Novel tacrine platinum(II) complexes display high anticancer activity via inhibition of telomerase activity, dysfunction of mitochondria, and activation of the p53 signaling pathway

Article abstract of DOI:10.1016/j.ejmech.2018.09.008

In this work, we designed and synthesized tacrine platinum(II) complexes [PtClL(DMSO)]?CH₃OH (Pt1), [PtClL(DMP)] (Pt2), [PtClL(DPPTH)] (Pt3), [PtClL(PTH)] (Pt4), [PtClL(PIPTH)] (Pt5), [PtClL(PM)] (Pt6) and [PtClL(en)] (Pt7) with 4,4′-dimethyl-2,2′-bipyridine (DMP), 4,7-diphenyl-1,10-phenanthroline (DPPTH), 1,10-phenanthroline (PTH), 2-(1-pyrenecarboxaldehyde) imidazo [4,5-f]-[1,10] phenanthroline (PIPTH), 2-picolylamine (PM) and 1,2-ethylenediamine (en) as telomerase inhibitors and p53 activators. Biological evaluations demonstrated that Pt1–Pt7 exhibited cytotoxic activity against the tested NCI–H460, Hep-G2, SK-OV-3, SK-OV-3/DDP and MGC80-3 cancer cell lines, with Pt5 displaying the highest cytotoxicity. Pt5 exhibited an IC₅₀ value of 0.13 ± 0.16 μM against SK-OV-3/DDP cancer cells and significantly reduced tumor growth in a Hep-G2 xenograft mouse model (tumor growth inhibition (TGI) = 40.8%, p < 0.05) at a dose of 15.0 mg/kg. Interestingly, Pt1–Pt7 displayed low cytotoxicity against normal HL-7702 cells. Mechanistic studies revealed that these compounds caused cell cycle arrest at the G2/M and S phases, and regulated the expression of CDK2, cyclin A, p21, p53 and p27. Further mechanistic studies showed that Pt5 induced SK-OV3/DDP cell apoptosis via dysfunction of mitochondria, inhibition of the telomerase activity by directly targeting the c-myc promoter, and activation of the p53 signaling pathway. Taken together, Pt5 has the potential to be further developed as a new antitumor drug.

Full text of DOI:10.1016/j.ejmech.2018.09.008

Accepted Manuscript
Novel tacrine platinum(II) complexes display high anticancer activity via inhibition of
telomerase activity, dysfunction of mitochondria, and activation of the p53 signaling
pathway
Qi-Pin Qin, Shu-Long Wang, Ming-Xiong Tan, Zhen-Feng Wang, Dong-Mei Luo, Bi-
Qun Zou, Yan-Cheng Liu, Peng-Fei Yao, Hong Liang
PII:
S0223-5234(18)30780-3
10.1016/j.ejmech.2018.09.008
EJMECH 10719
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 July 2018
Revised Date: 29 August 2018
Accepted Date: 4 September 2018
Please cite this article as: Q.-P. Qin, S.-L. Wang, M.-X. Tan, Z.-F. Wang, D.-M. Luo, B.-Q. Zou, Y.-
C. Liu, P.-F. Yao, H. Liang, Novel tacrine platinum(II) complexes display high anticancer activity
via inhibition of telomerase activity, dysfunction of mitochondria, and activation of the p53 signaling
pathway, European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.09.008.
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ACCEPTED MANUSCRIPT
Graphical abstract
Novel tacrine platinum(II) complexes display high anticancer
activity via inhibition of telomerase activity, dysfunction of
mitochondria, and activation of the p53 signaling pathway
a
Qi-Pin Qin a,d,1, Shu-Long Wang a,1, Ming-Xiong Tan a,*, Zhen-Feng Wang ,
Dong-Mei Luo a, Bi-Qun Zou b,d,*, Yan-Cheng Liu d, Peng-Fei Yao c,*, Hong Liang d,*
Pt5 induced SK-OV-3/DDP cell apoptosis via dysfunction of mitochondria, inhibition
of the telomerase activity and activation of the p53 signaling pathway.

ACCEPTED MANUSCRIPT
Novel tacrine platinum(II) complexes display high anticancer
activity via inhibition of telomerase activity, dysfunction of
mitochondria, and activation of the p53 signaling pathway
a
Qi-Pin Qin a,d,1, Shu-Long Wang a,1, Ming-Xiong Tan a,*, Zhen-Feng Wang ,
Dong-Mei Luo a, Bi-Qun Zou b,d,*, Yan-Cheng Liu d, Peng-Fei Yao c,*, Hong Liang d,*
a Guangxi Key Laboratory of Agricultural Resources, Chemistry and Biotechnology,
College of Chemistry and Food Science, Yulin Normal University, 1303 Jiaoyudong
Road, Yulin 537000, China. mxtan2018@126.com (M.-X. Tan); Telephone and Fax:
(086) 775-2623650.
b Department of Chemistry, Guilin Normal College, 21 Xinyi Road, Gulin 541001,
China. zoubiqun@163.com (B.-Q. Zou).
c
Guangxi Colleges and Universities Key Laboratory of Regional Ecological
Environment Analysis and Pollution Control of West Guangxi, College of Chemistry
and Environmental Engineering, Baise University, Baise, Guangxi 533000, China.
yaopengfei1986@163.com (P.-F. Yao).
d
State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal
Resources, School of Chemistry and Pharmacy, Guangxi Normal University, 15 Yucai
Road, Guilin 541004, China. hliang@gxnu.edu.cn (H. Liang).
1 These authors contributed equally to this work.
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ABSTRACT
In this work, we designed and synthesized tacrine platinum(II) complexes
[PtClL(DMSO)] CH3OH (Pt1), [PtClL(DMP)] (Pt2), [PtClL(DPPTH)] (Pt3),
[PtClL(PTH)] (Pt4), [PtClL(PIPTH)] (Pt5), [PtClL(PM)] (Pt6) and [PtClL(en)] (Pt7)
with
4,4'-dimethyl-2,2'-bipyridine
(DMP),
4,7-diphenyl-1,10-phenanthroline
(DPPTH), 1,10-phenanthroline (PTH), 2-(1-pyrenecarboxaldehyde) imidazo
[4,5-f]-[1,10] phenanthroline (PIPTH), 2-picolylamine (PM) and 1,2-ethylenediamine
(en) as telomerase inhibitors and p53 activators. Biological evaluations demonstrated
that Pt1−Pt7 exhibited cytotoxic activity against the tested NCI-H460, Hep-G2,
SK-OV-3, SK-OV-3/DDP and MGC80-3 cancer cell lines, with Pt5 displaying the
highest cytotoxicity. Pt5 exhibited an IC50 value of 0.13 ± 0.16 µM against
SK-OV-3/DDP cancer cells and significantly reduced tumor growth in a Hep-G2
xenograft mouse model (tumor growth inhibition (TGI)= 40.8%, p < 0.05) at a dose of
15.0 mg/kg. Interestingly, Pt1−Pt7 displayed low cytotoxicity against normal
HL-7702 cells. Mechanistic studies revealed that these compounds caused cell cycle
arrest at the G2/M and S phases, and regulated the expression of CDK2, cyclin A, p21,
p53 and p27. Further mechanistic studies showed that Pt5 induced SK-OV3/DDP cell
apoptosis via dysfunction of mitochondria, inhibition of the telomerase activity by
directly targeting the c-myc promoter, and activation of the p53 signaling pathway.
Taken together, Pt5 has the potential to be further developed as a new antitumor drug.
Keywords: platinum(II) complex, tacrine, p53, telomerase, anticancer activity,
apoptosis
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1. Introduction
Platinum complexes (e.g., cisplatin, carboplatin and oxaliplatin) are widely used as
antitumor drugs for treating a large number of human cancers and malignancies [1−7].
However, drug resistance and severe side effects of Pt-based drugs have motivated
inorganic chemists to design and synthesize less toxic, more effective, target-specific,
and novel Pt-based anticancer agents [8−20]. For example, Sadler and co-authors
reported that trans,trans,trans-Pt(N3)2(OH)2(py)2 was potently phototoxic towards
Hep-G2 (hepatoma), HaCaT (keratinocytes), OE19 (oesophageal adenocarcinoma),
A2780CIS (cisplatinresistant ovarian carcinoma) and A2780 (parental) cells [21−26].
Lippard and coworkers showed that cis-[Pt(NH3)2(quinoline)Cl]NO3 (quinoplatin) and
cis-[Pt(NH3)2(phenanthridine)Cl]NO3 (phenanthriplatin) destroyed cancer cells with
greater efficacy than that of cisplatin and oxaliplatin [27,28]. Chen described a
luminescent platinum(II) complex [Pt(6-phenyl-2,2’-bipyridyl)(C≡NtBu)]ClO4, which
exhibited potent cytotoxicity towards some tumor cell lines (i.e., KB, SH-5YSY,
NCI-H460 and SUNE1) and inhibited tumor growth in a NCI-H460 xenograft mouse
model [29]. Liu et al. showed that self-assembled cholesterol-asplatin-incorporated
nanoparticles (SCANs) were effective against cisplatin-resistant tumor cells [30].
A large number of acridine analogues, quindoline analogues, quinacridine
analogues and their platinum(II) complexes have been reported to inhibit the
telomerase activity and trigger cell senescence and apoptosis [31−48]. Kelland and
co-workers
showed
that
a
pentacyclic
acridine,
RHPS4
(3,11-difluoro-6,8,13-trimethyl(8H)-quino[4,3,2-kl]acridinium
methylsulfate),
induced tumor cell apoptosis via directly inhibiting the telomerase activity [36,37]. In
addition, the natural product quindoline has also been identified as a potent telomerase
tel
inhibitor, with IC50 values in the range of 6–16 µM [31,39,40]. It is interesting that
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N′-(10H-Indolo[3,2-b]quinolin-11-yl)-(2-morpholin-4-ylethyl)amine
and
its
derivatives showed stronger telomerase inhibitory activity compared with
disubstituted quindolines, with telIC50 values of 0.44–12.3 µM [31,41−44].
Furthermore, Teulade-Fichou reported that platinum(II) mono-para-quinacridine
(Pt-MPQ) can inhibit telomerase by preferentially trapping the antiparallel structure of
the 22AG G-quadruplex [31,46,47].
Several research groups have shown that tacrine, the first approved drug for the
Alzheimer's disease, is a potent nonselective inhibitor of acetylcholinesterase (AChE)
and butyrylcholinesterase (BuChE) [49−55]. Tacrine is a quindoline derivative with a
large planar aromatic conjugated system,[49−59] and its platinum(II) complexes
might be potential lead compounds for the development of new telomerase inhibitors
[1,2, 60−63].
In this work, we designed and synthesized seven organoplatinum(II) complexes
with structures as depicted in Figures 1 and 2, and investigated their abilities to act as
telomerase inhibitors and p53 activators in cisplatin-resistant SK-OV-3/DDP tumor
cells.
2. Results and discussion
2.1. Synthesis and characterization
9-Chloroacridine was synthesized with high efficiency (92.2% yield) by directly
heating the mixture of anthranilic acid (1.0 mmol) and cyclohexanone (1.0 mmol) in
100.0 mL POCl3 for 3 h (Fig. 1). Tacrine (H-L) was obtained from the reaction of
9-chloroacridine and concentrated ammonium hydroxide. Reaction of tacrine (H-L)
with cis-Pt(DMSO)2Cl2 in the presence of 10.0 mL methanol and 0.10 mL DMSO at
80 °C for 24 h led to the formation of a yellow complex, [PtClL(DMSO)] CH3OH
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(Pt1), [PtClL(DMP)] (Pt2), [PtClL(DPPTH)] (Pt3), [PtClL(PTH)] (Pt4),
[PtClL(PIPTH)] (Pt5), [PtClL(PM)] (Pt6) and [PtClL(en)] (Pt7) were synthesized by
the
reactions
of
Pt1
with
4,4'-dimethyl-2,2'-bipyridine
1,10-phenanthroline
(DMP),
(PTH),
4,7-diphenyl-1,10-phenanthroline
(DPPTH),
2-(1-pyrenecarboxaldehyde) imidazo [4,5-f]-[1,10] phenanthroline (PIPTH),
2-picolylamine (PM), and 1,2-ethylenediamine (en) in anhydrous CH3OH (25.0 mL)
at 65 °C for 6 h, respectively. The structures of tacrine (H-L) and its platinum(II)
complexes Pt1−Pt7 were characterized by ESI-MS, IR, elemental analysis, NMR, and
single crystal X-ray diffraction analysis (Figs. 1 and S1−S31). These results showed
that the Pt(II) center in Pt1−Pt7 adopts a four-coordinated square planar geometry.
Fig. 1. Synthesis of tacrine (H-L) and its platinum(II) complexes Pt1−Pt7. Reagents:
(a) POCl3, reflux, 3 h; (b) 3 eq. of concentrated ammonium hydroxide, pentanol,
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reflux, 18 h; (c) cis-Pt(DMSO)2Cl2, methanol/DMSO (10.0 mL/0.10 mL), 80 °C, 24 h;
(d) 1.0 mmol 4, 4'-dimethyl-2, 2'-bipyridine (DMP), 4,7-diphenyl-1,10-phenanthroline
(DPPTH), 1,10-phenanthroline (PTH), 2-(1-pyrenecarboxaldehyde) imidazo
[4,5-f]-[1,10] phenanthroline (PIPTH), 2-picolylamine (PM) or 1,2-ethylenediamine
(en), anhydrous CH3OH (25.0 mL), 65 °C, 6 h.
2.2. Crystal structure
The structure of Pt1 was determined by single crystal X-ray diffraction (Fig. 2).
The selected bond lengths and angles are listed in Tables S2−S4 in the Supporting
Information, and the crystallographic data are shown in Table S1 in the Supporting
Information. Pt1 showed a distorted square-planar geometry around the Pt(II) ion,
which is coordinated by the chelating tacrine ligand, one chlorido, and one DMSO
ligand. The values of the Pt1−S1, Pt1−C1, Pt1−Cl1 and Pt1−N1 bond distances were
determined to be 2.2071(16), 2.0230(5), 2.4014(15) and 2.0130(5) Å, respectively
(Tables S2), all of which are within the normal ranges [64–69]. The S1−Pt1−Cl1,
N1−Pt1−S1, N1−Pt1−Cl1 and N1−Pt1−C1 bond angles were determined to be
91.39(6), 178.75(12), 89.19(13) and 80.6(2)°, respectively (Table S3), all of which are
consistent with the corresponding values found in the Pt(dmba-R)LCl and
[Pt(diamine)(phen)]2+ crystal structures [64–69].
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Fig. 2. Single crystal X-ray crystal structure of Pt1.
2.3. Stability of Pt1−Pt7
The stability of Pt1−Pt7 (3.0 × 10–5 M) in a TBS buffer (pH 7.35, 10 mM Tris-HCl
with 1% DMSO) was determined using ESI-MS analysis.[64–69] The ESI-MS assay
investigated that the ESI-MS of Pt1−Pt7 (3.0 × 10–5 M) had the base peak for [M-H]−
at m/z = 504.9, [M-Cl]+ at m/z = 576.1, [M-Cl]+ at m/z= 724.2, [M-Cl]+ at m/z= 572.1,
[M-Cl]+ at m/z= 812.2, [M-Cl]+ at m/z= 500.1 and [M-Cl]+ at m/z= 452.1 in the TBS
buffer for 0 h, respectively. After Pt1−Pt7 (3.0 × 10–5 M) had been incubated in TBS
buffer for 48 h, these peaks at m/z = 504.3 for [M-H]−, m/z = 610.2 for [M-H]−, m/z=
724.2 for [M-Cl]+, m/z= 572.1 for [M-Cl]+, m/z= 812.3 for [M-Cl]+, m/z= 500.0 for
[M-Cl]+ and m/z = 486.1 for [M-H]− remained to be the strongest peaks in the ESI-MS
spectrum (Figs. S10, S13, S16, S19, S23, S27 and S31), suggesting that these
complexes are stable in the aqueous solution.
2.4. In vitro cytotoxicity
The cytotoxicity of the tacrine (H-L) ligand and its platinum(II) complexes
Pt1−Pt7 against various human tumor cell lines, including NCI-H460 (non-small cell
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lung cancer cell line), Hep-G2 (human hepatoma cell line), SK-OV-3 (human ovarian
cancer cells), SK-OV-3/DDP (cisplatin-resistant SK-OV-3 ovarian cancer cells),
MGC80-3 (human gastric mucous adenocarcinoma cells) and HL-7702 (human
hepatocyte cell line), was evaluated by the MTT assay. Cisplatin was used as a
positive control [70−72]. Pt1−Pt7 exhibited higher cytotoxicity against the five tumor
cell lines compared to the normal HL-7702 cell line (Tables 1 and S5). Among the
tumor cell lines, the SK-OV-3/DDP cell line showed the highest sensitivity towards
these Pt(II) complexes. Notably, among the seven Pt(II) complexes, Pt5 exhibited the
highest cytotoxicity against all five tumor cell lines. It was even more potent than
cisplatin in killing the tumor cells.
Table 1. IC50 values (µM)of Pt1−Pt7 against selected cell linesa.
NCI-H460
Hep-G2
SK-OV-3 SK-OV-3/DDP MGC80-3
HL-7702
>150
H-L
Pt1
Pt2
Pt3
Pt4
Pt5
Pt6
Pt7
>150
>150
>150
>100
>100
10.03±0.53
4.36±1.44
1.06±0.36
2.11±0.41
0.13±0.16
7.02±1.79
8.14±1.06
80.29±1.07
28.84±0.81 6.51±0.71 12.22±0.34
>100 >100 9.79±0.26
5.18±1.06 12.81±0.76 6.56±1.46
30.89±0.74 68.06±0.57
4.68±1.69 80.12±1.01
3.45±0.82 79.06±0.44
9.32±0.19 92.05±0.28
0.52±0.34 95.12±2.06
7.36±1.09 74.23±1.07
14.06±0.57 80.11±0.79
15.06±0.64 19.47±0.79
>100
5.01±0.41
15.96±1.32 7.82±1.58
36.26±0.73 >100
>100
2.38±0.95
>100
0.93±0.22
>100
10.22±0.42
Cisplatin b 17.41±0.94 15.16±1.21 13.36±1.05
a IC50 values are presented as mean ± SD (standard error of the mean) from five
b
independent experiments. The cells were incubated with each compound for 48 h.
The stock solution of cisplatin was prepared at a concentration of 1.0 mM with 0.154
M NaCl [70].
2.5. Cell apoptosis study with Pt1−Pt7
Cell apoptosis induced by Pt1−Pt7 in SK-OV-3/DDP tumor cells was performed
using the Annexin V-FITC and PI assays [72,73]. As shown in Fig. 3, upon treating
the cells with each individual Pt(II) complex at the corresponding IC50 concentration
for 24 h, the early apoptotic rate (Q3) increased significantly and the late apoptotic
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rate (Q2) also increased. The population of apoptotic cells (Q2+Q3) in the cisplatin
group is lower than those of Pt1−Pt7. Among Pt1−Pt7, Pt5 induced the most
significant degree of apoptosis.
Fig. 3. Flow cytometric analysis of SK-OV-3/DDP tumor cells treated with cisplatin
(80.0 µM), Pt1 (10.0 µM), Pt2 (4.0 µM), Pt3 (1.0 µM), Pt4 (2.0 µM), Pt5 (0.10 µM),
Pt6 (7.0 µM) and Pt7 (8.0 µM) for 24 h.
2.6. Cellular uptake
The cellular uptake of Pt1−Pt7 in SK-OV-3/DDP tumor cells was investigated
using ICP-MS, which can detect the level of platinum in the cells [74,75]. As shown
in Fig. 4A, the cellular uptake level of Pt5, i.e., 398 ± 5 ng of Pt per 106 cells, was
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17.8, 6.9, 1.3, 2.7, 14.4 and 14.8 times higher than that of Pt1, Pt2, Pt3, Pt4, Pt6 and
Pt7, respectively. In addition, these complexes exhibited different cellular distribution
(nuclear vs. mitochondria) patterns (Fig. 4B). Pt5 was mainly located in the nuclear
fraction, whereas cisplatin [48,60,62] and the other complexes showed preference
towards the mitochondria.
Fig. 4. Cellular uptake (A) and distribution (B) of Pt1-Pt7 in SK-OV-3/DDP tumor
after 24 h of incubation. The concentrations of Pt1-Pt7 used in this set of experiments
are the same as those described in Fig. 3.
2.7. Inhibition of the telomerase activity by Pt1-Pt7 in SK-OV-3/DDP tumor cells
Recent studies indicated that quindoline analogues can target telomere and affect
the telomerase activity, thereby inducing tumor cell apoptosis [76,77]. To investigate
whether Pt1-Pt7 can directly regulate the c-myc promoter (Pu27/c-myc G4 DNA),
affect the expression of hTRET, and consequently inhibit the telomerase function, the
TRAP-silver staining assay (TRAP assay), western blotting, RT-PCR, and a
transfection assay with a vector containing green fluorescent protein (EGFP) and
c-myc were performed using methods described previously [78−89]. The TRAP
profiles showed that a 24-hour treatment with Pt5 (inhibitory rate: 52.02%) is more
effective in inhibiting the telomerase function, compared to the results of Pt1
(inhibitory: 17.81%), Pt2 (inhibitory rate: 31.30%), Pt3 (inhibitory rate: 36.03%), Pt4
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(inhibitory rates: 32.90%), Pt6 (inhibitory rate: 28.96%) and Pt7 (inhibitory rate:
27.13%) in SK-OV-3/DDP tumor cells (Fig. 5). RT-PCR and western blot analysis
showed that the treatment with Pt5 induced the most significant decrease in the
mRNA and protein levels of c-myc and hTERT (Figs. 6 and 7), consistent with the
result of the TRAP assay. In addition, the transfection assay showed that Pt5 can
target the promoter of the c-myc gene and significantly inhibit the expression of
c-myc (Fig. 8).
Fig. 5. TRAP-silver staining assay on the effect of Pt1 (10.0 µM), Pt2 (4.0 µM), Pt3
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(1.0 µM), Pt4 (2.0 µM), Pt5 (0.10 µM), Pt6 (7.0 µM) and Pt7 (8.0 µM) in inhibiting
the telomerase activity in SK-OV-3/DDP tumor cells.
Fig. 6. Expression levels of the c-myc and hTERT genes in the SK-OV-3/DDP tumor
cells treated with Pt1 (10.0 µM), Pt2 (4.0 µM), Pt3 (1.0 µM), Pt4 (2.0 µM), Pt5 (0.10
µM), Pt6 (7.0 µM) and Pt7 (8.0 µM) for 24 h.
Fig. 7. Expression levels of c-myc and hTERT in the SK-OV-3/DDP tumor cells
treated with Pt1 (10.0 µM), Pt2 (4.0 µM), Pt3 (1.0 µM), Pt4 (2.0 µM), Pt5 (0.10 µM),
Pt6 (7.0 µM) and Pt7 (8.0 µM) for 24 h. (A) Western blot images. (B) Densitometric
analysis of the expression of c-myc and hTERT. The relative expression of each band
was determined by the density of each band divided by “density of each band/density
of actin band”. Mean SD was calculated from three independent measurements.
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Fig. 8. Transfection assay of a vector containing EGFP and c-myc. (A) The successful
transfection of the vector as indicated by the expression of EGFP in the transfected
SK-OV-3/DDP tumor cells. (B) The effect of Pt1−Pt7 on the c-myc promotor. The
transfected SK-OV-3/DDP tumor cells were treated with Pt1 (10.0 µM), Pt2 (4.0 µM),
Pt3 (1.0 µM), Pt4 (2.0 µM), Pt5 (0.10 µM), Pt6 (7.0 µM) and Pt7 (8.0 µM) for 24 h,
followed by the luciferase reporter gene assay to examine the expression level of
c-myc.
2.8. Pt1−Pt7 caused cell cycle arrest and regulated the expression of cell
cycle-related proteins
Recent studies indicated that inhibition of the telomerase activity by compounds
in tumor cells may be related to cell cycle arrest at the S and G2/M phases, thereby
inducing cell apoptosis [90−93]. Therefore, the status of cell cycle arrest for the
SK-OV-3/DDP cancer cells treated with Pt1−Pt7 for 24 h was analyzed. As shown in
Fig. 9, these compounds induced cell cycle arrest at the S and G2/M phases in the
tumor cells. Notably, Pt5 caused the most significant degree of cell cycle arrest
among Pt1−Pt7, i.e., 25.05% for the S phase and 15.57% for the G2/M phase. These
results confirmed that the antiproliferative mechanism induced by Pt1−Pt7 in the
SK-OV-3/DDP cancer cells is related to G2/M and S cell cycle arrest [90−95].
To further understand the mechanism of action of Pt1−Pt7, the expression levels
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of five cell cycle-related apoptotic proteins, i.e., CDK2, cyclin A, p21, p53 and p27,
were tested in the SK-OV-3/DDP cancer cells treated with Pt1-Pt7, using western blot
analysis (Fig. 10). The result suggested that Pt5 caused the most significant decrease
in the expression of cyclin A, but induced the most pronounced increase in the
expression of CDK2, p21, p53, and p27.
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Fig. 9. Cell cycle distribution of the SK-OV-3/DDP cancer cells treated with Pt1 (10.0
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µM), Pt2 (4.0 µM), Pt3 (1.0 µM), Pt4 (2.0 µM), Pt5 (0.10 µM), Pt6 (7.0 µM) and Pt7
(8.0 µM) for 24 h.
Fig. 10. Expresison levels of cell cycle-related proteins in the SK-OV-3/DDP cancer
cells treated with Pt1 (10.0 µM), Pt2 (4.0 µM), Pt3 (1.0 µM), Pt4 (2.0 µM), Pt5 (0.10
µM), Pt6 (7.0 µM) and Pt7 (8.0 µM) for 24 h. (A) Western blot images. (B)
Densitometric analysis of the expression of cyclin A, CDK2, p53, p27, and p21. The
relative expression of each band was determined by the density of each band divided
by “density of each band/density of actin band”. Mean SD was calculated from three
independent measurements.
2.9. Pt1−Pt7 induced mitochondrial dysfunction
It was acknowledged that mitochondrial integrity is highly related to the cell
viability [96,97]. Herein, we investigated whether Pt1−Pt7 induced mitochondrial
dysfunction by testing the mitochondrial membrane potential (MMP). The
SK-OV-3/DDP cells were treated with Pt1 (10.0 µM), Pt2 (4.0 µM), Pt3 (1.0 µM),
Pt4 (2.0 µM), Pt5 (0.10 µM), Pt6 (7.0 µM) and Pt7 (8.0 µM) for 24 h, and the MMP
values
were
measured
using
flow
cytometry
with
JC-1
(5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine) staining. As
shown in Fig. 11, there is an apparent loss of MMP in the SK-OV-3/DDP tumor cells
after the treatement with Pt1−Pt7, with Pt5 inducing the most significant MMP loss.
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Moreover, Pt5 caused the most significant decrease in the expression of bcl-2, but
induced the most pronounced increase in the expression of apaf-1, cytochrome c, and
bax (Fig. 12). In contrast, less regulation of the bcl-2, cytochrome c, and apaf-1
protein levels in SK-OV-3/DDP tumor cells was observed when treated with cisplatin
at the same conditions [60]. We also measured the effect of Pt1−Pt7 on the action of
caspase. Activation of caspase-3 and caspase-9 was observed in the cells treated with
Pt1−Pt7, with Pt5 inducing the most significant caspase activation (Figs. 13 and 14).
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Fig. 11. Status of MMP in the SK-OV-3/DDP tumor cells treated with Pt1 (10.0 µM),
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Pt2 (4.0 µM), Pt3 (1.0 µM), Pt4 (2.0 µM), Pt5 (0.10 µM), Pt6 (7.0 µM) and Pt7 (8.0
µM) for 24 h.
Fig. 12. Western blot analysis of the expression levels of apoptosis-regulating proteins
in the SK-OV-3/DDP cells treated with Pt1 (10.0 µM), Pt2 (4.0 µM), Pt3 (1.0 µM),
Pt4 (2.0 µM), Pt5 (0.10 µM), Pt6 (7.0 µM) and Pt7 (8.0 µM) for 24 h. (A) Western
blot images. (B) Densitometric analysis of the expression of bcl-2, apaf-1, cytochrome
c, bax. The relative expression of each band was determined by “density of each
band/density of actin band”. Mean SD was calculated from three independent
measurements.
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Fig. 13. Caspase-9 activation in the SK-OV-3/DDP cells after the treatment with Pt1
(10.0 µM), Pt2 (4.0 µM), Pt3 (1.0 µM), Pt4 (2.0 µM), Pt5 (0.10 µM), Pt6 (7.0 µM)
and Pt7 (8.0 µM) for 24 h.
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Fig. 14. Caspase-3 activation in the SK-OV-3/DDP cells after treatment with Pt1
(10.0 µM), Pt2 (4.0 µM), Pt3 (1.0 µM), Pt4 (2.0 µM), Pt5 (0.10 µM), Pt6 (7.0 µM)
and Pt7 (8.0 µM) for 24 h.
2.10. Pt5 induced cell apoptosis via activating the p53 signaling pathway in the
SK-OV-3/DDP cells
Previous studies have demonstrated that p53 is a key tumor suppressor protein at
the crossroads of cellular stress response pathways [98–106]. Upregulation of p53 can
lead to cell apoptosis, DNA repair, cell-cycle arrest, cellular senescence and
differentiation [107–111]. Furthermore, p53 is suggested to play a role in the
regulation of cell cycle arrest at the G2/M phase [111,112]. As the most active Pt(II)
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complexes in the cytotoxicity assay, Pt5 and Pt3 were chosen for the transfection
assay described below,[112–116] with Pt1 as a control group. As shown in Fig. 10,
Pt5 and Pt3 caused significant increase in the expression level of p53 in the
SK-OV-3/DDP cells, suggesting that upregulation of p53 may be an important
pathway for the cytotoxicity of these compounds [107–111]. To further understand
the mechanism of action of Pt5 and Pt3, we transiently depleted p53 via siRNA
interference. SK-OV-3/DDP tumor cells were transfected with the p53-siRNA-1
plasmid vector, the p53-siRNA-2 plasmid vector, the p53-siRNA-3 plasmid vector, or
siRNA-NC (the negative control plasmid vector), and harvested 48 h post-transfection
when the p53 depletion was at its maximum intensity and had been effective for at
least 24 h. The experimental results shown in Table S6 and Fig. 15 suggest that
p53-siRNA-3 is most effective in inhibiting the expression of p53. Fig. 16 shows that
the transfection did not cause significant morphologcal changes in the cells. After
being successfully transfected with the p53-siRNA-3 gene vector, the SK-OV-3/DDP
tumor cells were treated with Pt1 (10.0 µM), Pt3 (1.0 µM), or Pt5 (0.10 µM) for 24 h,
and then assayed by western blot. As shown in Fig. 17, Pt1, Pt3 and Pt5 did not cause
significant upregulation of p53 in the cells transfected with p53-siRNA-3.[107–116]
Nonetheless, Fig. 18 shows that treating the p53-siRNA-3-transfected cells with Pt5
caused an increase in the percentage of apoptotic cells (Q2+Q3). Taken together, it
can be concluded that Pt5 may trigger cell apoptosis through activation of
p53.[107–116]
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Fig. 15. Western blot analysis to determine the siRNA interference efficiency of
different p53-siRNR plasmids. (A) Western blot images. (B) Densitometric analysis of
the expression of p53. The relative expression of each band was determined by
“density of each band/density of actin band”. Mean SD was calculated from three
independent measurements.
Fig. 16. Fluorescence microscopy morphological analysis of the SK-OV-3/DDP tumor
cells transfected with the p53-siRNA-3 plasmid vectors. The SK-OV-3/DDP cells
were transfected with 2.0 µg p53-siRNA-3 plasmid and imaged using a Nikon
TE2000 scanning fluorescent microscope.
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Fig. 17. Effect of Pt1, Pt3 and Pt5 on the expression level of p53 in the
SK-OV-3/DDP tumor cells transfected with p53-siRNA-3. The transfected cells were
incubated with Pt1 (10.0 µM), Pt3 (1.0 µM) or Pt5 (0.10 µM) for 24 h. (A) Western
blot images. (B) Densitometric analysis of the expression of p53. The relative
expression of each band was determined by “density of each band/density of actin
band”. Mean SD was calculated from three independent measurements.
Fig. 18. Cell apoptosis induced by Pt1 (10.0 µM), Pt3 (1.0 µM) and Pt5 (0.10 µM) in
the SK-OV-3/DDP tumor cells previously transfected with the p53-siRNA-3 plasmid
(2.0 µg).
2.11. Interactions with BSA and GSH
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To understand the resistance mechanism of tacrine platinum(II) complexes in
SK-OV-3/DDP tumor cells, the interaction of Pt5 and Pt1 with glutathione (GSH) and
bovine serum albumin (BSA) were further studied through the method reported by
Patra and Liang [62,117]. As shown in the Fig. S32, the addition of Pt5 and Pt1 to the
solution of BSA resulted in a significant decrease of the fluorescent intensity of BSA
at 345 nm, with 98.23% and 37.33% quenching ratios, respectively, suggesting a
definite interaction of Pt5 and Pt1 with BSA protein [62,117]. In addition, the
absorbance of Pt5 (2.0×10-5 mol/L) at 225–600 nm exhibited a small decrease (Fig.
S33), demonstrating that Pt5 (2.0×10-5 mol/L) do not react with GSH in the
SK-OV-3/DDP tumor cells [62,117], thus its cytotoxicity in SK-OV-3/DDP tumor
cells was retained, which was agreed well with the fact that Pt5 exhibited high
activity against the SK-OV-3/DDP tumor cells (or the cisplatin-resistant SK-OV-3
ovarian cancer cells) [62,117].
2.12. Preliminary safety evaluation of Pt5 and Pt1
The safety of Pt5 was tested by administrating KM mice with a good practice
administration volume (i.e., 0.6 mL/20 g body weight) of the Pt5 solution (10% v/v
DMSO/saline) every two days (q2d) through intraperitoneal injection at a dose of 15
mg/kg and 10 mg/kg. During the 14 days of treatment, the mice showed no visible
signs of pain, distress, or discomfort，and their body weights kept increasing (Figs. 19
and S34). The average body weight of the untreated control group rose from 20.7 to
29.7 g; the average body weight of the mice receiving Pt5 at doses of 15 mg/kg and
10 mg/kg rose from 20.5 to 25.1 g and from 20.9 to 26.7 g, respectively. We also
performed safety test on Pt1 as a control. Due to the low solubility of Pt1, we chose
doses of 3 mg/kg and 2 mg/kg. The average body weight of the mice receiving Pt1 at
the doses of 3 mg/kg and 2 mg/kg rose from 19.7 to 26.0 g and from 20.7 to 28.3 g,
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respectively. At Day 14, all the animals were sacrificed, and no signs of peritonitis or
damage to organs were observed across all the groups.
Fig. 19. Average body weight of KM mice treated with Pt5 (A) and Pt1 (B).
2.12. Tumor growth inhibition by Pt5 and Pt1 in the Hep-G2 xenograft mouse model
When the tumors had grown to a volume of 90-160 mm3, the Hep-G2 xenograft
mice were randomized into vehicle control and treatment groups (n=6 per group) and
treated via IP injection with Pt5 at a dose of 15 mg/kg, Pt1 at a dose of 3mg/kg, or
cisplatin at a dose of 2 mg/kg every two days. As shown in Fig. 20, the average tumor
volume of the vehicle control group grew rapidly and reached a mean volume of
1082.7 mm3 on Day 18. In comparison, the average tumor volumes of the Pt5 and
cisplatin groups grew slowly, with a mean volume of 543.0 mm3 and 440.7 mm3 at
day 18, respectively, corresponding to relative tumor increment rates of 50.4% and
41.4%, respectively. The values of tumor weight were recorded after the mice were
killed on Day 18, and the tumor growth inhibition rates based on the tumor weight
were caculated. As shown in Fig. 20 and Tables S7−S9, the tumor growth inhibition
rate of Pt5 was determined to be 40.8%, slightly lower than that of cisplatin (58.5%).
Although cisplatin showed higher tumor growth inhibition, it displayed higher
toxicity as evidenced by the more significant loss of body weight compared to Pt5. As
showed in Fig. 20, the body weight of the animals treated with Pt5 showed a 14% loss
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in the body weight, less than the value of the cisplatin group (21.9%). Pt1 also
inhibited tumor growth but at a much lower inhibition rate compared to Pt5 (Fig. 21).
Consistent with the in vitro findings, we also demonstrated that Pt5 induced p53
activation and caused downregulation of the c-myc protein in the Hep-G2 tumor
xenograft mice (Fig. 22).
Fig. 20. In vivo anticancer activity of Pt5 in mice bearing Hep-G2 tumor xenograft.
(A) Effect of Pt5 (15 mg/kg/q2d), cisplatin (2 mg/kg/q2d), or vehicle (10% DMSO in
saline, v/v) on the tumor growth. Tumor growth was tracked by the mean tumor
volume (mm3) ± SD (n = 6) and calculated as the relative tumor increment rate (T/C,
%). (B) Body weight change of the Hep-G2 tumor xenograft mice turing treatment. (C)
Inhibition rates of tumor growth, calculated by the average tumor weight. (**) P <
0.05, treatment group vs. vehicle control. (D) Photographs of tumors from different
experimental groups.
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Fig. 21. In vivo anticancer activity of Pt1 in mice bearing Hep-G2 tumor xenograft.
(A) Effect of Pt1 (3 mg/kg/q2d), cisplatin (2 mg/kg/q2d), or vehicle (10% DMSO in
saline, v/v) on the tumor growth. Tumor growth was tracked by the mean tumor
volume (mm3) ± SD (n = 6) and calculated as the relative tumor increment rate (T/C,
%). (B) Body weight change of the Hep-G2 tumor xenograft mice turing treatment. (C)
Inhibition rates of tumor growth, calculated by the average tumor weight. (**) P <
0.05, treatment group vs. vehicle control. (D) Photographs of tumors from different
experimental groups.
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