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Ruthenium(II) polypyridyl complexes: Synthesis, characterization and anticancer activity studies on BEL-7402 cells.
Accepted Manuscript
Ruthenium(II) polypyridyl complexes: Synthesis, characterization
and anticancer activity studies on BEL-7402 cells
Dan Wan, Shang-Hai Lai, Chuan-Chuan Zeng, Cheng Zhang,
Bing Tang, Yun-Jun Liu
PII:
DOI:
Reference:
S0162-0134(17)30072-7
doi: 10.1016/j.jinorgbio.2017.04.026
JIB 10212
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
8 February 2017
24 April 2017
26 April 2017
Please cite this article as: Dan Wan, Shang-Hai Lai, Chuan-Chuan Zeng, Cheng Zhang,
Bing Tang, Yun-Jun Liu , Ruthenium(II) polypyridyl complexes: Synthesis,
characterization and anticancer activity studies on BEL-7402 cells. The address for the
corresponding author was captured as affiliation for all authors. Please check if
appropriate. Jib(2017), doi: 10.1016/j.jinorgbio.2017.04.026
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ACCEPTED MANUSCRIPT
Submitted to JIB
Ruthenium (II) polypyridyl complexes: synthesis, characterization
PT
and anticancer activity studies on BEL-7402 cells
RI
Dan Wana,1, Shang-Hai Laia,1, Chuan-Chuan Zenga, Cheng Zhanga, Bing Tanga,
School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou, 510006,
NU
a
SC
Yun-Jun Liua,b,*
b
MA
PR China
Guangdong Cosmetics Engineering & Technology Research Center, Guangzhou,
These authors contribute equally
AC
CE
PT
E
1
D
510006, PR China
* Corresponding author. E-mail address: lyjche@163.com(Y.J. Liu).
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Abstract Two new ligand PTTP (2-phenoxy-1,4,8,9-tetraazatriphenylene) and FTTP
(2-(3-fluoronaphthalen-2-yloxy)-1,4,8,9-tetraazatriphenylene)
polypyridyl
[Ru(N-N)2(FTTP)](ClO4)2
complexes
(N-N
=
dmb:
their
six
[Ru(N-N)2(PTTP)](ClO4)2
and
4,4′-dimethyl-2,2′-bipiridine;
dmp:
PT
ruthenium(II)
and
RI
2,9-dimethyl-1,10-phenanthroline; ttbpy: 4,4′-ditertiarybutyl-2,2′-bipyridine) were
SC
synthesized and characterized. The cytotoxic activity of the complexes against cancer
cells HeLa, BEL-7402, A549, HepG-2, HOS and normal cell LO2 was evaluated by
NU
MTT method. The IC50 values range from 1.5 ± 0.1 to 55.9 ± 7.5 µM. Complex 3
MA
shows the highest cytotoxic activity toward BEL-7402 cells (IC50 = 1.5 ± 0.1 µM).
Complex 5 displays most effective inhibition of the cell growth in A549 and HOS
D
cells with low IC50 values of 2.5 ± 0.6 and 2.6 ± 0.1 µM, respectively. The apoptosis,
PT
E
reactive oxygen species, mitochondrial membrane potential, DNA damage, autophagy
and anti-metastasis assay were investigated under a fluorescent microscope. The cell
CE
cycle arrest was assayed by flow cytometry, and the expression of caspases and Bcl-2
AC
family proteins was studied by western blot. The results obtained show that the
complexes induce apoptosis in BEL-7402 cells through a ROS-mediated
mitochondrial dysfunction pathway.
Keywords: Ru(II) polypyridyl complexes; apoptosis; anti-metastasis assay;
autophagy; ROS; western blot.
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1. Introduction
Transition metal complexes have received great attention as cancer therapy, due to the
clinical sources of cisplatin and its derivatives [1,2]. However, the side-effect of
cisplatin such as neurotoxicity and nephrotoxicity [3,4] limited the its clinical
PT
application. These drawbacks in platinum-based anticancer drugs have stimulated
RI
considerable attempts to replace cisplatin with suitable alternatives by other transition
SC
metal complexes. Ruthenium is the most attractive metal owing to its chemical and air
stability, structural diversity, low toxicity and ability to mimic iron binding in
NU
biological system, which finally supported them as highly potent anticancer agents
MA
other than platinum based drugs [5-9]. Recently, ruthenium compounds have received
increasing attention in the medicinal chemistry field, especially in relation to the
D
development of chemotherapeutics that present minimal side effects and immunity to
PT
E
the acquisition of drug resistance [10,11]. Presently, ruthenium complex NKP-1339
(trans-[tetrachloridobis(1H-indazole)ruthenate(III)]) has successfully entered into the
CE
clinical trials [12, 13]. NKP-1339 has been studied against solid tumors and showed
AC
promising results in a phase I clinical trial, most remarkably in patients with
gastrointestinal neuroendocrine tumors [14]. The ruthenium(II) complexes containing
dppz-like (dppz = dipyrido[3,2-a-2′,3′-c]phenanzine) show high anticancer activity.
Complex [Ru(bpy)2(dppn)]2+ (dppn = benzo[i]dipyrido[3,2-a:2′,3′-h]quinoxaline)
displays high inhibitory effect on the cell growth in HT-29 (IC50 = 6.4 ± 1.9 µM) and
MCF-7 (IC50 = 3.3 ± 1.2 µM) [15]. [Ru(bpy)(phpy)(dppz)]+ (phpy = 2-phenylpyridine)
was found to be rapidly taken up by cancer cells after a 2 h incubation [16].
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[Ru(phpy)(bpy)(dppn)]+ (bpy = 2,2′-bipyridine) is 6 times more active than the
platinum drug against HeLa cells, and it is able to disrupt the mitochondria membrane
potential
[Ru(phen)2(addppn)]2+
[17].
(addppn
=
acenaphtheno[1,2-b]-1,4-diazabenzo[i]dipyrido[3,2-a:2′,3′-c]phenazine) inhibits the
PT
cell growth in BEL-7402 cell (IC50 = 3.9 ± 0.4 µM) at G0/G1 phase and G2/M phase
=
phenantheno[1,2-b]-1,4-diazabenzo[i]dipyrido[3,2-a:2′,3′-c]phenazine)
SC
(pddppn
RI
in SK-BR-3 cell (IC50 = 5.1 ± 0.6 µM) [18]. Ru(II) complex [Ru(dmp)2(pddppn)]2+
exhibits very high cytotoxic activity toward BEL-7402 (IC50 = 1.6 ± 0.4 µM), MG-63
NU
(IC50 = 1.5 ± 0.2 µM) and A549 cells (IC50 = 1.5 ± 0.3 µM) [19]. Changes in the
MA
structure of main ligand could be used to attain diverse anticancer activity of
ruthenium(II) complexes. Therefore, extensive studies on different structure dppz-like
D
main ligand are necessary to further elucidate the anticancer effect and mechanism of
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Ru(II) complexes and discover some new potential anticancer reagents. In this article,
two new dppz-like ligands PTTP (PTTP = 2-phenoxy-1,4,8,9-tetraazatriphenylene)
ruthenium(II)
polypyridyl
AC
their
CE
and FTTP (FTTP = 2-(3-fluoronaphthalen-2-yloxy)-1,4,8,9-tetraazatriphenylene) and
(N-N
[Ru(N-N)2(FTTP)](ClO4)2
complexes
=
dmb:
[Ru(N-N)2(PTTP)](ClO4)2
and
4,4′-dimethyl-2,2′-bipiridine;
dmp:
2,9-dimethyl-1,10-phenanthroline; ttbpy: 4,4′-ditertiary butyl-2,2′-bipyridine, Scheme
1) were synthesized and characterized by elemental analysis, ESI-MS, IR, 1H NMR
and 13C NMR. The cytotoxicity in vitro, apoptosis, reactive oxygen species,
mitochondrial membrane potential, cell cycle distribution, cell invasion, autophagy
and Bcl-2 family proteins expression were investigated.
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2. Experimental
2.1. Materials and methods
All reagents and solvents were purchased commercially and used without further
DMSO,
4,4'-dimethyl-2,2'-bipyridine
(dmb),
4,4′-ditertiary
RI
experiments.
PT
purification unless otherwise noted. Ultrapure MilliQ water was used in all
(Roswell
Park
Memorial
Institute)
1640
SC
butyl-2,2′-bipyridine (ttbpy), 2,9-dimethyl-1,10-phenanthroline (dmp), and RPMI
were
purchased
from
Sigma.
NU
1,10-phenanthroline was obtained from the Guangzhou Chemical Reagent Factory.
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BEL-7402 (human hepatocellular carcinoma), A549 (human lung carcinoma), HeLa
(human cervical cancer), HepG2 (human hepatocellular carcinoma), HOS (human
D
osteosarcoma) and normal LO2 (human liver cancer) cell lines were purchased from
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the American Type Culture Collection. RuCl3·3H2O was purchased from the Kunming
Institution of Precious Metals.
CE
Microanalysis (C, H, and N) was carried out with a Perkin-Elmer 240Q elemental
AC
analyzer. Electrospray ionization mass spectra (ESI-MS) were recorded on a LCQ
system (Finnigan MAT, USA) using acetonitrile as mobile phase. The spray voltage,
tube lens offset, capillary voltage and capillary temperature were set at 4.50 KV, 30.00
V, 23.00 V and 200 oC, respectively, and the quoted m/z values are for the major peaks
in the isotope distribution. 1H NMR and 13C NMR spectra were recorded on a
Varian-500 spectrometer with DMSO-d6 as solvent and tetramethylsilane (TMS) as an
internal standard at 500 MHz at room temperature.
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2.2. Synthesis of ligands and complexes
2.2.1. 2-phenoxy-1,4,8,9-tetraazatriphenylene (PTTP)
1,10-phenthroline-5,6-dione
(0.210
g,
1.00
mmol)
[20],
PT
4-phenoxybenzene-1,2-diamine (0.200 g, 1.00 mmol) and citric acid (0.035g, 1.82
RI
mmol) were dissolved in 30 mL of ethanol and react at room temperature for 15 min.
SC
The brown precipitate was washed with water (3 × 30 mL) and brown powder was
obtained. Yield: 85%. Anal. Calcd for C24H14N4O: C, 76.99; H, 3.77; N, 14.96%.
NU
Found: C, 76.87; H, 3.86; N, 15.06%. FAB-MS: m/z = 375 [M + 1]. IR (KBr, cm-1):
MA
3373.3, 3056.4, 2964.1, 1626.9, 1599.9, 1509.4, 1453.9, 1437.6, 1404.4, 1358.1,
D
1323.3, 1222.1, 1159.4, 1071.2, 873.2, 742.8.
PT
E
2.2.2. 2-(3-fluoronaphthalen-2-yloxy)-1,4,8,9-tetraazatriphenylene (FTTP)
1,10-phenthroline-5,6-dione
(0.210
g,
1.00
mmol),
CE
4-(3-fluoronaphthalene-2-yloxy)benzene-1,2-diamine (0.268 g, 1 mmol) and citric
AC
acid (0.035g, 1.82 mmol) were dissolved in 30 mL of ethanol and react at room
temperature for 15 min. The brown precipitate was washed with water (3 × 30 mL)
and brown powder was obtained. Yield: 85%. Anal. Calcd for C28H15FN4O: C, 76.01;
H, 3.42; N, 12.66%. Found: C, 75.89; H, 3.55; N, 12.45%. FAB-MS: m/z = 443 [M +
1]. IR (KBr, cm-1): 3391.5, 3045.2, 1720.9, 1619.8, 1586.9, 1481.8, 1407.3, 1360.7,
1257.5, 1169.0, 1071.3, 858.7, 741.2.
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2.2.3. Synthesis of [Ru(dmb)2(PTTP)](ClO4)2 (1)
A mixture of cis-[Ru(dmb)2Cl2]·2H2O [21] (0.288 g, 0.50 mmol) and PTTP
(0.162 g, 0.50 mmol) in ethylene glycol (20 mL) was refluxed under argon for 8 h to
give a clear red solution. Upon cooling, a red precipitate was obtained by dropwise
PT
addition of saturated aqueous NaClO4 solution. The crude product was purified by
RI
column chromatography on neutral alumina with a mixture of CH3CN-ethanol (4:1,
SC
v/v) as eluent. The red band was collected. The solvent was removed under reduced
pressure and a red powder was obtained. Yield: 73%. UV/Vis (PBS): λmax (ɛ) = 284
NU
(68440), 401 (13520), 439 (12480). Anal. Calc for C48H38N8Cl2O9Ru: C, 55.28; H,
MA
3.67; N, 10.74%. Found: C, 55.41; H, 3.84; N, 10.53%. IR (KBr, cm-1): 3399.9,
3059.3, 2964.5, 1618.6, 1598.8, 1528.6, 1479.9, 1452.8, 1353.0, 1313.5, 1243.8,
D
1221.8, 1195.3, 1144.7, 821.5, 624.2. 1H NMR (DMSO-d6, Fig. S1a, supporting
PT
E
information): δ 9.56 (d, 2H, J = 7.0 Hz), 8.72 (d, 4H, J = 8.5 Hz), 8.54 (d, 1H, J = 8.5
Hz), 8.22 (dd, 2H, J = 5.0, J = 5.5 Hz), 8.03 (d, 1H, J = 6.5 Hz), 7.99 (d, 1H, J = 8.5
CE
Hz), 7.92 (d, 1H, J = 8.0 Hz), 7.62 (d, 4H, J = 5.5 Hz), 7.54 (t, 2H, J = 5.5 Hz), 7.51
AC
(d, 1H, J = 3.0 Hz), 7.43-7.37 (m, 5H), 7.19 (d, 2H, J = 5.5 Hz), 2.56 (s, 6H), 2.47 (s,
6H). 13C NMR: (DMSO-d6, 125 MHz, ppm): 156.27, 156.05, 153.31, 151.77, 151.01,
150.39, 150.18, 150.07, 149.69, 149.61, 140.46, 139.80, 139.19, 133.89, 132.61,
130.99, 129.78, 128.54, 128.32, 127.94, 127.53, 127.39, 127.13, 126.08, 125.05,
124.97, 120.08, 116.76, 20.75, 20.66. ESI-MS (CH3CN): m/z 422.5 ([M-2ClO4]2+).
2.2.4. Synthesis of [Ru(dmp)2(PTTP)](ClO4)2 (2)
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ACCEPTED MANUSCRIPT
This complex was synthesized in a manner identical to that described for 1, with
[Ru(dmp)2Cl2]·2H2O [22] in place of [Ru(dmb)2Cl2]·2H2O. Yield: 72%. UV/Vis
(PBS): λmax = 283 (54840), 396 (14800), 435 (12000). Anal. Calc for
C52H38N8Cl2O9Ru: C, 57.25; H, 3.51; N, 10.27%. Found: C, 57.11; H, 3.71; N,
PT
10.42%. IR (KBr, cm-1): 3481.3, 3057.4, 2923.7, 1624.8, 1597.6, 1509.0, 1480.1,
RI
1462.7, 1354.8, 1314.4, 1244.9, 1222.3, 1195.1, 1162.1, 856.7, 622.8. 1H NMR
SC
(DMSO-d6, Fig. S1b, supporting information): δ 9.38 (d, 2H, J = 7.0 Hz), 8.93 (dd,
2H, J = 8.0, J = 9.0 Hz), 8.46 (d, 4H, J = 7.0 Hz), 8.42 (d, 1H, J = 4.5 Hz), 8.26 (dd,
NU
2H, J = 5.5, J = 5.5 Hz), 7.99 (d, 3H, J = 5.0 Hz), 7.58 (d, 3H, J = 6.5 Hz), 7.54 (d,
MA
1H, J = 6.0 Hz), 7.49 (d, 2H, J = 5.5 Hz), 7.44 (d, 1H, J = 2.5 Hz), 7.41 (d, 1H, J =
2.5 Hz), 7.39 (d, 1H, J = 3.0 Hz), 7.37 (d, 1H, J = 5.0 Hz), 7.34 (d, 1H, J = 6.5 Hz),
D
7.32 (d, 1H, J = 2.5 Hz), 1.93 (s, 6H), 1.82 (s, 6H). 13C NMR (DMSO-d6, 125 MHz,
PT
E
ppm): 168.22, 166.95, 154.96, 153.98, 152.02, 151.16, 150.99, 150.89, 148.96,
147.74, 140.51, 139.89, 139.54, 139.31, 138.43, 136.88, 134.05, 133.77, 133.55,
CE
131.19, 129.71, 129.59, 128.14, 127.74, 127.61, 127.39, 127.32, 126.99, 126.70,
AC
126.53, 126.31, 120.16, 116.77, 113.91, 26.17, 25.83, 24.67, 19.05. ESI-MS (CH3CN):
m/z 993.0 ([M-ClO4]+), 890.2 ([M-2ClO4-H]+), 446.6 ([M-2ClO4]2+).
2.2.5. Synthesis of [Ru(ttbpy)2(PTTP)](ClO4)2 (3)
This complex was synthesized in a manner identical to that described for 1, with
[Ru(ttbpy)2Cl2]·2H2O [21] in place of [Ru(dmb)2Cl2]·2H2O. Yield: 70%. UV/Vis
(PBS): λmax = 285 (68760), 403 (16160), 437 (15600). Anal. Calc for
8
ACCEPTED MANUSCRIPT
C60H62N8Cl2O9Ru: C, 59.49; H, 5.16; N, 9.26%. Found: C, 59.78; H, 5.08; N, 9.14%.
IR (KBr, cm-1): 3500.4, 3072.8, 2958.5, 1614.7, 1540.3, 1509.2, 1480.3, 1462.7,
1413.3, 1362.2, 1314.6, 1245.8, 1222.7, 1194.5, 1161.9, 838.2, 622.3. 1H NMR
(DMSO-d6, Fig. S1c, supporting information): δ 9.58 (d, 2H, J = 8.0 Hz), 8.87 (d, 4H,
PT
J = 8.5 Hz), 8.53 (d, 1H, J = 9.0 Hz), 8.14 (d, 2H, J = 5.5 Hz), 8.03 (dd, 2H, J = 3.5, J
RI
= 2.5 Hz), 7.97 (d, 1H, J = 3.0 Hz), 7.64 (d, 4H, J = 6.5 Hz), 7.61 (d, 2H, J = 2.5 Hz),
SC
7.59 (d, 2H, J = 2.5 Hz), 7.50 (d, 1H, J = 2.5 Hz), 7.42 (d, 1H, J = 7.5 Hz), 7.38 (d,
4H, J = 5.5 Hz), 1.43 (s, 18H), 1.35 (s, 18H). 13C NMR (DMSO-d6, 125 MHz, ppm):
NU
161.94, 161.82, 156.49, 156.28, 153.12, 151.78, 151.26, 151.14, 150.67, 150.22,
MA
150.11, 140.46, 139.84, 139.34, 139.25, 133.89, 132.94, 132.69, 130.99, 129.88,
129.83, 128.88, 128.19, 127.95, 127.69, 127.54, 127.14, 126.08, 124.84, 124.35,
D
121.90, 121.81, 120.08, 116.76, 114.36, 114.22, 113.67, 35.54, 35.43, 30.08, 29.98.
PT
E
ESI-MS (CH3CN): m/z 1111.3 ([M-ClO4]+, 506.4 ([M-2ClO4]2+).
CE
2.2.6. Synthesis of [Ru(dmb)2(FTTP)](ClO4)2 (4)
AC
This complex was synthesized in a manner identical to that described for 1, with
FTTP in place of PTTP. Yield: 71%. UV/Vis (PBS): λmax = 283 (69000), 390 (13720),
441 (12280). Anal. Calc for C52H39N8FCl2O9Ru: C, 56.22; H, 3.54; N, 10.09%. Found:
C, 56.34; H, 3.66; N, 10.28%. IR (KBr, cm-1): 3433.4, 3067.0, 2960.3, 1618.4, 1586.9,
1547.3, 1485.8, 1468.7, 1414.7, 1354.9, 1307.5, 1217.4, 1208.6, 1175.8, 819.1, 622.3.
1
H NMR (DMSO-d6, Fig. S1d, supporting information): δ 9.55 (d, 1H, J = 7.0 Hz),
9.48 (d, 1H, J = 7.0 Hz), 8.72 (dd, 4H, J = 5.0, J = 5.5 Hz), 8.55 (d, 1H, J = 6.0 Hz),
9
ACCEPTED MANUSCRIPT
8.24 (dd, 2H, J = 5.0, J = 5.5 Hz), 8.20 (d, 1H, J = 5.5 Hz), 8.06 (d, 1H, J = 3.0 Hz),
8.01 (d, 1H, J = 5.5 Hz), 7.97 (d, 1H, J = 6.0 Hz), 7.93 (d, 1H, J = 2.5 Hz), 7.88 (d,
1H, J = 5.0 Hz), 7.67 (d, 1H, J = 8.5 Hz), 7.64-7.58 (m, 5H), 7.53 (dd, 2H, J = 6.0, J
= 5.5 Hz), 7.41 (d, 2H, J = 5.0 Hz), 7.19 (d, 2H, J = 6.0 Hz), 2.54 (s, 6H), 2.46 (s, 6H).
13
PT
C NMR (DMSO-d6, 125 MHz, ppm): 160.89, 156.29, 156.06, 154.26, 153.38,
RI
153.03, 151.03, 150.40, 149.91, 149.68, 149.59, 143.25, 140.32, 139.03, 138.52,
SC
133.03, 132.54, 131.58, 130.76, 130.07, 129.81, 128.54, 128.33, 127.54, 127.40,
126.26, 125.86, 125.05, 124.97, 120.91, 110.49, 20.76, 20.67. ESI-MS (CH3CN): m/z
MA
NU
1013.0 ([M-ClO4]+), 456.5 ([M-2ClO4]2+).
2.2.7. Synthesis of [Ru(dmp)2(FTTP)](ClO4)2 (5)
D
This complex was synthesized in a manner identical to that described for 2, with
PT
E
FTTP in place of PTTP. Yield: 72%. UV/Vis (PBS): λmax = 270 (58240), 389 (15080),
451 (10400). Anal. Calc for C56H39N8FCl2O9Ru: C, 58.04; H, 3.39; N, 9.67%. Found:
CE
C, 58.25; H, 3.31; N, 9.78%. IR (KBr, cm-1): 3400.6, 3069.3, 2923.6, 1623.2, 1587.7,
AC
1487.3, 1469.9, 1447.6, 1358.3, 1307.9, 1236.3, 1217.6, 1203.5, 860.7, 624.3. 1H
NMR (DMSO-d6, Fig. S1e, supporting information): δ 9.37 (d, 2H, J = 7.0), 8.90 (t,
2H, J = 7.5 Hz), 8.46 (d, 4H, J = 8.5 Hz), 8.41 (d, 1H, J = 4.5 Hz), 8.24 (dd, 2H, J =
8.5, J = 8.5 Hz), 8.15 (d, 1H, J = 8.5 Hz), 8.04 (d, 1H, J = 2.0 Hz), 7.95 (d, 3H, J =
6.5 Hz), 7.86 (d, 1H, J = 2.5 Hz), 7.63-7.57 (m, 6H), 7.51 (d, 1H, J = 1.0 Hz), 7.46 (d,
1H, J = 1.5 Hz), 7.39 (dd, 2H, J = 5.0, J = 5.0 Hz), 1.91 (s, 6H), 1.81 (s, 6H). 13C
NMR (DMSO-d6, 125 MHz, ppm): 167.99, 166.79, 160.69, 154.32, 153.78, 153.47,
10
ACCEPTED MANUSCRIPT
151.05, 150.59, 148.79, 147.55, 143.09, 140.31, 138.87, 138.55, 138.21, 136.64,
133.66, 133.16, 131.42, 130.71, 129.71, 129.52, 129.45, 127.52, 127.40, 127.13,
126.83, 126.46, 126.31, 126.09, 125.77, 120.77, 110.58, 25.98, 24.49. ESI-MS
PT
(CH3CN): m/z 480.1 ([M-2ClO4]2+).
RI
2.2.8. Synthesis of [Ru(ttbpy)2(FTTP)](ClO4)2 (6)
SC
This complex was synthesized in a manner identical to that described for 3, with
FTTP in place of PTTP. Yield: 71%. UV/Vis (PBS): λmax = 290 (58560), 391 (19200),
NU
442 (17080). Anal. Calc for C64H63N8FCl2O9Ru: C, 60.09; H, 4.96; N, 8.76%. Found:
MA
C, 60.27; H, 4.74; N, 8.89%. IR (KBr, cm-1): 3489.2, 3072.4, 2906.9, 1615.2, 1588.2,
1541.5, 1486.2, 1469.2, 1414.1, 1366.8, 1308.6, 1237.2, 1219.1, 1208.7, 1176.9,
D
1156.9, 843.5, 622.8. 1H NMR (DMSO-d6, Fig. S1f, supporting information): δ 9.57
PT
E
(d, 1H, J = 7.0 Hz), 9.51 (d, 1H, J = 7.5 Hz), 8.87 (d, 4H, J = 7.5 Hz), 8.55 (d, 1H, J =
6.5 Hz), 8.17 (d, 2H, J = 5.5 Hz), 8.14 (d, 1H, J = 5.0 Hz), 8.07 (d, 2H, J = 5.5 Hz),
CE
7.99 (d, 1H, J = 2.0 Hz), 7.93 (d, 2H, J = 5.5 Hz), 7.68 (d, 1H, J = 9.0 Hz), 7.65-7.59
AC
(m, 7H), 7.37 (d, 2H, J = 5.0 Hz), 7.26 (d, 1H, J = 1.5 Hz), 7.18 (d, 1H, J = 2.0 Hz),
1.43 (s, 18H), 1.33 (s, 18H). 13C NMR (DMSO-d6, 125 MHz, ppm): 161.95, 161.82,
160.92, 156.51, 156.29, 154.27, 153.21, 152.86, 151.29, 150.69, 150.42, 149.96,
143.28, 140.39, 139.05, 138.59, 133.13, 132.64, 131.60, 130.77, 130.18, 129.91,
127.66, 127.51, 126.26, 125.88, 124.85, 124.37, 121.91, 121.82, 120.93, 110.53,
35.55, 35.43, 30.08, 29.98. ESI-MS (CH3CN): m/z 1179.1 ([M-ClO4]+), 540.2
([M-2ClO4]2+).
11
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2.3. Stability of the complexes in buffer
All the complexes were first dissolved in a minimum amount of DMSO (0.5% of
the final volume) and then diluted with PBS to a required concentration. The stability
PT
was analyzed by monitoring the electronic spectra over 48 h on a Shimadzu
SC
RI
MPS-2000 spectrophotometer.
2.4. Cytotoxic activity evaluation in vitro
NU
3-(4,5-dimethylthiazole)-2,5-diphenyltetraazolium bromide (MTT) method [23]
MA
was used to determine the cytotoxic activity in vitro of the complexes. Cells were
placed in 96-well microassay culture plates (8 × 103 cells per well) and grown
D
overnight at 37 °C in a 5% CO2 incubator. The tested complexes were dissolved in
PT
E
DMSO and then added to the wells to achieve final concentrations ranging from 10–6
to 10–4 M. Control wells were prepared by addition of culture medium (100 μL). The
CE
plates were incubated at 37 °C in a 5% CO2 incubator for 48 h. Upon completion of
AC
the incubation, stock MTT dye solution (20 μL, 5 mg/mL–1) was added to each well.
After 4 h, buffer (100 μL) containing dimethylformamide (50%) and sodium dodecyl
sulfate (20%) was added to solubilize the MTT formazan. The optical density of each
well was measured with a microplate spectrophotometer at a wavelength of 490 nm.
The IC50 values were determined by the percentage of cell viability versus
concentration on a logarithmic graph and reading off the concentration at which 50%
of cells remain viable relative to the control. Each experiment was repeated at least
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three times to obtain the mean values.
2.5. Apoptosis assay by AO/EB staining method
BEL-7402 cells were seeded onto chamber slides in six-well plates at a density
PT
of 2 × 105 cells per well and incubated for 24 h. The cancer cells were cultured in
RI
RPMI 1640 and normal cell LO2 was cultured in DMEM supplemented with 10% of
SC
fetal bovine serum (FBS) and incubated at 37 C in 5% CO2. The medium was
removed and replaced with medium (final DMSO concentration, 0.05% v/v)
NU
containing the complexes (6.25 μM) for 24 h. The medium was removed again, and
MA
the cells were washed with ice-cold phosphate buffer saline (PBS), and fixed with
formalin (4%, w/v). Cell nuclei were counterstained with acridine orange (AO) and
D
ethidium bromide (EB) (AO: 100 µg/mL, EB: 100 µg/mL) for 10 min. The cells were
PT
E
observed and imaged under an inverted fluorescence microscope (Nikon, Yokohama,
CE
Japan) with excitation at 350 nm and emission at 460 nm.
AC
2.6. DNA damage assay
DNA damage was investigated by means of comet assay. BEL-7402 cells in
culture medium were incubated with 6.25 μM of complexes 1-6 for 24 h at 37 °C. The
cells were harvested by a trypsinization process at 24 h. A total of 100 μL of 0.5%
normal agarose in PBS was dropped gently onto a fully frosted microslide, covered
immediately with a coverslip, and then placed at 4 oC for 10 min. The coverslip was
removed after the gel had been set. A mixture of 50 μL of the cell suspension (200
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cells / μL) mixed with 50 μL of 1% low melting agarose was preserved at 37 °C. A
total of 100 μL of this mixture was applied quickly on top of the gel, coated over the
microslide, covered immediately with a coverslip, and then placed at 4 oC for 10 min.
The coverslip was again removed after the gel had been set. A third coating of 50 μL
PT
of 0.5% low melting agarose was placed on the gel and allowed to place at 4 oC for 15
RI
min. After solidification of the agarose, the coverslips were removed, and the slides
SC
were immersed in an ice-cold lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM
Tris, 90 mM sodium sarcosinate, NaOH, pH 10, 1% Triton X-100 and 10% DMSO)
NU
and placed in a refrigerator at 4 °C for 2 h. All of the above operations were
MA
performed under low lighting conditions to avoid additional DNA damage. The slides,
after removal from the lysis solution, were placed horizontally in an electrophoresis
D
chamber. The reservoirs were filled with an electrophoresis buffer (300 mM NaOH,
PT
E
1.2 mM EDTA) until the slides were just immersed in it, and the DNA was allowed to
unwind for 30 min in electrophoresis solution. Then the electrophoresis was carried
CE
out at 25 V and 300 mA for 20 min. After electrophoresis, the slides were removed,
AC
washed thrice in a neutralization buffer (400 mM Tris, HCl, pH 7.5). Cells were
stained with 20 μL of EB (20 μg∙mL−1) in the dark for 20 min. The slides were
washed in cold distilled water for 10 min to neutralize the excess alkali, air-dried and
imaged under an inverted fluorescent microscope.
2.7. Reactive oxygen species (ROS) detection
BEL-7402 cells were seeded into six-well plates (Costar, Corning Corp, New
14
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York) at a density of 2 × 105 cells per well and incubated for 24 h. The cells were
cultured in RPMI 1640 supplemented with 10% of FBS and incubated at 37 C in 5%
CO2. The medium was removed and replaced with medium (final DMSO
concentration, 0.05% v/v) containing complexes 1-6 (6.25 μM) for 24 h. The medium
PT
was removed again. The fluorescent dye DCHF-DA (10 μM) was added to the
RI
medium to cover the cells. The treated cells were then washed with cold PBS-EDTA
SC
(EDTA = ethylene diamine tetraacetic acid) twice, collected by trypsinization and
centrifugation at 1,500 rpm for 5 min, the cell pellets were suspended in PBS-EDTA
NU
and imaged under a fluorescent microscope. The DCF fluorescent intensity was
MA
determined by flow cytometry with excitation at 450 nm and emission at 520 nm.
D
2.8. Mitochondrial membrane potential assay
PT
E
BEL-7402 cells were treated with the complexes (6.25 μM) in 12-well plates for
24 h and then washed three times with cold PBS. The cells were detached with
CE
trypsin-EDTA solution. The collected cells were incubated for 20 min with 1 μg/mL
AC
of JC-1 in culture medium at 37 °C in the dark. The cells were immediately
centrifuged to remove the supernatant, and the cell pellets were suspended in PBS and
imaged under fluorescence microscope. The ratio of red/green fluorescence was
determined by FACSCalibur flow cytometry with excitation at 490 nm and emission
at 527 nm.
2.9. Cell cycle arrest by flow cytometry
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BEL-7402 cells were seeded into six-well plates (Costar, Corning Corp, New
York) at a density of 2 × 105 cells per well and incubated for 24 h. The cells were
cultured in RPMI 1640 supplemented with 10% of FBS and incubated at 37 °C in 5%
CO2. The medium was removed and replaced with medium (final DMSO
PT
concentration, 0.05% v/v) containing complexes 1-6 (6.25 μM). After incubation for
RI
24 h, the cell layer was trypsinized and washed with cold PBS and fixed with 70%
SC
ethanol. Twenty µL of RNAse (0.2 mg / mL) and 20 µL of propidium iodide (PI, 0.02
mg / mL) were added to the cell suspensions and they were incubated at 37 °C for 30
NU
min. Then the samples were analyzed with a FACSCalibur flow cytometry. The
MA
number of cells analyzed for each sample was 10000 [24].
D
2.10. Matrigel invasion assay
PT
E
The BD Matrigel invasion chamber was used to investigate the cell invasion
according to the manufacturer's instructions. BEL-7402 cells (4 × 104) in serum free
CE
media and the complexes (6.25 µM) were seeded in the top chamber of the two
AC
chamber Matrigel system. RPMI-1640 (20% FBS) was added as chemo-attractant into
the low chamber, and the cells were allowed to invade for 24 h. Then the
non-invading cells were removed from the upper surface and cells on the lower
surface were fixed with 4% paraformaldehyde and stained with 0.1% of crystal violet.
The membranes were photographed and the invading cells were counted under a light
microscope. The mean values were obtained from three independent experiments.
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2.11. Autophagy assay
BEL-7402 cells were seeded onto chamber slides in 12-well plates and incubated
for 24 h. The cells were cultured in RPMI 1640 supplemented with 10% of FBS and
incubated at 37 oC in 5% CO2. The medium was removed and replaced with medium
PT
(final DMSO concentration, 0.05% v/v) containing complexes 1-6 (6.25 µM) for 24 h.
RI
The medium was removed again, and the cells were washed with ice-cold PBS twice.
SC
The cells were stained with monodansylcadaverine (MDC) solution (50 µM) for 10
min and washed with PBS twice. Then the cells were observed and imaged under
NU
fluorescence microscope. The effect of the complexes on the expression of LC3 and
MA
Beclin-1 proteins was assayed by western blot (Bio-rad, Trans-Blot® TurboTM
D
System).
PT
E
2.12. The effect of autophagy on cell viability
The cell viability was determined using the MTT method. BEL-7402 cells were
CE
placed in 96-well microassay culture plates (8 × 104 cells per well) and cultured
AC
overnight at 37 oC in a 5% CO2 incubator. The cells were pretreated with
3-methyladenine (3-MA) for 3 h, followed by ruthenium complexes for 24 h. After
incubation, the cells were incubated with MTT (0.5 mg/ml) for 4 h at 37 oC. Upon
completion of the incubation, 100 μL DMSO was added to solubilize the MTT
formazan. The optical density of each well was then measured with a microplate
spectrophotometer at a wavelength of 490 nm. The viability (%) of cell growth was
calculated by the formula: (A490 (treatment group) / A490 (control)) × 100, A490 (treatment group) is
17
ACCEPTED MANUSCRIPT
the mean OD value of cells treated with the various ruthenium complexes and A490
(control) is the mean OD value of untreated cells. Each experiment was repeated at least
three times to obtain the mean values.
PT
2.13. The expression of caspases and Bcl-2 family proteins
RI
BEL-7402 cells were seeded in 3.5 cm dishes for 24 h and incubated with 6.25
SC
μM of the complexes in the presence of 10% FBS. The cells were harvested in lysis
buffer. After sonication, the samples were centrifuged for 20 min at 13,000 g. The
NU
protein concentration of the supernatant was determined by BCA (bicinchoninic acid)
MA
assay. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was done loading
equal amount of proteins per lane. Gels were then transferred to poly (vinylidene
D
difluoride) membranes (Millipore) and blocked with 5% non-fat milk in TBST [20
PT
E
mM Tris–HCl, 150 mM NaCl, 0.05% Tween (polyoxyethy-lene monolaurate
sorbaitan) 20, pH 8.0) buffer for 1 h. Then the membranes were incubated with
CE
primary antibodies at 1:5,000 dilutions in 5% non-fat milk overnight at 4 °C, and
AC
washed four times with TBST for a total of 30 min. After which the secondary
antibodies conjugated with horseradish peroxidase at 1:5,000 dilution for 1 h at room
temperature and then were washed four times with TBST. The blots were visualized
with the Amersham ECL Plus western blotting detection reagents according to the
manufacturer's instructions. To assess the presence of comparable amount of proteins
in each lane, the membranes were stripped finally to detect the β-actin. The gray
values were calculated with Bandscan.
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3. Results and discussion
3.1. Synthesis and characterization
The ligands PTTP and FTTP were synthesized by the reaction of
with
4-phenoxybenzene-1,2-diamine
or
PT
1,10-phenanthroline-5,6-dione
RI
4-(3-fluoronaphthalene-2-yloxy)benzene-1,2-diamine in ethanol using citric acid as
SC
catalyst at room for 15 min. The yellow ligands were obtained by filtration and
washed with water (3 × 30 mL). The complexes 1-6 were prepared by direct reaction
NU
of PTTP or FTTP with appropriate precursor complexes in ethylene glycol in
MA
relatively high yield. The desired Ru(II) complexes were isolated as the perchlorates
and purified by column chromatography. In the electrospray ionization mass spectra
D
of the complexes, the signals of [M-ClO4]+ and [M-2ClO4]2+ were observed. The
PT
E
observed molecular weights are consistent with the expected values. In the 13C NMR
spectra, the chemical shift values of 20.75, 20.66 for 1, 26.17, 25.83, 24.67, 19.05 for
CE
2, 30.08, 29.98 for 3, 20.76, 20.67 for 4, 25.98, 24.49 for 5 and 30.08, 29.98 for 6 are
AC
assigned to methyl group, 35.54, 35.43 for 3 and 35.55, 35.43 for 6 are attributed to
the tertiary butyl carbon atoms.
3.2. Stability studies of the complexes in PBS solution
The stability of the complexes in phosphate buffer solution (PBS) was
investigated by UV-Vis spectra. As shown in Fig. S2 (supporting information), no
obvious changes in absorption of the complex 1 were observed at 0 and 48 h (data not
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ACCEPTED MANUSCRIPT
presented for complexes 2-6), which indicated that the complexes are stable in PBS
solution.
3.3. Cytotoxic activity assay of the complexes toward cancer cells
PT
The MTT assay was used to evaluate the effects of the complexes 1-6 on
RI
cytotoxic activity against cancer cells BEL-7402, A549, HeLa, HepG2, HOS and
SC
normal LO2 cell lines. Cisplatin was used as a positive control. As shown in Table 1,
the IC50 values range from 1.5 ± 0.1 to 55.9 ± 7.5 µM. As a whole, the complexes 1-6
NU
show different inhibitory effect on the cell growth of the above selected cancer cell
MA
lines. Complex 2 displays the highest cytotoxic activity against HeLa and HepG2
cells with low IC50 values of 4.1 ± 0.3 and 2.0 ± 0.2 µM, respectively. According to
D
the IC50 values, we infer that the ruthenium(II) polypyridyl complexes containing dmp
PT
E
or ttbpy as ancillary ligands reveal higher cytotoxic activity than those complexes
with dmb as ancillary ligand. This may be caused by different hydrophobicity, in
CE
general, ancillary ligands dmp or ttbpy has larger hydrophobicity than dmb. Complex
AC
3 is the most effect on BEL-7402 cell growth among the complexes, and complex 5 is
sensitive to A549 with a low IC50 value of 2.5 ± 0.6 µM. Comparing the IC50 values,
complex 2 exhibits higher cytotoxicity than cisplatin against HeLa, BEL-7402 and
HepG2 cells, and complex 5 shows more effective inhibition on the cell growth of
A549 and HepG2 cells than cisplatin under the identical conditions. Comparing the
cytotoxicity in vitro, adding a benzene ring and a fluorine atom in the complexes 1-3,
complexes 4-6 show relative lower cytotoxic activity than those of complexes 1-3
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against BEL-7402 cells. The cytotoxic activity of complexes 2 and 3 against
BEL-7402 cells is comparable to those of [Ru(dmp)2(pddppn)]2+ (IC50 = 1.6 ± 0.4 µM)
[Ru(dmp)2(dptbt)]2+
[19],
(dptbt
=
12-(2,3-diphenyl-quinoxalin-6-yl)-4,5,10,13-tetraazabenzo[b]triphenylene, IC50 = 1.9
PT
± 0.3 µM) [25]. Regretfully, we found that the complexes 1-6 also show high
RI
cytotoxic activity against the normal cell line LO2. Therefore, it is very difficult to
SC
find a drug only to kill the cancer cells not to kill the normal cells. Since the
complexes displayed relative high effect on BEL-7402 cell growth, this cell line was
NU
selected for further investigation of the underlying mechanisms accounting for the
MA
action of ruthenium complexes.
D
3.4. Apoptosis studies with AO/EB staining method
PT
E
External aggression by a chemical compound sensed by the cells causes them to
undergo two major forms of death, necrosis or apoptosis, each with very distinct
CE
characteristics. The apoptosis of BEL-7402 cells was investigated with acridine
AC
orange (AO) and ethidium bromide (EB) double staining method. The AO/EB
staining is sensitive to DNA and was used to access changes in nuclear morphology.
As shown in Fig. 1, in the control (a), the living cells were stained bright green in
spots. After BEL-7402 cells were exposed to 6.25 µM of complexes 1 (b), 2 (c), 3 (d),
4 (e), 5 (f) and 6 (g) for 24 h, green apoptotic cells containing apoptotic features such
as nuclear shrinkage, chromatin condensation were found. Similar results can be
observed in other ruthenium (II) complexes [26,27]. To quantitatively compare to
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apoptotic effect induced by the complexes on BEL-7402 cells, the apoptosis was
assayed by flow cytometry. In the control (Fig. S3, supporting information), the
percentage in the apoptotic cells is 1.03%. After the treatment of the cells with 6.25
µM of complexes 1 (b), 2 (c), 3 (d), 4 (e), 5 (f) and 6 (g) for 24 h, the percentages of
PT
apoptotic cells are 15.25%, 13.34%, 6.39%, 6.50%, 9.26% and 3.66%, respectively.
RI
The apoptotic effect of complexes 1 and 2 is higher than that of complexes
SC
[Ru(phen)2(addppn)]2+ [18]. The apoptotic effect follows the order of 1 > 2 > 5 > 4 >
3 > 6. This may be caused by the different structures of the complexes. The apoptotic
NU
effect is not consistent with the cytotoxic activity of the complexes against BEL-7402
MA
cells (3 > 2 > 6 > 5 > 4 > 1). These data suggest that the complexes can induce
PT
E
3.5. DNA damage studies
D
apoptosis in BEL-7402 cells.
The proliferation of cancer cells needs DNA replication. DNA damage is a
CE
useful method to inhibit DNA replication. Moreover, DNA damage leads to DNA
AC
fragmentation, which is a hallmark of apoptosis, mitotic catastrophe or both [28]. To
investigate the effect of complexes 1-6 on the DNA damage, the single cell gel
electrophoresis assay known as comet assay was performed to asses DNA integrity. In
the control (Fig. 2 (a)), BEL-7402 cells didn′t show comet-like appearance. Treatment
of the cell with 6.25 µM of complexes 1 (b), 2 (c), 3 (d), 4 (e), 5 (f) and 6 (g) for 24 h
led to a formation with statistically significant and well-formed comets, and the length
of the comet tail represents the extent of DNA damage. The results indicate that the
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complexes can induce DNA damage, which is further evidence of apoptosis.
3.6. Reactive oxygen species (ROS) levels assay
Reactive oxygen species (ROS), including superoxide anion, hydrogen peroxide
PT
and hydroxyl radical, have been involved in the actions of many anticancer drugs
RI
through initiation of various apoptotic signaling pathways during chemotherapy [29].
SC
Reactive oxygen species (ROS) are known to affect mitochondrial membrane
potential and trigger a series of mitochondria associated events including apoptosis
NU
[30]. Many literatures reported that ruthenium complexes induce apoptosis through
evaluated
using
MA
the production of ROS [31-33]. The ROS levels induced by the complexes was
2′,7′-dichlorodihydrofluorescein
diacetate
(DCHF-DA)
as
form
2′,7′-Dichloro-3,6-fluorandiol
PT
E
non-fluorescent
D
fluorescence probe. DCFH-DA is cleaved by intracellular esterases into its
(DCFH).
Then
the
non-fluorescent substrate is oxidized by intracellular free radicals to produce a
CE
fluorescent product dichlorofluorescein (DCF) [34,35]. As is shown in Fig. 3A (a), in
AC
the control, owing to low ROS levels, it is difficult for DCHF-DA to be transferred
into fluorescent product DCF, no obvious fluorescence image spots are observed.
BEL-7402 cells exposure to Rosup (b, positive control) and 6.25 µM of complexes 1
(c), 2 (d), 3 (e), 4 (f), 5 (g), 6 (h) for 24 h, a lot of bright green image spots are found.
The findings show that the complexes can increase the ROS levels. This reveals that
the complexes 1-6, which selectively accumulates in carcinoma mitochondria,
activates the ROS-generating machinery and generates the highest amount of ROS
23
ACCEPTED MANUSCRIPT
when treated with cancer cells, leading to apoptosis. To quantitatively compare the
efficiency of ROS levels, DCF fluorescent intensity was determined by flow
cytometry. Fig. 3B shows that the DCF fluorescent intensity increases 1.5 for 1, 1.4
for 2, 3.3 for 3, 1.6 for 4, 1.7 for 5 and 11.4 times for 6 than the original, respectively.
PT
The DCF fluorescent intensity increase follows the order of 6 > 3 > 5 > 4 > 1 > 2.
RI
Complexes 6 and 3 show higher effect on the increasing the ROS levels than
SC
complexes 1, 2, 4 and 5. This may be caused by substituent tertiary butyl group in 3
and 6. Comparing the cytotoxicity in vitro, ROS generation induced by complexes 1-6
MA
NU
is not in line with the order of cytotoxicity of the complexes against BEL-7402 cells.
3.7. The changes assay in the mitochondrial membrane potential
D
Mitochondria act as a point of integration for apoptotic signals originating from
PT
E
both extrinsic and intrinsic apoptotic pathways [36,37]. Since ROS production is
closely related to mitochondrial dysfunction [38], the changes in mitochondrial
potential
associated
with
apoptosis
CE
membrane
were
iodide
using
(JC-1)
as
AC
5,5′,6,6′-tetrachloro-1,1′,3,3′-tetrethylbenzimidalylcarbocyanine
assayed
fluorescent probe. JC-1 exhibits potential-dependent accumulation in mitochondria,
indicated by a fluorescence emission shift from red (~590 nm) to green (~525 nm)
[39]. As shown in Fig. 4A (a), JC-1 forms aggregates and emits a red fluorescence
corresponding to high mitochondrial membrane potential. After the treatment of
BEL-7402 cells with 50 mM cccp (b, carbonyl cyanide 3-chlorophenylhydrazone,
positive control) and 6.25 µM of complexes 1 (c), 2 (d), 3 (e), 4 (f), 5 (g) and 6 (h) for
24
ACCEPTED MANUSCRIPT
24 h, JC-1 forms monomer and emits green fluorescence corresponding to low
mitochondrial membrane potential. The changes from the red to the green
fluorescence suggest that the complexes cause a decrease in the mitochondrial
membrane potential. The changes were also determined by the ratio of red/green
PT
fluorescent intensity by flow cytometry. In the control (Fig. 4B), the ratio of red/green
RI
fluorescence is 86.6. After BEL-7402 cells were exposed to 6.25 µM of complexes
SC
1-6 for 24 h, the ratios of the red/green fluorescence are 7.7, 5.6, 66.5, 14.5, 13.6 and
44.3, respectively. The reduction in the ratio of red/green indicates that the red
NU
fluorescence decreases and the green fluorescent intensity increases. The decrease in
MA
the mitochondrial membrane potential induced by 1-6 follows the order of 2 > 1 > 5 >
4 > 6 > 3. Complex 2 shows the highest efficiency among the complexes in inducing a
D
decrease in the mitochondrial membrane potential, complexes 3 and 6 exhibit relative
PT
E
lower effect on the mitochondrial membrane potential than other complexes. However,
in the assay of ROS levels, complexes 6 and 3 show the highest efficiency on the
CE
ROS levels among the complexes. This demonstrates that the complexes cause the
AC
order of changes in ROS levels is not consistent with the order of changes in
mitochondrial membrane potential. These results reveal that the complexes induced
apoptosis in BEL-7402 cells through mitochondria-mediated pathways.
3.8. The cell cycle arrest studies
To further investigate the inhibitory mechanism of the complexes against
BEL-7402 cell growth, the cell cycle distribution was studied by flow cytometry. As
25
ACCEPTED MANUSCRIPT
shown in Fig. 5, in the control, the percentage in the cell at G2/M phase is 15.64%.
After the cells were exposed to 6.25 µM of complexes 1-6 for 24 h, the percentages in
the cell at G2/M phase are 21.13% for 1, 25.44% for 2, 19.41% for 3, 18.64% for 4,
20.40% for 5 and 19.97% for 6, respectively. An increase of 5.49% for 1, 9.80% for 2,
PT
3.77% for 3, 3.00% for 4, 4.76% for 5 and 4.33% for 6 in the percentage in the cell at
RI
G2/M phase was observed, accompanied by corresponding reduction in the
SC
percentage in the cell at G0/G1 phase. The data indicate that the complexes inhibit the
cell growth in BEL-7402 cells at G2/M phase. Further the analyses find that the effect
NU
of the complexes on the cell cycle arrest follows the order of 2 > 1 > 5 > 6 > 3 > 4.
MA
Thus, it can be seen that the cytotoxic activity of the complexes has no inevitable
PT
E
3.9. Matrigel invasion assay
D
connection with the effect on the cell cycle arrest.
As a confirmatory test, the anti-invasive potential of the complexes towards
CE
BEL-7402 cells in the matrigel assay was evaluated. The results obtained from the
AC
study are shown in Table 2 and Table 3. In the presence of 6.25 µM of the complexes
1-6, the number of cell invasion decreased. Complexes 1-6 inhibiting the percentage
of cell invasion are 21.2%, 20.1%, 21.5%, 25.3%, 71.9% and 33.6% invasion,
respectively. The anti-metastatic effect of the complexes against BEL-7402 cells
followed the order of 6 > 5 > 4 > 3 > 1 > 2, this is not consistent with cytotoxic
activity of the complexes. To evaluate the effect of concentration on the cell invasion,
BEL-7402 cells were treated with different concentration of complex 6. As shown in
26
ACCEPTED MANUSCRIPT
Table 3, BEL-7402 cells were exposed to 6.25 µM of complex 6, the percentage of
inhibiting invasion cell is 33.6%. When the concentration of 6 is 50.0 µM, the
percentage of inhibiting invasion cell reaches 58.5%. Obviously, complex 6 shows a
concentration-dependent manner in the inhibiting invasion cell. These results
RI
PT
demonstrate that the complexes can effectively inhibit BEL-7402 cell invasion.
SC
3.10. Autophagy induced by the complexes
Autophagy is a life phenomenon and exists extensively in the eucell, and
NU
autophagy has been considered to be a third mode of cell death besides apoptosis and
MA
necrosis. To determine whether or not autophagy is truly triggered by the complexes,
BEL-7402 cells were treated with 6.25 µM of complexes 3 and 6 for 24 h, and the
D
cells were stained with monodansylcadaverine (MDC) as fluorescent probe to detect
PT
E
autophagic activity. As shown in Fig. 6A, compared with the control group, the
number of MDC-positive cells in BEL-7402 increased in the complexes-treated cells
CE
after 24 h incubation. LC3 is a hallmark, the conversion of LC3-I to LC3-II exhibits
AC
autophagy induction [40]. In addition, Beclin-1 protein is necessary to form
autophagosomes in autophagy. As shown in Fig. 6B, compared with the control, the
expression of LC3-II and Beclin-1 was increased. Comparing the effect of the
complexes on the expression of Beclin-1, complex 1 shows the highest autophagic
efficiency among the complexes. To investigate the effect of the autophagy on the cell
viability, BEL-7402 cells were treated by different concentrations of the complexes in
the presence or absence of autophagic inhibitor 3-MA. As shown in Fig. S4
27
ACCEPTED MANUSCRIPT
(supporting information), compared with the control, the cell viability caused by
complexes 3 and 6 decreases in the presence of 3-MA, which indicates that the
autophagy inhibits the cell death. The effect of the complexes on autophagy is not
consistent with the cytotoxic activity in vitro of the complexes against BEL-7402
RI
PT
cells.
SC
3.11. The expression of caspases and Bcl-2 family proteins studies
Caspases are known to mediate the apoptotic pathway [41,42]. Caspase 3 and 7
NU
are executioners of apoptosis as the processing of their substrates leads to
MA
morphological changes associated with apoptosis [43]. The activation of caspase 3
and 7 induced by the complexes was assayed by western blotting. The expression was
D
calculated by the ratio of the expression induced by the complexes/the expression in
PT
E
the control. As shown in Fig. 7, the expression of caspase 3 was upregulated, whereas
the level of expression of caspase 7 was downregulated. Bcl-2-related proteins are key
CE
regulators of the mitochondria-mediated apoptosis [44]. To further evaluate the effect
AC
of the complexes on the expression of Bcl-2 family proteins, BEL-7402 cells were
treated with 6.25 µM of complexes 1-6 for 24 h. As expected, the expression levels of
antiapoptotic proteins Bcl-2 and Bcl-x decreased, whereas the expression of
proapoptotic proteins Bak and Bid increased. As a result of these changes, the ratios
of Bcl-2/Bak and Bcl-x/Bid decreases significantly, leading to a generation of ROS, a
depletion of △ψm. Subsequently, the complexes induce activation of caspases 3 and 7.
All these findings indicate that mitochondrial pathways were involved in apoptosis
28
ACCEPTED MANUSCRIPT
driven by the complexes.
4. Conclusions
Two new ligands PTTP, FTTP and their six ruthenium(II) complexes were
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synthesized and characterized in detail. These complexes show relative high
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anticancer activity against the selected cell lines. Particularly, complex 2 displays
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strong inhibition of the cell growth toward HeLa, BEL-7402 and HepG-2 cells.
Complexes 1-6 can enhance the levels of ROS and induce a decrease in the
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mitochondrial membrane potential, and the complexes inhibit the cell growth in
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BEL-7402 cells at G2/M phase. In addition, the complexes can induce autophagy and
restrain the cell invasion with a concentration-dependent manner and regulate the
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Bcl-2 family proteins. In summary, the complexes induce apoptosis in BEL-7402 cell
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through an intrinsic ROS-mediated mitochondrial dysfunction pathway, which was
accompanied by the regulation of the expression of Bcl-2 family proteins. In addition,
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we also conclude that the complexes with high cytotoxicity in vitro against cancer cell
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may show low effect on ROS, mitochondrial membrane potential and other
bioactivity. In summary, we found that the ruthenium (II) complexes containing dmp
or ttbpy as ancillary ligands will effectively inhibit cancer cell growth. This work will
be helpful for design and synthesis of ruthenium (II) as potent anticancer drugs.
Acknowledgments
This work was supported by the Natural Science foundation of Guangdong
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Province (No. 2016A030313728), the National Nature Science Foundation of China
(No. 81403111) and the Project of innovation for enhancing Guangdong
Pharmaceutical University, provincial experimental teaching demonstration center of
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chemistry & chemical engineering.
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Captions for Schemes and Figures
Table 1 The IC50 values (µM) of ligand and the complexes toward the selected cell
lines
Scheme 1 Structures of Ru(II) dppz-like complexes
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Fig. 1 BEL-7402 cells were stained with AO/EB and observed under fluorescent
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microscope. BEL-7402 (a) exposed to 6.25 µM of complexes 1 (b), 2 (c), 3 (d),
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4 (e), 5 (f) and 6 (h) for 24 h.
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Fig. 2 Comet assay of EB-stained BEL-7402 cells (a) exposure to 6.25 μM of
complexes 1 (b), 2 (c), 3 (d), 4 (e), 5 (f) and 6 (g) for 24 h.
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Fig. 3 (A) Intracellular ROS was detected in BEL-7402 cells (a) exposure to Rosup (b,
positive control) and 6.25 µM of complexes 1 (c), 2 (d) , 3 (e), 4 (f), 5 (g) and
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6 (h) for 24 h. (B) The DCF fluorescent intensity was determined by flow
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cytometry after BEL-7402 (a) cells were exposed to 6.25 µM of complexes 1
(b), 2 (c), 3 (d), 4 (e), 5 (f) and 6 (g) for 24 h. I stands for the fluorescent
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Fig. 4 (A) Assay of BEL-7402 cells mitochondrial membrane potential with JC-1 as
fluorescent probe. BEL-7402 cellv (a) exposed to cccp (b, positive control)
and 6.25 μM of complexes 1 (c), 2 (d), 3 (e), 4 (f), 5 (g) and 6 (h) for 24 h. (B)
The ratio (R) of red/green fluorescent intensity was determined by flow
cytometry after BEL-7402 cells were treated with 6.25 μM of complexes 1-6
for 24 h.
Fig. 5 Cell cycle distribution of BEL-7402 cells exposure to 6.25 μM of complexes
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1-6 for 24 h.
Fig. 6 (A) Microscope images of invading BEL-7402 cells (a) that have migrated
through the Matrigel induced by 6.25 µM of complexes 1 (b), 2 (c), 3 (d), 4 (e), 5
(f) and 6 (g) for 24 h.
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Fig. 7 BEL-7402 cells were treated with 6.25 µM of the complexes for 24 h and the
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expression levels of caspases and Bcl-2 family proteins were examined by
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HOS
1
13.9 ± 1.0
19.8 ± 5.0
70.6 ± 4.4
16.8 ± 0.8
23.1 ± 1.3
11.0 ± 0.5
37.1 ± 3.3
2
4.1 ± 0.3
2.2 ± 0.2
78.2 ± 3.5
24.8 ± 8.6
2.0 ± 0.2
19.9 ± 0.7
29.9 ± 4.5
3
6.3 ± 0.2
1.5 ± 0.1
14.4 ± 0.8
15.8 ± 0.2
11.6 ± 1.1
4.9 ± 1.6
7.6 ± 0.2
4
55.9 ± 7.5
11.9 ± 2.0
127.0 ± 8.6
26.0 ± 5.7
9.8 ± 0.9
20.7 ± 0.1
52.9 ± 6.9
5
10.0 ± 0.7
6.1 ± 0.6
35.6 ± 1.6
2.5 ± 0.6
3.7 ± 0.3
2.6 ± 0.1
12.9 ± 1.7
6
6.7 ± 1.4
3.2 ± 0.1
12.8 ± 0.4
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Table 1 The IC50 (µM) values of complexes 1-6 toward the selected cell lines
8.2 ± 0.4
5.6 ± 0.9
4.9 ± 0.2
2.1 ± 0.1
Cisplatin
7.3 ± 1.4
11.5 ± 1.3
nd
7.5 ± 1.3
11.5 ± 1.2
nd
9.3 ± 1.5
BEL-7402
BEL-7402
48 h
A549
24 h
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HepG-2
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Table 2 The number of cell invasion and inhibition percentage of cell invasion by 6.25 µM of
complexes 1-6.
control
1
2
3
363
286
21.2
290
20.1
285
21.5
5
6
275
25.3
261
28.1
241
33.6
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Invasion cells number
Inhibition percentage (%)
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Concentration (µM)
6.25
12.5
25.0
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Inhibition percentage (%)
33.6
38.3
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43.5
58.5
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Table 3 Inhibition percentage of cell invasion by different concentration of complex 6
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Graphical abstract
Six new ruthenium(II) polypyridyl complexes were synthesized and characterized in
detail. The anticancer activity was investigated by cytotoxicity in vitro, apoptosis,
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reactive oxygen species (ROS), mitochondrial membrane potential, cell cycle arrest,
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cell invasion, autophagy and proteins expression.
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Highlights
Six new ruthenium (II) complexes were synthesized and characterized.
The
cytotoxicity
was
evaluated
by
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3-(4,5-dimethylthiazole)-2,5-diphenyltetraazolium bromide method.
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The apoptosis, comet assay, reactive oxygen species and cell invasion were
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investigated.
The mitochondrial membrane potential and cell cycle arrest were assayed.
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The expression of caspases and B-cell lymphoma-2 proteins was studied by
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western blot.
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