in hormone-refractory prostate cancer. Clin
Cancer Res 2005;11:6625–33.
In conclusion, our current results provide the first evidence that
substituted estrone oximes may selectively suppress cancer cell
proliferation by promoting apoptotic cell death and modulate the
cell cycle progression. Although relatively high concentrations
are needed to exert substantial activity, their cancer selectivity
[17] Chang FS, Chen W, Wang C, Tzeng CC, Chen YL. Synthesis and antiproliferative
evaluations of certain 2-phenylvinylquinoline (2-styrylquinoline) and 2-
furanylvinylquinoline derivatives. Bioorg Med Chem 2010;18:124–33.
[18] Krstic NM, Bjelakovic MS, Zizak Z, Pavlovic MD, Juranic ZD, Pavlovic VD.
Synthesis of some steroidal oximes, lactams, thiolactams and their antitumor
activities. Steroids 2007;72:406–14.

78
Á. Berényi et al. / Steroids 78 (2013) 69–78
[19] Mosmann T. Rapid colorimetric assay for cellular growth and survival:
[31] Poza J, Rega M, Paz V, Alonso B, Rodríguez J, Salvador N, et al. Synthesis and
evaluation of new 6-hydroximinosteroid analogs as cytotoxic agents. Bioorg
Med Chem 2007;15:4722–40.
application to proliferation and cytotoxicity assays.
1983;65:55–63.
J Immunol Methods
[20] Vermes I, Haanen C, Reutelingsperger C. Flow cytometry of apoptotic cell
death. J Immunol Methods 2000;243:167–90.
[32] Huang Y, Cui J, Chen S, Gan C, Zhou A. Synthesis and antiproliferative activity
of some steroidal lactams. Steroids 2011;76:1346–50.
[21] Ribble D, Goldstein NB, Norris DA, Shellman YG. A simple technique for
quantifying apoptosis in 96-well plates. BMC Biotechnol 2005;5:12.
[22] Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid
guanidinium thiocyanate–phenol–chloroform extraction. Anal Biochem
1987;162:156–9.
[33] Gupta A, Saha P, Descoteaux C, Leblanc V, Asselin E, Berube G. Design,
synthesis and biological evaluation of estradiol-chlorambucil hybrids as
anticancer agents. Bioorg Med Chem Lett 2010;20:1614–8.
[34] Roy S, Reddy BS, Sudhakar G, Kumar JM, Banerjee R. 17b-estradiol-linked
nitro-L-arginine as simultaneous inducer of apoptosis in melanoma and
[23] Gao Q, Tan J, Ma P, Ge J, Liu Y, Sun X, et al. PKC alpha affects cell cycle
progression and proliferation in human RPE cells through the downregulation
of p27kip1. Mol Vis 2009;15:2683–95.
[24] Matsuhashi N, Saio M, Matsuo A, Sugiyama Y, Saji S. Expression of p53 protein
as a predictor of the response to 5-fluorouracil and cisplatin chemotherapy in
human gastrointestinal cancer cell lines evaluated with apoptosis by use of
thin layer collagen gel. Int J Oncol 2004;24:807–13.
tumor-angiogenic vascular endothelial cells. Mol Pharm 2011;8:350–9.
[35] Frank É, Molnár J, Zupkó I, Kádár Z, Wölfling J. Synthesis of novel steroidal 17a-
triazolyl derivatives via Cu(I)-catalyzed azide–alkyne cycloaddition, and an
evaluation of their cytotoxic activity in vitro. Steroids 2011;76:1141–8.
[36] Saloranta T, Zupkó I, Rahkila J, Schneider G, Wölfling J, Leino R. Increasing the
amphiphilicity of an estradiol based steroid structure by Barbier-allylation –
ring-closing metathesis – dihydroxylation sequence. Steroids 2012;77:110–7.
[37] Tolomeo M, Simoni D. Drug resistance and apoptosis in cancer treatment:
development of new apoptosis-inducing agents active in drug resistant
malignancies. Curr Med Chem Anticancer Agents 2002;2:387–401.
[38] Kepp O, Galluzzi L, Lipinski M, Yuan J, Kroemer G. Cell death assays for drug
discovery. Nat Rev Drug Discovery 2011;10:221–37.
[39] Katsumi Y, Iehara T, Miyachi M, Yagyu S, Tsubai-Shimizu S, Kikuchi K, et al.
Sensitivity of malignant rhabdoid tumor cell lines to PD 0332991 is inversely
correlated with p16 expression. Biochem Biophys Res Commun 2011;413:
62–8.
[40] Sunley K, Butler M. Strategies for the enhancement of recombinant protein
production from mammalian cells by growth arrest. Biotechnol Adv
2010;28:385–94.
[25] Huang Q, Cheung AP, Zhang Y, Huang HF, Auersperg N, Leung PC. Effects of
growth differentiation factor 9 on cell cycle regulators and ERK42/44 in human
granulosa cell proliferation. Am
E1344–53.
J Physiol Endocrinol Metab 2009;296:
[26] Lee CH, Lim H, Moon S, Shin C, Kim S, Kim BJ, et al. Novel anticancer agent,
benzyldihydroxyoctenone, isolated from Streptomyces sp. causes G1 cell cycle
arrest and induces apoptosis of HeLa cells. Cancer Sci 2007;98:795–802.
[27] Shiratori O. Growth inhibitory effect of cardiac glycosides and aglycones on
neoplastic cells: in vitro and in vivo studies. Gann 1967;58:521–8.
[28] Stenkvist B. Is digitalis a therapy for breast carcinoma? Oncol Rep 1999;6:
493–6.
[29] Mijatovic T, Van Quaquebeke E, Delest B, Debeir O, Darro F, Kiss R. Cardiotonic
steroids on the road to anti-cancer therapy. Biochim Biophys Acta 2007;1776:
32–57.
[41] Nevins JR. The Rb/E2F pathway and cancer. Hum Mol Genet 2001;10:699–703.
[42] Borys D, Canter RJ, Hoch B, Martinez SR, Tamurian RM, Murphy B, et al. P16
expression predicts necrotic response among patients with osteosarcoma
receiving neoadjuvant chemotherapy. Hum Pathol. http://dx.doi.org/10.1016/
j.humpath.2012.02.003.
[30] Raju J, Bird RP. Diosgenin, a naturally occurring furostanol saponin suppresses
3-hydroxy-3-methylglutaryl CoA reductase expression and induces apoptosis
in HCT-116 human colon carcinoma cells. Cancer Lett 2007;255:194–204.