In vitro cytotoxic and apoptotic effect of vic-dioxime ligand and its metal complexes

Article abstract of DOI:10.1002/aoc.4818

In this study, vic-dioxime ligand, (1E,2E)-2-(hydroxyimino)-N′-[(1E)-2-oxo-2-phenylethylidene]ethanehydroximohydrazide (LH₂), and its Cu (II) and Ni (II) transition metal complexes were synthesized and characterized using analytical and spectro

Full text of DOI:10.1002/aoc.4818

Received: 16 November 2018
DOI: 10.1002/aoc.4818
Revised: 26 December 2018
Accepted: 7 January 2019
F U L L P A P E R
In vitro cytotoxic and apoptotic effect of vic‐dioxime ligand
and its metal complexes
Tülay Aşkın Çelik1
| Nursabah Sarikavakli2 | Özlem Sultan Aslantürk1
1 Department of Biology, Adnan Menderes
In this study, vic‐dioxime ligand, (1E,2E)‐2‐(hydroxyimino)‐N′‐[(1E)‐2‐oxo‐2‐
phenylethylidene]ethanehydroximohydrazide (LH2), and its Cu (II) and Ni
(II) transition metal complexes were synthesized and characterized using
analytical and spectroscopic techniques. Furthermore, in vitro cytotoxic and
apoptotic effects of this vic‐dioxime ligand and its Cu (II) and Ni (II) complexes
on Caco‐2 heterogeneous human epithelial colorectal adenocarcinoma cells
were evaluated. The effect of the vic‐dioxime ligand and its Ni (II) and Cu
(II) complexes in combination with Campto on the cells was also investigated.
The cytotoxicity test was carried using the MTT assay, and the apoptotic effect
was tested by DNA diffusion assay. Campto was used as a standard anti‐cancer
drug, Caco‐2 cancer cells treated with dimethylsulfoxide acted as solvent
control, and human peripheral lymphocytes were used as control.
University, 09010 Aydın, Turkey
2 Department of Chemistry, Adnan
Menderes University, 09010 Aydın,
Turkey
Correspondence
Tülay Aşkın Çelik, Department of Biology,
Adnan Menderes University, 09010 Aydın,
Turkey.
Email: tcelik@adu.edu.tr
Funding information
Aydın Adnan Menderes University Scien-
tific Research Foundation, Grant/Award
Number: FEF‐11005
The ligand and its complexes exhibit concentration‐dependent cytotoxic and
apoptotic behavior. The ligand induces the weakest cytotoxic and apoptotic
effects on both Caco‐2 cancer cells and lymphocytes. The Ni (II) complex of
ligand induces high cytotoxic and apoptotic effects on both Caco‐2 cancer cells
and lymphocytes. The Cu (II) complex of ligand has high cytotoxic and apopto-
tic effects on Caco‐2, but weak cytotoxic and apoptotic effects on lymphocytes.
The cytotoxic and apoptotic effects of the ligand and its Ni (II) and Cu (II)
complexes were found to be concentration dependent, i.e. the higher the
concentration is the more cytotoxic it will be. The present findings suggest that
Cu (II) complex has the potential to act as a promising anti‐cancer compound
against Caco‐2 colon cancer cells.
KEYWORDS
apoptosis, cytotoxic effect, DNA diffusion assay, metal complexes, vic‐dioxime ligand
1 | INTRODUCTION
from environmental and genetic factors. These factors
lead to disturbance in the genomic composition of a cell.
Cancer is a major public health problem worldwide and
is the second leading cause of death around the world.
The continuous decline in cancer death rates over
decades resulted in an overall drop of 26%, resulting in
approximately 2.4 million fewer cancer deaths during this
time period.[1] Cancer occurs as a result of contributions
The change in the cellular genome distorts the normal
physiology of the cells, for example, cell division, cell
differentiation, angiogenesis and cell migration, to cause
cancer.[2] Although a number of methodologies have
been developed and are developing to combat various
types of cancer, none of the routes/drugs is totally
Appl Organometal Chem. 2019;e4818.
wileyonlinelibrary.com/journal/aoc
© 2019 John Wiley & Sons, Ltd.
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https://doi.org/10.1002/aoc.4818

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ÇELIK ET AL.
effective and safe. Current anti‐cancer drugs have a
variety of side‐effects. An important restrictive factor of
chemotherapy is that this treatment also affects healthy
cells of the organism. Furthermore, present chemother-
apy drugs have undesirable side‐effects.[3] Therefore,
there is extensive research for new molecules that may
be used as an optimal treatment for cancer.[4] Oximes
and hydrazones are categories of Schiff base compounds,
and are an interesting class of coordinating ligands
possessing robust applications in analytical, biological
and chemical fields.[5] One of the striking features of this
class of ligands and its complexes is their resemblance
with bio‐molecules like proteins and nucleic acids.
Hydrazones have a wide variety of applications, such as
hole transporting agents[5] in organic photoconductor
layers, in the pharmaceutical industry as drugs for the
treatment of cancer, schizophrenia, leprosy, etc.[6,7] They
also have various biological activities, such as anti‐
microbial, analgesic, anti‐inflammatory, anti‐convulsant,
anti‐urease and anti‐tumor activities.[8–10]
Hydrazone and oxime ligands include a number of
probable bonding sites, such as carbonyl oxygen, imine
and azomethine nitrogen atoms. Therefore, these ligands
and their coordination compounds have been extensively
studied.[11–14] Especially, hydrazones possessing an
azomethine proton (‐NHN=CH‐) constitute an important
class of compounds that are extensively used and studied
in the area of novel drug development. It is shown that
hydrazone derivatives and its complexes inhibit DNA
synthesis and cell growth.[15,16]
Previous studies reported that different derivatives of
dioximes and their metal complexes possess anti‐tumor
activities against various cancer cell lines.[30,31] So, in
continuation of our work on hydrazones, we investigated
the cytotoxic and apoptotic effects of vic‐dioxime ligand,
(1E,2E)‐2‐(hydroxyimino)‐N′‐[(1E)‐2‐oxo‐
2phenylethylidene] ethanehydroximohydrazide (LH2),
and its Cu (II) and Ni (II) complexes on Caco‐2 heteroge-
neous human epithelial colorectal adenocarcinoma cells,
and compared them with those of anti‐cancer drug
Campto. Because the current drugs used for cancer treat-
ment also affect healthy cells of the organism, human
peripheral lymphocytes were used as control (healthy)
cells in the present study.
2 | EXPERIMENTAL
All of the starting materials and reagents were of standard
analytical grade obtained from Merck, Fluka and Aldrich,
and used without further purification. The chemicals
dimethylsulfoxide (DMSO), Dulbecco's modified Eagle's
medium (DMEM), fetal bovine serum (FBS), RPMI 1640
medium, Histopaque‐1077, phosphate‐buffered saline
(PBS), EDTA, SLS, Tris–HCl and NaOH were purchased
from Sigma Aldrich (St Louis, MO, USA). YOYO‐1 iodide
was purchased from Thermo Fisher Scientific (Massachu-
setts, USA). High‐resolution agarose and SFR agarose
were purchased from Thomas Scientific (New Jersey,
USA). Caco‐2 cells were purchased from American Type
Cell Culture Collection (ATCC HTB‐37).
Dioxime ligands are known to coordinate metal ions
as neutral dioximes and also as monoanionic dioximates
via dissociation of one oxime proton; they are also known
to act as bridging ligands via coordination through the
oxygens.[17,18] The chemistry of the bis‐dioximate
complexes of transition metal ions has attracted much
attention because of their importance as reference models
for vitamin B12,[19,20] dioxygen carriers,[21] catalysis in
chemical transformations,[22] intramolecular hydrogen
bonding and metal–metal interaction.[23,24] The excep-
tional stability and unique electronic properties of the
complexes can be attributed to their planar structure,
which is stabilized by hydrogen bonding. The high stabil-
ity of complexes prepared from vic‐dioxime ligands has
been used extensively for various purposes.[25] It is well
known that vic‐dioximes and their hydrazone derivatives
are capable of forming complexes with almost all the
metal ions because of the convenient synthetic procedures
and easy work‐up. The preparation and characterization
of complexes of different metal ions with vic‐dioximes
have already been reported.[26–28] Interestingly, 1:2
(metal–ligand ratio) complexes of the oxime and
hydrazone derivatives of oxime are also known.[29]
The melting points of the starting materials were
determined on a Buchi SPM‐20 apparatus in a sealed
capillary and are uncorrected. Infrared (IR) spectra were
obtained on a Varian 900 Fourier transform‐infrared
(FT‐IR) spectrometer using KBr pellets. 1H‐NMR and
13C‐NMR spectra were recorded at room temperature on
a Bruker 400 MHz spectrometer in DMSO. The absorption
spectra were taken on a Shimadzu UV‐1601 spectropho-
tometer. Elemental analyses were obtained using a Leco
CHNS‐932 analyzer. An Orion Expandable Ion Analyzer
EA 940 was used for the pH measurements. The
formula weights, colors, yields, melting point and molar
conductance magnetic susceptibilities were determined,
and elemental analyses UV–Vis, FT‐IR, 13C‐NMR, 1H‐
NMR of the ligand and its coordination complexes were
performed.
2.1 | Synthesis of the precursor, GH2
The
ethanehydroximohydrazide (GH2)[32] was synthesized by
reacting the (1Z,2E)‐N‐hydroxy‐2‐(hydroxyimino)
starting
material
(1Z,2E)‐2‐(hydroxyimino)

ÇELIK ET AL.
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ethanimidoyl chloride[33] and hydrazinium hydroxide.
Because the precursor, GH2, was not stable at room
temperature, it was used as obtained without further
purification.
FIGURE 1 Tautomeric forms of the ligand (LH2)
2.2 | Synthesis of the ligand (LH2)
The ligand LH2, (1E,2E)‐2‐(hydroxyimino)‐N′‐[(1E)‐
Histopaque). The blood was centrifuged at 400 g for 30
min at room temperature. The lymphocyte layer was
removed and washed twice in PBS at 250 g for 10 min.
Then, the cells were washed with RPMI‐1640 media.
2‐xo‐2‐phenylethylidene]
ethanehydroximohydrazide
was prepared by reacting (1Z,2E)‐2‐(hydroxyimino)
ethanehydroximohydrazide (GH2) and phenylglyoxal
monohydrate in 1:1 molar ratio (Scheme 1). Figure 1
shows the tautomeric forms of the ligand, LH2. The
ligand is soluble in common solvents such as CH2Cl2,
CHCl2, DMF, EtOH and DMSO.
The metal complexes of the ligand were prepared
under similar conditions in the presence of a strong base.
In general, the reaction of the LH2 with metal salts was
quick and high yielding, generating mononuclear
complexes corresponding to the general formula ML2.
Figure 2 represents the proposed square‐planar structure
for Ni (II) and Cu (II) complexes.[32]
Each culture contained 1 × 103 cells mL−1
. Isolated
lymphocytes were also treated with 10, 20 and 40 μM
ligand and its Cu (II) and Ni (II) complexes. Control cells
received only maintenance medium. Furthermore, the
anti‐cancer agent Campto (40 μg mL−1) was used as the
positive control, and 0.1% DMSO was used as the solvent
control. The plates were incubated at 37°C in a humidi-
fied incubator with 5% CO2 for 24 hr. At the end of the
treatment time, cell viability was determined by the
MTT assay.[35] The MTT assay is a colorimetric assay for
assessing cell metabolic activity. NAD(P)H‐dependent
cellular oxidoreductase enzymes may, under defined
conditions, reflect the number of viable cells present.
These enzymes are capable of reducing the tetrazolium
dye MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetra-
zolium bromide] to its insoluble formazan, which has a
purple color.[35]
Briefly, the cell culture was washed with 1 × PBS and
then 10 μL MTT solution was added to the culture. The
cultures were incubated at 37°C for 3 hr. The whole MTT
was removed with 1 × PBS, and 100 μL MTT solubilization
solution was added to each culture and incubated at room
temperature for 30 min until the cells were lysed and color
was obtained. The absorbance was read at 570 nm by using
2.3 | Cell culture and cytotoxicity assay
Human colon cancer cells (Caco‐2; ATCC HTB‐37) and
human peripheral lymphocytes were used in the cytotox-
icity assay. Caco‐2 cells were cultured in DMEM, supple-
mented with 20% FBS and penicillin–streptomycin under
humidified atmosphere of 5% CO2 at 37°C until conflu-
ent. The cells were trypsinized, and cytotoxicity assays
were carried out in 24‐well plates. Caco‐2 cells were
plated as 5 × 104 cells per plate in 24‐well plates and
incubated for 24 hr, during which a partial monolayer
formed. Cells were then treated with 10, 20 and 40 μM
ligand and its Cu (II) and Ni (II) complexes. Cells were
also treated with the vic‐dioxime ligand and its Cu (II)
and Ni (II) complexes in combination with Campto
(40 μg mL−1) for 24 hr.
a
microplate reader (Thermo Labsystems Multiscan
Spectrum).
Human peripheral lymphocytes, used as the control
(healthy) cells in the experiments, were isolated using
Histopaque‐1077 and cultured according to Boyum,
1968.[34] Blood was diluted 1:1 with PBS and layered onto
the Histopaque in the ratio of 4:3 (blood + PBS:
2.4 | DNA diffusion assay for apoptotic
and necrotic cell death evaluation
For the evaluation of the type of cell death after treat-
ments of vic‐dioxime ligand and its metal complexes,
SCHEME 1 (1E,2E)‐2‐(hydroxyimino)‐
N'‐[(1E)‐2‐oxo‐2‐phenylethylidene]
ethanehydroximohydrazide (LH2)

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ÇELIK ET AL.
FIGURE 2 A suggested structure
formulae of the square‐planar complex of
the ligand
DNA diffusion assay was performed following the proto-
col described by Singh (2005).[36] Agarose pre‐coated
slides were made by spreading 50 μL of 1% 3:1 high‐
resolution agarose on each slide, and drying them at
room temperature. Microgels were made on agarose pre‐
coated slides by mixing extract‐treated cells with 50 μL
0.6% high‐resolution agarose, and pipetting this mixture
onto the slide. The gel was immediately covered with
24 × 50 mm cover glasses. The slides were coded and
allowed to solidify for 5 min at room temperature. The
cover glasses were removed from the second layer of
microgel, and 200 μL of 2% SFR agarose was layered to
make a third layer, and immediately covered with 24 ×
50 mm cover glasses. After 2 min, cover glasses were
removed, and slides were immersed in a freshly made
lysing solution (1.24 M NaCl, 1 mM tetra sodium EDTA,
5 mM Tris–HCl, 0.01 sodium lauryl sarcosine, 0.2% DMSO
freshly added, 0.3 N NaOH freshly added) for 10 min at
room temperature. After lysis, the slides were twice
immersed in a neutralizing solution (50% ethanol, 1 mg
mL−1 spermine, 20 mM Tris–HCl, pH 7.4) for 30 min at
room temperature. The slides were air‐dried and stored
at room temperature. The slides were stained with 50 μL
of 1 μM YOYO‐1 and covered with a cover slip. One‐
thousand cells per slide were analysed. The cells that
were undergoing apoptosis or necrosis were distinguished
from normal cells. Apoptotic cell nuclei have a hazy or
undefined outline without any clear boundaries due to
nucleosomal‐sized DNA diffusing into the agarose.
Necrotic cell nuclei are bigger and poorly defined. They
have a clear, defined outer boundary of the DNA halo
and a relatively homogeneous halo appearance.[36]
the Kolmogorov–Smirnov Z‐test. The statistical differ-
ences between the control and treatment groups were
carried out using the non‐parametric Mann–Whitney test
(for independent samples). The correlations between
different variables were determined using the Spearman
Rank Correlation Test.
3 | RESULTS AND DISCUSSION
The
starting
material
(1Z,2E)‐2‐(hydroxyimino)
ethanehydroximohydrazide (GH2)[32] for this study was
prepared by reacting the (1Z,2E)‐N‐hydroxy‐2‐
(hydroxyimino) ethanimidoyl chloride and hydrazinium
hydroxide.[33] The compound (GH2) was not stable at
room temperature. Therefore, the compound (GH2) was
used as obtained without further purification. This vic‐
dioxime ligand, LH2, containing the hydrazone group
was obtained by the reaction of (1Z,2E)‐2‐(hydroxyimino)
ethanehydroximohydrazide (GH2)[32] and phenylglyoxal
monohydrate in 1:1 molar ratio. Figure 1 shows the tauto-
meric forms of the ligand, LH2. The metal complexes are
intensely colored and stable in air. Figure 2 represents the
proposed square‐planar structure for Ni (II) and Cu (II)
complexes. The metal complexes are intensely colored
and stable in air. The results of the compositional and
spectroscopic analyses are shown in Tables 1–4.
3.1 | NMR spectra
1
In the H‐NMR spectra, the OH protons of the oxime
groups are not equivalent, hence two peaks for these
protons are observed.[37,38] The characteristic oxime OH
proton is observed in between δ 14.11 and 11.82 ppm.
These chemical shifts are characteristic for hydrazones
and oximes.[39,40] On the basis of 1H‐NMR spectra, it
can be suggested that LH2 has the (E,E)‐structure. The
deuterium‐exchangeable protons of =N‐OH groups show
chemical shifts at δ 8.54 and 9.32 ppm. These singlets also
indicate the (E,E)‐structure of vic‐dioximes.[41,42] The
other singlet observed at δ 11.61 ppm can be assigned to
2.5 | Statistical analysis
Each experiment was carried out triplicate. Values are
expressed as means SD, and analyzed by one‐way
ANOVA (SPSS 13.0 software package program). Statisti-
cally significant difference was considered at the level of
P < 0.05. The normality of variables was evaluated using

ÇELIK ET AL.
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TABLE 1 Analytical and physical results for the newly synthesized compounds
Elemental analyses (%) (calcd/found)
μeff
(BM)a (°C)
M.p. (d)b
Yield
(%)
Compound
Color
Λc
C
H
N
M
M
C10H10N4O3 (LH2) Yellow
–
127
210
208
1.3
7.4
6.3
77
52
73
51.28 (51.52) 4.30 (4.20) 23.92 (23.28)
–
C20H18N8O6Ni
C20H18N8O6Cu
Dark‐brown Dia.
Dark‐ green 1.63
45.67 (43.57) 3.43 (3.47) 21.32 (21.28) 11.23 (11.51)
45.29 (44.94) 3.40 (3.74) 21.14 (20.79) 11.98 (12.33)
aμeff: magnetic moment; Dia.: diamagnetic.
bd: decomposition.
cMolar conductivity (Ω−1 cm2 mol−1).
TABLE 2 1H‐NMR spectra of the newly synthesized ligand and Ni (II) complex in DMSO‐d6 (δ, ppm)
Compounds
LH2
O‐H …. Oa
O‐Ha
N‐Ha
CH=NOH
CHArom.
–
14.11–11.82 (1H each, 2s)
11.61 (1H, s)
8.54–9.32 (1H each, 2s)
7.48 2H, d, J = 8.65
8.22 2H, d, J = 8.65
(L1H)2Ni
15.32 (2H, s)
–
–
–
–
the ‐N‐H signal, which can also be easily identified by
deuterium‐exchange (Table 2). The aromatic protons
would resonate as multiples between δ 7.48 and 8.22
ppm. In the 1H‐NMR spectra of LH2, two absorption
bands, which are assigned to the oxime ‐O‐H protons,
appear at a lower field, at δ 14.11–11.82. The chemical
shifts belonging to the ‐O‐H protons in vic‐dioxime disap-
IR spectrum of the ‐O‐H group of oxime shows stretching
vibrations at 3450 and 2600 cm−1 instead of a band at
about 3230 cm−1 because of an intermolecular hydrogen
bonding N···H–O.[46]
Also, the NH stretching band of LH2 was not observed
in the FT‐IR spectra probably due to overlapping with the
intermolecular hydrogen‐bonded ‐O‐H stretching fre-
quency. The stretching band of the imine group ‐C=N
of these compounds was observed at 1611 cm−1. The
stretching belonging to the ‐NH and ‐N‐O groups occur
at 3420 and 1005 cm−1, respectively. These values are in
agreement with the previously reported hydrazone and
oxime derivatives.[39,40] The FT‐IR spectra of Ni (II) and
Cu (II) have been compared with those for similar com-
pounds in order to establish the coordination modes of
the oxime ligands in these complexes.[32,43] The more rel-
evant ‐O‐H, ‐C=N and ‐N‐O absorption bands are given
in Table 3. The Ni (II) and Cu (II) complexes have FT‐
IR spectra very similar to those of the free ligand, except
for the absence of the ‐O‐H stretching vibrations. A weak
and low‐intensity band at 2340–2170 cm−1 indicates the
formation of the hydrogen bridge during complexation
of the ligand, as the hydrogen bridge reduces the strength
of the ‐O‐H bond. The occurrence of hydrogen bridges by
the loss of one oxime proton per oxime molecule during
the complexation of this ligand accounts for the two
non‐identical =N‐C‐ linkages, ‐C=N‐O‐H in the com-
plexes.[47] The stretching vibrations of the azomethine
groups appearing at 1610 cm−1 in the free ligand are
shifted to 1545–1540 cm−1 in the mononuclear Ni (II)
and Cu (II) complexes. At the same time, in the
complexes the bands observed near 1005 cm−1 in the free
ligand, which are assigned to the ν(N‐O), are shifted to
lower frequencies (990–980 cm−1) after complexation.
1
peared in the H‐NMR spectrum of the Ni (II) complex,
and the presence of a new resonance at a lower field at
δ 15.32 ppm was assigned by formation of the hydrogen
bridge, which could easily be identified by deuterium‐
exchange.[43] The geometric isomers of Ni (II) complex
can be inferred from the 1H‐NMR spectra, as the alterna-
tive chemical environment will show two O···H–O bridge
1
protons in the cis‐form, but also one H‐NMR resonance
in the trans‐ structure. The observed spectrum has only
one 1H‐NMR resonance, suggesting the trans‐ of the
complexes.[43]
A
13C‐NMR spectrum is one of the most convenient
ways to prove the true structure of vic‐dioxime. In the
13C‐NMR spectrum of LH2, the carbon resonances of the
oxime groups are observed at 146.57 and 145.79 ppm.
1
Observation of vic‐dioxime and ‐O‐H protons in the H‐
NMR, and of dioxime carbons in the 13C‐NMR spectra at
two different frequencies in each case, indicates that the
vic‐dioxime has an anti‐structure.[44] The equivalent car-
bon atoms, especially the adjacent hydroxyimino groups,
also confirm the (E,E)‐structure of vic‐dioximes.[45]
3.2 | Fourier transform‐infrared spectra
The tentative assignments of the most characteristic IR
bands were observed and are given in Table 3. The FT‐

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ÇELIK ET AL.
TABLE 3 Characteristic FT‐IR bands (cm−1) of the newly synthesized compounds as KBr pellets
Comp.
ν (NH)
ν (OH)
ν (CHar.)
ν (OH..O)
ν(C=Noxime
)
ν(C=Nhyd.
)
ν (NO)
ν(C=O)
LH2
3420 (b)
3245 (b)
3440 (b)
3230 (b)
–
3055 (w)
3055 (w)
3065 (w)
–
1610 (s)
1545 (s)
1540 (s)
1540 (m)
1590 (m)
1595 (m)
1005 (s)
980 (s)
990 (s)
1670 (m)
(L1H)2Ni
(L1H)2Cu
2170 (w)
2340 (w)
–
–
3340 (b)
b = broad, s = strong, m = medium, w = weak.
its mononuclear complexes is in the range of 1.3,
7.4–6.3 Ω−1 cm−2 mol−1. This result shows that the
complexes are non‐electrolytes.[43]
TABLE 4 Characteristic UV–Vis bands of the newly synthesized
compounds
Compounds
Solvent
Wavelength, λmax (nm)
LH2
ETOH
DMSO
DMSO
345, 295
(L1H)2Ni
(L1H)2Cu
678, 546, 488, 350
637, 450, 325, 280
3.6 | Cytotoxic effect of ligand and its Cu
(II) and Ni (II) complexes
The cytotoxic effects of the vic‐dioxime ligand and their
Ni (II), Cu (II) complexes were analyzed by culturing
the cells of human epithelial colorectal adenocarcinoma
(Caco‐2) and human peripheral lymphocytes in vitro.
The Caco‐2 cell line is homogeneous cells originally
derived from a colon carcinoma. The results of the
cytotoxicity test (MTT assay) are presented in Table 5.
The vic‐dioxime ligand induced a degree of cytotoxic
effect on the Caco‐2 cells and peripheral lymphocytes,
but this effect is not statistically significant. The ligand
and Campto combination induced higher cytotoxic effects
on Caco‐2 cells and lymphocytes when compared with
both the control and solvent control.
The Cu (II) complex of the ligand was significantly
increased with the cytotoxic effect dependent on
increased concentration in Caco‐2 cells. The Cu (II)
complex of the ligand did not cause a cytotoxic effect on
peripheral lymphocytes. The Cu (II) complex + Campto
combination induced a very high cytotoxic effect on
Caco‐2 cells when compared with control and solvent
control. The cytotoxic effect on lymphocytes was found
to be statistically significantly increased compared with
control and solvent control. Generally, Cu (II) of ligand
complex + Campto inhibited cell proliferation more than
positive control (Campto), and showed a higher cytotoxic
effect than Campto at both treatment periods. Increasing
concentrations caused a significant increase of cytotoxic
effects on peripheral lymphocytes. Also, the positive
control Campto induced cytotoxic effects on peripheral
lymphocytes (Table 5).
3.3 | Electronic spectra
The electronic spectra of the ligand and its complexes
were recorded in ethanol and DMSO (Table 4). In the
electronic spectra of the ligand and its metal complexes,
the 280–350 nm band seems to be due to both the
π → π* and n → π* transitions of ‐C=N and charge‐
transfer transition arising from p electron interactions
between the metal and ligand, which involve either a
metal‐to‐ligand or ligand‐to‐metal electron transfer.[48]
The electronic spectra of the Ni (II) and Cu (II) complexes
show absorption bands at 678 and 637 nm attributed to
1
1
2
2
the A1g → B1g for Ni (II) complex and T2g → Eg for
Cu (II) complex transitions, which are compatible with
the complexes having a square‐planar structure.[49]
3.4 | Magnetic susceptibility
The structures of the mononuclear complexes are sup-
ported by magnetic moment data. The Ni (II) is diamag-
netic as expected for d8 electronic configuration.[30] The
Cu (II) complex is paramagnetic and their magnetic
moment is 1.63 BM. The alternative chemical environ-
ments will give two (O···H–O) bridge protons in the cis‐
form, but only one in the trans‐form. Observation of the
1
(O···H–O) in the IR spectrum and H‐NMR spectra, at
one frequency in each case, indicate that the Ni (II)
complex is in the anti‐form media. According to the
above results, square‐planar geometry for the Ni (II) and
Cu (II) complexes is proposed.[32,43,44]
The Ni (II) complex of ligand induced significant cyto-
toxic effects on Caco‐2 cells at 40 μM concentration
(61.90%), and a low cytotoxic effect at 10 μM concentra-
tion (12.50%). The cytotoxic effect on lymphocytes was
found to be higher when compared with ligand. The
combination of Ni (II) complex + Campto induced a very
3.5 | Molar conductance
As seen from Table 1, the molar conductance measured
in DMSO solution (about 1 × 10−3 M) for the ligand and

ÇELIK ET AL.
7 of 11
TABLE 5 The cytotoxic effect of vic‐dioxime ligand on human
colon cancer cells (Caco‐2) and peripheral blood lymphocytes
in vitro cytotoxic activities of bivalent transition metal
hydrazone complexes of cobalt, nickel and copper.
Complexes interacted with DNA, and intercalative mode
of binding was revealed, whereas metal complexes
resulted in conformational change when interacted with
bovine serum albumin. The anti‐cancer activity of com-
plexes was tested against HeLa tumor cells and NIH
3 T3 normal cells. The results of the study showed that
the complexes are toxic only against tumor cells but not
to normal cells. Furthermore, they found that the
complexes with copper ion as a metal center were more
potent as compared with nickel and cobalt.[50] Li et al.
(2011a)[51] examined the cytotoxic behavior of Mn (II)
and Ni (II) complexes of thiosemicarbazones against
K562 leukemia cell line, and reported that Ni complexes
were more active than that of Mn. Sirajuddin et al.
(2015)[52] observed the in vitro cytotoxic potential of
carboxylic acid derivatives against H‐157, BHK‐21 and
HCEC cell lines. The derivatives were non‐toxic towards
normal cell lines, whereas significant inhibition was
found against cancer cell lines. The present results reveal
that cobalt complexes are more active than Ni complexes
against cancer cell lines. These complexes are more
potent inhibitors than ligand against H157 cancer cells,
whereas the complexes as well as ligands are non‐toxic
to normal cell lines. The non‐toxic nature of compounds
towards normal cells suggests their safe and significant
selectivity index.
% Cytotoxicity ( SD)
Groups
Caco‐2 cells
Lymphocytes
Control
0.00 0.00
0.00 0.00
Solvent control
(DMSO) (% 0.1)
2.13 0.179
2.21 0.024
Campto (40 μg mL−1
)
55.36 0.026*
8.18 0.090
23.52 0.015*
2.00 0.047
LH2–1
LH2–2
16.67 0.020*
24.39 0.073*
56.21 0.144*
60.61 0.044*
65.76 0.031*
66.61 0.007*
70.18 0.008*
76.13 0.002*
86.42 0.001*
90.32 0.006*
92.54 0.041*
12.50 0.022
26.49 0.049*
61.90 0.012*
56.25 0.013*
77.08 0.005*
82.74 0.017*
3.76 0.031
LH2–3
5.82 0.019
LH2–1 + Campto
LH2–2 + Campto
LH2–3 + Campto
LH2‐Cu1
51.17 0.007*
52.58 0.033*
53.52 0.021*
3.12 0.039
LH2‐Cu2
5.16 0.085
LH2‐Cu3
24.41 0.031*
49.77 0.001*
51.64 0.007*
52.58 0.041*
8.92 0.046
LH2‐Cu1 + Campto
LH2‐Cu2 + Campto
LH2‐Cu3 + Campto
LH2‐Ni1
LH2‐Ni2
13.15 0.036
36.15 0.030*
23.00 0.029*
38.03 0.010*
46.48 0.002*
LH2‐Ni3
LH2‐Ni1 + Campto
LH2‐Ni2 + Campto
LH2‐Ni3 + Campto
3.7 | Apoptotic and necrotic cell death
*P < 0.05 [LH2–1: 10 μM ligand; LH2–2: 20 μM ligand; LH2–3: 40 μM ligand;
LH2‐Cu1: 10 μM Cu (II) complex; LH2‐Cu2: 20 μM Cu (II) complex; LH2‐
Cu3: 40 μM Cu (II) complex; LH2‐Ni1: 10 μM Ni (II) complex; LH2‐Ni2:
20 μM Ni (II) complex; LH2‐Ni3: 40 μM Ni (II) complex].DMSO,
dimethylsulfoxide.
Chemical‐induced cell apoptotic and necrotic cell death
were determined usingDNA diffusion assay in this study,
and the results are presented in Table 6. The assay is a
simple, sensitive and reliable technique that allows the
assessment of programmed cell death or necrosis events
based on nuclear morphology.[53]
The vic‐dioxime ligand did not cause an apoptotic
effect on the Caco‐2 cells and peripheral lymphocytes.
When cells were treated with ligand and Campto
together, Caco‐2 cells and lymphocytes had higher
apoptotic effects than control and solvent control groups.
Despite their apoptotic activity on Caco‐2 cells, the rate of
necrotic cell death caused by ligand in both Caco‐2 cells
and lymphocytes was found to be low.
The Cu (II) complex of the ligand has been found to
have an increased apoptotic effect depending on
increased concentration of Caco‐2 cells. The Cu (II) com-
plex of the ligand did not cause a significant apoptotic
effect in peripheral lymphocytes (Table 6). When Cu (II)
complex was used in combination with Campto, the
apoptotic effect on Caco‐2 cells increased and reached
high cytotoxic effect on Caco‐2 cells when compared with
control, solvent control and Campto. Also, the cytotoxic
effect on lymphocytes was found to be statistically signif-
icantly increased compared with control, solvent control
and Campto. These results are also statistically significant
(Table 5). The present results reveal that Cu (II) complex
+ Campto and the Ni (II) complex + Campto showed
cytotoxic effects on Caco‐2 cells and lymphocytes, but
the Ni (II) complex + Campto has a less cytotoxic effect
compared with the Cu (II) complex + Campto against
lymphocytes.
Different biological activities like DNA binding and
cleavage studies of copper and nickel complexes
containing ferrocenyl hydrazone ligands were performed
by Krishnamoorthy et al. (2011).[50] They evaluated the
DNA binding, DNA cleavage, protein binding and

8 of 11
ÇELIK ET AL.
TABLE 6 The apoptotic effect of vic‐dioxime ligand on human colon cancer cells (Caco‐2) and peripheral blood lymphocytes
Caco‐2
Lymphocytes
Apoptotic
cells
Necrotic
cells
Apoptotic
cells
Necrotic
Groups
(% SD)
(% SD)
Total cells
(% SD)
cells (% SD)
Total cells
Control
0.00 0.00
2.00 0.10
0.00 0.00
0.10 0.01
3000
3000
0.00 0.00
2.00 1.00
0.00 0.00
0.10 0.10
3000
3000
Solvent control
(DMSO) (% 0.1)
Campto (40 μg mL−1
)
52.33 0.57*
6.33 0.57
1.67 0.56
2.67 0.56
1.33 0.57
2.67 1.54
3.33 0.57
5.67 0.56
5.00 0.00
2.33 1.54
3.00 1.00
2.67 0.56
5.00 1.00
3.33 0.57
4.67 1.54
3.33 0.56
5.00 0.00
7.67 0.57
4.33 1.54
6.00 0.00
7.67 0.56
3000
3000
3000
3000
3000
3000
3000
3000
3000
3000
3000
3000
3000
3000
3000
3000
3000
3000
3000
21.67 1.54*
2.33 0.56
0.3 0.06
0.2 0.10
0.5 0.30
0.8 0.00
4.67 0.57
5.33 0.56
6.67 0.57
0.27 0.06
0.33 0.06
0.67 0.57
2.00 0.00
1.33 0.56
2.67 0.57
1.33 1.73
1.67 1.57
2.00 0.00
1.00 1.00
1.00 0.00
1.67 1.57
3000
3000
3000
3000
3000
3000
3000
3000
3000
3000
3000
3000
3000
3000
3000
3000
3000
3000
3000
LH2–1
LH2–2
15.67 0.56*
21.33 0.57*
49.67 0.56*
53.33 1.54*
57.67 0.56*
61.67 0.57*
64.33 1.54*
70.00 0.00*
77.33 2.08*
81.00 0.56*
86.67 0.57*
9.00 1.00
3.67 0.57
LH2–3
4.33 0.56
LH2–1 + Campto
LH2–2 + Campto
LH2–3 + Campto
LH2‐Cu1
41.33 1.54*
41.67 0.56*
42.00 1.00*
3.00 0.00
LH2‐Cu2
4.67 0.56
LH2‐Cu3
17.33 0.57*
41.00 1.00*
44.67 0.57*
46.33 0.56*
7.00 0.00
LH2‐Cu1 + Campto
LH2‐Cu2 + Campto
LH2‐Cu3 + Campto
LH2‐Ni1
LH2‐Ni2
17.67 0.57*
48.33 0.56*
49.67 1.54*
65.00 0.00*
72.33 0.56*
9.33 0.56
LH2‐Ni3
31.00 1.00*
20.00 1.00*
27.67 0.57*
37.33 0.57*
LH2‐Ni1 + Campto
LH2‐Ni2 + Campto
LH2‐Ni3 + Campto
*P < 0.05 [LH2–1: 10 μM ligand; LH2–2: 20 μM ligand; LH2–3: 40 μM ligand; LH2‐Cu1: 10 μM copper (II) complex; LH2‐Cu2: 20 μM copper (II) complex; LH2‐
Cu3: 40 μM copper (II) complex; LH2‐Ni1: 10 μM nickel (II) complex; LH2‐Ni2: 20 μM nickel (II) complex; LH2‐Ni3: 40 μM nickel (II) complex].DMSO,
dimethylsulfoxide.
86.67% in the 40 μM Cu (II) complex and Campto mixture
group. When the Cu (II) complex of the ligand was used
with Campto, the apoptotic effect on lymphocytes was
found to be statistically significantly higher than
control and solvent control. The rate of necrotic cell death
in both Caco‐2 cells and lymphocytes was found to be
very low.
Campto, the apoptotic effect on lymphocytes was found
to be statistically significantly higher than control, sol-
vent control and Campto. The Ni (II) complex of ligand
caused a high apoptotic effect both on Caco‐2 cells and
in lymphocytes, while necrotic cell death is observed
with low levels of Ni (II) complex for Caco‐2 cell line
on lymphocytes.
The Ni (II) complex of ligand has not been found to
have an increasing apoptotic effect depending on
concentration of Caco‐2 cells and lymphocytes, but the
apoptotic effect on Caco‐2 cells and lymphocytes was
found to be significantly high in comparison with con-
trol and solvent control. When the Ni (II) complex was
used in combination with Campto, the apoptotic effect
on Caco‐2 cells increased and reached 72.33 0.56% in
the 40 μM Ni (II) complex and Campto mixture group.
When the Ni (II) complex of the ligand was used with
Apoptosis and necrosis are two major mechanisms of
cell death.[54] Apoptosis, in fact, is a programmed mecha-
nism for cell death, characterized by morphological and
biochemical changes. This evolutionarily conserved cell
death process is important for normal development and
tissue homeostasis.[55,56] It also occurs during pathologi-
cal conditions, as well as in response to many cancer
chemotherapeutic agents. Apoptosis is induced by a
variety of stimuli, such as cytotoxic compounds.[57] Cells
that are damaged by external injury undergo necrosis,

ÇELIK ET AL.
9 of 11
while cells that are induced to commit programmed
suicide because of internal or external stimuli undergo
apoptosis. An increasing number of chemo‐preventive
agents have been shown to stimulate apoptosis in pre‐
malignant and malignant cells in vitro or in vivo.[58]
The cytotoxicity of Cu (II) complexes may be caused
by their ability to bind and cleave DNA, which leads to
the arrest of the cell cycle and apoptosis, or the generation
of reactive oxygen species, followed by cell death.[59,60] In
light of the fact that copper is a bio‐essential element
responsible for numerous bioactivities in living organ-
isms, (CuL2) should be an excellent anti‐tumor agent for
applications.[61]
vic‐Dioximes are important ligands because of their
considerable attention in biology and chemistry.[62] Some
vic‐dioxime ligands show anti‐microbial properties.[62]
Previous studies suggested that hydrazone and oxime
derivatives and their metal complexes possess anti‐
proliferative effects against tumor cells, such as human
breast cancer cells, ovarian cancer cells, renal cancer
cells, hematological tumor cells, prostate cancer cells,
hepatocellular carcinoma cells, lung carcinoma cells and
HL‐60 human promyelocytic leukemia cells.[63–67] They
exhibit their anti‐cancer potential as anti‐proliferative,
cytotoxic, cell cycle arrest at G2/M, inducing apoptosis,
caspase activation, and by inhibiting tubulin polymeriza-
tion.[63–67]
more detailed studies have to be carried out using differ-
ent test systems.
4 | CONCLUSION
In this study, two new vic‐dioxime derivatives containing
hydrozone side groups and their metal complexes with
Cu (II) and Ni (II) were synthesized and evaluated, and
their cytotoxic, apoptotic and necrotic effects using MTT
assay and DNA diffusion assay on Caco‐2 heterogeneous
human epithelial colorectal adenocarcinoma cell line
and human peripheral lymphocytes were determined.
As a result of this study, among the test compounds
attempted Cu (II) complex of ligand showed higher
cytotoxic and apoptotic activity against tested cancer cells
than Ni (II) complex.
The results of the present work suggest that these
compounds are effective inhibitors, and may be promis-
ing agents for anti‐tumor testing with in vivo studies on
animals under ethical regulations. However, the exact
mechanism of apoptosis of the compounds under
consideration in cancer cells vs. normal cells still has to
be determined. These complexes may be suitable drugs,
and therefore beneficial and potential alternatives, and
hence should be investigated further in pharmacological
studies on how this ligand and its Cu (II) complex can
be used for developing new drugs for cancer treatment.
One of the salient characteristics of carcinogenesis is
that the dividing tumor cells fail to initiate apoptosis
following DNA damage.[68] Development of approaches
that reinstall the apoptotic machinery selectively within
tumor cells could be an effective measure of cancer con-
trol.[69] The primary mode of action of most anti‐cancer
drugs entails the induction of apoptosis in neoplastic
cells.
ACKNOWLEDGEMENT
The authors acknowledge the Adnan Menderes Univer-
sity Scientific Research Foundation for their financial
support (Project No: FEF‐11005).
Currently, cancer is the second cause of death around
the world. The incidence of cancer is increasing gradually
because of high population growth, and is expected to
reach 75% by 2030.[70] There are numerous medications
present for treatment of various cancer types, but there
is not any drug that is completely effective and safe.
Furthermore, most of the anti‐cancer drugs affect healthy
cells of patients, and produce other side‐effects.[3] There-
fore, researches are under way to develop new anti‐
cancer drugs to reduce cancer mortality rates and the
costs of cancer treatment, find the best cancer treatments,
and improve the lives of cancer patients. The results of
the present study suggest that the Cu (II) complex of
vic‐dioxime ligand represents a potential anti‐cancer
agent because of its high cytotoxic and apoptotic effect
against colon cancer cells, and its low effects on human
peripheral blood lymphocytes. However, in order to
elucidate the specific action mechanism of this complex,
ORCID
Tülay Aşkın Çelik https://orcid.org/0000-0001-6891-9089
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