Conjugation of substituted ferrocenyl to thiadiazine as apoptosis-inducing agents targeting the Bax/Bcl-2 pathway

Article abstract of DOI:10.1016/j.ejmech.2011.08.007

Ferrocene compounds are a class of biologically active compounds that has antitumour and antifungal properties. This study investigated the induction of apoptosis in human fibrosarcoma cells (HT1080) after treatment with a series of 6-ferrocenyl-3-subsituted7H-1,2,4-triazolo[3,4-b]- 1,3,4-thiadiazine (FTFs). We found that FTFs could suppress the viability of HT1080 cells. Cell cycle analysis showed that proliferative inhibition of HT1080 cells occurred through apoptosis, as the cells were blocked in G1 phase. Moreover, mitochondrial membrane staining assay demonstrated that FTFs exposure significantly decreased mitochondrial membrane potential. Finally, under the stress of FTFs, Bax/Bcl-2 ratio in HT1080 cells was significantly increased. These results suggested that FTFs-induced apoptosis in HT1080 cells may work dependent on a Bax/Bcl-2 pathway.

Full text of DOI:10.1016/j.ejmech.2011.08.007

European Journal of Medicinal Chemistry 46 (2011) 5000e5009
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Conjugation of substituted ferrocenyl to thiadiazine as apoptosis-inducing agents
targeting the Bax/Bcl-2 pathway
,
,
a,
Ruidong Miao a, Juan Wei a c, Minghua Lv a, Yan Cai b, Yuping Du a, Xinping Hui b , Qin Wang
*
**
a School of Life Sciences, Lanzhou University, Lanzhou 730000, PR China
b State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou 730000, PR China
c College of Food Science and Engineering, Gansu Agriculture University, Lanzhou 730070, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Ferrocene compounds are a class of biologically active compounds that has antitumour and antifungal
properties. This study investigated the induction of apoptosis in human fibrosarcoma cells (HT1080) after
treatment with a series of 6-ferrocenyl-3-subsituted7H-1,2,4-triazolo[3,4-b]- 1,3,4-thiadiazine (FTFs). We
found that FTFs could suppress the viability of HT1080 cells. Cell cycle analysis showed that proliferative
inhibition of HT1080 cells occurred through apoptosis, as the cells were blocked in G1 phase. Moreover,
mitochondrial membrane staining assay demonstrated that FTFs exposure significantly decreased
mitochondrial membrane potential. Finally, under the stress of FTFs, Bax/Bcl-2 ratio in HT1080 cells was
significantly increased. These results suggested that FTFs-induced apoptosis in HT1080 cells may work
dependent on a Bax/Bcl-2 pathway.
Received 11 March 2010
Received in revised form
31 July 2011
Accepted 4 August 2011
Available online 11 August 2011
Keywords:
1,2,4-Triazolo[3,4-b]-1,3,4-thiadiazine
Anti-tumor activity
Ferrocene
Ó 2011 Elsevier Masson SAS. All rights reserved.
Bax/Bcl-2 pathway
1. Introduction
molecular mechanisms underlying their anti-cancer effects are
inconclusive.
Since Kopfmaier and his colleagues [1,2] first discovered the
antiproliferative efficiency of ferrocene compounds by the example
of ferricenium salts, A number of detailed reviews committed to the
bio-organometallic chemistry of ferrocene compounds have been
published [3]. Incorporation of ferrocene fragment into the mole-
cule of an organic compound often leads to unexpected biological
activity, which is due to their different membrane permeation
properties and anomalous metabolism [4]. Moreover, ferrocene as
a redox mediator might involve in metabolism in organism [5e7].
Hence, ferrocene and its derivatives could be considered as
a potential agent and applied to medicine.
Some ferrocenium salts have exhibited anti-cancer activity [8],
and an experimental drug has been reported which is a ferrocenyl
version of tamoxifen [9]. Ferrocene derivatives are ideal for use in
drug design owing to its low toxicity [10,11]. On the other hand,
1,2,4-triazolo[3,4-b]-1,3,4-thiadiazine derivatives exhibit antimi-
crobial [12], antifungal, anti-cancer [13], anti-HIV-1 [14] and PDE4
inhibitory activities [15]. However, besides some sporadic reports
on the bioactivity role of ferrocenes and its derivatives, the detailed
The induction of apoptosis in malignant cells has recently become
an area of intensive study in cancer chemotherapy research [16]. In
recent years, considerable efforts have been devoted to searching for
anti-cancer drugs with high bioactivities and low toxicities [17].
In new drugs design, the development of hybrid molecules
through the combination of different pharmacophores in one frame
may lead to compounds with interesting biological profiles. In
order to search for novel heterocyclic derivatives containing
ferrocene moiety with potent biological activities, FTF1-3 (6-
ferrocenyl-3-subsituted 7H-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazine)
were successfully synthesized. In this study, we evaluated the anti-
tumor activities of these FTFs on human fibrosarcoma HT1080 cells.
We found that FTFs could inhibit tumor cell growth, induce cell
apoptosis, lead to collapse of the mitochondrial membrane poten-
tial and increase the ratio of Bax/Bcl-2. These results suggested that
FTFs might be potential anti-tumor reagents and FTF-induced
apoptosis might relate to intrinsic pathway activation.
2. Materials and methods
2.1. General
* Corresponding author. Tel.: þ86 931 891 5350.
Melting points were taken on an XT-4 melting point apparatus
and were uncorrected. 1H NMR and 13C NMR spectra were recorded
** Corresponding author. Tel.: þ86 139 1946 0373.
E-mail addresses: huixp@lzu.edu.cn (X. Hui), qwang@lzu.edu.cn (Q. Wang).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.08.007

R. Miao et al. / European Journal of Medicinal Chemistry 46 (2011) 5000e5009
5001
on Varian Mercury-400 or 300 MHz spectrometer with TMS as an
internal standard. IR spectra were obtained on a Nicolet NEXUS 670
FT-IR instrument. Elemental analyses were determined by
Elemental vario EL instrument.
2915, 1577, 1472, 1450, 1414, 1358, 1295, 1005, 966. Anal. Cacld. for
C21H18FeN4S: C, 60.88; H, 4.38; N, 13.52. Found: C, 60.79; H, 4.32; N,
13.60.
6-Ferrocenyl-3-(4-nitrophenyl)-7H-1,2,4-triazolo
thiadiazine (FTF3). Yield 71%, m.p. 187 ꢀC (dec.). 1H NMR
(300 MHz, CDCl3)
: 7.94 (d, J ¼ 8.4 Hz, 2H, ArH), 6.74 (d, J ¼ 8.4 Hz,
2H, ArH), 4.79 (s, 2H, CpH), 4.59 (s, 2H, CpH), 4.27 (s, 5H, CpH), 3.73
[3,4-b]-1,3,4-
4-Amino-5-aryl-1,2,4-triazol-3-thiones
(1aec)
and
2-
bromoacetylferrocene (2) were prepared according to literature
methods [18,19], respectively. All materials were obtained from
commercial suppliers and used without further purification.
FTFs were initially dissolved in dimethysulphoxide (DMSO) and
further diluted in culture medium. The final concentration of DMSO
in culture was always less than 0.4% (v/v) and did not cause any
toxicity by itself.
d
(s, 2H, SCH2). 13C NMR (100 MHz, DMSO-d6)
d: 156.3, 150.5, 148.6,
139.4, 127.7, 112.7, 112.4, 76.0, 70.7, 68.7, 66.6, 22.5. IR ymax (cmꢁ1):
3313, 3200, 2909, 1608, 1578, 1479, 1454, 1361, 1298, 1179, 1103,
1076, 1030, 1003. Anal. Cacld. for C20H15FeN5O2S: C, 53.95; H, 3.40;
N, 15.73. Found: C, 53.98; H, 3.24; N, 15.61.
The following antibodies were obtained from commercial
sources: polyclonal anti-human Bcl-2, polyclonal anti-human Bax
2.3. Cell lines and culture
and monoclonal anti-human
b-actin (Santa Cruz Biotechnology,
Santa Cruz, CA). DMEM medium was purchased from Gibco Co.,
USA. Fetal bovine serum (FBS) was purchased from Minhan Bio-
logical Engineering Materials Co., Ltd. (Lanzhou, China). All other
reagents were purchased from Sigma Chemical (St. Louis, MO),
except as noted.
HT1080 (human fibrosarcoma cells) and hTERT-BJ (inmortalized
human foreskin fibroblats) cells were obtained from the Second
Military Medical University (Shanghai, China) and cultured in DMEM
(Dulbecco’s Modified Eagle Medium) supplemented with 10% FBS
(Fetal Bovine Serum) in a humidified incubator at 37 ꢀC and 5% CO2.
2.4. MTT (Thiazolyl Blue Tetrazolium Bromide) assay
2.2. General procedure for preparation of 6-ferrocenyl-3-subsituted
7H-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazines (FTF1-3)
Effect of FTFs on the viability of HT1080 and hTERT-BJ cells was
determined by MTT assay. Briefly, ～1 ꢂ 104 cells of HT1080 and
hTERT-BJ per well were plated in 96-well plates and treated with
A
mixture of 4-amino-5-subsituted 1,2,4-triazol-3-thione
(0.005 mol), 2-bromoacetyl ferrocene (0.005 mol) in absolute
ethanol (40 ml) was refluxed for 8 h. The solvent was removed
under reduced pressure to give a red residue. The crude product
was purified by flash column chromatography to afford FTF1-3.
6-Ferrocenyl-3-phenyl-7H-1,2,4-triazolo [3,4-b]-1,3,4-thiadiazine
FTFs (FTF1 and FTF2: 20, 40, 80, 160 and 320
80 and 160 M) for 24, 48 and 72 h. At the end of stipulated time
following FTFs treatment, MTT (50 l of 5 mg/ml stock solution in
mM; FTF3: 5, 10, 20, 40,
m
m
PBS) was added into each well and incubated at 37 ꢀC for 4 h.
During this incubation period, water-insoluble formazan crystals
were formed, which were dissolved by the addition of 100 ul/well
DMSO. The optical density of each well was measured using a Bio-
Rad Model 3550 plate reader at 595 nm with reference at 655 nm.
Wells containing culture medium and MTT but no cells acted as
blanks. Cell survival was expressed as an absorbance (A) percentage
defined by (Adrug-blank/Acontrol-blank ꢂ 100).
(FTF1). Yield 72%, m.p. 210 ꢀC (dec.). 1H NMR (300 MHz, CDCl3)
d:
8.13e8.11 (m, 2H, ArH), 7.49e7.47 (m, 3H, ArH), 4.81 (s, 2H, CpH), 4.61
(s, 2H, CpH), 4.28 (s, 5H, CpH), 3.77 (s, 2H, SCH2). 13C NMR (100 MHz,
CDCl3) d: 157.3, 141.6, 130.0, 128.4, 128.0, 126.2, 72.3, 70.2, 67.9, 67.2,
24.1. IR ymax (cmꢁ1): 3065, 2913, 2854, 1575, 1471, 1455, 1435, 1381,
1362,1296,1232,1103, 1076, 1004, 966. Anal. Cacld. for C20H16FeN4S:
C, 60.01; H, 4.03; N, 14.00. Found: C, 60.22; H, 4.25; N, 13.89.
6-Ferrocenyl-3-p-tolyl-7H-1,2,4-triazolo [3,4-b]-1,3,4-thiadiazine
(FTF2). Yield 75%, m.p. 196 ꢀC (dec.). 1H NMR (300 MHz, CDCl3)
d:
2.5. Soft agar assay
7.95 (d, J ¼ 8.0 Hz, 2H, ArH), 7.22 (d, J ¼ 8.0 Hz, 2H, ArH), 4.73 (s, 2H,
CpH), 4.53 (s, 2H, CpH), 4.21 (s, 5H, CpH), 3.69 (s, 2H, SCH2), 2.35 (s,
After treatment with FTF2 (40 mM, 80 mM and 160 mM) and FTF3
3H, CH3). 13C NMR (100 MHz, CDCl3)
d: 157.1, 140.2, 129.1, 127.9,
(10
mM, 20
mM and 40
m
M) for 48 h, the HT1080 cells (1 ꢂ104) were
123.3, 77.1, 72.2, 70.2, 67.9, 24.1, 21.4. IR ymax (cmꢁ1): 3072, 2991,
cultured on a plate containing 0.5% base agar and 0.35% top agar in
N
NH
O
S
O
CS2, KOH
C2H5OH
NH2NH2 H2O
R
S
N
RCNHNH2
RCNHNHCS K
NH2
1a-c
S
N
COCH3
COCH2Br
N
NBS, (PhCO)2O2
1a-c
abs. ethanol
N
Fe
Fe
N
n-BuLi, TMSCl
Fe
R
2
FTF 1-3
R = C6H5 (FTF1), 4-CH3C6H4 (FTF2), 4-O2NC6H4 (FTF3)
Scheme 1. The cyclization of 4-amino-5-substituted 1,2,4-triazol-3-thione with 2-halogenocarbonyl compounds. Hydrazide reacted with CS2 in KOH-absolute ethanol solution to
give potassium aryl formyl-hydrazino dithioformate which was refluxed in excess hydrazine hydrate to obtain 4-amino-5-substituted 1,2,4-triazol-3-thione (1aec). Metalation of
acetyl ferrocene with LDA at ꢁ78ꢀC followed by sequential treatment with trimethylchlorosilane and an excess of NBS provided the 2-bromoacetylferrocene (2). As would be
anticipated, treatment of 1 with 2-bromoacetylferrocene (2) in refluxing absolute ethanol achieved the cyclic compounds FTF1-3 in moderate yields. The structures of the products
synthesized were characterized by 1H NMR, 13C NMR, IR and elementary analyses.

5002
R. Miao et al. / European Journal of Medicinal Chemistry 46 (2011) 5000e5009
Fig. 1. FTFs reduce the viability rate of BJ cells and HT1080 cells. Cells were treated by FTF1 (20e320
mM) for (A) 24 h, (B) 48 h and (C) 72 h; Cells were treated by FTF2 (20e320
mM)
for (D) 24 h, (E) 48 h and (F) 72 h; Cells were treated by FTF3 (10e160
experiments.
mM) for (G) 24 h, (H) 48 h and (I) 72 h. Results represent mean ꢃ S.E. of data from at least six individual

R. Miao et al. / European Journal of Medicinal Chemistry 46 (2011) 5000e5009
5003
DMEM with 10% FBS. After 21 days of incubation at 37 ꢀC in a 5% CO2
Table 1
IC50 values of FTF1, FTF2 and FTF3 on hTERT-BJ and HT1080 cells.
humidified atmosphere, colonies with﹥50 cells were counted.
IC50
(m
M) FTF1
FTF2
FTF3
hTERT-BJ HT1080 hTERT-BJ HT1080 hTERT-BJ HT1080
2.6. Morphological analysis
24 h
48 h
72 h
>320
154.4
120.4
>320
180.2
95.1
>320
203.5
209.5
108.2
178.0
130.2
>320
>320
79.8
202.8
23.0
15.4
HT1080 cells were plated in T25 flasks with a cell density of
mM, 80 mM and 160 mM)
and FTF3 (10 mM, 20 mM and 40 mM). After 48 h in culture, cells were
1 ꢂ 105 for 24 h and treated with FTF2 (40
observed and photographs were taken under a phase contrast
microscope (Nikon HFX, Japan).
2.10. Western blot analysis
Western blot analysis was done to determine the expression of
Bcl-2 and Bax in cells. HT1080 cells were treated with different
2.7. Cell cycle analysis
concentrations of FTFs (FTF1 and FTF2: 20, 40, 80, 160 and 320
mM;
Apoptotic cells can be recognized by their diminished stain-
ability with DNA specific fluorochromes [20], which could be
attributed to DNA degradation and its subsequent leakage from the
cell. The method for DNA labeling was as described previously with
minor modifications. Briefly, HT1080 cells at a density of 1 ꢂ 107
FTF3: 5, 10, 20, 40, 80 and 160 M) for 48 h. Cells were harvested,
m
washed with cold PBS (pH 7.4), and lysed with ice-cold lysis buffer
(50 mM TriseHCl, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 20 mM
NaF, 100 mM Na3VO4, 1% NP 40, 1 mM phenylmethylsulfonyl fluo-
ride (PMSF), pH 7.4) for 30 min, then centrifuged at 12,000 ꢂ g for
30 min at 4 ꢀC. The protein concentration of clear supernatant was
evaluated using Bradford method. Aliquots containing equal
amounts of proteins were subjected to SDS-PAGE electrophoresis.
Thereafter, proteins were electrophoretically transferred to
were treated with FTF2 (40
mM, 80
mM and 160 mM) and FTF3
(10 M, 20 M and 40
m
m
mM). After treatment for 48 h, cells were
prepared as a single cell suspension in 200 ml PBS, fixed with 2 ml
ice-cold 70% ethanol, and maintained at 4 ꢀC overnight. The cells
were harvested by centrifugation at 500 ꢂ g for 10 min, resus-
pended in 500 ml PBS supplemented with 0.1% Triton X-100 and
RNaseA (100
m
g/ml), incubated at 37 ꢀC for 30 min, and stained with
50
m
g/ml propidium iodide (PI) in the dark at 4 ꢀC for 30 min. The
red fluorescence of individual cells was measured with FACScan
(Becton Dickinson, Sunnyvale, USA), and the data were analyzed
using WinMDI2.8 software.
2.8. Acridine orange/ethidium bromide (AO/EB) double staining
assay
The cells were incubated in the presence of FTF2 (40
and 160 M) and FTF3 (10 M, 20 M and 40 M) for 48 h. The
attached cells were collected by centrifugation and then stained
with AO (100 g/ml) and EB (100 g/ml). Thereafter, 10 l cells were
mM, 80 mM
m
m
m
m
m
m
m
added onto the glass slides to test by fluorescence microscopy
(Olympus, Japan) using epi-illuminisation and a filter combination
suitable for observing fluorescein immediately with a magnifica-
tion of 100ꢂ. AO, which stains the nucleus through both intact and
damaged membranes, emits green fluorescence. EB, which stains
the nucleus only through damaged membrane, emits red fluores-
cence and predominated over AO. Thus, viable cells were observed
as having a uniform bright green nucleus and orange cytoplasm.
Early apoptotic cells still had green nuclei but chromatin conden-
sation was visible in the form of bright green patches. Late
apoptotic cells showed bright orange areas of condensed chromatin
in the nucleus, and necrotic cells showed a uniform bright orange
nucleus. Viable, apoptotic and necrotic cells were counted under
a fluorescence microscope.
2.9. Mitochondrial membrane staining
Cells were plated at 1 ꢂ 105 cells/flask and incubated for 48 h in
the presence of FTF2 (40
mM, 80
mM and 160
mM) and FTF3 (10
m
M,
20 mM and 40 mM). Cell cultures were exposed to Rh123 (10 mg/ml)
for 15 min in the dark at room temperature. Cells were then sus-
pended and centrifuged at 300 ꢂ g for 2 min and resuspended in
PBS. The cell suspensions were filtered through a nylon mesh
screen of 40 mm. Changes in Rh123 fluorescence intensity were
measured by FACScan flow cytometer (EPICS XL, Coulter, USA) with
excitation at 488 nm and emission at 525 nm.
Fig. 2. Colony-inhibition rates of FTF2 and FTF3 on HT1080 cells. After treatment
without or with different concentrations of FTF2 (40
mM, 80 mM and 160 mM) and FTF3
(10 M, 20 M and 40 M) for 48 h, cells were routinely plated and cultured for 21
m
m
m
days. One colony was defined to be an aggregate of >50 cells. (A) Cells were treated
without or with different concentration of FTF2; (B) Cells were treated without or with
different concentration of FTF3. Results represent mean ꢃ S.E. of data from at least
three individual experiments. *P < 0.05; **P < 0.01 versus control.

5004
R. Miao et al. / European Journal of Medicinal Chemistry 46 (2011) 5000e5009
Fig. 3. Morphological characteristics of HT1080 cells treated with FTF2 and FTF3. HT1080 cells were treated with increasing concentration of drug. (A) Control; (B)e(D) 40
80 M and 160 M of FTF2; (E)e(G) 10 M, 20 M and 40
M of FTF3. Cells were photographed after 48 h drug treatment (160ꢂ).
mM,
m
m
m
m
m
nitrocellulose membrane and non-specific sites were blocked with
5% skimmed milk in 1% Tween-20 in 20 mM TBS (pH 7.5) and
reacted with a primary polyclonal antibody Bax, Bcl-2 and mono-
was incubated by horseradish peroxidase-conjugated secondary
antibodies to anti-rabbit or anti-mouse immunoglobulins (Amer-
sham International) and specific bands were visualized by
enhanced chemiluminescence (ECL, Amersham International).
clonal antibody b-actin for 4 h at room temperature. The membrane

R. Miao et al. / European Journal of Medicinal Chemistry 46 (2011) 5000e5009
5005
2.11. Statistical analysis
To validate the induction of apoptosis by identifying morpho-
logical features, phase contrast microscopy was used and the
results showed that the dose-dependent loss in cell viability in
cultures of HT1080 cells treated with FTFs was accompanied by
morphological changes. After treatment with FTFs for 48 h, viable
cell numbers decreased as FTFs concentration increased (Fig. 3).
Moreover, the distinctive morphological features of cells including
detachment and cell shrinkage were observed. HT1080 cell death
induced by FTF3 was more obvious than FTF2.
Data were expressed as mean ꢃ S.E. All statistical analyses were
performed using Excel Software. In each case, P value < 0.05 was
considered statistically significant.
3. Results
3.1. Synthesis of title compounds
4-Amino-5-substituted 1,2,4-triazol-3-thiones (1aec), which
are required as starting materials, were prepared according to
literature method. The cyclization of 4-amino-5-substituted 1,2,4-
3.5. FTFs inhibit cells growth by causing G1 phase arrest followed
by apoptosis in HT1080 cells
triazol-3-thione with
the most useful method for the formation of the 1,2,4-triazolo[3,4-
b]-1,3,4-thiadiazine ring system. Treatment of with 2-
bromoacetylferrocene (2) in refluxing absolute ethanol, the cyclic
compounds FTF1-3 were successfully achieved in moderate yields
(Scheme 1).
a-halogenocarbonyl compounds has been
Many anti-cancer agents can inhibit proliferation and induce
apoptosis of cancer cells by blocking one or more stages of the cell
cycle. We thought that inhibition of cell viability by FTFs in HT1080
cells might be mediated through the initiation of apoptotic cascade.
Accordingly, we further examined the induction of apoptosis and
cell cycle arrest in HT1080 cells by FTFs. The cells were treated with
different concentrations of FTF2 or FTF3 for 48 h and analyzed for
cell cycle distribution by means of flow cytometry. As shown in
Fig. 4, when compared with that of the control, the percentage of
1
3.2. FTFs inhibit cell viability in HT1080 and hTERT-BJ cells
To determine whether FTFs can lead to loss of cell viability,
HT1080 and hTERT-BJ cells were treated with different concentra-
tions of FTFs for 24, 48 and 72 h (Fig. 1). The cytotoxitic effects of
FTFs were determined by MTT assay. FTFs exhibited a more
significant cytotoxic effect on human sarcoma cell HT1080 than
immortalized human foreskin fibroblasts hTERT-BJ. Our study
revealed that significant reduction in cell viability commenced at
80
(Fig. 1AeF). Whereas significant diminution of viable cell count
instigated at 20 M of FTF3 treatment on HT1080 cells (Fig. 1GeI).
However, during 72 h of incubation, the IC50 values of FTF1, FTF2
and FTF3 were 120.4 M, 209.5 M, 79.7 M in hTERT-BJ cells, and
95.1 M, 130.2 M and 15.4 M in HT1080 cells, respectively
mM of FTF1 and FTF2 treatment on HT1080 cells, respectively
m
m
m
m
m
m
m
(Table 1). Noticeable, inhibitory activities of FTF2 and FTF3 in
HT1080 cells were much higher than that in hTERT-BJ cells. The
results demonstrated that FTF2 and FTF3 held great potential for
therapeutic use. Based on cell viability assay in HT1080 cells after
treatment with different doses of FTFs, 40, 80, 160
mM doses of FTF2
and 10, 20, 40 M doses of FTF3 were selected for further studies.
m
3.3. FTFs inhibit colony formation of HT1080 cells
The soft agar assay for colony formation is an anchorage inde-
pendent growth assay in soft agar, which is considered the most
stringent assay for detecting malignant transformation of cells. As
shown in Fig. 2, when compared with control, treatment with
varying concentrations of FTFs for 48 h, the colony inhibition rates
of FTF2 (40, 80 and 160
mM) were 24.5%, 38.8% and 85.7% (Fig. 2A),
whereas the colony inhibition rates of FTF3 (10, 20, 40
mM) were
8.2%, 53.1% and 83.7% (Fig. 2B), respectively. The results demon-
strated that FTF2 and FTF3 both suppressed colony formation of
HT1080 cells in a dose-dependent manner. In particular, FTF3
exerted a stronger inhibitory activity than FTF2.
3.4. FTFs induce morphological changes of HT1080 cells
Cell apoptosis involves a series of biochemical events leading to
a characteristic cell morphology and death. In more specific terms,
a series of biochemical events that leads to a variety of morpho-
logical changes, including blebbing, changes to the cell membrane
such as loss of membrane asymmetry and attachment, cell
shrinkage, nuclear fragmentation, chromatin condensation, and
chromosomal DNA fragmentation.
Fig. 4. Cell cycle phase changes. HT1080 cells were treated with different concentra-
tions of FTF2 and FTF3 for 48 h (A) HT1080 cells treated with 40
mM, 80
mM and 160 mM
of FTF2; (B) HT1080 cells treated with 10
mM, 20 mM and 40 mM of FTF3. Results
represent mean ꢃ S.E. of data from at least three individual experiments. *P < 0.05;
**P < 0.01 versus control.

5006
R. Miao et al. / European Journal of Medicinal Chemistry 46 (2011) 5000e5009
cells in the G1 phase among HT1080 cells treated with 160
m
M FTF2
in a dose-dependent manner. These data indicated that entry into S
phase was inhibited. The apoptotic cells with degraded DNA appear
as cells with hypodiploid DNA content and are represented in so-
called "subG1" peaks on DNA histograms. The percentage of
and 20 M FTF3 increased from 44.8% to 69.2% and 66.1%, respec-
m
tively. Moreover, the percentage of cells in S phase among HT1080
cells treated with varying concentrations of FTF2 and FTF3 dropped
Fig. 5. AO/EB assay. A. HT1080 cells were treated with different concentrations of FTF2 and FTF3 for 48 h (a) HT1080 control; (b) HT1080 treated with 40
m
M of FTF2; (c) HT1080
M of FTF3; (g) HT1080 treated with 40
Necrotic cell. B. The analysis of apoptosis rates
treated with 80
mM of FTF2; (d) HT1080 treated with 160
mM of FTF2; (e) HT1080 treated with 10
m
M of FTF3; (f) HT1080 treated with 20
m
mM
of FTF3.（160ꢂ）
Normal cell
Early apoptotic cell
Late apoptotic cell
based on AO/EB assay. Apoptosis rates of HT1080 cells treated with different concentrations of FTF2 and FTF3 for 48 h were significantly changed. (a) HT1080 cells treated with
40 M, 80 M and 160 M of FTF2; (b) HT1080 cells treated with 10 M, 20 M and 40
M of FTF3. Results represent mean ꢃ S.E. of data from at least three individual experiments.
*P < 0.05; **P < 0.01 versus control.
m
m
m
m
m
m

R. Miao et al. / European Journal of Medicinal Chemistry 46 (2011) 5000e5009
5007
Fig. 5. (continued).
subG1 cells treated with FTF2 was 1.3%, 3.7%, 7.8% at the doses of 40,
80 and 160 M; while in case of FTF3, the percentage of subG1 cells
was 0.6%, 5.8%, 14.8% at the doses of 10, 20, 40 M, respectively.
From these results, we can infer that FTF3 as well as FTF2 led to cell
cycle arrest at the G1 phase followed by apoptosis in dose-depen-
dent manner.
permeabilization of mitochondrial membranes is a rate-limiting
step of apoptotic or necrotic cell demise. Rhodamine 123, whose
mitochondrial fluorescence intensity decreases quantitatively in
response to dissipation of mitochondrial transmembrane potential,
was used to evaluate disturbances in mitochondrial membrane
potential (DJm). As shown in Fig. 6, FTF2 and FTF3 both reduced the
mitochondrial membrane potential in a dose-dependent manner.
The relative membrane potential in the control group was taken as
m
m
Then we confirmed FTFs induced apoptosis by means of acridine
orange/ethidium bromide double staining method. Acridine
orange/ethidium bromide (AO/EB) staining was used to visualize
nuclear changes and apoptotic body formation that were charac-
teristic of apoptosis. The following four cellular states were recor-
ded under the microfluoroscope: (i) viable cells colored green with
normal nuclear structure (VN); (ii) early apoptotic cells stained
green with condensed or broken nuclear (VA); (iii) late apoptotic
cells that show orange color with condensed or broken nuclear
(NVA); and (iv) necrotic cells that appeared orange with a normal
nuclear structure (NVN). Apoptotic rate was calculated according to
the number of different cell types (apoptotic rate ¼ (VA þ NVA)/
(VN þ VA þ NVA þ NVN) ꢂ 100%) [21]. As shown in Fig. 5, treat-
100%. After treatment with 160 mM FTF2 and 40 mM FTF3 for 48 h, the
relative membrane potential was reduced to 10% and 8%, respec-
tively. These results demonstrated that mitochondrial pathway may
play a key role in modulating cell apoptosis caused by FTFs.
In the mitochondrial pathway of apoptosis, the Bcl-2 family of
proteins plays a critical role in controlling apoptotic machinery
in mammalian cells via its interacting pro- and anti- apoptotic
members [23], such as Bax and Bcl-2. Mitochondrial permeability
is believed to be affected by the up-regulation of Bax resulting
in the release of cytochrome c into the cytosol, which triggers
activation of caspase-9 and subsequently activation of caspase-3.
Bcl-2 inhibits apoptosis through stabilization of mitochondrial
membrane potential. Expressed on the outer mitochondrial mem-
branes, it blocks the release of apoptosis inducer proteins such as
cytochrome c from mitochondria to cytosol and thus inhibits the
subsequent apoptosis-executing signaling events [24,25]. Changes
in the Bax/Bcl-2 ratio suggest a corresponding change in mito-
chondrial permeability to begin the induction of apoptosis. To
assess the effects of FTFs on Bax and Bcl-2 expression in cells,
HT1080 cells were treated for 48 h with different concentrations of
FTF2 or FTF3, and the expression levels of Bcl-2 and Bax in cells
were determined by western blot analysis. As shown in Fig. 7, FTFs
treatment resulted in a significant increase in Bax/Bcl-2 ratio. These
ment of HT1080 cells with 40
increased the number of early apoptosis when compared with
control. Treatment with 160 M of FTF2 and 40 M of FTF3 showed
mM of FTF2 and 10 mM of FTF3 for 48 h
m
m
a significantly increasing number of apoptosis cells.
3.6. FTFs change mitochondrial membrane potential and expression
of Bcl-2 and Bax in HT1080 cells
The loss of mitochondrial membrane potential (MMP) is a hall-
mark of apoptosis. Cationic and lipophilic dye Rh123 was used to
permeate the negatively charged mitochondria, and then reflected
the mitochondrial membrane potential [22]. In many instances,
Fig. 6. Relative mitochondrial membrane potential of HT1080 cells treated with increasing concentration of FTF2 and FTF3 for 48 h (A) HT1080 cells treated with 40 mM, 80 mM and
160
m
M of FTF2; (B) HT1080 cells treated with 10
m
M, 20
mM and 40
m
M of FTF3. Results represent mean ꢃ S.E. of data from at least three individual experiments. *P < 0.05;
**P < 0.01 versus control.

5008
R. Miao et al. / European Journal of Medicinal Chemistry 46 (2011) 5000e5009
Fig. 7. (A) Western blot analysis of Bcl-2, Bax protein levels upon treatment with FTFs in different doses. HT1080 cells were treated with (40, 80 and 160
and 40 M) FTF3 for 48 h. Protein from the total cell lysate was subjected to SDS-PAGE and western blot using Bcl-2, Bax and -actin antibody. A representative blot from three
independent experiments with identical results is shown. Relative intensity of each band after normalization with the intensity of -actin in a blot (below each western blot) was
measured. (B) The Bax/Bcl-2 ratio was determined from three separate experiments by comparing the relative intensities of protein bands. *P < 0.05; **P < 0.01 versus control.
mM) FTF1, FTF2 or (10, 20
m
b
b
data suggested that FTFs exposure affected the components of the
Bcl-2 family of proteins, which would result in an alteration of the
Bax/Bcl-2 ratio in HT1080 cells.
compared to hTERT-BJ cells. Specially, side effect of FTF3 was less
than that of FTF2. These results suggested that FTF3 might be
a potential anti-tumor reagent. Differences in potency indicated
that the two compounds could be related to differences in
bioavailability and cellular uptake of the drug.
4. Discussion
Soft agar culture is closer to the in vivo situation than normal
culture. In the soft agar assay we observed that the malignant
potential of HT1080 cells was reduced after treatment with FTF2
and FTF3, respectively. Phase contrast microscope showed a dose-
dependent increase in the proportion of HT1080 cells having the
morphological features of cell death when treated with FTF2 and
FTF3, respectively, for 48 h. Subsequently, further tests were carried
out to investigate whether FTFs inhibited HT1080 cell growth by
inducing apoptosis.
The cell cycle is the series of events that take place in a cell
leading to its division and duplication. This process is tightly
controlled and coordinated to maintain normal tissue homeostasis.
The regulation of cell cycle is out of control in almost all tumor cells.
The proportion of cells which are in active cell division (versus
quiescent cells in G0 phase) in tumors is much higher than that in
Apoptosis is associated with a distinct set of biochemical and
physical changes involving the cytoplasm, nucleus and plasma
membrane. For cell homeostasis to be maintained, a balance
between the increase (by differentiation from precursors and by
proliferation) and decrease (by further differentiation and cell
death) in the number of a cell population has to be neatly balanced.
Tumor cells often have faulty apoptotic pathways. So, the induction
of tumor cell apoptosis by chemical and natural compounds is
considered as an efficiency anti-cancer therapy. FTFs are a series of
novel synthetic ferrocene derivatives. They were synthesized for
the first time and their activities have not previously been studied.
Introduction of FTFs to HT1080 cells led to enhancing cell death in
a time- and dose-dependent manner. FTF2 and FTF3 were more
effective in HT1080 cells with 50% reduction in cell viability

R. Miao et al. / European Journal of Medicinal Chemistry 46 (2011) 5000e5009
5009
normal tissue. At present, many anti-cancer drugs can induce
Acknowledgements
tumor cells cycle arrest at some phase followed by apoptosis. The
results of flow cytometry analysis showed that FTF2 and FTF3
induced cell cycle arrest at the G1 phase in HT1080 cells (Fig. 4).
FTF2 and FTF3 might disturb the function of G1/S checkpoint. Our
data also showed that FTF2 and FTF3 induced apoptosis of HT1080
by increasing population of cells in subG1 phase.
This work was supported by the International Cooperation Item
of The Ministry of Science and Technology of the People’s Republic
of China (No. 2009DFA30990，to Q. Wang), the Fundamental
Research Funds for the Central Universities (No. lzujbky-2009-45,
to R. Miao).
Apoptosis, which involves changes in the expression of a distinct
gene, represents a critical pathway that selectively allows certain
cells with damaged DNA to undergo cell death. Abnormalities in
this pathway may lead to uncontrolled cellular proliferation and
ultimately carcinogenesis [26]. In our study, the AO/EB double
staining assay confirmed further apoptosis induced by FTF2 and
FTF3 in HT1080 cells. Mitochondrial integrity is a fundamental issue
when dealing with the apoptosis resistance of tumor cell [27]. In
many apoptosis scenarios, the mitochondrial inner transmembrane
potential (DJm) collapses [28]. In our study, the mitochondrial
membrane potential of FTFs-treated HT1080 cells was decreased,
consistent with the previous results. Usually, Bcl-2 is bound to the
outer surface of the inner mitochondrial membrane and prevent
the release of cytochrome c into the cytoplasm [29]. Otherwise,
a family of Bcl-2 related genes is emerging that includes Bax,
a conserved homolog that heterodimerizes in vivo with Bcl-2 [30].
In normal cells, Bax, a mammalian cell death protein, normally
resides in the cytoplasm and translocates to mitochondria in
response to apoptotic stimuli, thereby promoting apoptosis by
disrupting the mitochondrial membrane barrier function through
formation of ion-permeable pores in mitochondrial membranes,
even when caspases are inactivated [31]. A pre-set ratio of Bax/Bcl-
2 appears to determine the survival or death of cells following an
apoptotic stimulus. Our data showed that the FTFs treatment of
HT1080 cells down-regulates Bcl-2 protein, and up-regulates the
protein levels of Bax. The increase of Bax expression might be
related with the mitochondrial inner transmembrane potential
References
[1] P. Kopfmaier, H. Kopf, E.W. Neuse, Angew. Chem. Int. Ed. 23 (1984) 456e457.
[2] P. Kopfmaier, H. Kopf, E.W. Neuse, J. Cancer Res. Clin. Oncol. 108 (1984)
336e340.
[3] D.R. van Staveren, N. Metzler-Nolte, Chem. Rev. 104 (2004) 5931e5985.
[4] E.E. Cable, H.C. Isom, Drug Metab. Dispos. 27 (1999) 255e260.
[5] N. Carolan, R.J. Forster, C. O’Fagain, Bioconjug. Chem. 18 (2007) 524e529.
[6] M. Mizuta, T. Terada, K. Seio, M. Sekine, Nucleic Acids Symp. Ser. (2004)
237e238.
[7] R. Kovjazin, T. Eldar, M. Patya, A. Vanichkin, H.M. Lander, A. Novogrodsky,
FASEB J. 17 (2003) 467e469.
[8] K. Kowalski, J. Zakrzewski, N.J. Long, N. Suwaki, D.J. Mann, A.J. White, Dalton
Trans. (2006) 571e576.
[9] O. Buriez, J.M. Heldt, E. Labbe, A. Vessieres, G. Jaouen, C. Amatore, Chemistry
14 (2008) 8195e8203.
[10] J. Spencer, A.P. Mendham, A.K. Kotha, S.C. Richardson, E.A. Hillard, G. Jaouen,
et al., Dalton Trans. (2009) 918e921.
[11] D. Dive, C. Biot, Chem. Med. Chem 3 (2008) 383e391.
[12] M.S. Karthikeyan, B.S. Holla, N.S. Kumari, Eur. J. Med. Chem. 43 (2008)
309e314.
[13] B.S. Holla, B.S. Rao, B.K. Sarojini, P.M. Akberali, N.S. Kumari, Eur. J. Med. Chem.
41 (2006) 657e663.
[14] F.P. Invidiata, D. Simoni, F. Scintu, N. Pinna, Farmaco 51 (1996) 659e664.
[15] A.P. Skoumbourdis, C.A. LeClair, E. Stefan, A.G. Turjanski, W. Maguire,
S.A. Titus, et al., Bioorg. Med. Chem. Lett. 19 (2009) 3686e3692.
[16] C. Burz, I. Berindan-Neagoe, O. Balacescu, A. Irimie, Acta Oncol. (2009) 1e11.
[17] P. Hersey, X.D. Zhang, N. Mhaidat, Adv. Exp. Med. Biol. 615 (2008) 105e126.
[18] J.R. Reid, N.D. Heindel, J. Heterocycl. Chem. 13 (1976) 925e926.
[19] A. Tarraga, P. Molina, D. Curiel, J.L. Lopez, M.D. Velasco, Tetrahedron 55 (1999)
14701e14718.
[20] C. Riccardi, I. Nicoletti, Nat. Protoc. 1 (2006) 1458e1461.
[21] R.R. Duke, J.J. Cohen, Current protocols in immunology. Johan wiley & Sons,
New York, 1992.
[22] R.D. Miao, Y. Han, L.Z. An, J.B. Yang, Q. Wang, Cell Biol. Int. 32 (2008) 217e223.
[23] R.J. Youle, A. Strasser, Nat. Rev. Mol. Cell Biol. 9 (2008) 47e59.
[24] J. Yang, X.S. Liu, K. Bhalla, C.N. Kim, A.M. Ibrado, J.Y. Cai, et al., Science 275
(1997) 1129e1132.
[25] T. Kuwana, D.D. Newmeyer, Curr. Opin. Cell Biol. 15 (2003) 691e699.
[26] B. Zhivotovsky, S. Orrenius, Carcinogenesis 27 (2006) 1939e1945.
[27] D.I. James, J.C. Martinou, Dev. Cell 15 (2008) 341e343.
[28] D. Arnoult, Trends Cell Biol. 17 (2007) 6e12.
[29] J.K. Brunelle, A. Letai, J. Cell. Sci. 122 (2009) 437e441.
[30] G.C. Shore, M. Nguyen, Cell 135 (2008) 1004e1006.
[31] P.F. Cartron, G. Bellot, L. Oliver, X. Grandier-Vazeille, S. Manon, F.M. Vallette,
FEBS Lett. 582 (2008) 3045e3051.
(DJm) collapses.
In conclusion, our data, for the first time, has shown a causal
connection between Bax and Bcl-2 modulation and induction of
apoptosis by FTFs. It has been suggested that Bcl-2 expression may
protect HT1080 cells from many different apoptotic stimuli,
including chemical reagents. Therefore, targeting at this survival
pathway constitutes a rational approach for the treatment of
cancer. Our study demonstrated that Bcl-2 and Bax were critical to
FTFs’ anti-cancer action. It will be interesting to further examine the
effects of FTFs on Bax/Bcl-2 pathway in HT1080 cells.