A monofunctional trinuclear platinum complex with steric hindrance demonstrates strong cytotoxicity against tumor cells

Article abstract of DOI:10.1016/j.jinorgbio.2014.06.006

Polynuclear platinum complexes constitute a special class of hopeful antitumor agents. In this study, a Y-type monofunctional trinuclear platinum complex (MTPC) with 1,3,5-tris(pyridin-2-ylmethoxy)benzene, ammine and chloride as ligands was synthesized and characterized by ¹H NMR and electrospray ionization mass spectrometry (ESI-MS). The DNA binding mode of MTPC was investigated using circular dichroism spectroscopy and gel electrophoresis, and the reactivity of MTPC towards glutathione was studied by ¹H NMR and ESI-MS. The results show that MTPC can affect the conformation of calf-thymus DNA (CT-DNA) significantly and tends to form 1,4-GG rather than 1,2-GG intrastrand crosslinks, which are different from the instance of cisplatin. MTPC reacts with glutathione quite slowly in comparison with cisplatin because of the steric hindrance. The cytotoxicity of MTPC was tested on the human breast cancer cell line MCF-7, the human non-small-cell lung cancer cell line A549, and the human ovarian cancer cell line Skov-3 by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. MTPC is more potent than or comparable to cisplatin. The cellular inhibition mode of MTPC was examined by flow cytometry using MCF-7 cells. MTPC arrests the cell cycle mainly in G2 or M phase, while cisplatin arrests the cell cycle in S phase. Similar to cisplatin, MTPC kills the cells predominantly through an apoptotic pathway.

Full text of DOI:10.1016/j.jinorgbio.2014.06.006

A monofuctional trinuclear platinum complex with steric hindrance demon-
strates strong cytotoxicity against tumor cells
Shangnong Wu, Xiaoyong Wang, Yafeng He, Zhenzhu Zhu, Chengcheng Zhu,
Zijian Guo
PII:
DOI:
Reference:
S0162-0134(14)00167-6
doi: 10.1016/j.jinorgbio.2014.06.006
JIB 9539
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
17 March 2014
5 June 2014
8 June 2014
Please cite this article as: Shangnong Wu, Xiaoyong Wang, Yafeng He, Zhenzhu Zhu,
Chengcheng Zhu, Zijian Guo, A monofuctional trinuclear platinum complex with steric
hindrance demonstrates strong cytotoxicity against tumor cells, Journal of Inorganic Bio-
chemistry (2014), doi: 10.1016/j.jinorgbio.2014.06.006
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ACCEPTED MANUSCRIPT
A monofuctional trinuclear platinum complex with steric hindrance
demonstrates strong cytotoxicity against tumor cells
Shangnong Wu a, Xiaoyong Wang b,*, Yafeng He a, Zhenzhu Zhu a, Chengcheng Zhu a,
Zijian Guo a,*
aState Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering,
Nanjing University, Nanjing 210093, PR China
bState Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences; State Key
Laboratory of Analytical Chemistry for Life Science, Nanjing University, Nanjing 210093, P. R. China
Abstract
Polynuclear platinum complexes constitute a special class of hopeful antitumor agents.
In this study, a Y-type monofunctional trinuclear platinum complex (MTPC) with
1,3,5-tris(pyridin-2-ylmethoxy)benzene, ammine and chloride as ligands was
1
synthesized and characterized by H NMR and electrospray ionization mass
spectrometry (ESI-MS). The DNA binding mode of MTPC was investigated using
circular dichroism spectroscopy and gel electrophoresis, and the reactivity of MTPC
1
towards glutathione was studied by H NMR and ESI-MS. The results show that
MTPC can affect the conformation of calf-thymus DNA (CT-DNA) significantly and
tends to form 1,4-GG rather than 1,2-GG intrastrand crosslinks, which are different
from the instance of cisplatin. MTPC reacts with glutathione quite slowly in
comparison with cisplatin because of the steric hindrance. The cytotoxicity of MTPC
was tested on the human breast cancer cell line MCF-7, the human non-small-cell
lung cancer cell line A549, and the human ovarian cancer cell line Skov-3 by the
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. MTPC is
more potent than or comparable to cisplatin. The cellular inhibition mode of MTPC
was examined by flow cytometry using MCF-7 cells. MTPC arrests the cell cycle
mainly in G2 or M phase, while cisplatin arrests the cell cycle in S phase. Similar to
cisplatin, MTPC kills the cells predominantly through an apoptotic pathway.
* Corresponding authors. Tel.: +86 25 83594549; fax: +86 25 83314502.
E-mail addresses: boxwxy@nju.edu.cn (X. Wang), zguo@nju.edu.cn (Z. Guo).
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Keywords: Antitumor drug, Polynuclear platinum complex, DNA binding,
Glutathione, Apoptosis
1. Introduction
Platinum-based antitumor drugs play an important role in the treatment of
various malignancies such as colorectal and testicular cancers [1]. As a leading
antitumor drug, cisplatin has been used in the clinic for more than three decades [2].
However, the efficacy of this drug has been greatly hampered by the drug resistance
and severe side effects such as nephrotoxicity, neurotoxicity and ototoxicity [3].
These imperfections motivate researchers to design novel platinum compounds in
order to procure new drugs superior to cisplatin [4]. Monofunctional polynuclear
platinum complexes have shown great potential to circumvent the drug resistance or
side effects, and in many cases, appreciable water solubility that is favorable for
clinical practice [5]. In addition, these complexes react with DNA rapidly and form
inter- or intrastrand cross-linking DNA adducts that are radically different from the
scenarios of cisplatin [6]. For instance, monofunctional polynuclear platinum complex
BBR3464 (Fig. 1) could react uniquely with DNA through electrostatic interaction
and hydrogen-bonding owing to its highly positive charge and am(m)ine ligand [7-9].
Unfortunately, this compound is vulnerable to glutathione (GSH), in that excess GSH
could fragment it into smaller species due to the trans-labilization of the linker upon
binding to the sulfur atom of GSH, which would hinder its application in vivo [10].
We reported earlier that a monofunctional trinuclear platinum complex
[Pt3(HPTAB)Cl3](ClO4)3 [HPTAB = N,N,N′,N′,N″,N″-hexakis(2-pyridylmethyl)-
1,3,5-tris(aminomethyl)benzene, Fig. 1] could form intra- and interstrand cross-
lingking DNA adducts and exhibit more potent cytotoxicity against P388 and A549
cell lines than cisplatin [11-13]. The relatively rigid and bulky ligand remarkably
reduces the reactivity of the complex to GSH, whereas lowers the water solubility at
the same time. In this work, we designed and synthesized a monofunctional trinuclear
platinum complex (MTPC, Fig. 1) which has a more flexible linker. We suppose that
the steric hindrance could slow down its reaction with GSH, and the platinum centers
could form DNA cross-links dissimilar to those formed by cisplatin; while the ether
bonds are conducive to the water solubility. To testify this assumption, the reactivity
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of MTPC to DNA and GSH in aqueous solutions was investigated and the
cytotoxicity towards tumor cells was studied.
[Fig. 1.]
2. Materials and methods
2.1. Reagents
All reagents and solvents were of commercial origin and used without further
purification unless otherwise noted. Cisplatin was purchased from Aldrich; GSH, calf-
thymus DNA (CT-DNA), and Tris were from Sigma. Annexin V conjugated with
fluorescein isothiocyanate (Annexin V-FITC) was purchased from KeyGen Biotech;
and propidium iodide (PI) was purchased from Beyotime. The human breast cancer
cell line MCF-7, the human non-small-cell lung cancer cell line A549 and the human
ovarian cancer cell line Skov-3 were originated from Cancer Institute & Hospital,
Chinese Academy of Medical Sciences.
2.2. Synthesis of the ligand
A mixture of 1,3,5-benzentriol (5 mmol, 0.61 g) and K2CO3 (40 mmol, 5.5 g) in
N,N-dimethylformamide (DMF, 50 mL) was stirred at room temperature for 30 min.
2-(Chloromethyl) pyridine hydrochloride (20 mmol, 3.24 g) was added to the mixture
and stirred at 353 K for 48 h. The products were added to icy water (500 mL) and
extracted with methylene chloride for three times. The ligand was obtained after
evaporating the solvent under reduced pressure and purifying by silica gel column
1
chromatography. Yield: 180 mg, 9%. H NMR (CD3OD, ppm): 8.55 (d, 3H), 7.89 (t,
3H), 7.59 (d, 3H), 7.39 (t, 3H), 6.34 (s, 3H), 5.15 (s, 6H). ESI-MS (positive mode,
m/z): 400.58, [M + H]+ (C24H22N3O3); 422.42, [M + Na]+ (C24H21N3O3Na).
2.3. Preparation of MTPC
Previously prepared cis-[Pt(NH3)2Cl(DMF)]NO3 (0.5mmol, 1 mL) was added to
the DMF solution of the ligand (0.15 mmol, 3 mL) [14]. The mixture was stirred at
323 K for 48 h in the dark. A yellow precipitate was obtained as the mixture was
added dropwise into acetone (100 mL) with stirring. MTPC was acquired after the
precipitate was filtrated and washed with acetone twice and acetonitrile thrice. Yield:
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140 mg, 68%. 1H NMR (CD3OD, ppm): 8.94 (d, 3H), 8.07 (t, 3H), 7.63 (d, 3H), 7.56
(t, 3H), 6.63 (s, 3H), 6.18(d, 3H), 5.85 (d, 3H), 4.58 (s, 9H), 4.38 (s, 9H). 195Pt NMR
(CD3OD, ppm): ‒2310.36. Elemental analysis found (calcd) for C24H39Cl3N12O12Pt3
(%): C, 20.47 (20.90); N, 11.73 (12.19); H, 3.13 (2.85). ESI-MS (positive mode, m/z):
397.83, [M
‒
3NO3]3+ (C24H39Cl3N9O3Pt3/3); 627.75, [M
‒
2NO3]2+
(C24H39Cl3N10O6Pt3/2); 1316.17, [M ‒ NO3]+ (C24H39Cl3N11O9Pt3).
2.4. Interaction with CT-DNA
Circular dichroism (CD) spectra of DNA in the presence of MTPC were
recorded on a Jasco J-180 spectropolarimeter in a cylindrical quartz cell (1 cm). Each
sample in Tris-HCl buffer (pH 7.4, 50 mM NaCl) was incubated at 310 K for 12 h
before measurement and scanned from 230 to 310 nm at a speed of 10 nm min‒1. The
concentration of CT-DNA in terms of per nucleotide was determined
spectrophotometrically at 260 nm using the molar extinction coefficient of 6600 M‒1
cm‒1 [15].
The unwinding of pUC19 plasmid DNA induced by MTPC was assessed by the
agarose gel electrophoretic mobility shift assay [16]. Samples of plasmid DNA were
incubated with MTPC at 310 K in the dark for 12 h in the molar ratio
([MTPC]/[DNA]) varying from 0 to 0.10. The reaction products were subjected to
electrophoresis on native agarose gel (1%) in TAE buffer (40 mM Tris-acetate, 1 mM
EDTA, pH 8.0) at 100 V for 90 min. The finished gel was stained with EtBr and
photographed with a transilluminator.
2.5. DNA binding mode
The 5′-FAM-labeled duplex was incubated with MTPC at 310 K for 12 h in the
molar ratio ([MTPC]/[DNA]) varying from 0 to 6. The reaction was quenched by
formamide loading buffer (5 µL) at 363 K for 2 min, and then cooled on ice. The
samples were followed by electrophoresis using 20% polyacrylamide gel under
denaturing conditions. Detailed procedures have been described in our previous report
[12].
2.6. Reaction with GSH
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1
The sample for H NMR was prepared by reacting MTPC with 3 equivalent of
GSH in phosphate buffer (25 mM, 90% H2O/10% D2O, pH 6.72) at 310 K for
1
different times in the dark. The status of the reaction was examined by H NMR
spectroscopy with a Bruker DPX 300 spectrometer at 298 K. The sample for ESI-MS
was prepared by reacting MTPC with 3 equivalent of GSH in Tris-HCl buffer (10 mM,
pH 7.2). The reaction was monitored by ESI-MS after the sample was incubated at
310 K for 4, 6 and 8 h, respectively, in the dark.
2.7. Cytotoxicity assay
MCF-7, A549 and Skov-3 cell lines were cultivated in RPMI-1640 medium
supplemented with 10% heat-inactivated fetal bovine serum under 5% CO2. The in
vitro cytotoxicity of MTPC was measured by the microculture tetrazolium (MTT)
assay [17]. Cisplatin was chosen as a positive reference. The detailed procedures of
the assay have been described in our early report [18]. The results are expressed as the
growth inhibition percentage calculated according to the ratio of (ODcontol
ODtest)/ODcontol × 100%.
‒
2.8. Cell cycle analysis
MCF-7 cells were exposed to MTPC at 310 K under 5% CO2 for 24 h and the
culture medium was collected carefully. Trypsin was added to the culture medium and
adherent cells were collected after mild centrifugation. The cells were washed with
PBS, fixed in ice-cold ethanol (70%) for 6 h, pelleted by centrifugation, washed again
with PBS, and stained with PI (1 mg mL‒1) in PBS for 30 min at 310 K in the dark.
The cells were analyzed using a BD LSRFortessa flow cytometry.
2.9. Detection of apopotosis
MCF-7 cells were exposed to different concentrations of MTPC at 310 K under
5% CO2 for 48 h, and harvested for apoptotic assay. The cells were resuspended in a
staining solution consisting of 10 mM HEPES/NaOH (pH 7.4), 140 mM NaCl, and
2.5 mM CaCl2. Annexin V-FITC (5 μL, 1μg mL‒1) and PI (5 μL, 1μg mL‒1) were
added to the suspension and incubated at room temperature in the dark for 15 min
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before analyzing by flow cytometry. At least 10,000 events were acquired for each
sample.
3. Results and discussion
3.1. Preparation and characterization of MTPC
Ligand L was designed and synthesized considering the need for steric hindrance
and coordination requirement of platinum. MTPC was obtained by reacting L with
cis-[Pt(NH3)2Cl(DMF)]NO3. The steric hindrance of the ligand in MTPC is supposed
to slow down the reaction of MTPC with GSH; on the other hand, the flexibility of
the linker may offer the possibility of forming multimodal DNA cross-linking adducts,
such as 1,3- and 1,4-GG intra- or interstrand crosslinks. In addition, the highly
positive charge of MTPC and the polar ether bond (‒O‒) could increase the speed of
forming DNA crosslinks and make the complex more soluble in water. These
characters may improve the bioavailability and bioactivity of MTPC. Both L and
1
MTPC were fully characterized by ESI-MS, H- and 195Pt-NMR (see Supplementary
Data). In the ESI-MS spectrum, a major peak was observed at m/z 397.83, which
could be attributed to the positively charged species [Pt3LCl3]3+. The result suggests
that the complex cation is stable in aqueous solution. In the 195Pt-NMR spectrum, only
one single peak was observed at ‒2310.36 ppm, suggesting that all the three Pt atoms
are in the same coordination environment.
3.2. Interaction with CT-DNA
The conformational changes of CT-DNA induced by MTPC were investigated
by CD spectroscopy. As shown in Fig. 2, the CD spectra of CT-DNA display two
bands: the positive band at 275 nm is due to the base stacking, and the negative band
at 248 nm is due to the helicity, which are characteristic of B-type DNA [19]. The
ellipticity of both positive and negative bands decreases as the [MTPC]/[DNA] molar
ratio increases from 0 to 0.08, which suggests that the CT-DNA helix can be unwound
by MTPC and the conformation of CT-DNA tends to change from B- to Z-type [20].
The results show that MTPC can affect the conformation of CT-DNA significantly at
low concentrations and the DNA binding mode is different from that of cisplatin,
which typically induces a slight increase in the ellipticity of the positive band [21].
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[Fig. 2.]
Further, the unwinding of pUC19 plasmid DNA induced by MTPC was explored
by agarose gel electrophoresis. It is known that the binding of unwinding agents to the
closed circular DNA can reduce its superhelical density and hence decrease its rate of
migration in the agarose gel [16]. As shown in Fig. 3, an obvious decrease in the
electrophoretic mobility for the supercoiled DNA (Form I) is observed after
incubation with MTPC, indicating that MTPC can induce the unwinding of DNA
duplex. The rate of the migration of the supercoiled DNA band (Form I) decreases
while the separation between the supercoiled and nicked DNA (Form II) bands
narrows with the increase of MTPC. As the molar ratio of MTPC to DNA reaches to
0.06 (lane 5), the band of Form I DNA comigrates with the band of Form II DNA.
This coalescence point corresponds to the amount of MTPC necessary for the
complete removal of all supercoils from the DNA. Cisplatin can also produce a
similar outcome, but the coalescence point is 0.076 [16], suggesting that MTPC is a
more effective perturbing agent for the tertiary structure of supercoiled DNA than
cisplatin. Beyond the coalescence point, the migration rate of the band begins to
increase again as positive supercoils are induced.
[Fig. 3.]
3.3. DNA binding mode
Considerable evidence has proved that the cytotoxicity of platinum complexes is
originated from their covalent binding to DNA after leaving the labile ligands [22].
Polynuclear platinum complexs could form a variety of combinative crosslinks such
as intra- and interstrand Pt-DNA adducts [23,24]. To investigate the possible DNA
binding modes of MTPC, we chose the following 18-mer duplex
to examine different crosslinks. The sequence of this duplex is derived from the breast
or ovarian cancer susceptibility gene BRCA1 (Genbank #U14680) where a somatic
mutation 15linsC occurs in exon 2 [25]. With 5′-FAM-labeling on strand OD1 or OD2,
this duplex is capable of probing the potential DNA binding modes for MTPC, that is,
1,4- or 1,3-GG intrastrand crosslinks or 1,3-GG interstrand crosslink. The
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electrophoresis patterns of the duplex in the denaturing polyacrylamide gel are shown
in Fig. 4. On the basis of their electrophoretic mobilities, the bands can be assigned to
monoadducts, di-monoadducts, 1,3- and 1,4-GG intrastrand crosslinks, respectively.
The di-monoadducts may be formed by two platinum centers of two MTPC molecules
through biding to different G bases in the duplex. Band E is darker than band D in
lanes 3 and 6, suggesting that 1,4-GG intrastrand crosslink is the main adduct.
Comparing lane 6 with lane 9, it is more evident that MTPC tends to form 1,4-GG
intrastrand crosslink rather than 1,3-GG intrastrand crosslink, which may be due to
the distance between the two platinum centers and steric hindrance are more favorable
for forming the 1,4-GG crosslink [26]. All the above attribution has been confirmed
by the matrix assisted laser desorption ionization-time of flight mass spectrometry
(MALDI-TOF MS) (see Supplementary Data). In this experiment, no interstrand
crosslink was observed. In general, the binding mode of MTPC to the DNA duplex
follows the characters previously delineated for the same sequence [12,26]. However,
the molecular details of the interactions between MTPC and DNA are not yet fully
understood.
[Fig. 4.]
3.4. Interaction with GSH
GSH, one of the most abundant intracellular sulfur-containing molecules, has
been demonstrated to be partially responsible for the drug resistance and toxic effects
of platinum drugs [27]. Introduction of a steric ligand to the platinum coordination
sphere could impede the substitution by GSH and hence reduce the reactivity of
complex to GSH, which has been proved by ZD0473 [28]. Therefore, we suppose that
the presence of 2-methylene pyridine in MTPC could decrease its reactivity to GSH,
which was examined by the following experiment.
1
The reaction of MTPC with GSH was first monitored by H NMR in phosphate
buffer (25 mM, pH 6.72) at 298 K. As shown in Fig. 5, after reaction for 4 h,
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resonances c at 2.9 ppm decrease apparently in intensity, while new resonances c′
appear at 2.4 ppm and overlap with resonances b. The high-field shift of resonances c
suggests that the sulphydryl group in GSH has bound to PtII, forming the Pt-SG
mono-adduct [29,30]. Resonances c keep decreasing with the time and almost
disappear at 12 h, while resonances c′ overlap with resonances b completely,
indicating that the reaction is nearly finished by this time. No more changes were
observed after 16 h. Interestingly, other resonances corresponding to protons a and d
also undergo some high-field shifting and broadening. This is understandable because
theoretically GSH has six potential binding sites for PtII, including a sulphydryl group,
a glutamate amino group, two carboxyl oxygen atoms, and two peptide N atoms.
Although the actual binding sites are dependent on the reaction conditions, the
resonance variations seem to suggest that most of these sites are more or less involved
in the interaction. Even so, sulphydryl is the predominant binding group. The
pronounced binding of MTPC to GSH occurs at 4‒8 h, which is much slower than the
binding of cisplatin to GSH in similar conditions (30 min ‒ 2.5 h) [29]. Judging by the
1H NMR spectra, the Pt-SG adducts formed by MTPC is simpler than that formed by
cisplatin.
[Fig. 5.]
The reaction of MTPC with GSH was further investigated after proceeding for 4,
6 and 8 h respectively using ESI-MS. The spectra are shown Fig. 6, and the
assignments of the peaks are summarized in Table 1. The results indicate that MTPC
can react with GSH to form three different kinds of adducts, namely, mono-adduct,
bi-adduct and tri-adduct. The abundance of mono- and bi-adduct peaks decreased
obviously at 4−6 h, suggesting these adducts can further combine with GSH to form
tri-adduct. Kinetic equilibrium was reached after 6 h with tri-adduct being the major
product. No more significant changes were observed for the bi- and tri-adduct peaks
henceforth. Oxidized GSH and hydrolyzed MPTC still exist in solution even at 8 h,
suggesting the reaction is incomplete yet. It is noteworthy that the linking ligand L did
1
not decompose during the reaction with GSH. In brief, both H NMR and ESI-MS
demonstrates that the reactivity of MTPC towards GSH is lower than that of cisplatin
and the reaction of MTPC with GSH is indeed retarded by the bulky ligand, implying
that the side-effects related to GSH might be reduced in vivo.
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[Fig. 6.]
[Table 1]
3.5. Cytotoxicity
The in vitro cytotoxicity of MTPC was tested against the human breast cancer
cell line MCF-7, the human non-small-cell lung cancer cell line A549, and the human
ovarian cancer cell line Skov-3 by the MTT assay. Cisplatin was chosen as a positive
reference. Detailed profiles are shown in the Supplementary Data. MTPC is more
cytotoxic than cisplatin agaist A549 and MCF-7 cell lines at all the tested
concentrations (10‒4‒10‒7 M), especially at low concentrations (< 10‒6 M). For
example, at 10‒7 M, MTPC still maintains an inhibition rate of 15.4% against A549
cells, while cisplatin is almost nontoxic. At high concentrations (≥ 10‒5 M), the
cytotoxicity of MTPC towards Skov-3 cell line is comparable to that of cisplatin; but
at low concentrations (≤ 10‒6 M), it is less effective than cisplatin. The half maximal
inhibitory concentration (IC50) of MTPC and cisplatin for these cells are listed in
Table 2. On the whole, MTPC shows a remarkable cytotoxicity to these tumor cells,
with A549 being the most sensitive cell line to this complex. We suppose that the
multimodal interactions between MTPC and cellular DNA are the main reasons for
the powerful activity. Since MTPC is an unconventional platinum complex, it might
be able to overcome some side effects related to cisplatin and its analogues.
[Table 2]
3.6. Arrest of cell cycle
The perturbation effect of MTPC on the cell cycle was investigated in MCF-7
cells using flow cytometry by detecting DNA content after staining with propidium
iodide (PI). Cisplatin was chosen as a positive control. A representative cell cycle
distribution and statistical analysis for different cell cycle phases are shown in Fig. 7.
MTPC disturbs the cell cycle remarkably as compared with the control. Specifically,
the cells in G0 or G1 phase decrease while those in G2 or M phase increase with the
increment of MTPC, and yet the cells in S phase only vary moderately (<10%).
Cisplatin also strongly disturbs the cell cycle, but it mainly arrests the cells in S phase
rather than in G2 or M phase. The different inhibition characters of MTPC (G2/M
phase arrest) and cisplatin (S phase arrest) may arise from their distinct DNA binding
modes. As demonstrated above, MTPC mainly forms 1,4- rather than 1,2-GG
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intrastrand DNA crosslinks, which may activate downstream signal pathways
differently from cisplatin and result in a special inhibition on the cell cycle.
Interestingly, we found that some dinuclear platinum complexes that chiefly form 1,3-
or 1,4-intrastrand DNA crosslinks also induce G2 or M phase arrest in COC1 cells
[31].
[Fig. 7.]
3.7. Cytostatic pathway
Anticancer drugs usually induce cancer cells to undergo apoptosis and necrosis,
particularly the former. Cells in early apoptosis display phosphatidylserine exposure
on the outer surface of the plasma membrane, which can be detected by flow
cytometry after staining with Annexin V-FITC and PI [32]. According to the
variation of Annexin V, apoptotic cells (Annexin V+) can be distinguished from the
intact cells (Annexin V−); and PI can further discriminate early apoptotic cells
(Annexin V+/PI−) from the late apoptotic or necrotic (Annexin V+/PI+) cells. The
cellular density plots of MCF-7 cells in the presence of MTPC or cisplatin were
shown in Fig. 8. As compared with the control, MTPC can significantly prompt the
phosphatidylserine exposure (Annexin V+) and loss of plasma membrane integrity
(PI+) of MCF-7 cells. Although cisplatin can also spur the cells to undergo similar
changes, it is less efficient than MTPC in inducing the late apoptosis or necrosis
(Annexin V+/PI+). The percentages of MCF-7 cells at different stages in the presence
of MTPC or cisplatin are listed in Table 3. As the data shown, the cells in late
apoptosis or necrosis for cisplatin are only 5.6% at 2 µM, while those for MTPC are
19.9% at the same concentration. As a result, the cells remain intact (Annexin V−/PI−)
for cisplatin are 71.8% at 2 µM, while those for MTPC are 46.3% at the same
concentration. The percentage of cells in early apoptosis induced by MTPC is also
higher than that induced by cisplatin. Obviously, MTPC displays a stronger impact on
MCF-7 cells than cisplatin does at the tested concentrations. In spite of the difference,
the influence of MTPC and cisplatin on the phosphatidylserine exposure and loss of
plasma membrane integrity seems to vary only in extent, thus their cytostatic pathway
should be similar. It has been shown that phosphatidylserine exposure usually
precedes the loss of plasma membrane integrity during apoptosis, while these events
tend to coincide in necrosis [33]. Since MTPC and cisplatin both induce MCF-7 cells
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to pass through the intermediate Annexin V+/PI− stage instead of going directly to the
upper right quadrant (Annexin V+/PI+), the predominant cell death mode in this case
is apoptosis.
[Fig. 8.]
[Table 3]
4. Conclusion
A novel Y type monofunctional trinuclear platinum complex MTPC was
synthesized in this work. The complex affects the conformation of DNA more
efficiently and reacts with GSH much slower than cisplatin does. The DNA adducts
formed by MTPC are 1,4-GG, and to a less extent, 1,3-GG intrastrand cross-links,
which are different from that formed by cisplatin. The cytotoxicity of MTPC is higher
than that of cisplatin against MCF-7 and A549 tumor cell lines, and is comparable to
that of cisplatin against Skov-3 cell line at high concentrations (>10‒6 M). Unlike
cisplatin, MTPC arrests the cell cycle mainly in G2 or M phase, though shares a
similar apoptotic pathway to kill the tumor cells. Many studies have shown that
platinum complexes that bind to DNA in a manner fundamentally different from that
of cisplatin can change the spectrum and intensity of the antitumor activity. MTPC
can form either monoadducts or non-1,2-GG intrastrand crosslinks with DNA, which
provide a firm basis for its remarkable cytotoxicity. By comparison with our early
[Pt3(HPTAB)Cl3](ClO4)3, the non-leaving ammine ligand and high water solubility of
MTPC make it more accessible to DNA. Considering the cationic nature and the
stability of linking ligand in the presence of GSH, MTPC is worthy of further studies
on general toxicity in vivo and interactions with other biomolecules. This compound
may also be useful in nucleic acid research as an effective GG cross-linking agent.
5. Abbreviations
CD circular dichroism
CT-DNA calf-thymus DNA
DMF N,N-dimethylformamide
FITC fluorescein isothiocyanate
GSH glutathione
HEPES 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid
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MALDI-TOF MS
spectrometry
matrix assisted laser desorption ionization-time of flight mass
MTPC monofunctional trinuclear platinum complex
MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
PBS phosphate buffered saline
PI propidium iodide
Acknowledgements
We are grateful for the financial support from the National Basic Research
Program of China (Grant 2011CB935800) and the National Natural Science
Foundation of China (Grants 90713001, 21271101, 21131003, 21021062).
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Table 1 Assignments of the peaks observed in the ESI-MS spectra for the reaction of
MTPC with GSH (see Fig. 6).
Observed m/z
713.58
Species
Formula
[MPTC + GSH − 2Cl − 3H − 3NO3]2+
[MPTC + 2GSH − 3Cl + OH − 3H − 3NO3]2+
[MPTC + 3GSH − 3Cl − NH3 − 4H − 3NO3]2+
[(GS)2 + H]+
C34H53N12O9ClSPt3
C44H71N15O16S2Pt3
C54H83N17O21S3Pt3
C20H33N6O12S2
C24H38N7O8Pt3
858.33
993.83
613.50
1138.33
[MPTC − 3Cl − 2NH3 + 5OH − 3NO3]+
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Table 2 The IC50 (μM) values of MTPC and cisplatin for MCF-7, A549, and Skov-3
cell lines at 48 h.
Complex
MTPC
MCF-7
A549
Skov-3
2.0 ± 0.3
5.7 ± 0.1
1.5 ± 0.1
6.7 ± 0.6
4.5 ± 0.5
2.2 ± 0.3
cisplatin
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Table 3 Percentage of MCF-7 cells at different stages after incubation with MTPC or
cisplatin for 48 h and staining with Annexin V and PI analyzed by flow cytometry
(see Fig. 8). Data are expressed as mean (%) ± S. D. of at least three separate
experiments and are analyzed by Student’s t-test.
Complex
MTPC
Conc. (μM)
Intact
Early apoptosis Late apoptosis
0.5
2.0
2.0
72.0 ± 0.6
46.3 ± 0.5
71.8 ± 0.8
15.5 ± 0.4
27.9 ± 0.5
21.6 ± 0.3
8.5 ± 0.3
19.9 ± 0.9
5.6 ± 0.6
MTPC
cisplatin
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Figure Legends
Fig. 1. Structures of monofunctional trinuclear platinum complexes BBR3464,
[Pt3(HPTAB)Cl3](ClO4)3 and MTPC.
Fig. 2. CD spectra of CT-DNA in the presence of MTPC at different [MTPC]/[DNA]
molar ratios.
Fig. 3. Agarose gel electrophoresis patterns of supercoiled pUC19 plasmid DNA
incubated with MTPC at 310 K for 24 h. Lane 1, DNA contol; lanes 2‒8,
[MTPC]/[DNA] = 0.01, 0.02, 0.04, 0.06, 0.07, 0.09, 0.10, respectively.
Fig. 4. Denaturing polyacrylamide gel electrophoresis patterns of different 5′-FAM-
labeled duplexes after incubation with MTPC at 310 K for 12 h in 2, 4, 6 molar ratios
([MTPC]/[DNA]), respectively. Lane 0, duplex control; lanes 1‒3, OD1 and OD2
both labeled duplex; lanes 4‒6, OD1 labeled duplex; lanes 7‒9, OD2 labeled duplex.
Band assignments: A, unreacted duplex; B, monoadducts; C, di-monoadducts; D, 1,3-
GG intrastrand cross-link; E, 1,4-GG intrastrand crosslink.
1
Fig. 5. Time-dependent H NMR spectra and resonance assignments for the reaction
of MTPC with GSH in a 1:3 molar ratio in phosphate buffer (25 mM, pH 6.72)
1
determined at 298 K. The resonance of proton e is overlapped by the residual H2O
resonance and is not shown.
Fig. 6. ESI-MS spectra for the reaction of MTPC with GSH recorded at 310 K at 4, 6
and 8 h.
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Fig. 7. Impact of MTPC or cisplatin on the cell cycle of MCF-7 cells after incubation
for 24 h. The statistical data for different cell cycle phases are mean (%) of at least
three separate experiments.
Fig. 8. Flow cytometric analysis of MCF-7 cells after incubation with MTPC or
cisplatin for 48 h and subsequent staining with Annexin V and PI. Dots with Annexin
V‒/PI‒, Annexin V+/PI‒, and Annexin V+/PI+ characters represent intact, early
apoptotic, and late apoptotic cells, respectively.
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Fig. 1.
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Fig. 2.
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Fig. 3.
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Fig. 4.
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Fig. 5.
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Fig. 6.
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Fig. 7.
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Fig. 8.
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Graphical Abstract
Polynuclear platinum complex MTPC reacts with DNA to produce 1,4-GG intrastrand
adduct and bring potent cytotoxicity against cancer cells. Unlike cisplatin, MTPC
arrests the cell cycle mainly in G2 or M phase.
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