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In Vitro and in Vivo Anticancer Activity of Copper


Bis(thiosemicarbazone) Complexes
Duraippandi Palanimuthu,† Sridevi Vijay Shinde,‡ Kumaravel Somasundaram,*,‡
and Ashoka G. Samuelson*,†

Department of Inorganic and Physical Chemistry, and ‡Department of Microbiology and Cell Biology, Indian Institute of Science,
Bangalore, India 560012
*
S Supporting Information
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ABSTRACT: Neutral and cationic copper bis(thiosemicarbazone) complexes bearing methyl, phenyl, and hydrogen, on the
diketo-backbone of the ligand have been synthesized. All of them were characterized by spectroscopic methods and in three cases
by X-ray crystallography. In vitro cytotoxicity studies revealed that they are cytotoxic unlike the corresponding zinc complexes.
Copper complexes Cu(GTSC) and Cu(GTSCHCl) derived from glyoxal-bis(4-methyl-4-phenyl-3-thiosemicarbazone)
(GTSCH2) are the most cytotoxic complexes against various human cancer cell lines, with a potency similar to that of the
anticancer drug adriamycin and up to 1000 fold higher than that of the corresponding zinc complex. Tritiated thymidine
incorporation assay revealed that Cu(GTSC) and Cu(GTSCHCl) inhibit DNA synthesis substantially. Cell cycle analyses
showed that Cu(GTSC) and Cu(GTSCHCl) induce apoptosis in HCT116 cells. The Cu(GTSCHCl) complex caused distinct
DNA cleavage and Topo IIα inhibition unlike that for Cu(GTSC). In vivo administration of Cu(GTSC) significantly inhibits
tumor growth in HCT116 xenografts in nude mice.

■ INTRODUCTION
The discovery of cisplatin ushered in an era of cancer therapy
pathways such as inhibition of DNA and RNA synthesis and
disruption of ATP production, and surprisingly none of them
that included inorganic metal complexes extensively.1 Though function through ROS generation.
cisplatin is widely used as an anticancer drug to cure many More recently, some copper bis(thiosemicarbazone) com-
cancers, severe side effects and acquired resistance due to plexes have shown selectivity to cells having low levels of
prolonged treatment have spurred investigators to find oxygen compared to normal cells.27−29 A good example is
alternatives for circumventing drug resistance.2 With this aim, Cu(ATSM) which selectively accumulates in hypoxic cancer
several classes of metal complexes have been synthesized using cells.29 This might be attributed at least partly, to irreversible
various ligands and metal ions, and their anticancer activity has reduction of Cu(II) to Cu(I) and subsequent trapping of
been successfully evaluated both in vitro and in vivo.3−8 copper within the cells whereas in normal cells, Cu(I) is likely
Transition-metal-based thiosemicarbazone complexes are one to be reoxidized to Cu(II), because of the high oxygen tension
such class of complexes that have been investigated intensely present in these cells, and suffer expulsion from the cell. Short
over five decades. 9 − 1 3 Among them, copper bis- synthesis times have enabled synthesis of radiolabeled copper
(thiosemicarbazone) complexes have attracted more attention, complexes that are theranostic, permitting diagnosis and
because many of them displayed promising anticancer therapy simultaneously.25 Thus radiolabeled 64Cu(ATSM) has
activity.14−20 been used as a radiotherapy agent against hamsters bearing
The mono(thiosemicarbazone) complexes of copper con- human GW39 colon cancer tumors, significantly increasing
taining 1 or 2 equiv of the ligand are well understood, and they their survival time (6 fold compared to controls).30 Besides its
manifest cytotoxicity mainly through ROS generation.21−23 therapeutic utility, it has also shown promise as a positron
However, the mechanism of action of copper bis- emission tomography-computed tomography (PET/CT) agent
(thiosemicarbazone) complexes is less studied though signifi- to diagnose the progress of cervical carcinoma and has
cant progress has been achieved in elucidating the mechanistic successfully cleared clinical human trial phase II recently.31,32
pathway of copper biacetyl-bis(4-methyl-3-thiosemicarbazone) Following the successful story of 64Cu(ATSM), a number of
[Cu(ATSM)], copper 2-ethoxy-2-ketobutyraldehyde-bis- copper bis(thiosemicarbazone) complexes with different radio-
(thiosemicarbazone) [Cu(KTS)] and copper glyoxal-bis(4-
methyl-3-thiosemicarbazone) [Cu(GTSM)].24−26 These com- Received: July 3, 2012
plexes exhibit their anticancer activity by activating multiple Published: January 15, 2013

© 2013 American Chemical Society 722 dx.doi.org/10.1021/jm300938r | J. Med. Chem. 2013, 56, 722−734
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labeled nuclides, 60Cu, 61Cu, 62Cu, and 64Cu, have been tested used for the preparation of the neutral complexes) in ethanol or
as PET/CT imaging agents.33−37 in a ethanol−chloroform (1:1) mixture for 4 h. The resulting
Recently, we have shown that the ligand bis- copper complexes were characterized by spectroscopic
(thiosemicarbazone) of glyoxal (GTSCH2) can be used for techniques such as UV−visible and IR spectroscopy, ESI-MS,
live cell imaging in conjunction with zinc.38 We have now and elemental analysis. In addition, the complexes were
directed our attention toward the corresponding copper subjected to cyclic voltammetry, to molar conductivity, and,
complexes and their anticancer activity. A series of new copper when single crystals were available, to X-ray crystallography.
bis(thiosemicarbazone) complexes derived from biacetyl-bis(4- The electronic absorption and infrared spectra of these
pyrrolidinyl-3-thiosemicarbazone) (ATSCH2), benzil-bis(4-pyr- compounds are consistent with the N2S2 coordination
rolidinyl-3-thiosemicarbazone) (BTSCH2), and glyoxal-bis(4- proposed with a possible coordination from a chloride or the
methyl-4-phenyl-3-thiosemicarbazone) (GTSCH2) ligands counterion as in the case of perchlorate. The presence of the
have been synthesized and characterized. Their cytotoxicity perchlorate counterion could be deduced from the molar
was estimated in different human cancer cell lines, where the conductance in DMF (1 mM). Thus, complexes Cu(ATSCH)-
copper complexes derived from GTSCH2 were found to be ClO4 and Cu(BTSCH)ClO4 showed molar conductances of 62
most cytotoxic in all the cell lines tested. Further, we have and 66 S m2 M−1 respectively, indicating that both complexes
studied their cellular accumulation and distribution in HCT116 were 1:1 electrolytes. For comparison, the well-known complex
cells and observed significant accumulation of copper in bis(1,10-phenanthroline)copper(II) nitrate {[Cu(phen)2]-
cytoplasm when compared to the nucleus in 1 h. Tritiated (NO3)2}, a 1:2 electrolyte, was shown to have a molar
thymidine incorporation assay in HCT116 cells with Cu- conductance of 115 S m2 M−1 under the same conditions. In
(GTSC) and Cu(GTSCHCl) showed a reduction in DNA contrast, Cu(ATSC), Cu(BTSC), Cu(GTSC), and Cu-
synthesis. Cell cycle analyses results further suggest that these (GTSCHCl) show molar conductances of 3, 18, 6, and 13 S
complexes induce significant apoptosis. Biophysical studies such m2 M−1 respectively, confirming the neutral nature of the
as DNA interaction, DNA cleavage, and Topo IIα inhibition complexes.
throw light on the mechanistic aspects of their anticancer The molecular structures of the three complexes Cu(ATSC),
activity. In vivo cytotoxicity studies in mice bearing colon Cu(BTSC), and Cu(GTSC) are depicted in Figure 2, and their
cancer (HCT116) showed impaired tumor formation in crystallographic data are given in Table S1 of Supporting
response to Cu(GTSC) treatment.


Information. In all structures, the Cu(II) center adopted a
distorted square planar geometry by coordinating to two sulfur
RESULTS AND DISCUSSION and two nitrogen atoms. The ligand is present in a syn-
Synthesis and Characterization. The bis- conformation at the diimine core and wraps itself around the
(thiosemicarbazone) ligands and their copper complexes were metal center. In the case of Cu(GTSC), the structure is quite
synthesized using the previously reported procedure with minor different from the zinc complex characterized earlier which
modifications (Figure 1).39−42 The ligands were synthesized by exhibited a trimeric structure, [Zn(GTSC)]3, and the ligand has
an anti-conformation at the diimine.38 The C−S bond length in
all neutral complexes is 1.75 Å, which clearly indicates thiolate
coordination.43
The reduction potential of Cu(II) in copper bis-
(thiosemicarbazone) complexes is an important parameter
that modulates their cellular retention and hypoxia selectivity.35
Recently, Dearling and Fujibayashi illustrated through in vitro
and in vivo experiments that complexes which are difficult to
reduce, with an E1/2 that is lower than −0.50 V, can show
hypoxia selectivity whereas the easily reducible complexes do
not show such selectivity.28,35 We have measured the E1/2 using
cyclic voltammetry for the newly synthesized complexes in dry
DMF vs SCE, and the results are given in Table 1. A
representative cyclic voltammogram for Cu(GTSC) complex (5
mM) is shown in Figure S1 of Supporting Information. These
half-wave potentials (E1/2) were assignable to the Cu(II)/Cu(I)
redox couple. It is known that small modifications on the
diketone backbone changes the redox potential to a large
Figure 1. Proposed chemical structures of bis(thiosemicarbazone) extent.29 We also find that the substitution of pyrrolidine
ligands and their copper complexes. moiety on the N-terminus of thiosemicarbazide, as in
Cu(ATSC) or Cu(ATSCH)ClO4, results in a reduction
potential close to that of Cu(ATSM). On the other hand, the
refluxing thiosemicarbazide and a 1,2-diketone in 2:1 mol ratio replacement of electron-donating methyl groups in Cu(ATSM)
with a few drops of concentrated sulfuric acid (Scheme S1, with phenyl groups in Cu(BTSC) and Cu(BTSCH)ClO4 or
Supporting Information). The ligands were characterized by hydrogens in Cu(GTSC) on the 1,2-diketone backbone of
multinuclear (1H and 13C) NMR spectroscopy. The copper bis(thiosemicarbazone) markedly increases the reduction
complexes, in both neutral and cationic forms, were prepared potential to −0.34, −0.36, and −0.18 V, respectively. Thus,
by refluxing 1 equiv of the ligand with the appropriate copper complexes Cu(ATSC) and Cu(ATSCH)ClO4 are likely to be
salts (copper(II) perchlorate salt was used for cationic complex hypoxia selective, as they have a large negative redox potential,
preparation, and copper(II) chloride or copper(II) acetate was but they do not exhibit significant cytotoxicity like that of
723 dx.doi.org/10.1021/jm300938r | J. Med. Chem. 2013, 56, 722−734
Journal of Medicinal Chemistry Article

Figure 2. ORTEP representations of (A) Cu(ATSC), (B) Cu(BTSC), and (C) Cu(GTSC) complexes. Thermal ellipsoids are shown at the 40%
probability level, and solvent molecules, if any, are omitted for clarity.

Table 1. Cyclic Voltammetry Data for Copper Cu(GTSC), vide infra, making them less interesting for
Bis(thiosemicarbazone) Complexesa therapeutic applications. The five-coordinated complex Cu-
(GTSCHCl) is unique as it is cytotoxic, vide infra, and at the
no. complex E1/2 (V)b ΔEp (mV)c |ipa|/|ipc|d
same time, showed a completely irreversible reduction potential
1 Cu(ATSM) −0.58 91 0.99 at −0.66 V which is conducive to hypoxia selectivity.
2 Cu(ATSC) −0.53 90 1.03 In Vitro Cytotoxicity. The cytotoxic activity of the
3 Cu(ATSCH)ClO4 −0.54 94 1.29 bis(thiosemicarbazone) ligands and their copper complexes
4 Cu(BTSC) −0.34 73 1.02 against various human cancer cell lines such as SiHa (cervical
5 Cu(BTSCH)ClO4 −0.36 95 1.06 cancer), MCF-7 (breast cancer), PC-3 (prostate cancer), A-
6 Cu(GTSC) −0.18 93 0.99 2780 (ovarian cancer), and HepG2 (hepatocellular liver cancer)
7 Cu(GTSCHCl)e − − − were evaluated. For comparison, the corresponding zinc
a
All reduction potentials are with respect to SCE, and a 10 mV s−1 complexes were synthesized according to the literature
scan rate was used for all the measurements. bHalf-wave reduction procedures, and their cytotoxicity was evaluated.38,44 Cultured
potential is given by E1/2 = (Epa + Epc)/2. cThe difference in potential cancer cells were treated with compounds with different
is given by ΔEp = Epa − Epc, where Epa and Epc are the anodic and
cathodic peak potentials, respectively. dRatio of anodic peak current
concentrations for 48 h, and the percentage of cell growth
over the cathodic peak current. eIt showed an irreversible reduction inhibition was determined by sulforhodamine-B (SRB) assay.45
peak at −0.66 V with a ΔEp of 330 mV and | ipa|/| ipc| of 0.30. Adriamycin was used as a positive control. The GI50 values are
given in Table 2. The copper complexes of BTSCH2 were
found to be inactive (>100 μM) in almost all the cell lines
724 dx.doi.org/10.1021/jm300938r | J. Med. Chem. 2013, 56, 722−734
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Table 2. Cell Growth Inhibition of Bis(thiosemicarbazone) Ligands and Their Copper and Zinc Complexesa
GI50b (μM)
ligands/complexes SiHA MCF-7 PC-3 A-2780 HepG2
BTSCH2 >100 >100 >100 >100 >100
Cu(BTSC) >100 >100 >100 >100 27.50
Cu(BTSCH)ClO4 >100 >100 >100 >100 2.36
Zn(BTSC) >100 >100 >100 >100 29.80
ATSCH2 >100 −c >100 >100 1.45
Cu(ATSC) >100 2.60 >100 >100 28.40
Cu(ATSCH)ClO4 >100 2.50 >100 0.12 <0.1
Zn(ATSCHCl) >100 >100 >100 0.13 <0.1
GTSCH2 51.30 >100 80.40 <0.1 <0.1
Cu(GTSC) 0.19 0.17 2.03 0.18 0.13
Cu(GTSCHCl) 2.10 0.13 0.15 0.17 <0.1
[Zn(GTSC)]3 >100 2.27 >100 >100 >100
adriamycind 0.15 <0.1 1.80 <0.1 1.00
a
Data are presented as a mean of three independent experiments. bRefers to the amount of drug (in μM) required to inhibit 50% cell growth in 48 h.
c
GI50 could not be obtained, as the compound exhibited poor solubility. dAdriamycin was included for comparison.

tested, except Cu(BTSCH)ClO4 complex which is highly toxic Table 3. IC50 Values for Cu(GTSC) and Cu(GTSCHCl)
(2.36 μM) to HepG2 cells. Complexes of ATSCH2 having a
IC50a (μM)
low redox potential (E1/2 ∼ −0.54 V) showed anticancer
activity against certain cell lines: Cu(ATSC) in MCF-7 (2.60 complexes MDA-MB-231 HCT116 HaCaT
μM) and Cu(ATSCH)ClO4 in MCF-7 (2.50 μM), A-2780 Cu(GTSC) 1.45 ± 0.07 1.23 ± 0.27 0.65 ± 0.07
(0.12 μM) and HepG2 (<0.1 μM) cell lines (Table 2). Cu(GTSCHCl) 2.05 ± 0.35 1.59 ± 0.28 1.55 ± 0.21
Interestingly, the copper complexes derived from the GTSCH2 a
Cytotoxicity are expressed as IC50 in 48 h. Data represented are the
ligand are cytotoxic against all the cell lines analyzed, with GI50 average of three independent experiments with ± SD.
values ranging from 2.1 μM to <0.1 μM, which is similar to
adriamycin. The copper complex of the ligand GTSCH2 shows
cytotoxicity 1000 fold higher than that of the zinc analogue, cellular uptake and distribution of copper within HCT116 cells.
which has a unique trimeric structure [Zn(GTSC)]3 (Table The treatment of HCT116 cells with the complexes (25 μM)
2).38 The ligand BTSCH2 is relatively less toxic to the cancer for 60 min led to a substantial increase in the cellular copper
cells than their copper complexes, and the ligand ATSCH2 is concentration compared to the untreated control, suggesting
cytotoxic only to the HepG2 cell line. However, GTSCH2 facile internalization of complexes within 60 min. As shown in
displayed significant cytotoxicity against A-2780 and HepG2 Figure 4A, Cu(GTSC) (0.29 ± 0. 0.09 nmol Cu/106 cells) was
cells (<0.1 μM). taken up by the cell about 1.5 times more efficiently than
The cytotoxicity for the most active complexes Cu(GTSC) Cu(GTSCHCl) (0.19 ± 0. 0.03 nmol Cu/106 cells). Recently,
and Cu(GTSCHCl) (structures are shown in Figure 3) were White et al. probed the mechanisms underlying copper
bis(thiosemicarbazone) complex uptake in cancer cells, where
they found that the Cu(ATSM) and Cu(GTSM) complexes
internalized into human neuron M17 and glioblastoma U87MG
cells via combined passive diffusion and protein-carrier
mediated pathways.46 It is possible that uptake of Cu(GTSC)
and Cu(GTSCHCl) complexes in HCT116 cells occur through
similar pathways.
To examine the cellular distribution of the complex, the
copper concentration was measured in the cytoplasmic and
nuclear extracts fractionated from HCT116 cells after the
Figure 3. Most active copper bis(thiosemicarbazone) complexes. exposure of cells to Cu(GTSC) and Cu(GTSCHCl) complexes
for 60 min. The results, shown in Figure 4B, suggest a
evaluated in a few more human cell lines which included significant increase in the total copper concentration in the
HCT116 (colorectal cancer), MDA-MB-231 (breast cancer), treated cells. A large amount (91% to 97%) of the total copper
and HaCaT (immortalized keratinocyte) cells by MTT assay is localized in the cytoplasm compared to the concentration of
(Table 3). Analyses of cytotoxicity data showed that the IC50 of copper in the nucleus (9% to 3%). Our observations are similar
both complexes was below 2.05 μM in these three cell lines. to those of White et al., who have also studied the localization
Upon confirmation of the excellent cytotoxicity properties, of copper bis(thiosemicarbazone) complexes in U87MG (glial
further cellular (in HCT116 cells) and biophysical studies were cells) and M17 (neuron cells) by measuring the copper
performed to understand their mechanism of action. accumulation in cell membrane and cytosol. They found that
Cellular Uptake and Distribution of Copper in Cells. treatment of U87MG cells with one of the copper bis-
The copper bis(thiosemicarbazone) complexes are nonfluor- (thiosemicarbazone) complexes, Cu(ATSM), for 60 min
escent in nature, and so inductively coupled plasma-optical significantly increased copper concentration, and ∼70% was
emission spectrometry (ICP-OES) was used to estimate the found in the cytosol and 30% in the cell membrane.46
725 dx.doi.org/10.1021/jm300938r | J. Med. Chem. 2013, 56, 722−734
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Figure 4. HCT116 cells were treated with Cu(GTSC) and Cu(GTSCHCl) complexes for 60 min at 37 °C. Copper content in whole cell (A) and in
cytoplasm and nucleus (B) was measured by ICP-OES. Control cells were treated with vehicle (1% DMSO). Data shown are mean values ±
standard deviations of three independent measurements for each experiment.

Figure 5. Effect of Cu(GTSC) and Cu(GTSCHCl) on DNA synthesis was evaluated using [3H]-thymidine incorporation assay. (A) HCT116 cells
were incubated with Cu(GTSC) and Cu(GTSCHCl) at 1.23 and 1.59 μM (IC50 concentrations), respectively, for 24, 48, and 72 h at 37 °C. (B)
HCT116 cells were treated with increasing concentrations of Cu(GTSC) and Cu(GTSCHCl) for 48 h at 37 °C. DNA synthesis was measured as
[3H]-thymidine incorporation. Data shown are means ± SD of results from three experiments.

Figure 6. Effects of Cu(GTSC) and Cu(GTSCHCl) complexes on cell cycle of HCT116 cells after the treatment for 24, 48, and 72 h, where 1% of
DMSO and adriamycin were used as negative and positive controls, respectively. Cell cycle progression was analyzed by FACS and quantified using
Summit 4.3v software. The percentage of cell cycle population (A) and subG1 populations (B) are shown after the treatment of complexes at their
IC50 concentrations. Data shown are the mean values of two independent experiments ± SD.

The cellular cytotoxicity of these compounds is also in accord the uptake of complexes which in turn is influenced by their
with their lipophilicity as measured by the traditional “shake lipophilicity.
Mechanism of Cell Cytotoxicity by Cu(GTSC) and
flask” method. The observed log Poctanol values for Cu(GTSC)
Cu(GTSCHCl) Complexes. Several metal complexes including
and Cu(GTSCHCl) complexes are 1.58 ± 0.15 and 1.44 ± copper bis(thiosemicarbazone) are known to exhibit their
0.12, respectively. The cytotoxicity of the two closely related cytotoxicity by inhibiting DNA synthesis.24,47−49 None of the
complexes in HCT116 cells could be due to the difference in copper bis(thiosemicarbazone) complexes are known to exhibit
726 dx.doi.org/10.1021/jm300938r | J. Med. Chem. 2013, 56, 722−734
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anticancer activity through ROS generation like the closely Table 4. Results of the Binding Constants of Ligands and
related copper mono(thiosemicarbazone) complexes.24,25 In Their Copper Complexes for CT-DNAa
our studies, we found that Cu(GTSC) and Cu(GTSCHCl) are
no. ligands/complexes Kappb (× 105 M−1)
the most cytotoxic among the complexes tested. To examine
whether these complexes have any effect on the DNA synthesis, 1 BTSCH2 ND
we performed a [3H]-thymidine incorporation assay by treating 2 Cu(BTSC) 3.21 ± 0.51
3 Cu(BTSCH)ClO4 7.70 ± 0.41
cultured HCT116 cells with Cu(GTSC) and Cu(GTSHCl) at
4 ATSCH2 0.14 ± 0.01
their IC50 concentrations (1.23 μM and 1.59 μM, respectively)
5 Cu(ATSC) 0.62 ± 0.02
for 24, 48, and 72 h. The amount of incorporated [3H]-
6 Cu(ATSCH)ClO4 3.73 ± 0.20
thymidine is directly proportional to the extent of DNA
7 GTSCH2 0.20 ± 0.01
synthesis. The treatment of HCT116 cells with Cu(GTSC) and
8 Cu(GTSC) 1.70 ± 0.28
Cu(GTSCHCl) significantly reduced the DNA synthesis in a
9 Cu(GTSCHCl) 4.19 ± 0.57
time- and concentration-dependent manner (Figure 5A and a
5B). Interestingly, both Cu(GTSC) and Cu(GTSCHCl) Each value is the average of two sets of experiments. bApparent
binding constant (Kapp) was measured by an ethidium bromide
showed almost the same extents of reduction in [3H]- displacement assay.
thymidine incorporation, which were in line with cytotoxicity
observed. On the basis of these studies, we inferred that
Cu(GTSC) and Cu(GTSCHCl) induce cytotoxicity, possibly results indicate that the cationic complexes interact with DNA,
by inhibiting DNA synthesis. at least initially, through an electrostatic interaction.
Because HCT116 cells treated with Cu(GTSC) and Thermal stability and viscosity changes of CT-DNA in the
Cu(GTSCHCl) complexes showed reduced [3H]-thymidine presence of metal complexes were carried out to probe the
incorporation, we wanted to check the effect of copper nature of binding. The melting temperature (Tm), a measure of
complexes on cell cycle progression and cell death. The cells the thermal stability of DNA, increases in the presence of the
were treated with Cu(GTSC) and Cu(GTSCHCl) at their IC50 complexes Cu(GTSC) and Cu(GTSCHCl), implying stabiliza-
concentrations for 24, 48, and 72 h and analyzed by flow tion of double-stranded DNA.
cytometry after staining with propidium iodide (Figure 6A and The mode of DNA binding was further assessed by viscosity
6B). Adriamycin was used a positive control. No significant experiments. Copper complexes display a steady increase in the
change in cell cycle profile was seen in cells treated with copper specific viscosity, with increasing concentration; however, the
complexes, which suggested that the complexes are not change was less pronounced than that observed with ethidium
inducing cell cycle arrest at any phase of the cell cycle (Figure bromide, a known intercalator, but significantly greater than
6A). As expected, the positive control adriamycin (0.9 μM) what is caused by Hoechst33258, a minor groove binder
showed 31%, 50%, and 53% increase in the population of G2/M (Figure S3, Supporting Information). On the basis of these
phase with concomitant reduction in G0/G1 phase at 24, 48, studies, it might be concluded that these complexes interact
and 72 h, suggesting a G2/M phase arrest (Figure 6A). It was with DNA via partial intercalation that stabilizes the double-
also noticed that the population of sub G1, an indicator for stranded duplex.
DNA Cleavage Property of Copper Complexes. The
apoptosis, increases in Cu(GTSC)- and Cu(GTSCHCl)-
ability of copper complexes to cause DNA cleavage was studied
treated cells (Figure 6B). Interestingly, the subG1 population
by agarose gel electrophoresis using pBR322 DNA (Figure 7,
of Cu(GTSC) was found to gradually increase from 2 to 7.7
and Figures S4−S6, Supporting Information). The extent of
fold with respect to controls from 24 to 72 h. However,
conversion of supercoiled (SC) DNA to nicked-circular (NC)
Cu(GTSCHCl) treated cells showed 2.6 to 3.0 fold increase
DNA is indicative of the cuts/nicks in DNA. The pBR322 DNA
under the same condition. As expected, the positive control was incubated with copper complexes at 100 μM for 4 h at 37
adriamycin also induced apoptosis. Put together, these results °C. Complex [Cu(phen)2](NO3)2 was used as a positive
suggest that [3H]-thymidine incorporation is the result of control (20 μM). The activity was quantified and is displayed in
apoptosis induction.50 So we conclude that the mechanism of Table 5 in the form of IC50 (concentration required to convert
cytotoxicity by these copper complexes may primarily involve 50% SC DNA to NC DNA). Surprisingly, all copper
apoptosis induction. complexes, except Cu(GTSCHCl), showed less than 50% of
DNA Interaction Studies. Mechanistic studies with DNA cleavage at 100 μM concentration (Table 5, and Figures
Cu(GTSC) and Cu(GTSCHCl) at cellular level prompted us S4−S6, Supporting Information). The Cu(GTSCHCl) com-
to carry out DNA interaction studies to understand the mode plex showed 50% DNA cleavage at 39 μM (Figure 7A and 7B),
of DNA binding at the molecular level. To assess the binding and the extent of DNA cleavage increased with increasing
efficacy of bis(thiosemicarbazone) ligands and their copper concentration of Cu(GTSCHCl). It might be recalled that the
complexes with DNA, competitive binding experiments with Cu(GTSCHCl) displayed 2.5 fold higher binding constant
respect to ethidium bromide (EB) were performed on CT- compared to the closely related Cu(GTSC). Further, DNA
DNA. Monitoring the quenching of emission from highly cleavage is not quenched under argon atmosphere (Figure 7C,
fluorescent EB-DNA adducts with successive addition of the and Figure S7, Supporting Information), suggesting that
complex (Figure S2, Supporting Information) gave apparent reactive oxygen species (ROS) are not involved. Hence, it is
binding constants, and they are listed in Table 4. The apparent likely that a hydrolytic pathway is operative in the DNA
binding constants (Kapp) obtained for all compounds are in the cleavage effected by this complex. Thus, Cu(GTSCHCl) is
range of 0.14 × 105 to 7.7 × 105 M−1. The Kapp of both neutral likely to cleave DNA to a greater extent unlike other copper
and cationic complexes are 4.4 to 21 fold higher than that of bis(thiosemicarbazone) complexes reported in this study.
their ligands. In addition, the cationic complexes have a higher Topoisomerase IIα Inhibition by Copper Complexes.
propensity to bind DNA compared to neutral complexes. These Some of the copper thiosemicarbazone complexes have been
727 dx.doi.org/10.1021/jm300938r | J. Med. Chem. 2013, 56, 722−734
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Figure 7. (A) Agarose gel electrophoresis of pBR322 DNA (36 μM) treated with increasing concentrations of Cu(GTSCHCl) complex (lanes 4 to
16) for 4 h at 37 °C. Lane 1, DNA control; lane 2, DNA + 3.8% DMF; lane 3, DNA + [Cu(phen)2](NO3)2 (20 μM); lanes 4 to 16, DNA +
Cu(GTSCHCl) at 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 75 μM, respectively. Percentage of DNA cleavage is given in the form of nicked circular
(NC). (B) Quantification of DNA cleavage. (C) The graph is derived from cleavage of pBR322 DNA by Cu(GTSCHCl) complex with increasing
concentrations under anaerobic (argon) or aerobic conditions (oxygen). Data shown are mean values ± standard deviations of two measurements.

Table 5. In Vitro pBR322 DNA Cleavage and Topo IIα


Inhibition by Copper Bis(thiosemicarbazone) Complexes at
100 μM
no. complex % DNA cleavagea % Topo IIα inhibitionb
1 Cu(ATSC) 27 23
2 Cu(ATSCH)ClO4 30 18
3 Cu(BTSC) 41 49
4 Cu(BTSCH)ClO4 39 60
5 Cu(GTSC) 24 52
6 Cu(GTSCHCl) 86 90
a
DNA alone showed an average cleavage of 18%. bTopo IIα inhibition
percentage was set to zero (100% activity of Topo IIα) for the
untreated control.

reported to induce cell death by inhibiting Topo IIα


activity.51−53 So we investigated the efficacy of these complexes
for human Topo IIα enzyme inhibiting ability using a Topo
IIα-mediated DNA relaxation assay (Figure 8, Table 5, and
Figure S8, Supporting Information). All copper complexes were
incubated (100 μM) with Topo IIα−DNA mixture for 30 min
at 37 °C. Anthracyclin-based anticancer compound adriamycin Figure 8. (A) Effect of Cu(GTSCHCl) complex on human Topo IIα-
(5 μM), a known Topo IIα inhibitor, was used as a positive mediated DNA relaxation activity. Supercoiled pBR322 DNA (lane 1)
control.54 Copper complexes of BTSCH2 and the Cu(GTSC) was incubated with Topo IIα in the absence of adriamycin (lane 2) or
in the presence of 5 μM of adriamycin (lane 3) and 10, 25, 50, and 100
complex showed moderate inhibition at 100 μM concentrations μM of Cu(GTSCHCl) at 15 min (lane 4 to 7) and 30 min (lane 8 to
(inhibition ranging from 49% to 60%, Table 5, and Figure S8, 11) of incubation, respectively. The supercoiled and nicked-circular
Supporting Information). The appearance of a DNA ladder in DNA are represented by I and II. (B) Percentage of Topo IIα
the gel confirmed that these complexes act as Topo IIα poisons. inhibition (SC DNA/total DNA) by Cu(GTSCHCl) complex after 15
Both cationic and neutral copper complexes of ATSCH2 and 30 min of incubation at 37 °C.
analogues were found to be inactive or less active (18% to
23% inhibitors at 100 μM, Table 5, and Figure S8, Supporting
728 dx.doi.org/10.1021/jm300938r | J. Med. Chem. 2013, 56, 722−734
Journal of Medicinal Chemistry Article

Information). Cu(GTSCHCl) complex was found to be the that received Cu(GTSC) complex show tumor growth
most active complex in this series (90% inhibition at 100 μM, inhibition with time but the mice that received vehicle show
Figure 8 and Table 5). To explore further, we carried out the exponential increase in tumor growth. The tumor growth was
time-dependent (15 and 30 min) and concentration (10, 25, inhibited significantly by 95 ± 3.9% in Cu(GTSC) complex-
50, and 100 μM)-dependent Topo IIα inhibition for Cu- treated mice, compared with control mice at the end of the
(GTSCHCl) (Figure 8). The complex shows enzyme inhibition 26th day (Figure 9). This result confirmed that the Cu(GTSC)
in a dose-dependent manner and showed a minor increase in complex possesses excellent antitumor activity in vivo.
the inhibition with time from 15 to 30 min. All these results
indicate that the DNA cleaving complex Cu(GTSCHCl) shows
significant Topo IIα inhibition activity compared to other
■ CONCLUSION
A series of bis(thiosemicarbazone) ligands and their copper
complexes. On the basis of our experimental results, we complexes were synthesized, and their anticancer activity was
propose that, like the other Topo IIα poisons (e.g., evaluated. Among all these compounds, Cu(GTSC) and
adriamycin), the copper complexes may ligate and stabilize Cu(GTSCHCl) were found to be most cytotoxic. Their
the DNA−Topo IIα cleavage complex, which in turn cause cytotoxicity was almost on par with the well-known anticancer
permanent double-strand breakage of supercoiled pBR322 drug adriamycin. The treatment of Cu(GTSC) and Cu-
DNA. However, in cells, such a double-strand breakage leads to (GTSCHCl) in HCT116 cells significantly increased the
apoptosis or necrosis.55 These results are surprising because copper concentration largely in cytoplasm in 60 min. Reduction
many copper thiosemicarbazone complexes are known to act as of the [3H]-thymidine incorporation observed both in a dose-
catalytic inhibitors of Topo IIα by ligating ATP hydrolysis and time-dependent manner suggested inhibition of DNA
domain and hampering ATP hydrolysis.53 Cu(GTSC) and synthesis. Further analysis of the cell cycle indicated no major
Cu(GTSCHCl) showed almost similar cytotoxicity in all the change in the cell cycle but a significant increase in the subG1
cell lines, but the Topo IIα inhibition shown by Cu- population, a clear indicator of apoptosis. At the molecular
(GTSCHCl) is 90% while Cu(GTSC) showed only 52% level, it is seen that the complexes interact with DNA through
Topo IIα inhibition. While we cannot ignore inhibition of partial intercalation. However, the mode of action is likely to be
Topo IIα by copper bis(thiosemicarbazone) complexes, it different in these complexes, as Cu(GTSCHCl) showed
cannot be projected as the only mechanism by which these significant DNA cleavage whereas Cu(GTSC) did not. Also,
complexes manifest their cytotoxicity. Cu(GTSCHCl) inhibits Topo IIα very effectively compared to
In Vivo HCT-116 Xenograft Studies. In vitro studies with Cu(GTSC). Despite the increased activity of Cu(GTSCHCl)
copper complexes suggest that the newly synthesized Cu- in carrying out DNA cleavage and inhibiting Topo IIα activity,
(GTSC) and Cu(GTSCHCl) complexes exhibit significant and Cu(GTSC) appears to be equally cytotoxic, in HCT116 cells.
consistent cytotoxicity in several human cancer cell lines. To This could be due to increased lipophilicity and better cellular
test the efficacy of these complexes to inhibit tumor growth in uptake of Cu(GTSC) compared to Cu(GTSCHCl). The
vivo, an experiment was carried out with human colorectal excellent cytotoxicity and cellular uptake properties of Cu-
tumor xenografts (Figure 9). Cu(GTSC) (5 mg/kg/d) was (GTSC) in vitro prompted us to examine its in vivo activity in
injected intraperitoneally (ip) into one group of female nude nude mice. In mice with colorectal carcinoma xenograft
mice for 7 days, and only vehicle was injected into another (HCT116 cancer cells), Cu(GTSC)-treated mice display
group of female nude mice, as a control. Interestingly, the mice augmented tumor regression compared to mice that received
only the vehicle control. These complexes could be further
modified with cleavable linkers, making them suitable for
targeted delivery which is now our current interest.

■ EXPERIMENTAL SECTION
Materials and Methods. All the reagents and chemicals were
purchased from Merck, SD Fine-Chem Limited, or Spectrochem
Limited, unless otherwise noted, and used without further purification.
1
H and 13C{1H} NMR spectra were recorded using a Bruker AMX 400
instrument operating at 400 MHz for 1H and at 100 MHz for 13C
NMR with tetramethylsilane as an internal standard. Elemental
analyses were carried out using a Thermo Finnigan Flash EA 1112
CHNS analyzer. All the complexes tested have a purity of >95% as
determined by elemental analysis. ESI-MS of the complexes/ligands
were recorded in a Thermo Finnigan LCQ Deca Plus instrument.
Infrared spectra were recorded on a Bruker ALPHA10 FT-IR
spectrometer operating in ATR mode. All the UV−visible experiments
were carried out on a PerkinElmer Lambda 35 instrument.
Fluorescence measurements were carried out with a Horiba Jobin
Figure 9. In vivo anticancer activity of Cu(GTSC) complex in mice Yvon Fluoromax-4 spectrofluorimeter. Molar conductivity was
bearing HCT116 xenografts. Mice received either Cu(GTSC) (5 mg/ measured using a Control Dynamics conductivity meter, and viscosity
kg/d; n = 8) complex or vehicle (DMSO; n = 7) daily for 7 days after on a Schott Gerate AVS310 automated viscometer equipped with a
one day of tumor implantation by ip injection. Tumor volumes were thermostat. Copper was estimated by inductively coupled plasma
measured at different time points and plotted. Tumor volumes are optical emission spectroscopy (ICP-OES) (PerkinElmer Optima 2000
expressed with ± standard error. Statistical comparison was made DV). The copper standard for ICP-OES measurements was purchased
between control and treated groups by two-way ANOVA test in from Fluka Analytical. Supercoiled pBR322 plasmid DNA (cesium
GraphPad Prism 5.01 software. Overall significance obtained was chloride purified) was purchased from Bangalore Genie (India). Zinc
0.0001. A p value <0.05 was considered to be statistically significant. complexes of BTSCH2, ATSCH2, and GTSCH2 were prepared

729 dx.doi.org/10.1021/jm300938r | J. Med. Chem. 2013, 56, 722−734


Journal of Medicinal Chemistry Article

according to the literature procedure and recrystallized before use.38,44 71%. Mass data: ESI+ m/z: calcd for CuC14H23N6S 2, 402.08 [M −
All buffers used for DNA cleavage experiments were prepared using ClO4¯]+; found, 402.20. Anal. Calcd (%) for CuC14H22S2N6ClO4: C
standard procedures.56 Calf-thymus (CT) DNA, ethidium bromide, 33.53, H 4.42, N 16.76, S 12.79. Found: C 33.87, H 5.01, N 16.61, S
glycerol, bromophenol blue, and the reagents used for the cell culture 12.82. IR , cm −1: 3264 (w, N−H), 2960 (w), 2865 (w), 1572 (m,),
study were purchased from Sigma (St. Louis, MO). Tris-HCl and 1450 (s, CN), 1271 (s, thioamide), 1226 (s), 1093 (vs, ClO4), 1039
agarose (molecular biology grade) were procured from RANKEM (s), 840 (s, C−S), 621 (m). UV−visible in DMF [λmax, nm (ε, M−1
(India). Human Topo IIα enzyme kit for the relaxation assay was cm−1)]: 321 (36 800), 368 (25 500), 515 (10 000), 567 (8900).
obtained from TopoGEN (Port Orange, FL). DMEM media, Copper Benzil-bis(4-pyrrolidinyl-3-thiosemicarbazone): Cu-
penicillin, and streptomycin were obtained from Sigma (St. Louis, (BTSC). Cu(BTSC) was prepared following the same procedure used
MO). Fetal calf serum (FCS) and MTT were used for growth for preparation of Cu(ATSC). The product was isolated as a dark
inhibition assays obtained Sigma Chemical Co. (India). Sterile 96-well brown solid. Crystals suitable for single crystal X-ray crystallography
flat-bottom tissue-culture plates, tissue-culture plates, and other plastic were grown in dichloromethane. Yield: 83%. Mass data: ESI+ m/z:
wares were purchased from Tarsons (India). All solutions and buffers calcd for CuC24H27S2N6, 526.11 [M + H+]+; found, 526.20. Anal.
were prepared using purified Milli-Q Biocel water from Millipore. Calcd (%) for CuC24H26S2N6·0.5 H2O: C 53.86, H 5.09, N 15.70, S
Synthesis of Bis(thiosemicarbazone) Ligands and Their 11.98. Found: C 54.08, H 5.23, N 15.03, S 11.94. IR, cm−1: 2986 (w),
Copper Complexes. Bis(thiosemicarbazone) ligands and their 2863 (w), 1428 (vs, CN), 1260 (s, thioamide), 877 (m), 689 (m).
precursors were prepared by following the literature procedur- UV−visible in DMF [λmax, nm (ε, M−1 cm−1)]: 318 (28 100), 354 (18
e.38−40Copper bis(thiosemicarbazone) complexes were synthesized 600), 490 (11 000), 541 (8000).
following a slightly modified procedure reported previously.41,42 Copper Benzil-bis(4-pyrrolidinyl-3-thiosemicarbazone) Per-
Details are given below. chlorate: [(CuBTSCH)ClO4]. To a solution of BTSCH2 (85 mg, 0.183
Copper Glyoxal-bis(4-methyl-4-phenyl-3-thiosemicarba- mmol) in chloroform (10 mL) was added Cu(ClO4)2·6H2O (69 mg,
zone): Cu(GTSC). To a suspension of GTSCH2 (230 mg, 0.60 0.186 mmol) in ethanol (10 mL). After the mixture was refluxed for 4
mmol) in ethanol (15 mL) was added copper acetate (121 mg, 0.61 h, the precipitate that formed was filtered, washed with ethanol and
mmol). The reaction mixture was refluxed for 4 h and cooled to room diethyl ether, and then dried in vacuo. This afforded 90 mg of a dark
temperature. The brown-colored precipitate was filtered and washed brown-colored product. Yield: 78%. Mass data: ESI+ m/z: calcd for
with copious amounts of ethanol, followed by diethyl ether, and then CuC24H27S2N6, 526.11 [M − ClO4¯]+; found, 526.33. Anal. Calcd (%)
dried in vacuo to give 200 mg of the product. Yield; 75%. Thin needle- for CuC24H27S2N6ClO4: C 46.00, H 4.34, N 13.41. Found: C 46.42, H
shaped crystals suitable for X-ray diffraction were grown by diffusing a 4.12, N 13.34. IR, cm−1: 2969 (w), 2871 (w), 1575 (m), 1495 (m),
chloroform solution of the compound with toluene. Mass data: ESI+ 1436 (s, CN), 1235 (s, thioamide), 1093 (vs, ClO4), 745 (m), 622
m/z: calcd for CuC18H18N6S2Na, 468.02 [M + Na+]+; found, 467.53. (m). UV−visible in DMF [λmax, nm (ε, M−1 cm−1)]: 319 (27 000),
Anal. Calcd (%) for CuC18H18S2N6·0.5H2O: C 47.51, H 4.21, N 18.47. 3524 (18 000), 490 (11 800), 541 (8000).
Found: C 47.31, H 3.65, N 18.07. IR, cm−1: 2945 (vs), 1581(w), 1448 Caution: Copper perchlorate salts in the presence of organic compounds
(s, CN), 1379 (vs, thioamide),1279 (s), 1173 (s), 770 (s, C−S), in the dry state could be explosive and should be handled in small
695 (s), 543 (m) (vs, very strong; s, strong; m, medium; w, weak). quantities with adequate precautions, though no incidents were encountered
UV−visible in DMF [λmax, nm (ε, M−1 cm−1)]: 323 (22 000), 381 (16 in this study.
200), 512 (10 900), 567 (7600). Crystal Structure Determination. Crystals suitable for single
Copper Glyoxal-bis(4-methyl-4-phenyl-3-thiosemicarba- crystal X-ray diffraction were grown as mentioned in the synthetic
zone) Chloride: Cu(GTSCHCl). Copper chloride (67 mg, 0.40 procedure. A suitable crystal was mounted on a glass fiber, and the
mmol) was added to a suspension of GTSCH2 (150 mg, 0.39 mmol) data were collected at 293 K on a Bruker APEX CCD diffractometer
in ethanol (10 mL) and refluxed for 4 h. The resulting gray-colored (Bruker, Madison, WI) using graphite-monochromatized Mo Kα
precipitate was filtered and washed with ethanol, followed by diethyl radiation (λ = 0.71073). The structure was solved by direct methods
ether, and dried in vacuo to yield 117 mg of the product. Yield: 62%. with the SIR-92 program available in the WINGX software suite and
Mass data: ESI+ m/z: calcd for CuC18H19N6S2, 446.05 [M − Cl¯]+; refined by full-matrix least-squares on F2 with SHELXL-97, using
found, 446.53. Anal. Calcd (%) for CuC18H19S2N6Cl·H2O: C 43.19, H anisotropic displacement parameters for non-hydrogen atoms.57,58
4.23, N 16.79, S 12.81. Found: C 43.23, H 4.00, N 16.00, S 12.85. IR, Hydrogen atoms were located or fixed at their ideal position using
cm−1: 3442 (w, O−H), 3334 (w, N−H), 1565 (m), 1495 (vs, CN), SHELXL-97.59
1398 (s, thioamide), 1353 (s), 1123 (m), 1022 (m), 842 (m, C−S), Cyclic Voltammetry. Cyclic voltammogram of copper bis-
743 (m), 699(m). UV−visible in DMF [λmax, nm (ε, M−1 cm−1)]: 314 (thiosemicarbazone) complexes were recorded on a CH Instruments
(11 000), 405 (21 000), 510 (2100), 565 (1500). electrochemical analyzer attached to a three-electrode cell setup with a
Copper Biacetyl-bis(4-pyrrolidinyl-3-thiosemicarbazone): platinum working electrode, platinum counter electrode, and KCl-
Cu(ATSC). A solution of ATSCH2 (280 mg, 0.82 mmol) in saturated Ag/AgCl reference electrode (saturated KCl). All measure-
chloroform (20 mL) was added to a solution of copper chloride ments were carried out under nitrogen atmosphere at room
(140 mg, 0.82 mmol) in ethanol (10 mL), and the resulting reaction temperature. Copper complexes were dissolved in degassed DMF (5
mixture was stirred at room temperature for 4 h. During the reaction, mM) containing 0.1 M n-tetrabutylammonium perchlorate
golden brown crystals were formed which were collected by filtration (nBu4NClO4) as the supporting electrolyte. Ferrocene was used as
and washed with ethanol, followed by diethyl ether, and dried in vacuo an internal reference with E1/2 = 0.64 V (ΔEp = 82 mV; |ipa|/|ipc| =
to afford 157 mg of the pure product. Yield: 48%. Mass data: ESI+ m/ 1.13) in DMF against SCE under similar conditions. The reported E1/2
z: calcd for CuC14H23N6S2, 402.08 [M + H+]+; found, 401.47. Anal. for ferrocene is 0.51 V in DMF (1 mM, 0.1 M nBu4NClO4, platinum as
Calcd (%) for CuC14H22S2N6: C 41.82, H 5.52, N 20.90, S 15.95. working and counter electrodes, Ag/AgCl as reference electrode) at 25
Found: C 42.03, H 5.75, N 19.97, S 15.42. IR, cm−1: 2964 (m), 2858 °C.60
(m), 1537 (w), 1432 (vs, CN), 1285 (vs), 1217 (s), 841 (m, C−S), Lipophilicity Measurement. The lipophilicity (log Poct) was
621 (m). UV−visible in DMF [λmax, nm (ε, M−1 cm−1)]: 321 (32 measured in triplicate for Cu(GTSC) and Cu(GTSCHCl) complexes
300), 368 (22 000), 515 (8700), 566 (8200). by the standard “shake-flask” method.61 As these complexes are
Copper Biacetyl-bis(4-pyrrolidinyl-3-thiosemicarbazone) insoluble in both 1-octanol and water, the partition coefficient was
Perchlorate: [(CuATSCH)ClO4]. A solution of Cu(ClO4)2·6H2O measured in CHCl3−H2O (log Pchloroform) mixtures. log Poct was
(280 mg, 0.76 mmol) in ethanol (10 mL) was added to ATSCH2 derived using the regression equation Poctanol = (1.343 + log
ligand (232 mg, 0.68 mmol) in chloroform (20 mL), and the reaction Pchloroform)/1.126.62 UV−visible spectroscopy was used to measure
mixture was refluxed for 4 h and cooled to room temperature. The the partition coefficient of copper complexes in a CHCl3−H2O
precipitate formed was filtered, washed with ethanol and diethyl ether, mixture. In brief, 1 mg of the complex was dissolved in 6 mL of dry
and dried in vacuo to give 243 mg of a brown-colored product. Yield: chloroform. Three milliliters of the chloroform solution was added to

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3 mL of milli-Q water and stirred at room temperature for 4 h. gradual addition of CT-DNA until maximum fluorescence was
Aliquots taken from the chloroform layer were centrifuged at 9000 achieved from the formation of an intercalative DNA−EB adduct.
rpm for 30 min. Absorbance was measured for the chloroform layer Addition of increasing amounts of the copper complex to the DNA−
(A) and the remaining 3 mL stock chloroform solution (A0). An EB adduct quenched the fluorescence. The apparent binding constant
absorption difference, A0 − A gave the concentration of copper (Kapp) was calculated from the following equation: KEB × [EB] = Kapp
complex dissolved in water. × [complex], where the [complex] denotes the complex concentration
MTT Assay. The MTT assay was carried out as described required for 50% reduction in fluorescence (KEB = 1.0 × 107 M−1,
previously to measure cell viability.63 Three thousand cells in 100 [EB] = 2.6 μM).68
μL of growth media were seeded in the wells of a 96-well plate. After DNA Melting Experiments. DNA melting experiments were
24 h, 100 μL of various concentrations of copper bis- carried out on a Perkin-Elmer Lambda 40 UV−visible spectropho-
(thiosemicarbazone) complexes was added and incubated for 48 h at tometer attached to a Peltier temperature controller. The melting of
37 °C in a CO2 incubator. At the 48th hour of incubation, MTT (20 CT-DNA in 5 mM Tris-HCl (5 mM NaCl, pH 7.2) was measured, in
μL of 5 mg/mL) was added to the plate. The contents of the plate the absence/presence of copper complexes from 40 °C to 90 °C. The
were pipetted out carefully, the formazan crystals formed were melting temperature (Tm) of DNA was determined from the first-
dissolved in 200 μL of DMSO, and the absorbance was measured at derivative plot of absorbance at 260 nm with respect to temperature.
550 nm in a microplate reader (Molecular Devices, Spectramax M5e). DNA Viscosity Studies. Viscosity experiments were carried out on
A graph of the concentration versus percentage cell viability was a Ubbelodhe viscometer at 37 °C. DNA solutions (150 μM) were
plotted, and the concentration at which 50% cell death occurred was prepared in 5 mM Tris-HCl (5 mM NaCl) buffer at pH 7.2. Aliquots
used as the IC50 value. of solutions containing the copper complexes (1.5 mM), ethidium
Inductively Coupled Plasma−Optical Emission Spectrome- bromide, and Hoechst33258 were prepared in DMF and added to
try (ICP-OES). HCT116 cells (∼10 million cells) were treated with 25 DNA such that the concentration increased gradually from 0 to 150
μM of Cu(GTSC) and Cu(GTSCHCl) complexes for 1 h at 37 °C in μM. The flow time was measured three times, after 5 min of
a humidified 5% CO2 incubator. The spent media was removed, and incubation, with each addition of the copper complex, and the average
the cells were washed with 5 mL of PBS, scraped, and collected in 5 flow time was taken for calculation of relative viscosity. Relative
mL of PBS. The scrapped cells were spun down, by centrifuging at viscosity was calculated from the following equation: η = (t− t0)/t0,
2500 rpm for 10 min. The cell pellet so obtained was dissolved in 1 M where t is the flow time of DNA with or without complex, and t0 is the
NaOH (1 mL) and diluted with 2% (v/v) HNO3 (5 mL) for flow time for the buffer. Finally, the relative specific viscosity, (η/η0)1/3
determining whole cell copper content. Another set was treated was plotted against [complex]/[DNA] ratio in which η is the relative
similarly, the nuclear and cytoplasmic fractions were isolated as viscosity of the DNA with addition of copper complex and η0 denotes
described by Schreiber et al.,64 and the final solution made up to 5 mL the relative viscosity of DNA alone.69,70
using 2% (v/v) HNO3. The instrument was calibrated for copper using DNA Cleavage Experiments. DNA cleavage experiments were
standard solutions containing 10, 50, 100, 500, and 1000 ppb copper. carried out using supercoiled pBR322 plasmid DNA using agarose gel
[3H]-Thymidine Incorporation Assay. The [3H]-thymidine electrophoresis. Stock solutions of DNA were diluted using milli-Q
incorporation experiment was performed as previously described.65−67 water (final DNA concentration ∼36 μM), and solutions of the copper
Briefly, HCT116 cells were seeded in a 96-well plate (3000 cells/well) complex were prepared in freshly made 20 mM HEPES buffer, pH 7.2
and treated with Cu(GTSC)and Cu(GTSCHCl) at their IC 50 (concentration range from 0 to 100 μM). Reactions were performed
concentrations for 24, 48, and 72 h at 37 °C. The cells were pulsed by incubating the DNA (4 μL), in the absence/presence of increasing
with [3H]-thymidine (1 × 105 cpm) 6 h before the end of the concentrations of the copper complexes at 37 °C. [Cu(phen)2](NO3)2
experiment. Cells were harvested at the indicated time points and complex was used as a positive control. After a 4 h incubation, the
loaded on a glass fiber filter paper. Filters were dried, and the amount reaction mixture was quenched by adding 4 μL of loading dye (10 mM
of incorporation of radioactivity into newly synthesized DNA was Tris-HCl, 0.1 M EDTA, 0.25% bromophenol blue) and then frozen at
measured using a liquid scintillation beta-counter (Beckman −20 °C for 30 min. The reaction mixture was loaded on the agarose
LS6000IC). The measured cpm (counts per minute) which is directly gel (0.9%), and the electrophoresis was carried out in a dark room at
proportional to [3H]-thymidine incorporation was converted to 50 V for 3 h in TAE buffer (40 mM Tris base, 20 mM acetic acid, 1
percentage. The incorporation in the control cells (0.5% DMSO) mM EDTA). Agarose gel was stained with ethidium bromide solution
was taken as 100%. In another experiment, 3000 cells/well were (1 μg/mL) for 30 min followed by destaining for 4 h in milli-Q water.
incubated with Cu(GTSC) and Cu(GTSCHCl) at 0.5, 1.0, 2.0, 3.0, Cleaved DNA bands were visualized under UV light and photo-
and 4.0 μM concentrations for 48 h at 37 °C. After 8 h of complex graphed. The percentage of cleaved DNA was estimated from the
treatment, the cells were pulsed with [3H]-thymidine (1 × 105 cpm) intensities of supercoiled (SC) and nicked-circular (NC) DNA bands
and processed, and radioactivity was measured. All experiments were using BioRad Gel Doc XR software.
done in triplicate with their respective controls. The DNA cleavage caused by the Cu(GTSCHCl) complex was
Cell Cycle Analysis. HCT116 cells were treated with Cu(GTSC) measured under argon atmosphere (anaerobic condition) and oxygen
and Cu(GTSCHCl) complexes at their IC50 concentrations for 24, 48, atmosphere (aerobic condition) for mechanistic discrimination. All
and 72 h. At indicated time points, the cells were washed twice with solutions of the anaerobic reaction were degassed and used
PBS and harvested using 1X trypsin−EDTA. The cells were washed immediately. Reaction mixtures (8 μL) were purged with argon gas
again with PBS and fixed with cold ethanol 70% overnight at −20 °C. for 15 min, subsequently closed, and incubated for 4 h at 37 °C.
The cells were washed with PBS once and then incubated with 40 μg/ Topoisomerase IIα Inhibition Assay. Human Topo IIα kit was
mL ribonuclease A (Sigma) for 1 h at 37 °C. Propidium iodide was purchased from TopoGEN. Topo IIα activity was measured by the
then added to the cell suspension to achieve a final concentration of 20 pBR322 DNA relaxation assay following the protocol provided by
μg/mL and incubated for another 1 h at 37 °C. The cells (30 000 TopoGEN. Briefly, 20 μL of the reaction mixture was prepared which
events for each sample) were analyzed by a flow cytometer (Beckman contains 0.2 μg (6 μL) of supercoiled pBR322 plasmid DNA, varying
Coulter CyAn) and were quantified using the Summit 4.3v software. amounts of the copper complex (5 μL), relaxation buffer (4 μL
DNA Binding Experiment. DNA binding propensity of copper containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10 mM MgCl2,
complexes were measured by the fluorescence-based ethidium 0.5 mM ATP, 0.5 mM dithiothreitol), and 1 unit of Topo IIα (5 μL).
bromide displacement. A typical assay was carried out as follows. Reaction mixtures were incubated for 30 min or for 15 and 30 min in
The concentration of CT-DNA was determined from the absorbance the kinetic experiments as indicated at 37 °C. The reaction mixtures
at 260 nm (ε = 6600 M−1 cm−1). Emission spectra of EB, with were quenched by adding the stopping buffer (5% sarkosyl, 0.125%
addition of DNA (in 400 mM NaClO4), were recorded from 560 to bromophenol blue, 25% glycerol) and loaded on a 0.9% agarose gel in
700 nm (excitation at 546 nm). EB alone showed minimal TAE buffer (40 mM Tris base, 20 mM acetic acid, 1 mM EDTA) and
fluorescence, and the fluorescence was enhanced greatly with the electrophoresed for 3 h at 50 V. The gel was stained (1 μg/mL) for 15

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min and destained for 30 min (milli-Q water) subsequently, and gels pyrrolidinyl-3-thiosemicarbazone) chloride; Cu(BTSC), copper
were photographed and analyzed using a BioRad Gel Doc XR benzil-bis(4-pyrrolidinyl-3-thiosemicarbazone); (CuBTSCH)-
software. Topo IIα inhibition percentage was obtained from the ratio ClO4, copper benzil-bis(4-pyrrolidinyl-3-thiosemicarbazone)
of supercoiled DNA to total DNA in each well. Nonhomogeneous perchlorate; Zn(BTSC), zinc benzil-bis(4-pyrrolidinyl-3-thio-
backgrounds led to a large error of ±10% of the reported values in
quantification.
semicarbazone); Zn(BTSCHCl), zinc benzil-bis(4-pyrrolidinyl-
Tumorigenicity Assay. Anticancer activity of Cu(GTSC) was 3-thiosemicarbazone) chloride; Cu(KTS), copper 2-ethoxy-2-
examined in HCT116 colon xenograft model as previously ketobutyraldehyde-bis(thiosemicarbazone); Cu(GTSM), cop-
described.71 Briefly, 5 million HCT116 cells were injected per glyoxal-bis(4-methyl-3-thiosemicarbazone); Topo IIα,
subcutaneously into the right posterior flank of each female nude topoisomerase IIα; EB, ethidium bromide; CT, calf-thymus;
mouse aged 4−5 weeks. Mice were divided into two groups with seven NC, nicked-circular; SC, supercoiled; MTT, 3-(4,5-dimethylth-
animals in one and eight in the other. After one day of tumor iazol-2-yl)-2,5-diphenyltetrazolium bromide; DMEM, Dulbec-
implantation, one group of animals (eight mice) was injected co’s Modified Eagle’s Medium; FCS, fetal calf serum; SRB,
intraperitoneally (ip) with 5 mg/kg body weight Cu(GTSC) (90 sulforhodamine B; phen, 1,10-phenanthroline
μg/18 g body weight), and the other was injected with an equal
volume of vehicle (0.65% DMSO in DMEM media) for seven
consecutive days. From the sixth day onward, the tumor volume was
measured. The tumor volume (Tv) was calculated using the formula:
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The authors declare no competing financial interest.


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