Stabilization of G-quadruplex DNA, inhibition of telomerase activity, and tumor cell apoptosis by organoplatinum(II) complexes with oxoisoaporphine

Article abstract of DOI:10.1021/jm5012484

Two G-quadruplex ligands [Pt(L^a)(DMSO)Cl] (Pt1) and [Pt(L^b)(DMSO)Cl] (Pt2) have been synthesized and fully characterized. The two complexes are more selective for SK-OV-3/DDP tumor cells versus normal cells (HL-7702). It was found that both Pt1 and Pt2 could be a telomerase inhibitor targeting G-quadruplex DNA. This is the first report demonstrating that telomeric, c-myc, and bcl-2 G-quadruplexes and caspase-3/9 preferred to bind with Pt2 rather than Pt1, which also can induce senescence and apoptosis. The different biological behavior of Pt1 and Pt2 may correlate with the presence of a 6-hydroxyl group in L^b. Importantly, Pt1 and Pt2 exhibited higher safety in vivo and more effective inhibitory effects on tumor growth in the HCT-8 and NCI-H460 xenograft mouse model, compared with cisplatin. Taken together, these mechanistic insights indicate that both Pt1 and Pt2 display low toxicity and could be novel anticancer drug candidates.

Full text of DOI:10.1021/jm5012484

Article
pubs.acs.org/jmc
Stabilization of G‑Quadruplex DNA, Inhibition of Telomerase
Activity, and Tumor Cell Apoptosis by Organoplatinum(II) Complexes
with Oxoisoaporphine
Zhen-Feng Chen,* Qi-Pin Qin, Jiao-Lan Qin, Yan-Cheng Liu, Ke-Bin Huang, Yu-Lan Li, Ting Meng,
Guo-Hai Zhang, Yan Peng, Xu-Jian Luo, and Hong Liang*
State Key Laboratory Cultivation Base for the Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry
and Pharmacy, Guangxi Normal University, Yucai Road 15, Guilin 541004, P. R. China
*
S Supporting Information
a
b
ABSTRACT: Two G-quadruplex ligands [Pt(L )(DMSO)Cl] (Pt1) and [Pt(L )(DMSO)Cl] (Pt2) have been synthesized and
fully characterized. The two complexes are more selective for SK-OV-3/DDP tumor cells versus normal cells (HL-7702). It was
found that both Pt1 and Pt2 could be a telomerase inhibitor targeting G-quadruplex DNA. This is the first report demonstrating
that telomeric, c-myc, and bcl-2 G-quadruplexes and caspase-3/9 preferred to bind with Pt2 rather than Pt1, which also can
induce senescence and apoptosis. The different biological behavior of Pt1 and Pt2 may correlate with the presence of a 6-
b
hydroxyl group in L . Importantly, Pt1 and Pt2 exhibited higher safety in vivo and more effective inhibitory effects on tumor
growth in the HCT-8 and NCI-H460 xenograft mouse model, compared with cisplatin. Taken together, these mechanistic
insights indicate that both Pt1 and Pt2 display low toxicity and could be novel anticancer drug candidates.
INTRODUCTION
In addition, the G-quadruplex structure has recently been
found not only in telomeres but also in promoter regions of
certain genes such as c-myc, bcl-2, and c-kit, and these G-
■
Telomere structures contain highly repetitive guanine (G) rich
DNA sequences and are essential for maintaining the stability
of eukaryotic chromosomes. Human telomeric DNA is
composed of a repeated double-stranded [TTAGGG-
CCCTAA]n sequence except for the 3′-terminal region,
which consists of a single-stranded tandem [TTAGGG]
repeated sequence over several hundred bases. Recent studies
demonstrate that G-quadruplex structures are stable and
detectable in human cells. G-quadruplexes participate in
7
quadruplexes have their own specific structures. The formation
or stabilization of G-quadruplexes in these regions may play a
key regulatory role. In c-myc oncogene, G-rich sequences can
form c-myc G-quadruplexes and regulate the transcription of c-
myc oncogene. The c-myc functions as a gene-specific
transcription factor through its protein product that controls
a variety of genes responsible for a wide range of human
1
2
8
cancers. Particular interest has been focused on c-myc, another
9
several key biological events, especially those associated with
aging and diseases. They are involved in the regulation of
telomerase activity that is activated in 80−90% of human
tumors and can serve as specific tumor-selective targets for
important quadrome gene involved in cell senescence,
transcriptionally activating the catalytic subunit of telomerase
Received: August 14, 2014
3
−6
chemotherapy.
©
XXXX American Chemical Society
A
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Article
a
b
Figure 1. Synthetic routes for the preparation of the 1-azabenzanthrone (L ), 6-hydroxyloxoisoaporphine alkaloid (L ), and their platinum(II)
complexes Pt1 and Pt2. Reagents are the following: (a) ethanol (reflux); (b) anhydrous AlCl /NaCl (220−230 °C); (c) concentrated H SO (230−
3
2
4
2
40 °C); (d) ethanol/water (10:1) (reflux); (e) NaOH (room temperature 24 h); (f) methanol/water (25:1) (reflux).
b
Figure 2. Crystal structures of L (left) and Pt1 (middle) and Pt2 (right) showing atom labeling.
1
0
hTERT and consequently influencing the elongation of
telomere. Another apoptosis-related human bcl-2 oncogene
also contains a GC-rich sequence which is located upstream
from P1 promoter and has the potential to form G-quadruplex
cleaving agents. Similar to organic compounds, they can
stabilize the G-quadruplex via π−π stacking and electrostatic
interactions and have the ability to interact with nucleic acids
(including quadruplex DNA) through alternative and/or
additional modes, such as direct coordination to bases or the
1
1
structures and regulate the transcription of bcl-2. Therefore,
targeting G-quadruplexes in gene promoters may constitute a
novel anticancer strategy.
During the past decade, a number of small molecules have
been reported to efficiently stabilize G-quadruplex DNA, inhibit
16
phosphate backbone. Because of the great potential of the
metal complexes as G-quadruplex binders, more and more
metal complex G-quatruplex DNA binders have been reported
12
1
7
18,19
20
23
recently, such as nickel(II), platinum(II),
palladium(II),
1
1,13
21
22
the telomerase activity, or regulate the gene expression.
ruthenium(II), copper(II), cobalt(II), and zinc(II)
Most of these compounds are organic compounds with a large
planar aromatic conjugated system, which in general can
stabilize the G-quadruplex structure mainly by π−π stacking
and electrostatic interactions with G-quartet plane of G-
complexes. Among them, the platinum(II) complex G-
quadruplex binders are most widely exploited for their great
18
potential as anticancer agents. Therefore, we synthesized two
organoplatinum(II) complexes with structures as depicted in
Figures 1 and 2 and investigated their abilities to act as selective
and effective G-quadruplex binders. The rationale is to maintain
the planar π-conjugation of the 1-azabenzanthrone or 6-
hydroxyloxoisoaporphine alkaloid scaffolds through the for-
mation of platinum(II) complexes, which have a more extended
π-conjugated planar aromatic structure. This approach can
significantly improve their π−π interactions with G-quadruplex
and can also covalently bind to guanine of G-quadruplex. These
1
4
quatruplex DNA. Some G-quadruplex binders that are
capable of binding to the groove and loop have also been
1
5
reported recently. On the other hand, metal complexes have a
very broad range of structural and electronic properties that can
be successfully exploited when designing quadruplex DNA
binders. Furthermore, the metal complexes possess interesting
optical, magnetic, or catalytic properties and could in principle
be exploited for the development of quadruplex probes and
B
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Article
a
b
Table 1. IC50 (μM) Values of L , L , Pt1, and Pt2 for the Selected Cells for 48 h
compd
T-24
Hep-G2
SK-OV-3
SK-OV-3/DDP
BEL-7404
NCI-H460
HCT-8
>100
HL-7702
La
Lb
16.8 ± 2.0
38.4 ± 1.5
4.8 ± 1.7
26.7 ± 1.9
28.1 ± 0.7
17.4 ± 4.6
20.5 ± 1.3
14.3 ± 1.6
14.4 ± 2.0
9.5 ± 0.4
>100
36.8 ± 1.5
13.9 ± 2.2
4.8 ± 0.9
4.5 ± 0.4
65.9 ± 1.5
36.7 ± 0.1
54.7 ± 0.2
15.6 ± 0.3
26.2 ± 0.8
12.4 ± 0.4
>100
>100
>100
66.9 ± 0.5
38.4 ± 0.9
10.7 ± 0.6
18.9 ± 0.5
60.2 ± 0.7
36.5 ± 0.4
13.2 ± 0.5
15.4 ± 0.6
64.5 ± 0.4
>100
1
37.6 ± 3.6
30.5 ± 2.4
15.6 ± 1.7
2
>100
a
cisplatin
15.6 ± 0.3
a
Cisplatin is the clinical medicine.
Figure 3. SK-OV-3/DDP cells were treated with Pt1 and Pt2 for 8 h at 37 °C. Platinum content in whole cell (A) and in different fraction (B) was
measured by ICP-MS. Control cells were treated with vehicle (1% DMSO). Data shown are mean values ± standard deviations of three independent
measurements for each experiment.
compounds can induce tumor cell apoptosis through multiple
mechanisms, especially in cisplatin-resistant tumor cells SK-
OV-3/DDP. Meanwhile, using cisplatin as positive control, we
further investigated the in vivo antitumor efficacy of Pt1 and
Pt2 utilizing HCT-8 and NCI-H460 xenograft models.
Pt2 may lead to an increase in the membrane permeability and
the affinity to the molecular targets, which is responsible for its
high cytotoxicity.
Stability of Pt1 and Pt2 in Solution. L , L , Pt1, and Pt2
were tested for their stability in physiological conditions (Tris-
KCl buffer solution with pH value of 7.35, containing 1%
DMSO) by means of UV−vis spectroscopy. As shown in Figure
S18 (Supporting Information), the time-dependent (at 0, 2, 4,
a
b
RESULTS AND DISCUSSION
■
Synthesis and Characterization. The ligands 1-azaben-
8
, 12, and 24 h) UV−vis spectra of each compound indicated
a
b
zanthrone (L ) and 6-hydroxyloxoisoaporphine alkaloid (L )
a b
that L , L , Pt1, and Pt2 were stable in TBS (Tris buffer
solution) for 24 h at room temperature. Their stabilities were
further confirmed by HPLC experiments. As shown in Figure
S19 (Supporting Information), all four molecules were stable
for 24 h in DMSO stock solution.
were synthesized according to the reported procedures (Figure
2
4
1
1
). Their structures were characterized by IR, ESI-MS, H
13
and C NMR spectrometry, and elemental analyses. In
addition, L was structurally characterized by single-crystal X-
b
a
b
ray diffraction analysis. Its crystal structure is shown in Figure 2.
In Vitro Cytotoxicity. The in vitro cytotoxicity of L and L
b
Selected bond lengths (Å) and bond angles (deg) for L are
and their platinum complexes Pt1 and Pt2 (cisplatin as positive
control) against seven human tumor cells T-24, Hep-G2, SK-
OV-3, SK-OV-3/DDP, BEL-7404, NCI-H460, and HCT-8 and
one normal liver cell HL-7702 was assessed by MTT assay. The
inhibitory rates are listed in Table S3 (Supporting Informa-
tion). For comparison, cisplatin and Pt(DMSO) Cl were used
reported in Table S1 (Supporting Information), and crystal
b
data and structure refinement parameters for L are listed Table
b
S2 (Supporting Information). As shown in Figure 2, L has a
25
planar structure similar to 10-chloro-1-azabenzanthrone.
Crystal Structures of Pt1 and Pt2. Pt1 and Pt2 were
2
2
a
b
prepared via the reaction of cis-Pt(DMSO) Cl with L and L
2
2
as controls. After incubation of the cells with the compounds at
a concentration of 20 μM for 48 h under identical conditions,
these compounds exhibited different antitumor activity and
certain extent of selectivity. Against the tested tumor cells, the
in the presence of ethanol (or methanol) and water under
solvothermal conditions (Figure 1). Their structures were
1
13
characterized by IR, H and C NMR, ESI-MS (Supporting
Information, Figures S1−S17), and elemental analysis. The
single-crystal X-ray diffraction analyses for Pt1 and Pt2 revealed
that in each case the platinum(II) center is four coordinated by
a
b
inhibitory rates of Pt1 and Pt2 were higher than that of L , L ,
a
b
and Pt(DMSO) Cl , respectively. Interestingly, L , L , Pt1, and
2
2
Pt2 displayed low inhibitory rates against normal live cell HL-
7702. The in vitro antitumor activities of Pt1 and Pt2 were
further quantified by determining the corresponding IC50
values. As shown in Table 1, Pt1 and Pt2 exhibited significantly
∧
a
∧
b
the corresponding chelating ligand (C N-L or C N-L ) via the
heterocyclic nitrogen and carbon atom, one DMSO ligand via
the sulfur atom, and one chlorine, to form a square-planar
geometry (Figure 2). Selected bond lengths (Å) and bond
angles (deg) are reported in Table S1 (Supporting
Information), and crystal data and structure refinement
parameters are listed Table S2 (Supporting Information).
a
b
enhanced cytotoxicity compared with free L and L in general.
a
For example, Pt1 was 2.5 times stronger than L against T-24,
and Pt1 and Pt2 were more cytotoxic to SK-OV-3/DDP
cisplatin-resistant tumor cells than to SK-OV-3. They showed
low IC50 values (4.5−4.8 μM) on SK-OV-3/DDP, more than
b
a
Because L contains a 6-hydroxyl group and different from L ,
C
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.5−7 times more cytotoxic than the corresponding free L and
Article
a
2
b
L , and even 13 times more cytotoxic than cisplatin alone. Pt2
was more toxic than Pt1 and cisplatin toward NCI-H460 and
HCT-8. According to Table S3 (Supporting Information) and
Table 1, Pt1 and Pt2 displayed a synergistic effect upon the
combination of platinum with corresponding oxoisoaporphine
a
b
alkaloids L and L .
Cellular Uptake and Distribution of Platinum in
Cancer Cells. Since Pt1 and Pt2 exhibited the best in vitro
cytotoxicity on SK-OV-3/DDP cells, we selected these cells to
investigate the cellular uptake and distribution of platinum in
cancer cells, using the inductively coupled plasma mass
spectrometry. Treating SK-OV-3/DDP cells with the com-
plexes (10.0 μM) for 8 h resulted in a substantial increase in the
cellular platinum concentration compared to the untreated
control, suggesting facile internalization of complexes within 86
h. As shown in Figure 3A, Pt2 ((5.90 ± 0.68 nmol of Pt)/10
cells) was taken up by the cells approximately 2.44 times and
1
1
.5 times more efficiently than Pt1 ((2.42 ± 0.30 nmol of Pt)/
Figure 4. Confocal microscopy of the SK-OV-3/DDP cells treated
with 5.0 μM Pt1, Pt2 for 12 h. Images were acquired using a CarlZeiss
LSM 710 microscope (magnification 400×).
6
6
0 cells) and cisplatin ((4.11 ± 0.59 nmol of Pt)/10 cells),
respectively.
To examine the cellular distributions of Pt1 and Pt2, the
platinum concentrations were determined in the nuclear
fraction, nuclear proteins, membrane proteins, cytoplasmic
protein, and other extracts fractionated from SK-OV-3/DDP
cells after the exposure of the cells to Pt1 and Pt2 for 8 h
according to the reported method. As shown in Figure 3B,
Pt1 and Pt2 all accumulated to a high extent in the nuclear
fraction and membrane fraction, while cisplatin only accumu-
lated in the membrane fraction. The percentage of platinum
from Pt2 in the nuclear fraction was slightly higher than that
from Pt1 and cisplatin. It is also notable that there was a certain
amount of accumulation of platinum from the complexes in the
other fractions, including the total soluble proteins. The
differences in cellular platinum distribution can be attributed
to the different cellular pathways involved in the uptake and
efflux of the platinum complexes, which may be related to the
apoptotic pathways they activated.
seen. Treatment with 5.0 μM Pt1 slightly changed the nuclear
morphology (Figure 5B), and more nuclei of Pt1-treated SK-
OV-3/DDP tumor cells appeared hypercondensed (brightly
stained) at 10.0 μM (Figure 5C). It should be noted that the
numbers of apoptotic nuclei containing condensed chromatin
increased significantly as the result of treatment with 20.0 μM
Pt1 (Figure 5D). Similar results were observed with Pt2
treatment, but it was interesting to find that treatment with 5.0
μM Pt2 induced significant apoptosis (Figure 5F). With the
increased concentrations of Pt2, the number of apoptotic nuclei
increased more significantly than treatment with Pt1.
26
Senescence Induction. To evaluate the long-term effects
of Pt1 and Pt2 on SK-OV-3/DDP cancer cells, subcytotoxic
concentrations (0.5 μM) of both compounds were employed to
avoid acute cytotoxicity and other nonspecific events that could
lead to difficulty in interpretation of results. Seven days after
treatment with 0.5 μM Pt1 and Pt2, SK-OV-3/DDP cells
displayed increased proportion of flat and giant cells with
phenotypic characteristics of senescence as revealed by the
senescence-associated β-galactosidase (SA-β-Gal) assay (Figure
Confocal Morphological Examination. The AO/EB
27
molecular probes were used to detect apoptotic cells. As
shown in Figure 4, apparent changes of cell morphology took
place after treating the SK-OV-3/DDP tumor cells with 5.0 μM
Pt1 and Pt2 for 12 h. Pt1 caused a small number of SK-OV-3/
DDP cells to shrink and undergo apoptosis. In contrast, Pt2
caused a large number of SK-OV-3/DDP cells to shrink and
undergo apoptosis. The apoptotic body was observed
obviously. The results primarily suggested that both Pt1 and
Pt2 could induce apoptosis in SK-OV-3/DDP cells at 5.0 μM
dosage after 12 h of treatment. However, Pt2 exhibited stronger
ability to induce apoptosis than Pt1, which might be attributed
28
6). The results indicated that Pt2 might induce senescence
mainly due to the shortening of telomere length, whereas the
effect of Pt1 on SK-OV-3/DDP cells was not so obvious,
implying that dysfunctional telomeres could initiate cellular
senescence or apoptosis to suppress tumorigenesis.
Cell Cycle and Apoptosis. In order to confirm the cell
morphological analytical results, we further investigated the cell
cycle and apoptosis using SK-OV-3/DDP tumor cells. The cell
cycle analysis of untreated and treated SK-OV-3/DDP cells for
48 h with 5.0 μM Pt1 and Pt2 showed that both compounds
induced evident perturbation of the cell cycle and blocked the
cells in G2 and G1 phases, respectively (Figure 7 and Figure
S20, Supporting Information). G2 population of 36.24% was
observed in Pt1 group, while the corresponding G2 population
of 8.23% was observed in untreated control cells, which was
different from the cycle G1 arrest in Pt2-treated cells, where the
G1 population was 70.07%, compared with a G1 population of
51.81% in the control group.
b
to the 6-hydroxyl group in the L of Pt2.
The ability of Pt1 and Pt2 to induce apoptosis was further
confirmed by analyzing the nuclear morphology of the
compound-treated SK-OV-3/DDP tumor cells. SK-OV-3/
DDP tumor cells were treated with Pt1 and Pt2 for 12 h and
stained with membrane-permeable blue Hoechst 33258 to
detect apoptosis morphologically. As shown in Figure 5, the
Hoechst 33258 fluorescent photomicrographs of cultured SK-
OV-3/DDP tumor cells treated with 5.0, 10.0, and 20.0 μM Pt1
and Pt2 for 12 h, respectively, indicated that in the control
cultures, nuclei of SK-OV-3/DDP tumor cells appeared with
regular contours and were round, and SK-OV-3/DDP tumor
cells with smaller nuclei and condensed chromatin were rarely
We further investigated the ability of Pt1 and Pt2 to induce
apoptotic cell death in SK-OV-3/DDP tumor cells by flow
cytometry, and the results indicated that Pt2 induced apoptotic
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Figure 5. Morphological changes in the nuclei (typical of apoptosis) of cultured SK-OV-3/DDP cancer cells. SK-OV-3/DDP cancer cells treated
with 5.0, 10.0, and 20.0 μM Pt1 and Pt2 for 12 h, respectively, and stained with Hoechst 33258. Selected fields illustrating occurrence of apoptosis
were shown. Cells with condensed chromatin (brightly stained) were defined as apoptotic SK-OV-3/DDP cancer cells. Images were acquired using a
Nikon Te2000 deconvolution microscope (magnification 200×).
Figure 6. Expression of SA-β-Gal in SK-OV-3/DDP cells after continuous treatment with Pt1 and Pt2. SK-OV-3/DDP cells were treated with 0.5
μM Pt1, Pt2 or 0.1% DMSO (control) continuously for 7 days. Then the cells were fixed, stained with SA-β-Gal staining kit, and photographed.
Images were acquired using a Nikon Te2000 deconvolution microscope (magnification 100×).
eukaryotic chromosomes. Mammalian telomeres consist of
several kilobases of tandem TTAGGG repeats bound by the
29
related telomere-specific proteins, TRF1 and TRF2. TRF1
negatively controls telomere length by cis-inhibiting telomerase
activity. Overexpression of wild type TRF1 leads to telomere
shortening, whereas inhibition of TRF1 with a dominant-
30
negative mutant allele leads to telomere lengthening. TRF2 is
a key component of human telomere protein complex. TRF2
31
also binds to duplex telomeric repeats as a homodimer. Like
TRF1, overexpression of intact TRF2 results in progressive
32
telomere shortening too. Meanwhile, TRF2 deletion leads to
telomere dysfunction and TIF formation. TIF is formed by the
association of damaged telomeres with DNA damage response
factors, such as 53BP1, ATM, γ-H2AX, Rif1, and the Mre11
3
3a,b
Figure 7. Effect of cell cycle of SK-OV-3/DDP treated with 5.0 μM of
Pt1, Pt2 for 48 h compared with untreated cells. The percentages of
cells in the different phases of cell cycle are reported inside the relative
histogram.
complex.
Furthermore, 53BP1 binds to the tumor
suppressor protein p53 and has a potential role in DNA
33c
damage responses. Therefore, to clarify the effects of Pt1 and
Pt2 on telomere dysfunction, we preferred to detect the
expression of 53BP1, TRF1, and TRF2 in SK-OV-3/DDP cells
by immunofluorescence and Western blotting (Figures 9−11).
As shown in Figures 9 and 10, compared to the control group,
the treatment of cells with Pt2 (4.5 μM) resulted in remarkable
overexpression of 53BP1 (green in the merged images), TRF1
(fuchsia in the merged images), and TRF2 (fuchsia in the
merged images), suggesting that Pt2 significantly induced DNA
damage (overexpression of 53BP1) and telomeres damage
(overexpression of TRF1 and TRF2) in SK-OV-3/DDP cells.
Moreover, it was reported that TRF1 and TRF2 were two
cell death in a dose-dependent manner, as demonstrated by
combined staining with annexin V-FITC (which detects
phosphatidylserine residues translocated from the inner to the
outer cell membrane in early apoptotic stages) and propidium
iodide (PI, which stains necrotic cells) (Figure 8). In contrast,
Pt1 failed to induce significant early stage apoptosis under the
same conditions.
Detection of 53BP1, TRF1, and TRF2 by Immuno-
fluorescence and Western Blotting. Telomeres are
heterochromatic structures found at the ends of linear
33d
negative regulators of telomere length. In the present study,
E
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Figure 8. Effect of cell apoptosis of SK-OV-3/DDP treated with Pt1 and Pt2 for 12 h compared with the untreated cells. (A) The induction of
apoptosis by Pt1 and Pt2 was examined by FACS analysis with PI and FITC-annexin V staining. (B) Populations for the apoptotic cells treated by
Pt1 and Pt2.
Figure 9. Binding of 53BP1 and TRF1 to uncapped telomeres. SK-OV-3/DDP cells were treated with 4.5 μM Pt1 and Pt2 for 12 h, respectively, and
then were processed for 53BP1 (green) and TRF1 (red). The nuclei were stained (DAPI, blue) in the merged images. Images were acquired using a
CarlZeiss LSM 710 microscope (magnification 400×).
F
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Figure 10. Binding of 53BP1 and TRF2 to uncapped telomeres. SK-OV-3/DDP cells were treated with 4.5 μM Pt1 and Pt2 for 12 h, respectively,
and then were processed for 53BP1 (green) and TRF2 (red). The nuclei were stained (DAPI, blue) in the merged images. Images were acquired
using a CarlZeiss LSM 710 microscope (magnification 400×).
Figure 11. (A) Western blot to determine the translation of 53BP1, TRF1, and TRF2 in SK-OV-3/DDP cells treated with Pt1 (4.5 μM) and Pt2
(
4.5 μM) for 12 h. (B) Densitometric analysis of 53BP1, TRF1, and TRF2 fragment normalized to β-actin band from part A. The relative expression
of each band is (density of each band)/(density of actin band). Mean and SD values were from three independent measurements.
Figure 12. Expression level of caspase-3/9 proteins in SK-OV-3/DDP cells with 10.0 μM Pt1 or Pt2 for 8.0 h. Caspase-3/9 was assessed by the
CasPGLOW fluorescein activite caspase-3/9 staining kits.
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Figure 13. Representative photos of SK-OV-3/DDP cancer cells by JC-1 staining: polarized mitochondria are marked by punctuate orange-red
fluorescence staining. On depolarization, the orange-red punctuate staining is replaced by diffuse green monomer fluorescence. Apoptotic cells,
showing primarily green fluorescence, are easily differentiated from healthy cells, which show red and green fluorescence. Parts A and E represent the
control without apoptosis. Parts B, C, D represent the 5.0, 10.0, 20.0 μM Pt1-treated samples with apoptotic SK-OV-3/DDP cancer cells,
respectively. Parts F, G, H represent three concentrations of Pt2 with apoptotic SK-OV-3/DDP cancer cells, respectively. Images were acquired using
a Nikon Te2000 deconvolution microscope (magnification 200×).
a
b
a
Table 2. FID Assay for L , L , Pt1, and Pt2 on DNA
La
Pt1
Lb
Pt2
Pu27DC50 (ctDNADC /Pu27DC50#)
6.37 (−)
1.35 (10.33)
5.76 (2.42)
3.49 (4.00)
2.50 (5.58)
4.04 (3.45)
4.23 (3.30)
6.02 (2.32)
7.43 (1.88)
13.95 (1.00)
15.13 (−)
>100 (−)
>100 (−)
16.65 (−)
>100 (−)
>100 (−)
>100 (−)
>100 (−)
>100 (1.00)
0.55 (58.84)
2.42 (13.37)
3.46 (9.35)
1.31 (24.69)
1.52 (21.29)
11.23 (2.88)
1.94 (16.68)
6.24 (5.19)
32.36 (1.00)
50
c‑kit‑1DC50 (ctDNADC50/c‑kit‑1DC50)
c‑kit‑2DC50 (ctDNADC50/c‑kit‑2DC50)
HTG21DC50 (ctDNADC50/HTG21DC50)
Pu39DC50 (ctDNADC / DC )
>100 (−)
>100 (−)
40.58 (−)
>100 (−)
60.98 (−)
>100 (−)
>100 (−)
>100 (1.00)
Pu39
50
50
mutPu39DC50 (ctDNADC50/mutPu39DC50)
Pu22DC50 (ctDNADC / DC )
Pu22
50
50
ds26DC50 (ctDNADC / DC )
ds26
50
50
ctDNADC50 (ctDNADC50/ctDNADC50)
a
ctDNA
G4
35a
“−” indicates that no data can be calculated. # stands for G4-FID selectivity, defined as
DC / DC ratio. DC50 obtained in μM.
50 50
it was found that the percentage of TRF1- and TRF2-induced
telomeres damage by Pt2 was 72.33%. In contrast, less
telomeres damage induction (48.26%) on SK-OV-3/DDP
cells was observed when treated with Pt1 under the same
conditions. These results suggested that the DNA damage
response foci at telomeres induced by Pt2 were over other
chromosomal regions. The selectivity of Pt2 for telomeric
regions is better than that of Pt1.
Furthermore, the expression of 53BP1, TRF1, and TRF2
protein in the SK-OV-3/DDP cells treated with Pt1 and Pt2
was further identified using Western blotting. As shown in
Figures 11, compared with the control group, Pt2 led to 151.04
diseases, most notably cancer. Caspase-3/9, the executioner
caspase, is able to directly degrade multiple substrates including
34
structural and regulatory proteins. Thus, therapeutic strategies
designed to stimulate apoptosis by activating caspase-3/9 may
help in combating cancer caused by deficient apoptosis. Some
small molecules have been developed to selectively activate
caspase-3/9. We thus investigated whether the effector caspase-
3/9 is activated when SK-OV-3/DDP cells are exposed to Pt1
and Pt2. As shown in Figure 12, cells treated with Pt1 and Pt2
exhibited a significant increase in caspase-3/9 activity, as
indicated by the activation of caspase-3 in Pt1- and Pt2-teated
cells (by 37.0% and 77.4%, respectively) and by the activation
of caspase-9 in Pt1 and Pt2 groups (by 34.6% and 56.2%,
respectively), compared with the control group. The results
suggested that Pt1 and Pt2 are efficient activators of caspase-3/
9, and Pt2 is more effective than Pt1. The activation of caspase-
3/9 is an important pathway in Pt1- and Pt2-induced SK-OV-
3/DDP apoptosis.
Assessment of Changes in Mitochondrial Membrane
Potential. The loss of mitochondrial membrane potential is a
characteristic of apoptosis. In nonapoptotic cells, JC-1 exists as
a monomer in the cytosol (green) and accumulates as
aggregates in the mitochondria (orange-red). In apoptotic
and necrotic cells, JC-1 exists in monomeric form and stains the
cytosol green. The control cells exhibited punctate orange-red
staining (Figure 13A), indicating coupled mitochondria with a
±
8.07%, 1522.92 ± 14.76%, and 632.584 ± 10.25% increase of
53BP1, TRF1, and TRF2 protein expression level, respectively.
The results indicate that Pt2 can induce telomere dysfunction
and DNA damage. Comparatively, the Pt1 induced a weak
increase of the expression of 53BP1, TRF1, and TRF2 protein
on SK-OV-3/DDP cells under the same conditions (the
accumulation of 53BP1, TRF1, and TRF2 of 106.02 ± 12.37%,
682.55 ± 9.91%, and 376.47 ± 9.91%, respectively). The results
agreed with DNA damage of immunofluorescence in the SK-
OV-3/DDP cells.
Caspase-3/9 Activation Assay. The well characterized
caspase family plays a crucial role in modulating programmed
cell death, which is a genetically regulated and evolutionarily
conserved process with numerous links to many human
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normal Δψ. As shown in Figure 13B, a few SK-OV-3/DDP
cancer cells showed green fluorescence, which indicated that
they were apoptotic cells with 5.0 μM Pt1 treatment. However,
Figure 13C and Figure 13D show increased numbers of SK-
OV-3/DDP cancer cells with green fluorescence, suggesting
that Pt1 induced apoptosis in SK-OV-3/DDP cancer cells.
Similar results were also observed with Pt2-treated SK-OV-3/
DDP cancer cells. All Pt1/2-treated SK-OV-3/DDP cancer
cells appeared green at 20 μM (Figure 13D and Figure 13H),
but as shown in Figure 13F and Figure 13G, Pt2 induced more
obvious apoptosis of SK-OV-3/DDP cancer cells compared
with Pt1.
and Pt2 produced the ΔTm values of 6.83 and 8.54 °C for
F21T, 12.83 and 14.54 °C for FPu18T, respectively. Under 0.5
μM Pt1 and Pt2, the ΔTm values for FMidG4T were 4.45 and
6.77 °C, respectively. However, under the same conditions, the
ΔT values for duplex DNA (F32T + H20M) generated by Pt1
m
a
b
and Pt2 were 2.74 and 2.32 °C, respectively. Therefore, L , L ,
Pt1, and Pt2 exerted a weaker stabilizing effect of the duplex
DNA (F32T + H20M) than F21T, FPu18T, and FMidG4T G-
quadruplex DNA (Figure S35−S39). Moreover, Pt2 with a 6-
b
hydroxyl group in L has higher selectivity for the quadruplex-
DNA over duplex-DNA than Pt1. Taken together, Pt2 was
favored to bind with the human telomeric G-quadruplex and G-
quadruplex in the promoter region of c-myc and bcl-2, showing
a relatively high thermodynamic stability, high binding
constants, and a strong ability to induce conformational change
in G-quadruplex. In addition, the ESI-MS results indicated that
Pt1 and Pt2 covalently coordinated to guanine of G4-DNA
Selectivity for Binding of G-Quadruplex and Duplex
a
b
DNA by L , L , Pt1, and Pt2 Determined by Spectro-
a
b
scopic Methods. The binding ability of L , L , Pt1, and Pt2 to
G-quadruplex and duplex DNA was studied using FID assay,
fluorescence titration analysis, circular dichroism (CD) spectral
analysis, FRET assay, and ESI-MS (for detailed results,
discussion, and experiments, see Supporting Information). As
shown in Table 2, the FID assay results indicated that Pt1 and
Pt2 exhibited better selectivity for HTG21 and Pu27 G-
quadruplex DNAs than to other DNAs, which are the most
3
5f,g
(
Figure S40).
Interactions with GSH. As one of the most widely
distributed intracellular reductants, GSH also plays an
36
important role in metal detoxification. To date, a quantitative
study has shown that GSH is a crucial contributor to protect
against platinum cytotoxicity, and it may function in concert
Pu27
efficient TO displacers ( DC50 down to 1.35 and 0.55 μM
37
HTG21
with or independently of glutathione or metallothionein. At
with Pt1 and Pt2,
DC50 down to 2.50 and 1.31 μM with
the same time, in some platinum-resistant cancer cells,
glutathione and metallothionein levels are relatively high,
which can neutralize the activated platinum effectively in the
cytoplasm before DNA binding occurs. Therefore, we further
explored the interaction of Pt1 and Pt2 with GSH. As shown in
Figure S41 (Supporting Information), the absorbance of Pt2 at
Pt1 and Pt2, respectively, also as shown in Table S4 and
Figures S21 and S22). The selectivity of Pt1 and Pt2 for G4-
ctDNA
G4
DNA over duplex DNA is moderate, with
DC / DC
50 50
3
5a
ratio (or Est. Sel.) within the range of 1.88- and 10.33-fold
and 2.88- and 58.84-fold, respectively. As expected, Pt2 with a
b
6
-hydroxyl group in L has higher selectivity for quadruplex-
4
63 nm exhibited a small decrease while the absorbance at
DNA than Pt1. The data from fluorescence titration analysis
about 245 nm increase upon the addition of GSH. Compared
with Pt2, only obvious absorption changes at about 245 nm
were observed in the UV spectra upon addition of GSH to the
solution of Pt1. Interestingly, the result is closely correlated
with the cytotoxicity of the two complexes. The GSH-reactive
Pt1 and Pt2 are biologically active in tumor cells. Upon
entering tumor cells, Pt1 and Pt2 do not react with GSH;
therefore, their cytotoxicity in tumor cells is retained, which
agrees well with the fact that Pt1 and Pt2 exhibited high activity
against the cisplatin-resistant SK-OV-3/DDP cells.
and circular dichroism (CD) spectral analysis show that the
a
b
binding of L , L , Pt1, and Pt2 with G-quadruplex DNA might
increase the stability of the structure of G4-DNA and hence
interfere with the DNA function. The results of fluorescence
titration analysis and CD indicated that HTG21, Pu39, and
mutPu39 DNAs preferentially folded into an intramolecular
3
5b−d
+
mixed G-quadruplex
in the solutions containing K , while
35e
the Pu27 DNA preferred to form a parallel conformation (as
shown in Tables S5 and S6 and Figures S23−S34). As shown in
Table 3, the FRET results demonstrated clearly that 2 μM Pt1
Transcription and Translation of hTERT and c-myc
Gene. Telomerase catalyzes the elongation of telomere, which
involves complicated processes, such as the association and
dissociation of catalytic enzyme. G-quadruplex ligands may
affect the event mainly via two pathways, regulation of hTERT
and c-myc, or stabilization of the telomeric G-rich end to block
Table 3. ΔT Data (°C) of G-Quadruplex DNA Obtained by
m
RT-PCR
compd
concn of compd
DNA (0.5 μM) Tm (°C)
ΔTm (°C)
La
2.0 μM (0.5 μM)
F21T
53.65
70.72
58.70
29.42
53.65
70.72
58.70
29.42
53.65
70.72
58.70
29.42
53.65
70.72
58.70
29.42
4.45 (2.62)
11.95 (4.25)
2.69
3
the association with the catalytic enzyme. Meanwhile, hTERT
1
0
2
.5 μM (0.5 μM)
FPu18T
is the key catalytic domain of the telomerase enzyme, which is
closely related to telomerase function and regulated by c-myc
.5 μM
FMidG4T
F32T + H20M
F21T
38
a
b
.0 μM (0.5 μM)
2.27 (0.67)
6.83 (2.68)
12.83 (4.31)
4.45
protein. Because of the weak inhibitory activity of L and L
Pt1
Lb
2.0 μM (0.5 μM)
in telomerase activity assay, only Pt1 and Pt2 were selected for
quantitative RT-PCR and Western blot assay. Total RNA from
SK-OV-3/DDP cells was extracted and reversely transcribed
into cDNA to investigate the effect of the two Pt(II) complexes
on c-myc and hTERT gene transcription. The cDNA was then
used as a template in the specific amplification of c-myc and
hTERT sequence by real-time PCR, with glyceraldehyde
phosphate dehydrogenase (GAPDH) as internal control.
As shown in Figure 14A, generally, both Pt1 and Pt2 could
increase the related protein products of hTERT with the
increase of Pt1 and Pt2 concentrations. To further study the
translation of hTERT gene, whole cell lysate of SK-OV-3/DDP
1
0
2
.5 μM (0.5 μM)
FPu18T
.5 μM
FMidG4T
F32T + H20M
F21T
.0 μM (0.5 μM)
2.74 (0.24)
4.48 (2.65)
12.30 (4.28)
2.75
2.0 μM (0.5 μM)
1
0
2
.5 μM (0.5 μM)
FPu18T
.5 μM
FMidG4T
F32T + H20M
F21T
.0 μM (0.5 μM)
1.82 (0.76)
8.54 (3.42)
14.54 (5.15)
6.77
Pt2
2.0 μM (0.5 μM)
1
0
2
.5 μM (0.5 μM)
FPu18T
.5 μM
FMidG4T
F32T + H20M
.0 μM (0.5 μM)
2.32 (0.39)
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5
Figure 14. qRT-PCR to determine the expression of hTERT, c-myc, and bcl-2 in the SK-OV-3/DDP cell line treated with Pt1 and Pt2. 5 × 10 SK-
2
OV-3/DDP cells were treated in a 25 cm flask with medium (no drug), 5.0 μM of Pt1 or Pt2 for 48 h, and the total RNA was extracted and
subjected to reverse transcription, followed by PCR for hTERT (A), c-myc (B), or bcl-2 (C) and GAPDH (control).
Figure 15. (A) Western blot to determine the translation of hTERT in SK-OV-3/DDP cell line treated with 2.5, 5.0, 10.0 μM Pt1, Pt2 and cisplatin
for 48 h. (B) Densitometric analysis of 122 kDa cleaved hTERT fragment normalized to 42 kDa β-actin band from part A. The relative expression of
each band is (density of each band)/(density of actin band). Mean and SD values were from three independent measurements.
Figure 16. (A) Western blot to determine the translation of c-myc in SK-OV-3/DDP cells treated with 2.5, 5.0, 10.0 μM Pt1, Pt2 and cisplatin for 48
h. (B) Densitometric analysis of 60 kDa cleaved c-myc fragment normalized to 42 kDa β-actin band from part A. The relative expression of each
band is (density of each band)/(density of actin band). Mean and SD values were from three independent measurements.
cells was prepared, and hTERT protein was detected in
Western blot analysis, with β-actin as loading reference. Figure
consistent with the previously reported association between
myc overexpression and the induction of telomerase activity.
39
1
5 shows that both Pt1 and Pt2 were able to up-regulate the
However, Pt2 could down-regulate the transcription of c-myc,
which still further up-regulated the expression of hTERT in SK-
OV-3/DDP cells. The mechanisms may be that after c-myc
binds to E-box, it directly activates the expression of hTERT
gene, and such activation is not correlated with cell
proliferation and independent of de novo synthesis of proteins.
Thus, although Pt2 inhibited c-myc expression, the gene
transcription and translation of oncogene hTERT in concen-
tration-dependent mode; furthermore, Pt2 was more effective
than Pt1. These results were further confirmed by RT-PCR and
Western blot methods.
The inhibitory activity of Pt1 and Pt2 on the expression of c-
myc gene was also studied using RT-PCR and Western blot
assay. As shown in Figure 14B, Pt1 increased the protein
products of c-myc compared with the control, whereas Pt2
down-regulated the transcription and translation of oncogene c-
myc. Such observations were further confirmed by the Western
blot results of the translation of c-myc in SK-OV-3/DDP cells
treated with Pt1 and Pt2, respectively (Figure 16). All these
results confirmed that binding of Pt1 to c-myc promoter region
increased the promoter stability and up-regulated c-myc
transcription, which further up-regulated the expression of
hTERT, as detected by real-time RT-PCR. The same results
40
hTERT was still overexpressed in cells.
Transcription and Translation of bcl-2 Gene. RT-PCR
was carried out to investigate the effect of Pt1 and Pt2 on bcl-2
gene transcription in SK-OV-3/DDP cells. As shown in Figure
14C, Pt2 down-regulated the transcription of bcl-2; on the
contrary, Pt1 increased the RNA level of bcl-2. While 10 μM
Pt2 led to a 65% decrease of bcl-2 mRNA level, Pt1 generated
an opposite effect, suggesting that Pt1 and Pt2 exerted
differential effects on bcl-2 gene transcription. These
observations were further confirmed by Western blot analysis.
As shown in Figure 17, Pt2 dose-dependently decreased the
39
were also observed by other groups; e.g., our results are also
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Figure 17. (A) Western blot to determine the translation of Bcl-2 in SK-OV-3/DDP cell lines treated with 2.5, 5.0, 10.0 μM Pt1, Pt2 and cisplatin
for 48 h. (B) Densitometric analysis of 26 kDa cleaved bcl-2 fragment normalized to 42 kDa β-actin band from part A. The relative expression of
each band is (density of each band)/(density of actin band). Mean and SD values were from three independent measurements.
Figure 18. Transfections of EGFP (A) and c-myc (B) plasmid vector in SK-OV-3/DDP cells.
protein products of bcl-2, whereas Pt1 up-regulated the
translation of bcl-2. These results verified that Pt2 could
inhibit the activity of bcl-2 gene via its special interaction with
11
the promoter region of Pu39, but Pt1 did not undergo such
pathway.
Transfection. In order to verify whether Pt1 and Pt2 can
directly regulate c-myc promoter (Pu27) and the expression of
hTRET in SK-OV-3/DDP cells, consequently inhibiting the
telomerase function, we constructed EGFP and c-myc gene
41
vectors according to the methods reported by Chalfie and
4
2
He. After successful transfection of SK-OV-3/DDP cells, an
amount of 5.0 μM Pt1 and Pt2 was added to these cells,
respectively, and incubated for 24 h. Subsequent fluorescent
microcopy study showed that (Figure 18A) after EGFP plasmid
transfection, SK-OV-3/DDP cells displayed green fluorescence,
indicating a successful transfection. As shown in Figure 18B, in
comparison with the control, Pt1 up-regulated the hTRET
expression in SK-OV-3/DDP cells, resulting in inhibition of the
activity of telomerase via activation of c-myc promoter, whereas
Pt2 achieved these effects by down-regulating the activity of c-
myc promoter. It is interesting that such different effects may
Figure 19. Inhibitory rates (%) and TelIC50 of L , L , Pt1, and Pt2 on
telomerase.
a
b
the telomerase inhibition properties of Pt1 and Pt2 were
significantly improved upon oxoisoaporphine alkaloids ligating
Pt(II). It is interesting that the inhibitory potential on
telomerase was enhanced with a hydroxy group at the 6-
b
correlate with the 6-hydroxy group at the 6-position of L in
Pt2, which is in agreement with the results mentioned above.
Telomerase Inhibition (TRAP Assay). Many studies have
shown that stabilization of G-quadruplexes or ligand-induced
quadruplex formation by the telomeric G-rich strand can inhibit
b
position of L in Pt2.
a
b
In order to further determine the ability of L , L , Pt1, and
Pt2 to inhibit the telomerase in SK-OV-3/DDP tumor cells, we
8,18a,43
the activity of telomerase.
Telomerase is overexpressed in
a
b
treated SK-OV-3/DDP cells with 5.0 μM L , L , Pt1, and Pt2,
respectively, for 48 h. The telomerase activity was assayed using
a PCR-TRAP ELISA kit (Roche, USA) according to the
manufacturer’s guide. As shown in Figure 20, the cellular
inhibitory effect of Pt1 and Pt2 on telomerase was obviously
8
5−90% of the human tumor cells but has undetectable activity
44
in most of the normal somatic cells. Therefore, we conducted
TRAP assay to further characterize telomerase activity.
Solutions of L , L , Pt1, and Pt2, respectively, were added to
a
b
a
b
a telomerase reacting mixture containing extract from Hep-G2
higher than that of L and L ; moreover, the telomerase
inhibitory property of Pt2 was significantly better than that of
Pt1, which agrees well with the results of FRET and CD assays.
Together the above studies indicated that Pt1 could exert
4
5
cells, which express high levels of telomerase. The inhibition
a
b
ratios of L , L , Pt1, and Pt2 were summarized in Figure 19 and
Table S7 (Supporting Information). As indicated in Table S7,
K
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intrinsic characteristic amino acids of the protein, i.e.,
tryptophan and tyrosine. Changes in the emission spectra of
tryptophan are common in response to protein conformational
transitions, subunit associations, substrate binding, or denatura-
tion. Therefore, the intrinsic fluorescence of BSA can provide
considerable information on their structure and dynamics and is
often utilized in the study of protein folding and association
reactions. The quenching of emission intensity of BSA was
observed in the presence of the complex because of the changes
in tryptophan environment resulting from possible changes in
47
BSA protein secondary structure.
The interactions of BSA with Pt1 and Pt2 were studied by
fluorescence spectroscopy at 296, 303, and 310 K. The BSA
solution (0.5 μM) was titrated with various concentrations of
Pt1 and Pt2 (0−8 μM), respectively. The effects of Pt1 and Pt2
on the fluorescent emission spectra of BSA are shown in
Figures S42 and S43 and Table S9 (Supporting Information).
The addition of Pt1 and Pt2 to the solution of BSA resulted in
a significant decrease of the fluorescent intensity of BSA at 345
nm, suggesting a definite interaction of Pt1 and Pt2 with BSA
a
b
Figure 20. Inhibitory rates (%) of L , L , Pt1, and Pt2 on telomerase
in SK-OV-3/DDP tumor cells.
effects by up-regulating the transcription and translation of c-
myc, hTERT, and bcl-2 genes, while Pt2 might play its role via
other pathways in SK-OV-3/DDP tumor cells.
48
protein.
Protein Binding Studies. Anticancer activity may be
strongly affected by drug−protein interactions in the blood-
stream. Serum albumin, the most abundant and important
protein in the plasma, is the major transport protein. Albumin is
capable of binding many endogenous and exogenous drugs
reversibly. In addition, it may aid in the selective delivery of
drugs to the tumor region and facilitate drug access into the
cell. The tryptophan emission quenching experiments were
carried out to investigate the interaction of Pt1 and Pt2 with
proteins by using bovine serum albumin (BSA) as model
protein. Generally, the fluorescence of BSA is derived from two
In the case of Pt1, the Stern−Volmer constants were
calculated from the slope of the plot F /F versus [Pt1] (Figures
0
5
−1
S42 and S43), and the values were found to be 0.80 × 10 M
5
−1
5
−1
(296 K), 0.87 × 10 M (303 K), and 1.33 × 10 M (310 K),
respectively (Table S9). However, the Ksv of Pt2 was not
suitable to calculate by Stern−Volmer equation because the
correlation between F /F and [Pt2] was nonlinear. These
0
4
6
results suggested that Pt2 exhibited higher binding affinity than
Pt1, and the quenching of both Pt1 and Pt2 was dynamic.
Growth Inhibition of HCT-8 and NCI-H460 Xenograft
in Vivo. To test the efficacy of Pt1 and Pt2 to inhibit tumor
Figure 21. In vivo anticancer activity of Pt1 and Pt2 in mice bearing HCT-8 xenograft. (A) Effect of Pt1, Pt2 ((6 mg/kg)/2 days), cisplatin ((2 mg/
3
kg)/2 days), or vehicle (5% DMSO in saline, v/v) on growth of tumor xenograft. Tumor growth is tracked by the mean tumor volume (mm ) ± SD
(
n = 6−7) and calculated as % tumor growth inhibition (%TGI). (B) Body weight change (presented as % change from initial weight). (C) Tumor
weight was recorded after the mice were killed: (∗∗∗) P < 0.001, (∗∗) P < 0.01, (∗) P < 0.05. (D) Photographs of tumor from treatment groups and
vehicle group.
L
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Figure 22. In vivo anticancer activity of Pt1 and Pt2 in mice bearing NCI-H460 xenograft. (A) Effect of Pt1, Pt2 ((6 mg/kg)/2 days), cisplatin ((2
mg/kg)/2 days), or vehicle (5% DMSO in saline, v/v) on growth of tumor xenograft. Xenograft was established sc in anthymic mice and allowed to
3
reach a volume of 100−300 mm before treatment. Date represent tumor volumes as mean ± SD (n = 6) and calculated as % tumor growth
inhibition (%TGI). (B) Body weight change (presented as % change from initial weight). (C) Tumor weight was recorded after the mice were killed:
(∗) P < 0.05. (D) Photographs of tumor from treatment groups and vehicle group.
growth in vivo, the HCT-8 and NCI-H460 tumor xenograft
nude mice were used. In the two models, mice with tumors at
the volume of 100−300 mm were randomized into vehicle
control and treatment groups (n = 6−7/group) and treated via
ip injection with Pt1, Pt2, and cisplatin, respectively, every
other day. As shown in Figure 21A and Figure 21B, treatment
groups were injected with Pt1 and Pt2 at two doses (6 or 3
complexes exhibited better antitumor activity than cisplatin
(TGI, 31.8%; IR, 25.5%).
3
Throughout the treatment period, there was no significant
difference in body weight change among the control group and
Pt1- and Pt2-treated groups (Figure 21B), and the animals
appeared healthy, with no visible signs of pain, distress, or
discomfort. No signs of peritonitis or damage to organs were
observed (Figure S44 in Supporting Information). Although
cisplatin treatment also caused tumor growth inhibition, its
toxicity was apparent, as evidenced by loss of body weight
compared to vehicle control (Figure 22B).
(mg/kg)/2 days) for 16 days in the HCT-8 tumor model and
at one dose (6 (mg/kg)/2 days) in NCI-H460 model; cisplatin
was injected at doses of 2 (mg/kg)/2 days, and control mice
received the vehicle (5% DMSO in saline, v/v).
In the human colorectal tumor HCT-8 xenograft model, as
shown in Figure 21 and Tables S10−S12 (Supporting
Information), the treatment with Pt1 and Pt2 at 6 mg/kg
resulted in a statistically significant reduction in tumor volume
with tumor growth inhibition (TGI) of 48.4% and 42.9%
respectively (P < 0.001 and P < 0.01, respectively, vs vehicle
control group), which was greater than that of cisplatin (TGI,
The preliminary results indicated that Pt1 and Pt2 exhibited
high safety in vivo and were even more effective in inhibiting
tumor growth in the HCT-8 and NCI-H460 xegograft mouse
models than cisplatin.
Structure−Activity Relationships. On the basis of the
results mentioned-above on the biological activity and
anticancer mechanism studies, some SAR trends between the
a
b
35.7%). Similar results were observed in the tumor weight
L , L , Pt1, Pt2 and the differences in biological activity can be
change (Figure 21C); i.e., at a dose of 6 mg/kg, Pt1 (inhibition
rate, IR, 38.5%) and Pt2 (IR, 37.7%) displayed better antitumor
activity than cisplatin (IR, 30.4%). In addition, the data showed
that treatment with Pt1 presented a dose-dependent inhibition
of tumor growth.
In the human lung cancer NCI-H460 xenograft model, as
shown in Figure 22 and Tables S13−S15 (Supporting
Information), after 16 days of treatment, Pt2 at a dose of 6
mg/kg caused a substantial reduction in both tumor volume
and weight with a TGI of 50.1% and IR of 44.8% (P < 0.05). By
contrast, Pt1 displayed a lower activity on NCI-H460 tumor
growth in vivo (TGI, 38.1%; IR, 35.9%). However, both
established. First, in general, the combination of platinum with
corresponding oxoisoaporphine alkaloids L and L appears to
a
b
produce an increase biological activity with respect to free
a
b
ligands L and L . Second, excluding some exceptions, the
homologous series of complexes Pt1, Pt2, differing only in the
ligands, also show some structure−activity relationships. For
example, against NCI-H460 and SK-OV-3/DDP, the presence
of a 6-hydroxyl group in ligand (Pt2) seems to generate an
increase in cytotoxicity with respect to the ligand lacking such
hydroxyl group (Pt1). From the in vivo anticancer activity of
Pt1 and Pt2 in mice bearing NCI-H460 xenograft, such SARs
trend also was observed. Furthermore, the anticancer action
M
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Figure 23. Proposed antitumor mechanisms for Pt1 and Pt2.
Table 4. Sequences of Oligomers (Primers) Used in This Work
oligomer
sequence
c-kit-1
c-kit-2
HTG21
Pu22
5′-CGGGCGGGCACGAGGGAGGGT-3′
5′-CGGGCGGGCGCTAGGGAGGGT-3′
5′-GGGTTAGGGTTAGGGTTAGGG-3′
5′-TGAGGGTGGGTAGGGTGGGTAA-3′
FMidG4T
FPu18T
F21T
5′-FAM-CGGGCGCGGGAGGAAGGGGGCGGGAGC-TAMRA-3′
5′-FAM-AGGGTGGGGAGGGTGGGG-TAMRA-3′
5′-FAM-GGGCTAGGGCTAGGGCTAGGG-TAMRA-3′
5′-CAATCGGATCGAATTCGATCCGATTG-3′
5′-TGGGGAGGGTGGGGAGGGTGGGGAAGG-3′
5′-AGGGGCGGGCGCGGGAGGAAGGGGGCGGGAGCGGGGCTG-3′
5′-AGGGGCAAACGCAAAAGGAAAAAAACGGGAGCGGGGCTG-3′
Ts: 5′-TGGTGCTCCATGAGGAGACA-3′
ds26
Pu27
Pu39
mutPu39
c-myc
Cx: 5′-GTGGCACCTCTTGAGGACCT-3′
GAPDH
hTERT
Ts: 5′-GCCTCTTGCACGACCAACTG-3′
Cx: 5′-CGGAAGGCCATGCCT GTCAG-3′
Ts: 5′-TGGTCTCCACGAGCCTCCGAGCG-3′
Cx: 5′-CATCCACATAGAGGCCACCACGT-3′
5′-FAM-CCGCATCTCTACATTCAAGAACTGGCATGCGG-TAMRA-3′
5′-GCCAGTTCTTGAATGTAGAG-3′
F32T
H20M
mechanism studies also confirmed that such SARs might
originate from different biological behavior of Pt1 and Pt2. It
could be demonstrated that Pt2 with 6-hydroxy group in Lb
exhibited stronger induction of cell senescence and apoptosis
and telomeric region and led to more severe telomeres damage
in SK-OV-3/DDP cells than Pt1.
CONCLUSION
■
To develop effective G-quadruplex DNA binders exerting
inhibitory effect on telomerase activity, two oxoisoapopine-
organoplatinum(II) complexes (Pt1 and Pt2) were prepared
a
than Pt1 without 6-hydroxy group in L ; Pt2 displayed stronger
telomerase inhibition ability and better selectivity for G4-DNA
N
DOI: 10.1021/jm5012484
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
and evaluated for antitumor activity in vitro and in vivo. The
binding experimental results from various biological assays,
including FID assay, FRET assay, fluorescence titration, and
CD, revealed that Pt2 had relatively different but stronger
binding affinity to G-quadruplex DNA in telomere and
promoters of c-myc and bcl-2 than Pt1. Further cellular assays,
including uptake assay, flow cytometry, RT-PCR, Western
blotting, immunofluorescence, transfection, and TRAP assay,
gave evidence that Pt1 and Pt2 exerted its antitumor effect on
tumor cells. As shown in Figure 23, we propose the action
mechanisms that the effect of Pt1 and Pt2 on tumor cells may
involve the major pathways. Both complexes could activate
caspase-3/9 to initiate cell apoptosis, and the effect of Pt2
appeared stronger. Pt2 arrested cell growth at G1 phase in
contrast to Pt1 (at G2/M phase). Pt2 exerted its antitumor
mechanism mainly via its inhibition of telomerase activity and
interaction with telomeric, c-myc, and bcl-2 G-quadruplexes,
and inducing senescence as well as apoptosis finally caused SK-
OV-3/DDP tumor cells’ death. Compared with Pt2, Pt1 also
can induce senescence and apoptosis of SK-OV-3/DDP cells,
for which the activity was weaker than that of Pt2, but its
process was mainly mediated by apoptotic pathway owing to
interaction with telomeric G-quadruplex, and inhibition
telomerase activity. The different biological activities of Pt1
and Pt2 may be correlated with the structural feature of
Tris-HCl buffer. All the spectroscopic experiments were performed at
room temperature.
Instrumentation. Infrared spectra were obtained on a PerkinElm-
er FT-IR spectrometer. Elemental analyses (C, H, N) were carried out
on a PerkinElmer series II CHNS/O 2400 elemental analyzer. NMR
spectra were recorded on a Bruker AV-500 NMR spectrometer.
Fluorescence measurements were performed on a Shimadzu RF-5301/
PC spectrofluorophotometer. ESI-MS spectra were performed on
Thermofisher Scientific Exactive LC−MS spectrometer (Thermal
Elctronic, USA). The circular dichroic spectra of DNA were obtained
on a JASCO J-810 automatic recording spectropolarimeter operating
at 25 °C. The region between 200 and 400 nm was scanned for each
sample. MTT assay and fluorescent intercalator displacement (FID)
assay were performed on M1000 microplate reader (Tecan Trading
Co. Ltd., Shanghai, China). Cell cycle analysis results were recorded
on FACS Aria II flow cytometer (BD Biosciences, San Jose, CA, USA).
FRET assay was performed on 7500fast RealTime PCR (ABI Co. Ltd.,
USA)
a
a
Synthesis of 1-Azabenzanthrone (L ) and Pt1. L was
24
synthesized by using modified method reported by Tang et al.
The extract was condensed under reduced pressure, and the resultant
precipitate was washed, dried, and sublimed at 130−140 °C at a
a
1
pressure of 1 mmHg to give the product L as a light yellow solid. H
NMR (500 MHz, DMSO-d ) δ 8.78 (dd, J = 5.6, 3.3 Hz, 2H), 8.52
6
(dd, J = 7.2, 1.1 Hz, 1H), 8.41 (dd, J = 8.2, 1.0 Hz, 1H), 8.25 (dd, J =
7.9, 1.2 Hz, 1H), 8.06−8.03 (m, 1H), 8.02−8.01 (m, 1H), 7.89 (td, J =
13
7.6, 1.4 Hz, 1H), 7.74−7.71 (m, 1H). C NMR (126 MHz, DMSO-
) δ 182.71, 147.96, 144.49, 136.59, 135.28, 134.76, 134.62, 132.20,
131.50, 131.10, 130.19, 128.54, 127.47, 125.37, 122.39, 121.92. ESI-
d
6
b
oxoisoapopine ligand in Pt2, for L contains a 6-hydroxyl
+
+
a
MS m/z: 278.2 [M + CH CH OH + H ] . IR (KBr): 3434, 3056,
3 2
group, which is absent in the L of Pt1. Remarkably, our
3
9
005,1983, 1667, 1613, 1596, 1580, 1438, 1393, 1292, 1218, 1205,
49, 851, 795, 754, 702, 650, 584 cm . Elemental analysis calcd (%)
findings illustrated that Pt1 and Pt2 exhibited high safety in
vivo and even more effective inhibitory effect on tumor growth
in the HCT-8 and NCI-H460 xenograft mouse model than
cisplatin. Taken together, the two organoplatinum(II) com-
plexes Pt1 and Pt2 exhibited antitumor effects on SK-OV-3/
DDP cells in vitro via both cell senescence- and cell apoptosis-
mediated mechanisms. Therefore, Pt1 and Pt2 may have the
potential to be further developed into safe and effective
anticancer agents.
−1
for C H NO: C 83.10, H 3.92, N 6.06. Found: C 83.21, H 3.90, N
16
9
5
.93.
The complex [PtL (DMSO)Cl] (Pt1) was prepared by treating L
(0.0231 g, 0.1 mmol) with Pt(DMSO) Cl (0.0422 g, 0.1 mmol) in
a
a
2
2
a
ethanol/water (10:1) under solvothermal conditions. L deprotonation
took place upon the addition of Pt(II) ion to the ligand solution to
form the chelated complex in the presence of pyridine (Figure 1).
Brown block crystals suitable for X-ray diffraction analysis were
1
harvested. Yield (0.0455 g, 75%). H NMR (500 MHz, DMSO-d ) δ
6
8
.86−8.83 (m, 2H), 8.58 (d, J = 7.2 Hz, 1H), 8.30 (d, J = 7.7 Hz, 1H),
EXPERIMENTAL METHODS
8.17−8.14 (m, 1H), 7.94−7.91 (m, 1H), 7.85−7.83 (m, 1H), 7.43−
■
7
1
1
.40 (m, 1H). 13C NMR (125 MHz, DMSO-d ) δ 182.8, 182.3, 147.5,
6
Materials. All chemical reagents were commercially available and
used without further purification, unless noted specifically. The purity
of all target compunds used in the biophysical and biological studies
was ≥95%. All compounds were toutinely checked by HPLC. Thiazole
orange (TO), Tris, RNase A, and propidium iodide (PI) were
purchased from Sigma. 53BP1, TRF1, TRF2, bcl-2, hTERT, and c-myc
were purchased from Abcam. DNA oligomers (highly polymerized
stored at 4 °C; long-term storage at −20 °C) are listed in Table 4 and
were obtained from Shanghai Sangon Biological Engineering
Technology & Services (Shanghai, China). The DNA concentration
per pair was determined based on the absorbance value at λ = 260 nm
44.0, 137.5, 134.3, 134.2, 131.1, 130.6, 129.7, 128.1, 127.0, 125.4,
−
24.9, 122.3, 121.4, 40.4. ESI-MS m/z: 497.9 [M − DMSO + Cl] . IR
(KBr): 3474, 3071, 3000, 2912, 1659, 1515, 1585, 1564, 1226, 1124,
−1
1
291, 1032, 1017, 979, 941, 785, 720, 685, 650 cm . Elemental
analysis calcd (%) for C H ClNO PtS: C 40.12, H 2.62, N 2.60.
18
14
2
Found: C 40.35, H 2.75, N 2.55.
b
Synthesis of 6-Hydroxyloxoisoaporphine Alkaloid (L ) and
Pt2. A mixture of phthalic anhydride (30 g, 0.2 mol) and 4-bromine
phenylethylamine (40.5 g, 0.2 mol) in anhydrous ethanol was refluxed
for 6 h. After cooling to 0−5 °C, the formed crystals of 4-
bromophenylethylphthalimide (49.5 g, 75%) were filtered and washed
with ethanol. The product was pure enough for the next reaction.
To a mixture of anhydrous aluminum chloride (75 g, 0.56 mol) and
sodium chloride (15 g, 0.26 mol), 4-bromophenylethylphthalimide (33
g, 0.1 mol) was slowly added at 180 °C for 30 min. The reaction was
allowed to continue at 220−230 °C for 3.5 h. The product was cooled,
finely ground, and poured slowly into concentrated sulfuric acid (650
mL) at 90 °C. The mixture was stirred and heated at 230−240 °C for
3.5 h. After being cooled, the solution was placed on ice. Sodium
hydroxide was added until pH 2−3 was obtained, and the resultant
precipitate was filtered and washed in turn with dilute aqueous sodium
hydroxide and water to give the crude product 6-bromine-
oxoisoaporphine, which was extracted with acetic acid.
5
−1
−1
(
ε260 = 3.81 × 10 M (strand) 9cm for DNA oligomers by using
4
UV/vis absorption spectroscopy. Unless otherwise stated, spectro-
scopic titration experiments were carried out in 10 mM Tris-HCl (pH
7
.35) containing 100 mM KCl. Telomerase PCR ELISA kits were
purchased from Roche (USA). The total RNA isolation kit and the
two-step RT-PCR kit were purchased from TIANGEN. All tumor cell
lines were obtained from the Shanghai Institute for Biological Science
(
China).
Stock solutions of all the compounds (2 mM) were made in
DMSO. Further dilutions to working concentrations were made with
corresponding buffer. The formation of all intramolecular G-
quadruplexes was analyzed as follows: the oligonucleotide samples,
dissolved in Tris-KCl-HCl buffer, were heated to 95 °C for 10 min,
gently cooled to room temperature, and then incubated at 4 °C
overnight. The human telomeric was extemporaneously prepared in
The extract was condensed under reduced pressure, and the
resultant precipitate was washed, dried, and sublimed at 130−140 °C
at a pressure of 1 mmHg to give the 6-bromine-oxoisoaporphine (9.55
O
DOI: 10.1021/jm5012484
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
g, 31%) as a light yellow solid. The crystal of 6-bromine-
oxoisoaporphine was prepared in methanol under solvothermal
humidified atmosphere of 5% CO /95% air. After removal of the
2
supernatant, DMSO (100 μL) was added to dissolve the formazan
crystals. The absorbance was read on a microplate reader at 490/630
nm. The cytotoxicity was evaluated based on the percentage of cell
survival compared with the negative control. The final IC50 values were
calculated by the Bliss method (n = 5). All tests were repeated in at
least three independent trials.
1
conditions. H NMR (500 MHz, CDCl -d ) δ 8.94 (d, J = 7.9 Hz,
3
1
1
H, 2-H−Ar), 8.82 (d, J = 5.6 Hz, 1H, 11-H−Ar), 8.60 (s, 1H, 4-H−
Ar), 8.44 (d, J = 7.7 Hz, 1H, 8-H−Ar), 8.16 (s, 1H, 5-H−Ar), 7.86 (t, J
7.5 Hz, 1H, 10-H−Ar), 7.70 (m, 2H, 8-H−Ar and 9-H−Ar). IR
KBr): 3060, 1667, 1595, 1396, 1283, 1190, 1092, 897, 705, 642, 595
=
(
−1
cm .
To a stirred refluxing suspension of 6-bromine-oxoisoaporphine
3.1 g, 0.01 mol) in 80 mL of EtOH/H O (5:3), NaOH (4.0 g, 0.1
Uptake of Platinum Complexes in Cells. SK-OV-3/DDP cells
(∼10 million cells) were treated with 10 μM Pt1 and Pt2, respectively,
(
for 8 h at 37 °C in a humidified 5% CO incubator. The medium was
2
2
mol) was added. The mixture was stirred at reflux for 12 h, cooled to
room temperature, filtered, and washed with ether and water. The
crude solid was purified by column chromatography with petroleum
ether/ethyl acetate (100:1) elution to afford a yellow solid 6-hydroxyl-
then removed, and the cells were washed with 5 mL of PBS, scraped,
and collected in 5 mL of PBS. The scrapped cells were collected by
centrifuging at 2500 rpm for 10 min. The obtained cell pellet was
dissolved in 1 M NaOH (1 mL) and diluted with 2% (v/v) HNO (5
3
b
1
oxoisoaporphine alkaloid (L ) (1.24 g, 50%). H NMR (500 MHz,
mL) for determining the whole cell platinum content. Another set was
treated similarly to isolate nuclear fraction, nuclear proteins,
membrane proteins, and cytoplasmic protein as described by Schreiber
DMSO-d ) δ 15.01 (s, 1H), 8.88 (d, J = 8.0 Hz, 1H), 8.80 (d, J = 5.2
6
Hz, 1H), 8.38 (d, J = 9.1 Hz, 1H), 8.35 (d, J = 7.9 Hz, 1H), 8.00 (d, J
2
6
=
5.2 Hz, 1H), 7.96−7.94 (m, 1H), 7.80−7.77 (m, 1H), 7.57 (d, J =
et al. The final solution was made up to 5 mL using 2% (v/v) HNO3,
and the amount of platinum taken up by the cells was determined by
ICP-MS. The instrument was calibrated for platinum using standard
solutions containing 10, 50, 100, 500, and 1000 ppb platinum.
Confocal Morphological Examination. The AO/EB molecular
9
1
1
3
1
.1 Hz, 1H). 13C NMR (125 MHz, DMSO-d ) δ 186.1, 168.5, 144.0,
6
43.8, 139.7, 136.7, 134.8, 130.7, 130.5, 123.0, 126.6, 125.2, 122.6,
21.2, 108.1. ESI-MS m/z: 473.0 [M − 2Cl + CH OH] . IR (KBr):
423, 3065, 2950, 1636, 1657, 1448, 1423, 1380, 1352, 1267, 1242,
+
3
−1
27
163, 1141, 889, 852, 790, 754, 729, 589, 551 cm . Elemental analysis
probes were used to detect apoptotic cells. To examine whether Pt1
calcd (%) for C H NO : C 77.72, H 3.67, N 5.67. Found: C 77.65, H
and Pt2 induce apoptosis in SK-OV-3/DDP cell line, cells were plated
in six-well plates and treated with 5.0 μM Pt1 and Pt2 for 12 h,
respectively. The cells were then trypsinized, harvested, suspended in
PBS, stained with 100 μg/mL AO and 100 μg/mL EB for 10 min at
room temperature, respectively, and visualized immediately by
confocal microscopy (CarlZeiss LSM 710, Germany) with excitation
wavelength of 488 nm (AO) or 510 nm (EB). Apoptotic cells were
defined based on the nuclear morphology changes such as chromatin
condensation and fragmentation.
16
9
2
3
.58, N 5.74.
The complex [PtL (DMSO)Cl] (Pt2) was prepared by treating L
0.0246 g, 0.1 mM) with Pt(DMSO) Cl (0.0422 g, 0.1 mM) in
b
b
(
2
2
b
methanol/water (25:1) under solvothermal conditions. L deprotona-
tion took place upon the addition of Pt(II) ion to the ligand solution
to form the complex in the presence of pyridine (Figure 1). Brown
block crystals suitable for X-ray diffraction analysis were harvested.
1
Yield (0.0499 g, 90%). H NMR (500 MHz, DMSO-d ) δ 14.57 (s,
6
1
H), 9.01 (d, J = 7.8 Hz, 1H), 8.91 (d, J = 5.2 Hz, 1H), 8.56 (d, J = 7.6
Fluorescence Morphological Examination. To examine
whether Pt1 and Pt2 induce apoptosis in SK-OV-3/DDP cancer
cells, the cells were plated in six-well plates and treated with 5, 10, and
20 μM Pt1 and Pt2 for 12 h, respectively. After the treatment, the cells
were washed with PBS and fixed for 10 min at room temperature. The
cells were rinsed twice in PBS and stained with a fluorescent dye,
Hoechst 33258 fluorescent dye (Beyotime, China), at room
temperature in the dark for 10 min. The cells were then washed
twice with PBS, examined, and immediately photographed under a
fluorescent microscope (Nikon Te2000, Japan) with excitation
wavelength of 330−380 nm. Apoptotic cells were defined on the
basis of nuclear morphology changes such as chromatin condensation
and fragmentation.
SA-β-Gal Assay. After long-term culture of cells with Pt1 and Pt2,
the growth medium was aspirated, and the cells were fixed in 2%
formaldehyde/0.2% glutaraldehyde for 15 min at room temperature.
The fixing solution was removed; the cells were gently washed twice
with PBS and then stained using β-Gal staining solution containing 1
mg/mL 5-bromo-4-chloro-3-indolyl-β-D-galactoside overnight at 37
°C. The staining solution was removed, and the cells were washed
three times with PBS and viewed under an optical microscope and
photographed (Nikon Te2000, Japan).
Hz, 1H), 8.14 (m, 2H), 7.85 (t, J = 7.5 Hz, 1H), 7.50 (t, J = 7.5 Hz,
+
1
3
1
H). ESI-MS m/z: 473.0 [M − DMSO − Cl + CH OH] . IR (KBr):
3
418, 2972, 2923, 1626, 1602, 1563, 1476, 1421, 1382, 1256, 1223,
−1
086, 1045, 990, 877, 847, 806, 790, 738 cm . Elemental analysis
calcd (%) for C H ClNO PtS: C 38.96, H 2.54, N 2.52. Found: C
18
14
3
3
8.83, H 2.43, N 2.58.
X-ray Crystallography. The data collection of single crystals of Lb
and Pt1 and Pt2 was performed on a SuperNova CCD diffractometer
equipped with graphite monochromated Mo Kα radiation (λ =
0
direct methods and refined using SHELX-97 programs. The non-
hydrogen atoms were located in successive difference Fourier
synthesis. The final refinement was performed by full-matrix least-
squares methods with anisotropic thermal parameters for no-hydrogen
.710 73 Å) at room temperature. The structures were solved with
50
2
atoms on F . The hydrogen atoms were added theoretically and riding
on the concerned atoms. The paramerters used intensity collection
and refinements are summarized in Tables S1 and S2 together with the
crystal data.
Cytotoxicity Assay and Cellular Morphology. The cell culture
was maintained in RPMI-1640 medium supplemented with 10% fetal
bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL
streptomycin in 25 cm2 culture flasks at 37 °C in a humidified
Flow Cytometric Analysis. In cell cycle analysis, the SK-OV-3/
atmosphere with 5% CO . All cells to be tested in the following assays
DDP cells were maintained with 10% fetal calf serum in 5% CO at 37
2
2
had a passage number of 3−6.
°C. Cells were harvested by trypsinization and rinsed with PBS. After
5
6
T-24, HepG2, SK-OV-3, SK-OV-3/DDP, BEL-7404, NCI-H460,
centrifugation, the pellet (10 −10 cells) was suspended in 1 mL of
PBS and then fixed by dropwise addition of 9 mL of precooled (4 °C)
70% ethanol under violent shaking. After treatment, cells were
collected and fixed with ice-cold 70% ethanol at −20 °C overnight.
Fixed cells were resuspended in 0.5 mL of PBS containing 50 μg/mL
propidium iodide and 100 μg/mL RNase A. The cell cycle distribution
was analyzed by FACS Calibur flow cytometer (BD) and calculated
using ModFIT LT software (BD).
HCT-8, and HL-7702 cells were seeded in 96-well plates ([(5.0 ×
3
1
0 )/180 μL]/well). Cells were grown for 12 h to reach 70%
confluence before treatment, and an amount of 20 μL of tested various
concentrations of compounds was added to each well. The final
concentrations of the tested compounds were 1.25, 2.5, 5.0, 10.0, and
2
0.0 μM, respectively. The resultant solutions were subsequently
added to a set of wells. Control wells contained media with 0.5%
DMSO. The microtiter plates were incubated at 37 °C in a humidified
Apoptosis Analysis. Apoptosis was detected by flow cytometric
atmosphere of 5% CO /95% air for another 2 days. At the end of each
analysis of annexin V staining. Annexin V-FITC/PI assay was
2
5
1
incubation period, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide (MTT) solution (10 μL, 5 mg/mL) was added to
each well, and the cultures were incubated further for 6 h at 37 °C in a
performed as previously described. Briefly, adherent SK-OV-3/
DDP cells were harvested and suspended in the annexin-binding buffer
5
(5 × 10 cells/mL). Then cells were incubated with annexin V-FITC
P
DOI: 10.1021/jm5012484
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Journal of Medicinal Chemistry
Article
and PI for 1 h at room temperature in the dark and immediately
analyzed by flow cytometry. The data are presented as biparametric
dot plots showing PI red fluorescence vs annexin V-FITC green
fluorescence.
vs complex concentration; [Q] is the total concentration of quencher;
K is the bimolecular quenching constant; and τ is the average lifetime
q
0
−8
of protein in the absence of quencher (its value is 10 s).
6
Telomerase Inhibition (TRAP Assay). A total of 5 × 10 tumor
cells untreated or treated with compound were pelleted and lysed for
30 min on ice. The lysate was centrifuged at 13 000 rpm for 30 min at
4 °C; the supernatant was collected and stored at −80 °C before use.
Telomerase inhibition efficiency of the compound was measured by
telomeric repeat amplification protocol enzyme-linked immunosorbent
Immunofluorescence. SK-OV-3/DDP cells were grown on
polylysine-coated coverslips, rinsed in phosphate-buffered saline,
fixed in cold methanol for 20 min, permeabilized for 10 min in 0.5%
Triton X-100 on ice, and blocked in 5% BSA for 30 min at room
temperature. The coverslips were incubated with rabbit monoclonal
anti-53BP1, mouse monoclonal anti-TRF1 and anti-TRF2 (Abcam)
primary antibodies for 3.0 h at 25 °C. The coverslips were washed and
incubated with fluorescein conjugated goat anti-mouse (1:1000) and
rhodamine conjugated goat anti-mouse (1:1000) secondary antibodies.
Finally, the cells stained with 0.1 mg/mL DAPI. Fluorescence images
were captured using confocal microscopy (CarlZeiss LSM 710,
Germany).
5
3
assay (TRAP-ELISA). The procedure was the same as for the TRAP
assay. The telomerase product was detected using an enzyme-linked
probe. Absorbance read by the microplate reader was reported as
A450nm against the blank (reference, A690nm). For RNase treatment, the
maximum value of absorbance for the negative control (2.5 μL of the
corresponding RNase-treated or heated cell extract) should be lower
than 0.25(A450nm − A690nm) units. The absorbance for the positive
control examined in 30 min reaction (cell extract was prepared from
the immortalized telomerase-expressing human kidney cells, and
lyophilizate extract was supplied with the kit) should be higher than
Determination of Caspase-3 and Caspase-9 Activity by Flow
Cytometric Analysis. The measurement of caspase-3 and caspase-9
activity was performed by CaspGLOW fluorescein active caspase-3
and caspase-9 staining kit. After treatment with 10.0 μM Pt1 and Pt2
for 8 h, respectively, the cells in RPMI 1640 medium with 10% FBS at
1
.5(A450nm − A690nm) units.
RNA Extraction. Cell pellets harvested from each well of the
6
a density of 1 × 10 cells/mL were harvested, washed with PBS three
culture plates were lysed in RZ lysis solution. RNA was extracted with
RNAsimple total RNA kit (TIANGEN) according to manufacturer’s
protocol and eluted in distilled, deionized water with 0.1% diethyl
pyrocarbonate (DEPC) to a final volume of 50 μL. RNA was stored at
times, and then resuspended in 300 μL volume; 1 μL of FITC-DEVD-
FMK or FITC-LEHD-FMK was consequently added and incubated
for 1.0 h at 37 °C in 5% CO incubator. The cells were then examined
2
by a FACSAria II flow cytometer equipped with a 488 nm argon laser.
The results were represented as the percent change on the activity
comparing with the control.
−
80 °C before use.
Reverse Transcription Polymerase Chain Reaction (RT-PCR).
Total RNA was used as the template for reverse transcription using the
following protocol: each 20 μL reaction contained 2.0 μL of 10× RT
mix, 2.0 μL of dNTP (2.5 mM), 2.0 μL of oligo-dT15 primer, 1.0 μL
Measurement of Mitochondrial Membrane Potential. The
loss of mitochondrial membrane potential (Δψ) was assessed using a
lipophilic cationic fluorescent probe, JC-1 (5,5′,6,6′-tetrachloro-
of Quant reverse transcriptase, 10 μL of DEPC-H O, and 2 μg of total
2
1
,1′,3,3′-tetraethylbenzimidazolylcarbocyanine; Beyotime). Cells trea-
RNA. The mixture was incubated at 37 °C for 60 min and then
ted with 5.0, 10.0, and 20.0 μM Pt1 and Pt2 for 12 h were incubated
with 5 μg/mL JC-1 for 20 min at 37 °C and examined by fluorescence
microscopy. The emission fluorescence for JC-1 was monitored at 530
and 590 nm, under the excitation wavelength at 488 nm. The orange-
red emission of the dye is attributable to a potential-dependent
aggregation in the mitochondria, which reflects the Δψ. Green
fluorescence reflects the monomeric form of JC-1, appearing in the
cytosol after mitochondrial membrane depolarization.
immediately placed on ice. The synthesized cDNA was stored at −80
°
C. Real-time PCR was performed on 7500 fast RealTime PCR (ABI
Co. Ltd., USA) by using 2.5× Real Master Mix/20× SYBR solution
(
TIANGEN), according to the manufacturer’s protocol. The primer
sequences are shown in Table 4 (c-myc, hTERT, bcl-2, and GAPGH).
The total volume of 20 μL of real-time RT-PCR reaction mixtures
contained 9.0 μL of 2.5× Real Master Mix/20× SYBR solution, 0.25
μM each of forward and reverse primers, 1.0 μL of cDNA, and
nuclease-free water. The program used for all genes consisted of a
denaturing cycle of 3 min at 95 °C, 45 cycles of PCR (95 °C for 20 s,
Spectral Studies of Interactions with GSH. The UV/vis
spectroscopic titrations were carried out at room temperature by
−
5
−5
adding increasing amounts of GSH (0, 2.0 × 10 , 4.0 × 10 , 6.0 ×
5
8 °C for 30 s, and 68 °C for 30 s), a melting cycle consisting of 95 °C
for 15 s, 65 °C for 15 s, and a step cycle starting at 65 °C with a 0.2
C/s transition rate to 95 °C. The specificity of the real-time RT-PCR
−
5
−5
−4
−4
−4
1
0 , 8.0 × 10 , 1.0 × 10 , 1.2 × 10 , and 1.4 × 10 M,
respectively) to a solution of Pt1 or Pt2 at a fixed concentration (2.0 ×
°
−
5
1
0
M) contained in a quartz cell. The time span between two
product was confirmed by melting curve analysis. The PCR product
sizes were confirmed by agarose gel electrophoresis and ethidium
bromide staining. Three replications were performed, and the bcl-2
mRNA, hTERT mRNA or c-myc mRNA level was normalized against
successive spectra was 10 min.
Protein Binding Studies. All the experiments involving the
binding of the complex with BSA were carried out in double distilled
water with trisodium citrate (Tris, 10 mM) and sodium chloride (100
GAPDH mRNA level for each sample. Results of real-time PCR were
−
3
mM) and adjusted to pH 7.35 with hydrochloric acid. The 1 × 10
M
−ΔΔCT
analyzed using the 2
method in the program Origin 8.0 to
BSA stock solution was stored at 4 °C for no more than 5 days before
use. Pt1 and Pt2 were both prepared as 1 × 10 M DMSO stock
compare the transcriptional levels of bcl-2, hTERT or c-myc genes in
each sample relative to untreated control.
−3
solutions for protein binding studies.
Western Blot. Cells harvested from each well of the culture plates
were lysed in 150 μL of extraction buffer consisting of 149 μL of RIPA
lysis buffer and 1 μL of PMSF (100 mM). The suspension was
centrifuged at 10 000 rpm at 4 °C for 10 min, and the supernatant (10
μL for each sample) was loaded onto 10% polyacrylamide gel and then
transferred to a microporous polyvinylidene difluoride (PVDF)
membrane. Western blotting was performed using anti-bcl-2, anti-
hTERT, anti-c-myc, anti-53BP1, anti-TRF1, anti-TRF2, or anti-β-actin
primary antibody and horseradish-peroxidase-conjugated anti-mouse
or anti-rabbit secondary antibody. The protein bands were visualized
using chemiluminescence substrate.
−7
The solution of 5.0 × 10 M BSA was distilled by adding 1.5 μL of
× 10 M BSA in buffer (100 mM NaCl and 10 mM trisodium
−3
1
citrate at pH 7.35). The binding of the protein with the complex was
studied by adding a certain amount of a solution of the complex
−7
stepwise (0−8 μM) into the solution of 5.0 × 10 M BSA. The
emission spectra were monitored by keeping the excitation of BSA at
2
80 nm, and the emission was monitored in the range of 290−450 nm.
The slit width was set as 5 nm/5 nm for E /E , respectively, and the
x
m
emission was observed at 345 nm.
Commonly, fluorescence quenching can be described by the
52
following Stern−Volmer equation.
5
Transfection. SK-OV-3/DDP cells (8.0 × 10 ) were grown in 3
cm Petri dishes, and after 24 h, DNA transfections were performed as
F /F = 1 + k τ [Q] = 1 + K [Q]
0
q 0
SV
4
1
42
follows. First, 2.0 μg of EGFP plasmid or 2.0 μg c-myc plasmid
where F and F are the steady-state fluorescence intensities in the
were co-tranfected into cells using Lipofectamine 2000 (Invitrogen).
Then, Pt1 and Pt2 were added into medium after 6 h of transfection,
respectively. After another 24 h of drug treatment, the cells were
0
absence and presence of quencher, respectively; KSV is the Stern−
Volmer quenching constant, obtained from the slope of the plot F /F
0
Q
DOI: 10.1021/jm5012484
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
imaged using Nikon TE2000 (Japan) scanning fluorescent microscope
and studied by luciferase reporter gene assay kit.
Natural Science Foundation of Guangxi Province (Grants
2
012GXNSFDA053005 and 2012GXNSFDA385001) as well
In Vivo Antitumor Activity toward HCT-8 and NCI-H460
Human Tumor. The in vivo antitumor studies were carried out at the
Institute of Biomedical Engineering of the Chinese Academy of
Medical Sciences (Tianjin, China). The handling of animals and the
experimental design were approved by the Ethnics Committee and
Animal Care Committee of the Institute. Six-week-old athymic BALB/
cA nu/nu female mice (18−20 g for NCI-H460 model) were
purchased from the Institute of Laboratory Animal Resources of
National Institutes for Food and Drug Control of China (Beijing).
Animals were housed in a sterile environment with conditions of
constant photoperiod (12 h light/12 h dark at 23−24 °C and 65−85%
humidity). HCT-8 and NCI-H460 cells were harvested and injected
as “BAGUI Scholar” program of Guangxi Province of China.
ABBREVIATIONS USED
FID, fluorescent intercalator displacement; G4, G-quadruplex;
■
TO, thiazole orange; T , melting temperature; SD, standard
m
deviation; FRET, fluorescence resonance energy transfer; PI,
propidium iodide; TGI, tumor growth inhibition; IR, tumor
growth inhibition rate
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2
length (l) and width (w) and calculating volume (V = lw /2) as
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±
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ASSOCIATED CONTENT
Supporting Information
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AUTHOR INFORMATION
Corresponding Authors
Z.-F.C.: telephone, 086-773-2120998; fax, 086-773-21209958;
■
*
e-mail, chenzf@gxnu.edu.cn.; Hong Liang, PhD Telephone:
H.L.: telephone, 086-773-2120958; fax, 086-773-21209958; e-
*
mail, hliang@gxnu.edu.cn.
1
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Notes
Montagnac, A.; Qui, K.-H.; Temime-Smaali, N.; Trentesaux, C.;
Mergny, J.-L.; Gueritte, F.; Riou, J.-F. A new steroid derivative
stabilizes G-quadruplexes and induces telomere uncapping in human
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported by the National Natural Science
Foundation of China (Grants 21271051, 81473102,
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