Design, synthesis and investigation of procaine based new Pd complexes as DNA methyltransferase inhibitor on gastric cancer cells

Article abstract of DOI:10.1016/j.inoche.2021.108846

Procaine is a specific inhibitor of DNA methyltransferase (DNMT). In the present work, the Schiff base ligands were synthesized from procaine with the addition of salicylaldehyde and naphthaldehyde as metal precursor for procaine-based transition metal complexes (L¹ and L²) with palladium (Pd). The compounds were checked and characterized for identity and purity using elemental analysis, SEM, melting points, FT-IR, and NMR spectral data. Among the compounds, L¹-Pd and L²-Pd were found to display significant cytotoxicity with IC₅₀ values of 10.21 μM and 10.79 μM against human gastric cancer cell line MKN-45. Novel synthesized procaine derivatives target molecules and their palladium complexes had an inhibitory effect on colony formation and wound healing on MKN-45 cells. Furthermore, these compounds were evaluated for their apoptotic activity and DNMT activity in MKN-45 cells. Western blotting and qPCR studies demonstrated that compound L¹-Pd and L²-Pd induced apoptosis and a slight decrease in anti-apoptotic Bcl-2 protein/gene expression and highly increased pro-apoptotic Bax protein/gene expression in MKN-45 cells treated. Also, total DNMT enzyme activity and protein expression (DNMT1, DNMT3a, and DNMT3b) levels were decreased in L¹-Pd and L²-Pd treated cells. The L¹-Pd and L²-Pd complexes showed that they are potential new DNMT inhibitors to prevent cancer-induced DNA hypermethylation and can be used in the treatment of gastric cancer.

Full text of DOI:10.1016/j.inoche.2021.108846

Inorganic Chemistry Communications 132 (2021) 108846
Contents lists available at ScienceDirect
Inorganic Chemistry Communications
journal homepage: www.elsevier.com/locate/inoche
Design, synthesis and investigation of procaine based new Pd complexes as
DNA methyltransferase inhibitor on gastric cancer cells
,
*
Salih Pas¸aa, Omer Erdoganb, Ozge Cevikb
a Afyon Kocatepe University, Faculty of Education, Department of Science, Afyonkarahisar 03200, TURKEY
b Aydin Adnan Menderes University, School of Medicine, Department of Biochemistry, Aydin 09010, TURKEY
A R T I C L E I N F O
A B S T R A C T
Keywords:
Procaine is a specific inhibitor of DNA methyltransferase (DNMT). In the present work, the Schiff base ligands
were synthesized from procaine with the addition of salicylaldehyde and naphthaldehyde as metal precursor for
procaine-based transition metal complexes (L1 and L2) with palladium (Pd). The compounds were checked and
characterized for identity and purity using elemental analysis, SEM, melting points, FT-IR, and NMR spectral
data. Among the compounds, L1-Pd and L2-Pd were found to display significant cytotoxicity with IC50 values of
10.21 µM and 10.79 µM against human gastric cancer cell line MKN-45. Novel synthesized procaine derivatives
target molecules and their palladium complexes had an inhibitory effect on colony formation and wound healing
on MKN-45 cells. Furthermore, these compounds were evaluated for their apoptotic activity and DNMT activity
in MKN-45 cells. Western blotting and qPCR studies demonstrated that compound L1-Pd and L2-Pd induced
apoptosis and a slight decrease in anti-apoptotic Bcl-2 protein/gene expression and highly increased pro-
apoptotic Bax protein/gene expression in MKN-45 cells treated. Also, total DNMT enzyme activity and protein
expression (DNMT1, DNMT3a, and DNMT3b) levels were decreased in L1-Pd and L2-Pd treated cells. The L1-Pd
and L2-Pd complexes showed that they are potential new DNMT inhibitors to prevent cancer-induced DNA
hypermethylation and can be used in the treatment of gastric cancer.
Procaine
Schiff Ligand
Pd Complex
DNMT Inhibitor
Anticancer
Apoptosis
1. Introduction
rapidly gained momentum in recent years. As a result, many different
investigations and approaches are now being used to develop new
Cancer is a brutal disease that is caused by several structural de-
teriorations at the cellular level. There are still no effective treatments
for several types of cancer, however much research has been done to
provide a greater understanding of the processes involved in the pro-
gression of the disease. Cancer cells have the ability to grow rapidly and
form colonies or tumors within the living tissue. The colony formation
assay has been the standard procedure to assess the effects of cytotoxic
agents on cancer cells [1]. In addition to that, wound healing assay is a
simple, inexpensive and effective method for monitoring the migration
of cells. This method is constructed to observe cell migration into a
“wound” that is created on a cell monolayer [2]. Advanced abilities such
as invasion, migration, survival, and colony formation are required in
the metastasis of cancer cells. The main handicap for cancer treatments
is the lack of protection of the healthy cells in the body. Besides, the
chemotherapy drugs are also highly toxic to normal cells, not specif-
ically to cancer cells. Drugs synthesized to interrupt the various cancer
mechanisms are more effective and attempts in this direction have
therapeutics.
DNA methyltransferase (DNMT) inhibitors are chemical components
for hypomethylation of genome in multiple processes of DNA methyl-
ation (e.g., DNA imprinting and X-chromosome inactivation) and have
recently been used as anticancer treatments [3–6]. DNMT enzymes may
cause epigenetic modifications in the modulation of gene expressions by
catalyzing some regions of DNA methylation. To date, three different
DNMT (DNMT1, DNMT3a, and DNMT3b) that are responsible for the
methylation of mammalian cells have been found. Essentially, most
DNMTs have the capacity of catalyzing the addition of a methyl group to
the fifth carbon atom of the cytosine residues at the CpG sites by using S-
adenosyl methionine as a methyl donor [7]. The control of the DNMTs is
crucial since the hypermethylation of tumor suppressor genes creates an
aggressive process for cancer formation. Therefore, one of the ap-
proaches for cancer treatments is the development of DNA methyl-
transferase inhibitors. Procaine, azacytidine, decitabine, zebularine,
etc., are currently used as DNMT inhibitors for cancer treatment. Due to
* Corresponding author.at: Aydin Adnan Menderes University, School of Medicine, Department of Biochemistry, Efeler, Aydin 09100, TURKEY.
E-mail address: ozge.cevik@adu.edu.tr (O. Cevik).
https://doi.org/10.1016/j.inoche.2021.108846
Received 21 April 2021; Received in revised form 1 August 2021; Accepted 4 August 2021
Available online 8 August 2021
1387-7003/© 2021 Elsevier B.V. All rights reserved.

S. Pas¸a et al.
Inorganic Chemistry Communications 132 (2021) 108846
the lack of data on many aspects of DNMT inhibitors a great deal of effort
has been devoted to develop DNMT inhibitors for both specific cancers
and for cancer in general [8]. DNMT inhibitors are generally classified
into two different classes: nucleoside inhibitors and non-nucleoside in-
hibitors [9]. Nucleoside inhibitors are a class of nucleoside analogues
that consist of cytidine derivatives which can be incorporated into DNA
and can be effective by the formation of a suicidal covalent complex with
DNMT. Non-nucleoside molecules are classified as compounds that have
different chemical structures, these molecules generally bind directly to
DNMT and result in changes in the mechanism of their actions [9].
Furthermore, DNA methylation is a stable epigenetic mark in humans
since it takes place at the C5 location of cytosines, especially in a CpG
dinucleotide context and in non-CpG regions of stem cells [10–12].
However, the use of these DNMT inhibitors is limited due to their dis-
advantages, such as poor structural stability, relative toxicity, and drug
resistance [13]. Therefore, it is necessary to develop new DNMT in-
hibitors in cancer treatment.
water. Yellow product was recrystallized in ethanol. The obtained pre-
cipitate was recovered by filtration, washed and air-dried. (Fig. 1) M.P:
170–174 ◦C, Yield: 93%. Elemental Analysis: [C20H24N2O3] (340.42 g/
mol): C, 70.56; H, 7.11; N, 8.23; O, 14.10. IR(cmꢀ 1, KBr): 1616 HC = N,
3406O-H, 1112 ArC-N, 3051 Ar-CH, 2976 Aliph-CH, 1712C = O, 1274C-
O, 2484 hydroxyl stretching bond [25], 2582 atmospheric CO2 [26]. 1H
NMR (DMSO‑d6): δ 9.02 ppm (1H, s, CH = N), δ 1.27 ppm (6H, t, J = 8
Hz, N-(CH2-CH3)2), δ 3.37 ppm (4H, q, J = 12 Hz, N-(CH2-CH3)2), δ 4.52
ppm (4H, t, J = 4 Hz, –CH2-CH2–), δ 12.61 ppm (1H, s, –OH), δ
6.57–7.70 ppm (8H, m, Ar-CH). 13C NMR (DMSO‑d6): δ 160 ppm (1H,
CH = N), δ 161 ppm (1H, Ar-C-OH), δ 9 ppm (N-(CH2-CH3)2), δ 49 ppm
(N-(CH2-CH3)2), δ 58 ppm (O-CH2-CH2-N), δ 59 ppm (O-CH2-CH2-N), δ
165 ppm C = O), δ 117–136 ppm (Ar-C).
L2: 2-(diethylamino) ethyl 4-(((2-hydroxynaphthalen-1-yl)methylene)
amino) benzoate: The synthesis of L2 was performed according to the
literature procedure [24]. A solution of 2-hydroxynapthaldehyde
(0.172 g, 1 mmol) in 30 mL ethanol and a solution of procaine (0.236
g, 1 mmol) in 30 mL ethanol were mixed and refluxed for 6 h. After the
Transition metals, called as d-block elements, are essential for many
biological functions. Properties such as electron mobility in the valance
shells and the ability to be coordinated by many ligands having electron
donor elements, such as N, O, and S, makes them important in the areas
of protein structural stability and functionality. In particular, palladium
and platinum complexes such as cisplatin [14–16], have found a great
deal of use as actual or potential anticancer agents.
◦
mixture was cooled to 25 C, the solvents (EtOH and occurred water)
were evaporated. The remained yellowish solid precipitate was recrys-
tallized in ethanol. Then it was filtered and washed. It was yielded
approximately 88% after dried in a ventilated oven. (Fig. 2) M.P:
183–185 ◦C, [C24H26N2O3] (390.47 g/mol): C, 73.82; H, 6.71; N, 7.17;
O, 12.29. IR(cmꢀ 1, KBr): 1624 HC = N, 3406O-H, 1153 ArC-N, 3057 Ar-
CH, 2976 Aliph-CH, 1714C = O, 1271C-O. 1H NMR (DMSO‑d6): δ 9.68
ppm (1H, s, CH = N), δ 1.28 ppm (6H, t, J = 8 Hz, N-(CH2-CH3)2), δ 3.24
ppm (4H, q, J = 4 Hz, N-(CH2-CH3)2), δ 4.62 ppm (4H, –CH2-CH2–), δ
10.81 ppm (1H, s, –OH), δ 6.98–8.96 ppm (m, Ar-CH). 13C NMR
(DMSO‑d6): δ 154 ppm (1H, CH = N), δ 172 ppm (1H, Ar-C-OH), δ 8 ppm
(N-(CH2-CH3)2), δ 47 ppm (N-(CH2-CH3)2), δ 49 ppm (O-CH2-CH2-N), δ
50 ppm (O-CH2-CH2-N), δ 165 ppm C = O), δ 122–138 ppm (Ar-C).
L1-Pd: The ligand (L1, 0.0357 g, 0.105 mmol) was first dissolved in
acetone:chloroform (10 mL + 10 mL) mixture by heating. A solution of
palladium acetate (Pd(AcO)2, 0.0112 g, 0.05 mmol) in 15 mL acetone
was added dropwise into ligand solution. The reflux reaction was
continued for 12 h. Subsequently, it was cooled to room temperature
and the solvent was evaporated. Brown solid product was separated and
washed with distilled water and ethanol. Then it was used without
further purification. (Fig. 3) M.P: >250 ◦C. Yield: 71%. Elemental
Analysis: [C40H46N4O6Pd] (785.24 g/mol): C, 61.18; H, 5.90; N, 7.14; O,
12.23; Pd, 13.55. IR(400–4000 cmꢀ 1, KBr): 1600 HC = N, 1178 ArC-N,
3032 Ar-CH, 2916 Aliph-CH, 1716C = O, 1274C-O, 2848 symmetric CH2
stretching transition [27], 464 Pd-O, 582 Pd-N. 1H NMR (DMSO‑d6): δ
8.32 ppm (CH = N), δ 1.23 ppm (N-(CH2-CH3)2), δ 3.35 ppm (N-(CH2-
CH3)2), δ 4.40 ppm (–CH2-CH2–), δ 7.14–8.22 ppm (Ar-CH).
In the current study, the effort has been made to enlighten the
employment of Pd complexes, obtained from procaine-derived Schiff
compounds, in DNA methylation to repress cancer growth. Procaine-
originated structures have been frequently used for this purpose and
attract great attention to be as a significant agent in DNMT inhibitors
[17–22]. Therefore, ligands were selected from the previous studies
[23,24] to investigate the best biological effect with their Pd complexes
on Gastric Cancer Cells. The possible synergetic effect comprised of the
Schiff base ligands (L1 and L2) and their transition metal complexes as
L1-Pd and L2-Pd with the employment of procaine hasn’t been investi-
gated for DNMT inhibitors yet. Therefore, this work focuses on DNMT
investigations with the newly synthesized Pd complexes containing
Schiff base ligands to develop alternative drugs for gastric cancer
treatments.
2. Experimental
2.1. Reagents and methods
All the reagents, solvents, and materials were commercially pur-
chased from Sigma-Aldrich and Merck. The compounds and solvents
were appropriately purified, if necessary. All cell culture supplements
and medium for cell culture studies were purchased from Gibco Com-
pany. 13C NMR and 1H NMR spectra were recorded for obtained ligands
and complexes in d6-DMSO solution on a Bruker DPX 400 MHz spec-
trometer at 300 K. The morphological analyses were investigated by
SEM-EDX (Scanning Electron Microscope-Energy Dispersive X-ray
Spectroscopy) with LEO 1430 VP instrument working with tungsten
filament lamp. The SEM images were recorded after 5000 times
magnification. The stretching vibrations were obtained after 30 scans
with 4 cmꢀ 1 resolution by Shimadzu LabSolutions IR 8000 with KBr
L2-Pd: A solution of ligand (L2, 0.0488 g, 0.05 mmol) in 20 mL
ethanol was set in a reflux system. 0.0056 g, 0.025 mmol Pd(AcO)2 was
dissolved in 15 mL acetone and added drop by drop into the reaction
flask. The reaction was refluxed for 12 h and subsequently cooled to
room temperature. The evaporating solvents from the reaction mixture
afforded a dark brown solid precipitate. It was washed with plenty of
ethanol and distilled water to purify it from the unreacted compounds.
The solid product was then dried and used without further purifications.
(Fig. 4) M.P: >250 ◦C, Yield: 68%. [C48H50N4O6Pd] (885.35 g/mol): C,
65.12; H, 5.69; N, 6.33; O, 10.84; Pd, 12.02. IR(400–4000 cmꢀ 1, KBr):
1600 HC = N, 1269 ArC-N, 3053 Ar-CH, 2918 Aliph-CH, 1716C = O,
1269C-O, 472 Pd-O, 592 Pd-N, 2848 symmetric CH2 stretching transi-
tion [27]. 1H NMR (DMSO‑d6): δ 8.70 ppm (CH = N), δ 1.23 ppm (N-
(CH2-CH3)2), δ 3.31 ppm (N-(CH2-CH3)2), δ 4.60 ppm (–CH2-CH2–), δ
6.11–8.26 ppm (m, Ar-CH and napht.–CH).
pellet in the scale of 400–4000 cmꢀ 1
.
2.2. Synthesis of ligands and Pd(II) complexes
L1: 2-(diethylamino) ethyl 4-((2-hydroxybenzylidene) amino) benzoate:
The synthesis of L1 was performed according to the literature [23]. A
procaine solution (0.236 g, 1 mmol) was dissolved properly in 30 mL
ethanol in two necked round bottom flask. The salicylaldehyde (0.122 g,
1 mmol) was then diluted in 20 mL ethanol to add into the flask drop by
drop. It was continued stirring for 3 h. The resulting yellow solid was
filtered off and respectively washed with 10 mL cold ethanol and cold
2.3. Cell culture
MKN-45 human gastric cancer cell lines were used in this study. Cells
were cultured in RPMI-1640 medium supplemented with 10% fetal
bovine serum FBS, 100 U/mL penicillin and 100 μg/mL streptomycin
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Inorganic Chemistry Communications 132 (2021) 108846
Fig. 1. The synthetic route of L1 by condensation with procaine and salicylaldehyde.
Fig. 2. The synthetic route of L2 by condensation with procaine and 2-hydroxy-1-naphthaldehyde.
2.4. Cell viability assay
The effects of procaine derivatives and their palladium complexes on
MKN-45 cells viability were determined using a tetrazolium-based
microplate assay with MTT, which was previously applied in our
study. [28]. Briefly, the MKN-45 cells were seeded into a 96-well plate at
a density of 1 × 104 cells/well in the 100 µL medium. After incubating
the cells for 24 h, the dilutions of procaine derivatives and its palladium
complexes at different doses (0.1–100 μg/mL) were added and incu-
bated for 24 h. The percentage of cell viability was measured on ELISA
reader (Biotek Co., USA) at a wavelength of 570 nm. The % cell viability
was calculated using the formula given below in Eq. (1). The images of
MKN-45 cells before and after treatment of procaine derivatives and its
palladium complexes were also assessed using an inverted microscope
attached to the camera system (Olympus NP40).
Fig. 3. The structural illustration of L1-Pd complex.
% Cell Viability= (OD test sample/OD control) X 100 (1)
2.5. Colony formation assay
The colony formation experiment was conducted according to the
literature [29,30]. IC50 doses of procaine Pd complexes were added and
incubated with MKN-45 cells for 24 h. After treatment, the medium
containing procaine derivatives and its palladium complexes was
removed and replaced with a pure medium. The medium was changed
every 3 days for 10 days until visible colonies were formed. The stained
cells were examined with an inverted microscope (Olympus CX40,
Japan) and an imaging system. The number of colonies in each well was
counted and analyzed [29,30].
2.6. Wound healing assay
Fig. 4. The structural illustration of L2-Pd complex.
The wound healing process was carried out according to the litera-
ture [31]. Cells were treated with procaine derivatives and its palladium
complexes for 24 h. After an incubation period of 24 h, the cells migrated
into the scratched area were photographed under a phase-contrast
inverted microscope (Olympus CX40, Japan). The distance that cells
had migrated into the cell-free space was measured by Image J software
(NIH, USA). The width of each migrated area was used to calculate the
and 2 mM L-glutamine. The cultures were incubated at 37˚C in a hu-
midified atmosphere with 5% CO2. The culture medium was replaced
with fresh medium every 2 days until reaching suitable confluency of
about 90%. All experiments were repeated multiple times.
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Inorganic Chemistry Communications 132 (2021) 108846
relative proportion of wounded at time zero [31].
3. Results and discussion
3.1. Chemical identification
2.7. Western blotting for protein expression
1
The H NMR spectra of L1 and L2 showed expected signals for the
imine group (CH = N) at 9.02 ppm for L1 and 9.68 ppm for L2 [36].
These peaks in the L1-Pd and L2-Pd complexes have changed to 8.32 ppm
and 8.70 ppm, respectively, as a result of coordination via the imine
nitrogen atoms [37]. Signals due the hydroxyl protons, observed at 6.10
ppm and 6.08 ppm for the free ligands, disappeared on complexation,
indicating deprotonation of the ligands. Peaks for the aliphatic and ar-
omatic protons were also shifted upon complexation (Supplementary
Material Figs. 1-8).
Bax, Bcl-2, DNMT1, DNMT3a and DNMT3b protein expression were
determined with western blotting. For this purpose, the procedures
worked before were followed [29,32,33]. The total proteins were
extracted from 5x106 cells that had been treated with procaine de-
rivatives and its palladium complexes. The protein concentrations of
lysates were determined by the bicinchoninic acid method (71285-
Merck Millipore, Germany) [34]. Equal amount of protein was separated
by 12% polyacrylamide gels and then transferred onto PVDF membranes
(sc-3723, Santa Cruz, USA). The membranes were blocked with 2.5%
BSA at 4˚C overnight and then incubated with specific primary antibodies
(Bax (sc-7480), Bcl-2 (sc-492), DNMT1(sc-271729), DNMT3a (sc-
373905), DNMT3b (sc-376043) and β-actin (sc-47778) from Santa Cruz
Biotechnology, USA. After washing with Tris-buffered saline with 0.1%
Tween-20 (TBST), the membranes were incubated with the corre-
sponding HRP-conjugated secondary antibodies in room temperature.
β-actin was used as a housekeeping control for normalization. The
expression levels of proteins were visualized with an imaging system
(SynGene G-BOX Chemi XRQ) and analyzed using the software.
The infrared spectra show vibrations for three significant groups:
imine (CH = N), metal–ligand (Pd-N, Pd-O) and hydroxyl (O-H). It was
found that CH = N stretching vibrations of the ligands have been shifted
to lower frequency in the complexes, as a consequence of coordination
between the Pd and the imine nitrogen. The newly observed peaks at
582 cmꢀ 1 and 592 cmꢀ 1 correspond to Pd-N stretching vibrations. The
absence of broad hydroxyl stretching (O-H) in the complexes, found at
around 3200 cmꢀ 1 and 3400 cmꢀ 1 in the ligands, confirms the coordi-
nation between the metal atom and deprotonated oxygen atom [38].
The Pd-O vibrations were found at 464 cmꢀ 1 and 472 cmꢀ 1 [37–43].
The surface morphology (SEM images) of L1-Pd and L2-Pd looks like
agglomerated particles (Fig. 5a) with stacked layers and small spherical
particles, respectively (Supplementary Material Fig. 9-10). The
elemental mapping images (Fig. 5a and Fig. 5b) of procaine Schiff-based
Pd complexes clearly showed that both of the newly obtained com-
pounds have Pd metals in their structure.
2.8. QPCR for gene expression
The total RNA was extracted from 5x106 MKN-45 cells as previously
described [35]. A total of 1
μg RNA was reverse transcribed as the
template for cDNA synthesis using high capacity cDNA Reverse Tran-
scription Kit (Applied Biosytem). Quantitative real-time PCR was per-
formed for Bax, Bcl-2 and GAPDH using primers. The primers sequence
was: Bax: 5′-GCCCTTTTGCTTCAGGGTTT-3′(forward),5′-TCCAATGTC-
CAGCCCATGAT-3′(reverse); Bcl-2: 5′-GACAGAAGATCATGCCGTCC-3′
(forward), 5′-GGTACCAATGGCACTTCAAG-3′(reverse); GAPDH: 5′-
3.2. Effects of procaine derivatives on cell viability
The cytotoxic efficiencies of the compounds were investigated
against MKN-45 gastric cancer cell lines and IC50 values were evaluated.
The 50% inhibitory concentrations (IC50) were determined after 24-hour
exposure to 0.1–100 µM doses of procaine derivatives and the palladium
complexes, and those concentrations (IC50) were used in subsequent
experiments. Treatment of MKN-45 gastric cancer cells with procaine
derivatives and the palladium complexes inhibited the proliferation of
the cells (Fig. 6). By looking at the morphological images of the MKN-45
cells (Fig. 6a), it was observed that the cells treated with procaine, L1, L1-
Pd, L2, and L2-Pd demonstrated the signs of apoptosis, with the blebbing
of the plasma membrane and reduction in cell size. On the other hand,
especially the L1-Pd and L2-Pd compounds at IC50 doses, the cells moved
away from each other and floated in the medium by rising from the well
surface [44,45]. The IC50 concentrations of L1-Pd and L2-Pd complexes
against the MKN-45 cells were calculated as 10.21 ± 2.21 and 10.79 ±
1.48, respectively. Tanaka et al. reported IC50 doses of glycoconjugated
palladium complexes on MKN-45 and MKN-28 gastric cancer cells as
61.2 and 78.9 µM [46]. Compared with this study, all the synthesized
procaine compounds exhibited a greater cytotoxic effect on MKN-45
cells than procaine, while palladium complexes exhibited a better
cytotoxicity than the ligands. As a study similar to our results, Halby
et al. [50] reported that they synthesized the new procainamide de-
rivatives and some of these compounds exhibited cytotoxic effects on
DU-145 prostate cancer cells and HCT-116 colon cancer cells [50].
Furthermore, many metal-based drugs undergo redox processes un-
like to the most of organic chemotherapeutics in the cellular environ-
ment. These cases significantly affect and modify the physicochemical
properties of such complexes due to geometry, charge, and reactivity.
Hence, the knowledge of the redox potential can be crucial for the un-
derstanding of the mode of action underlying the anticancer activity of
metal compounds [47,48]. It has been reported that, compared to meta-
and para-counterparts, the presence of functional groups in the ortho
position of ligand attached to palladium may increase cytotoxic ability.
In the L1-Pd and L2-Pd, palladium is attached to the hydroxyl group in
AGGGCTGCTTTTAACTCTGT-3′(forward),
5′-CCCCACTTGATTTTG-
GAGGA-3′(reverse). One hundred nanograms of cDNA were amplified
using Sybr Green PCR Master Mix (Applied Biosytem) on the ABI Ste-
pOne Plus detection system, programmed for 95 ◦C for 10 min, then 40
cycles of: 95 ◦C for 15 s, 60 ◦C for 1 min. The amplification results were
analyzed using StepOne Software v2.3 (Applied Biosystems, Foster City,
CA) and the genes of interest were normalized to the corresponding
GAPDH results. Data were expressed as fold induction relative to the
control.
2.9. DNMT activity assay
The impact of the procaine derivatives and their palladium com-
plexes on the methylation level was evaluated in MKN-45 gastric cancer
cells using a colorimetric DNMT activity assay. Cells (1x106 cells/well)
were seeded into a 60 mm culture dish and were allowed to grow
overnight. Then, cells were treated with IC50 doses of procaine de-
rivatives and its palladium complexes during 24 h. After incubating the
cells for 24 h, cells were washed with 1x PBS solution and collected via
trypsin. Nuclear extracts from cells were isolated using a commercial kit
(Nuclear Extraction Kit 2900, Millipore). These extracts were then used
for DNMT activity measurement. Experiments were carried out ac-
cording to the commercial kit (DNMT Activity Quantification Kit
ab113467, Abcam).
2.10. Statistical analysis
Data from three independent experiments are presented as mean ±
SD. Differences between groups were analyzed by t-test of results. Sta-
tistical analysis was performed using GraphPad Prism version 5.0 soft-
ware. Statistical significance is defined as follows: *, p ≤ 0.05; **, p ≤
0.01; ***, p ≤ 0.001.
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Inorganic Chemistry Communications 132 (2021) 108846
Fig. 5. The EDX pictograms of obtained complexes.
Fig. 6. The effect of procaine derivatives and the palladium complexes in MKN-45 gastric cancer cells. a) Cell morphological changes after treatment with IC50 doses
of procaine derivatives and its palladium complexes in MKN-45 cell lines for 24 h. b) Graphical illustration of % cell survival rate of MKN-45 cells after treatment with
IC50 doses of procaine derivatives and its palladium complexes.
Fig. 7. Effects of procaine derivatives and its palladium complexes on cell migration in MKN-45 cells. a) Colony formation assay in MKN-45 cells. Cells were treated
with IC50 doses of procaine derivatives and their palladium complexes and were cultured for 14 days and were stained with crystal violet. Colonies showed as
overview images (up) and detailed images.
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Inorganic Chemistry Communications 132 (2021) 108846
Fig. 8. Effects of procaine derivatives and its palladium complexes on apoptotic proteins in MKN-45 gastric cancer cells. a) Western blot analysis of expression levels
of Bax and Bcl-2 proteins in IC50 doses of procaine, ligands and palladium complexes in MKN-45 cells. Each protein band was normalized to the intensity of β-actin
used. b) Western blot densitometry analysis of the ratio of Bax/β-actin protein expression levels in MKN-45 cells (*p < 0.05, ***p < 0.001, compared to control cells)
for the three biological replicates within each group. c) Western blot densitometry analysis of the ratio of Bcl-2/β-actin protein expression levels in MKN-45 cells. (*p
< 0.05, compared to control cells) for the three biological replicates within each group. d-e) Effects of procaine, ligands and palladium complexes on Bax and Bcl-2
gene expression in MKN-45 cells (*p < 0.05 and ***p < 0.001 compared to control in PC3 cells).
Fig. 9. Methylation status of MKN-45 cells after exposure to procaine derivatives and its palladium complexes using western blot and methylation analysis of
DNMT1, 3a/3b. A) Western blot bands of expression levels of DNMT1, 3a/b proteins in procaine derivatives and its palladium complexes (10 μM) for 24 h treatment
in MKN-45 cells. Each protein band was normalized to the intensity of β-actin used. B) Effects of procaine, ligands and palladium complexes on DNMT enzyme
activity (10 μM) for 24 h treatment in MKN-45 cells C) Western blot densitometry analysis of the ratio of DNMT1/β-actin protein expression levels in MKN-45 cells.
(*p < 0.05, **p < 0.01, compare to control cells) for the three biological replicates within each group. D) Western blot densitometry analysis of the ratio of DNMT3a/
β-actin protein expression levels in MKN-45 cells. (*p < 0.05, **p < 0.01, ***p < 0.001 compared to control cells) for the three biological replicates within each
group. E) Western blot densitometry analysis of the ratio of DNMT3b/β-actin protein expression levels in MKN-45 cells. (**p < 0.01 compare to control cells) for the
three biological replicates within each group.
the ortho position from salicylaldehyde with a coordination bond. For
this reason, palladium complexes exhibited better cytotoxic effects in
MKN45 cells than ligands.
3.3. Clonogenic and wound healing assay
First approach was the examination of the effects of procaine de-
rivatives and their palladium complexes treatment on colony formation
ability against MKN-45 cells. As shown Fig. 7a, the number of colonies
are reduced by the exposure of MKN-45 cells with procaine, L1, L1-Pd, L2
and L2-Pd,. Especially in IC50 doses of L1-Pd and L2-Pd, colony devel-
opment drastically decreased by 40% and 35%, respectively, compared
The cytotoxicity parameters for MKN-45 cells, in terms of IC50 ob-
tained after 24 h of exposure, are listed in Table 1.
6

S. Pas¸a et al.
Inorganic Chemistry Communications 132 (2021) 108846
activities are detected and activity increase are obtained by the treat-
ment of the L1 and L2 compared to control (Fig. 9b). The effects of
procaine derivatives and the palladium complexes on the levels of
DNMT protein expression were performed via western blotting using
specific antibodies against DNMT1, DNMT3a, and DNMT3b (Fig. 9a).
Our findings showed that the expression level of DNMT1, DNMT3a and
DNMT3b was decreased for the procaine, L1-Pd and L2-Pd while the
expression level of DNMT1, DNMT3a and DNMT3b was increased for
the L1 and L2 after 24 h of treatment (Fig. 9c, 9d and 9e). 3a and 3b have
been shown in literature for various types of cancer for overexpression of
DNMT1[57,58]. In light of this information, new inhibitors that reduce
the methylation activity of DNMTs are being developed in cancer
treatment [7]. For this aim, Pechalrieu et al. [50] synthesized some of
the novel 3-halo-3-nitroflavones and stated that compounds 3a and 3b
provide inhibition on DNMT and inhibition level is obtained at a
micromolar concentration by the treatment of 3a. In a similar study,
Pellerito et al. [59] synthesized the dibutyltin (IV) complex of caffeic
acid and demonstrated that these compounds reduce protein levels of
DNMT1 in MDA-MB 231 and HCT116 cells by western blot techniques
[59]. Similar in this study, especially procaine, L1-Pd and L2-Pd signif-
icantly reduced the protein expression levels of DNMT-1,3a/3b in our
study. It also shows that DNA methylation-mediated regulation of genes
involved in apoptosis may be a different mechanism in cancer by
escaping tumor cells from apoptosis. These changes are either hypo-
methylation to reactivate anti-apoptotic genes or hypermethylation to
suppress pro-apoptotic genes [60]. Genes involved in mitochondrial
apoptosis in glioblastoma cells are associated with DNA methylation and
involved in metastasis in cancer [61].
Table 1
The IC50 values of MKN-45 cells treated with procaine
derivatives and the Palladium complexes.
Compounds
IC50 Values (µM)
Procaine
L1
35.06 ± 3.01
13.19 ± 1.07
10.21 ± 2.21
13.15 ± 1.06
10.79 ± 1.48
L1-Pd
L2
L2-Pd
to the control group (Fig. 7b). Similar to colony formation assay,
treatment of MKN-45 cells with procaine, L1 L1-Pd, L2 and L2-Pd
,
resulted in a significant decrease in cell motility in the scratch area
compared to control after 24 h of scratch formation (Fig. 7c). In Fig. 7d,
L1-Pd and L2-Pd respectively showed 63% and %56 inhibition of tumor
cell motility through wound healing in MKN-45 cells. The data sug-
gested that procaine, L1 L1-Pd, L2, and L2-Pd diminish the metastasis
,
effect of MKN-45 gastric cancer cells and highly effective in inhibiting
migration. Our results indicate that the novel synthesized procaine de-
rivatives and their palladium complexes had an inhibitory effect on
colony formation and wound healing on MKN-45 cells which would
have the effect on reducing metastasis of cancers such as gastric cancer.
3.4. Apoptotic proteins and genes expression
Apoptosis is an efficient pathway for the control of cancer cells and
there are two different pathways for this to occur, based on the initiator
signal occurring within the cells. The extrinsic pathway is initiated in the
cell membrane, through death receptors, while the intrinsic pathway is
controlled through the Bcl-2 protein family and affects the permeability
of the mitochondrial outer membrane. For apoptosis to occur, it is
important that drug molecules are able to enter the cell and activate the
mitochondrial pathway [49]. Measurement of the expression levels of
Bax and Bcl-2 are most commonly used as apoptosis markers. Increased
levels of Bcl-2 expression in cancer cells prevent apoptosis and lead to
tumor progression, while a decrease in Bcl-2 and an increase in Bax
expression induce cell death and eliminate tumor cells [50–52]. In order
to determine apoptotic effects of procaine, ligands, and palladium
complexes on MKN-45 cells, we examined to expression levels of anti-
apoptotic protein Bcl-2 and pro-apoptotic protein Bax (Fig. 8a). The
obtained data showed that for the procaine, L1-Pd, and L2-Pd the
expression level of pro-apoptotic protein Bax was increased, while the
expression of anti-apoptotic protein Bcl-2 was dramatically reduced
after 24 h of treatment. However, the levels of Bcl-2 expression in L1 and
L2 treated MKN-45 cells increased as compared to the control group
(Fig. 8b and Fig. 8c). On the other hand, by looking at gene expression
data, the findings indicated that the expression levels of Bax (pro-
apoptotic gene) were increased significantly (Fig. 8d) in MKN-45 cells
treated with L1-Pd and L2-Pd while the expression levels of Bcl-2 (anti-
apoptotic gene) were notably decreased relative to the control (Fig. 8e).
The results clearly showed that especially L1-Pd and L2-Pd complexes
induced cell death by triggering apoptotic pathways. In a similar study,
Valentini et al. stated that palladium complexes of Curcumin activate
apoptosis by increasing Bax levels and decreasing Bcl-2 levels in LNCaP,
PC3 and DU145 prostate cancer cells [53].
4. Conclusion
In this study, the DNMT inhibitory potential of newly synthesized
procaine derivatives and their palladium complexes (L1, L1-Pd, L2, and
L2-Pd) was investigated on MKN-45 cells. Consequently, in this study,
MKN-45 cells treated with synthesized L1-Pd and L2-Pd compounds were
stimulated to apoptosis by suppressing both anti-apoptotic Bcl-2
expression and inducing pro-apoptotic Bax expression. Furthermore, it
was also found that the tested compounds showed an inhibitory effect on
colony formation and wound healing. In addition, since the palladium
complexes of the synthesized ligands provide inhibition on DNMT1, 3a/
3b enzymes, the activation of tumor suppressor genes was triggered by
preventing DNA methylation. Therefore, this study highlights the
importance of discovering and developing new drug derivatives used in
the treatment of gastric cancer and sheds light on new research.
CRediT authorship contribution statement
Salih Pas¸a: Conceptualization, Investigation, Methodology, Formal
analysis. Omer Erdogan: Investigation, Software, Validation, Data
curation. Ozge Cevik: Conceptualization, Resources, Investigation,
Writing – original draft, Writing – review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
3.5. DNMT activity
Acknowledgements
One of the current approaches in cancer treatment is the develop-
ment of DNMT inhibitors. Recently, scientists have studied on the
development of DNMT inhibitors [50,54,55]. DNMT1, DNMT3a and
DNMT3b are the main enzymes responsible for methylation in mammals
[56]. In this regard, we observed DNMT cellular enzymatic activity at
the IC50 concentration of the test compounds. The examination of DNMT
enzyme activity values revealed that in the case of L1-Pd and L2-Pd lower
We thank ADU-BILTEM for providing laboratory and facility support
for our research. This work was supported by the Aydin Adnan Menderes
University Scientific Research Foundation with the project number TPF-
20012 to Dr. Ozge Cevik.
7

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Inorganic Chemistry Communications 132 (2021) 108846
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assignments for a series of n-alkyl carboxylic acids, J Chem Phys 70 (1979)
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Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
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