Novel thiosemicarbazone derivative 17B interferes with the cell cycle progression and induce apoptosis through modulating downstream signaling pathways

Article abstract of DOI:10.1016/j.genrep.2019.100578

Thiosemicarbazones (TSCs) are interesting group of chemical compounds that received significant levels of attention due their wide range of pharmacological effects including antibacterial, antiviral, and especially antitumor activities. Several thiosemicarbazone derivatives have been extensively reported recently with their antitumor properties but designing and developing novel thiosemicarbazone derivatives with more potent chemotherapeutic activities is of great interest for cancer future cancer therapy. Thus, here we aimed to demonstrate as yet undetermined anti-cancer properties of novel thiosemicarbazone derivative 17B. Viability of cells was determined using MTT assay and LDH activities were analyzed using lactate dehydrogenase activity assay. Apoptosis were assayed using Annexin V-FITC and PI double staining method and cell cycle analysis was achieved by using PI staining with fluorescence-activated cell sorting and migration capacities of cells were determined by wound healing assay. As a result, 17B limited cell viability and showed cytotoxic effects in a dose-dependent manner in A549, MCF7 and U2OS cells. In addition, it inhibited progression through cell cycle by interfering with the G1/S transition and triggered apoptosis by modulating expression levels of pro-apoptotic and anti-apoptotic mediators in MCF7 and U2OS cells. Also, 17B significantly impaired the migration of cancer cells and delayed wound healing in all cells. Consequently, findings of the present study have strongly indicated that 17B might be a novel anti-cancer agent for the treatment of breast cancer and osteosarcoma but not for lung cancer. Our results have provided mechanistic insights into anti-cancer properties of a novel thiosemicarbazone derivative 17B.

Full text of DOI:10.1016/j.genrep.2019.100578

Journal Pre-proof
Novel thiosemicarbazone derivative 17B interferes with the cell
cycle progression and induce apoptosis through modulating
downstream signaling pathways
Esra Bozgeyik, Demet Tasdemir-Kahraman, Kaifee Arman,
Ibrahim Bozgeyik, Aysegül Karakucuk-Iyidogan, Ecir Ali
Cakmak
PII:
S2452-0144(19)30220-1
DOI:
https://doi.org/10.1016/j.genrep.2019.100578
GENREP 100578
Reference:
To appear in:
Gene Reports
Received date:
Revised date:
Accepted date:
21 August 2019
29 November 2019
10 December 2019
Please cite this article as: E. Bozgeyik, D. Tasdemir-Kahraman, K. Arman, et al., Novel
thiosemicarbazone derivative 17B interferes with the cell cycle progression and induce
apoptosis through modulating downstream signaling pathways, Gene Reports (2019),
https://doi.org/10.1016/j.genrep.2019.100578
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Novel thiosemicarbazone derivative 17B interferes with the cell cycle progression and
induce apoptosis through modulating downstream signaling pathways
Esra Bozgeyik*,a, Demet Tasdemir-Kahramanb, Kaifee Armanc,d, Ibrahim Bozgeyike, Aysegül
Karakucuk-Iyidoganf, Ecir Ali Cakmakg
aNamık Kemal University, Faculty of Medicine, Department of Medical Biology, Tekirdag,
Turkey.
bGaziantep University, Faculty of Medicine, Biochemistry, Gaziantep, Turkey
cInstitut de recherches cliniques de Montreal (IRCM), Montreal, H2W1R7, QC, Canada
dDivision of Experimental Medicine, McGill University, Montreal, QC, Canada
eAdiyaman University, Faculty of Medicine, Department of Medical Biology, Adiyaman,
Turkey.
fGaziantep University, Faculty of Arts and Science, Department of Chemistry, Gaziantep,
Turkey.
gSakarya University, Faculty of Medicine, Department of Medical Biology, Sakarya, Turkey.
*Corresponding Author:
Esra BOZGEYİK
Tekirdag Namik Kemal University
Faculty of Medicine, Department of Medical Biology,
Tekirdag, Turkey
e-mail: gyk.esra@gmail.com, ebozgeyik@nku.edu.tr
Phone: +90 282 250 5942
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ABSTRACT
Thiosemicarbazones (TSCs) are interesting group of chemical compounds that received
significant levels of attention due their wide range of pharmacological effects including
antibacterial, antiviral, and especially antitumor activities. Several thiosemicarbazone
derivatives have been extensively reported recently with their antitumor properties but
designing and developing novel thiosemicarbazone derivatives with more potent
chemotherapeutic activities is of great interest for cancer future cancer therapy. Thus, here we
aimed to demonstrate as yet undetermined anti-cancer properties of novel thiosemicarbazone
derivative 17B. Viability of cells was determined using MTT assay and LDH activities were
analyzed using lactate dehydrogenase activity assay. Apoptosis were assayed using Annexin
V-FITC and PI double staining method and cell cycle analysis was achieved by using PI
staining with fluorescence-activated cell sorting and migration capacities of cells were
determined by wound healing assay. As a result, 17B limited cell viability and showed
cytotoxic effects in a dose-dependent manner in A549, MCF7 and U2OS cells. In addition, it
inhibited progression through cell cycle by interfering with the G1/S transition and triggered
apoptosis by modulating expression levels of pro-apoptotic and anti-apoptotic mediators in
MCF7 and U2OS cells. Also, 17B significantly impaired the migration of cancer cells and
delayed wound healing in all cells. Consequently, findings of the present study have strongly
indicated that 17B might be a novel anti-cancer agent for the treatment of breast cancer and
osteosarcoma but not for lung cancer. Our results have provided mechanistic insights into
anti-cancer properties of a novel thiosemicarbazone derivative 17B.
Keywords: Anti-cancer agent, Cancer, Cytotoxicity, Thiosemicarbazone Derivative, 17B
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INTRODUCTION
According to recent findings of Global Cancer Statistics, it was estimated that 18.1 million
new cancer cases and 9.6 million cancer related deaths will occur in 2018 (Bray et al., 2018).
Also, lung cancer was identified to be the most frequently encountered type of cancer with a
ratio of 11.6 % (among all cases) and responsible from the 18.4 % of the all cancer-related
deaths (Bray et al., 2018). Female breast cancer was identified to be the second mostly
diagnosed cancer after lung cancer. Additionally, it was shown that the most commonly
diagnosed cancer and the leading cause of cancer death varies widely between countries and
within each country depending on the socioeconomic status in both genders (Bray et al.,
2018). Moreover, although recent progress in cancer therapy is promising, it is not sufficient
enough to fight against cancer. Besides, existing chemotherapeutic agents are not very
effective and they have adverse side effects that are very difficult to cope with. Also, patients
develop resistance to several chemotherapeutics (Kara et al., 2015). Accordingly, developing
novel therapeutic agents with strong, selective anticancer effects and low toxicity are of great
interest
Thiosemicarbazones (TSCs) are interesting group of chemical compounds that display a
diverse range of biological activities including antitumor, antibacterial, antifungal, anti-
amoebic, antiviral and analgesic activities (Afrasiabi et al., 2004; Patel et al., 2013). Also, it
was well-reported that thiosemicarbazone derivatives are involved in the inhibition of
ribonucleotide reductase, which is a rate-limiting enzyme in DNA synthesis, reducing
ribonucleotides to deoxyribonucleotides. Thus, they interfere with the nucleic acid synthesis
in cells (Li et al., 2001). Although mechanism of ribonucleotide reductase inhibition was not
ascertained at first, now we know that metal chelating activities of thiosemicarbazones are the
chief factor in their antineoplastic effects (Kalinowski et al., 2009). Furthermore, recent
studies have indicated that increased activity of ribonucleotide reductase leads to the
destruction of hemostatic balance of normal cells and speeds up malignant transformation (Yu
et al., 2009). The thiosemicarbazone derivative compounds, which are ribonucleotide
reductase antagonists, exhibit antineoplastic activity by inhibiting DNA synthesis and repair
(Alvero et al., 2006). Another activity of thiosemicarbazone derivatives is to render topo-II
DNA complex unstable by alkylating thiol residues, showing anticancer activities (Liu et al.,
1992; Ferrari et al., 1999). These substances bind to DNA by producing an electrophilic alkyl
cation that can react with free electron pairs or nucleophilic centers carrying negative charges.
Thus, making covalent bond with DNA and blocking DNA replication. This produces DNA
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damage and if the damage is not repaired, the cell cycle is blocked and the cell dies (Liu et al.,
1992; Ferrari et al., 1999).
Although antitumor properties and mechanism of the action of several thiosemicarbazone
derivatives have been extensively reported, designing and developing novel
thiosemicarbazone derivatives with high potential to be used in clinic is of great interest.
Accordingly in the present study, we aimed to demonstrate the anticancer activities of a
thiosemicarbazone derivative, 17b. Previously, we have shown that 17b have significant anti-
proliferative activities in HGC-27 human gastric carcinoma cells in contrast to tested
reference antineoplastic agent, Paclitaxel as reported in (Tasdemir et al., 2015). However, the
effect of 17b on proliferation, migration and apoptosis of cancer cells remained muchly
elusive. Therefore, here we demonstrate significant anticancer activity of a thiosemicarbazone
derivative 17b.
MATERIAL AND METHODS
Synthesis of 17b and preparation of solutions
Synthesis
of
17b
((S)-(+)-2-[(4-piperidin-1-il)benzilidin])-N-(1-
feniletil)hidrazinkarbotiyoamid) (C21H26N4S (366.52 g/mol)) was achieved as previously
described (Karaküçük-İyidoğan et al., 2011; Tasdemir et al., 2015). Briefly, 1 mmol of chiral
phenylethylamine was slowly dissolved in 100 ml of chloroform and thiophosgene was added
dropwise in a 1: 1 ratio. 3 mmol of sodium hydroxide was dissolved in very small amount of
water and added to the reaction medium. After stirring the reaction at 0 ° C overnight, the
resulting product was extracted with distilled water and the organic phase was collected and
dried with sodium sulfate and purified by column chromatography. The resulting 1 mmol
chiral isothiocyanate derivative compound was dissolved in diethylether and 1 mmol
hydrazine monohydrate dissolved in the same solvent was added dropwise. The reaction
mixture was allowed to stir at room temperature overnight. The resulting viscous product was
washed with petroleum ether. 1.53 mmol of 4- (piperidin-4-yl) benzaldehyde was dissolved in
20 mL of methanol and added on 1.53 mmol (S) - (+) - N- [phenylethyl]
hydrazinecarbotioamide dissolved in 10 mL of hot methanol. The precipitated solid material
was crystallized in ethanol. Synthesized compound (0.40 g, yield; 72 %) was yellow in color
and melting point was 79-80 ºC.
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Cell culture
In the cell culture experiments, A549 (ATCC CCL-185), MCF-7 (ATCC HTB-22) U2-OS
(ATCC HTB-96) cancer cell lines were commercially obtained from ATCC (American Type
Culture Collection). Cells were maintained using Dulbecco’s modified eagle’s medium
(DMEM) containing 10 % fetal calf serum (FCS) (all from Sigma-Aldrich, MO, USA) in a
humidified carbon dioxide incubator (Thermo Fisher Scientific Inc.,Wilmington, USA)
adjusted to following conditions; 37°C and 5 % CO2. When the cell density in the culture
dishes was about 80-90 %, 0.25 % Tripsin-EDTA solution was used to detach cell from the
culture plates.
Cell viability assay
To analyze cell viability of cancer cells, colorimetric MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-
Diphenyltetrazolium Bromide) (from Sigma-Aldrich, MO, USA) assay was used. MTT is a
staining technique based on the reduction of mitochondrial enzymes by MTT substance. In
this assay, water-soluble MTT dye is converted to insoluble purple formazan particles within
cells. Briefly, cells were seeded to 24-well cell culture plates and incubated until they reach
80-90 % confluency. Subsequently, they were incubated with different concentrations (25, 50
,100 and 200 µM) of 17b for a period of 24 hours. After incubation period, wells were rinsed
with HBSS (Hank's Balanced Salt Solution) and treated with 1 mg/ml MTT solution for a
period of 45-60 minutes at 37 ºC. Following incubation, MTT was removed and the formazan
particles were solubilized with 500 µl DMSO and corresponding concentrations were
determined by using Thermo Scientific™ Multiskan™ GO Microplate Spectrophotometer at
570 nm.
LDH activity assay
For the measurements of the LDH activity in living cells, Lactate Dehydrogenase Activity
Assay Kit (#MAK066, Sigma-Aldrich) were used and the manufacturer's recommendations
were followed. Briefly, prior to MTT assay, supernatants were saved for the LDH activity
assay. Subsequently, for each sample, a total of 50 µl of mixture was prepared with 48 µl of
LDH assay buffer and 2 µl LDH Substrate Mix. Then, 50 µl of samples were loaded on 96-
well plates and mixed with 50 µl of the prepared reaction mixture. The first reading was
performed at 450 nm and readings were repeated every 5 min. Plates were kept at 37 ºC
during experiments.
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Apoptosis assay
Induction of apoptosis in cells treated with 17b was analyzed by Annexin V-FITC and PI
double staining method by using with Annexin V-FITC Early Apoptosis Detection Kit (Cell
Signaling Technology, #6592) by following the instructions of the manufacturer. Briefly, cells
were seeded to 6-well culture plates with a concentration of 1x106 cells per well and
incubated overnight and subsequently treated with the 17b. Following 24 hours incubation,
cells were detached using Trypsin-EDTA (0.25%) (from Sigma), centrifugated and rinsed
with cold HBSS. Cells were later incubated with 12.5 µl PI (propidium iodide) and 1 µl
Annexin V for 15 minutes and 140 µl 1X Annexin V Binding Buffer were added prior to
analysis. Positive cells were detected and quantified by fluorescence-activated cell sorting
(FACS) analysis by using Gallios Flow Cytometer (Beckman Coulter).
Cell cycle analysis
For the analysis of cell cycle, cells were seeded to 6-well culture plates with a concentration
of 1x106 cells per well and incubated overnight and subsequently treated with the 17b for 24
hours. After incubation, cells were trypsinized with Trypsin-EDTA and washed with HBSS
and subsequently fixed with 70 % cold ethanol solution. Ethanol was removed following
fixation and cells were then washed with PBS and centrifugated at 1500 rpm for 5 minutes.
Pellet were later resuspended in 200 µl Guava® Cell Cycle Reagent (Guava Technologies,
#4500-0220) and incubated in a dark place for 30 minutes at room temperature. Following
incubation, analysis was performed in Gallios Flow Cytometer (Beckman Coulter).
Real-time PCR
Isolation of total RNA was achieved by using High Pure RNA Isolation Kit (#11828665001)
from Roche with an additional RNase treatment step. Subsequent synthesis of cDNA from
obtained RNA samples were achieved by using Maxima H Minus First Strand cDNA
Synthesis Kit (#K1681) according to the recommendations of the manufacturer. Sequences
and features of the primer sequences used in quantitative amplification reactions were
provided in Supplementary Table 1. qPCR were held in Rotor Gene Q (Qiagen Sample &
Assay Technologies, Germany) instrument by using Maxima SYBR Green/ROX qPCR
Master Mix (#K0221) recommendations of the manufacturer were followed.
Wound healing assay
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For the analysis of wound closure, cells were seeded to 6-well culture plates and allowed to
form cell monolayer. Subsequently, cells were incubated with the 17b for 24 hours and rinsed
with HBSS following incubation. Wounds were then generated using 100 µl pipette tips and
washed twice with HBSS to eliminate cell fractionations and first images recorded under light
microscope. An image was taken every 24 hours until wounds are closed completely. The
gaps were then measured using ImageJ software.
RESULTS
17B restricts cell viability and exerts cytotoxicity in a dose-dependent manner
To observe the inhibitory effects of 17b on viability of A549, MCF7 and U2OS cancer cells
and determine the inhibitory concentrations, we used MTT assay in which living cells
transform the water-soluble MTT to insoluble purple formazan particles by mitochondrial
succinate dehydrogenase as a function of redox potential. Consistent with our previous
observations (Tasdemir et al., 2015), treatment with 17b led to a significant inhibition of cell
viability in a dose-dependent manner in all three cell lines. Inhibitory concentrations of 17b
were determined as 99.8 µM, 213 µM and 203.7 µM in A549, MCF7 and U2OS cells,
respectively. DMSO treated group was considered as a control group in comparisons.
Furthermore, since lactate dehydrogenase (LDH) is an oxidative enzyme that catalyzes the
interconversion of pyruvate and lactate and released as a result cell damage, LDH activity
assay provides information about the cellular toxicity in living cells. Accordingly, we further
performed LDH assay to measure cellular toxicity upon 17B treatments. In consistent with the
anti-proliferative activity of 17B, a dose-dependent elevation of cellular toxicity was observed
in cells treated with 17B. Particularly, LDH activities in A549, MCF7 and U2OS cells treated
with 17B were 26.23±15.84, 111.3±25.02 and 116.1±22.43, respectively.
17B induces apoptosis and activates caspase-mediated killing
Given that 17B is a novel anti-proliferative agent in A549, MCF7 and U2OS cells, we further
wondered whether it induces cellular apoptosis and might be involved in the cellular apoptotic
pathways in these cells. To elucidate whether 17B triggers apoptosis in A549, MCF7 and
U2OS cells, we used Annexin V-FITC and propidium iodide (PI) double staining with
fluorescence-activated cell sorting (FACS) analysis in which translocation of
phosphatidylserine was monitored by the help of recombinant annexin V conjugated to green-
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fluorescent FITC dye and dead cells using PI. Consistent with the anti-proliferative and
cytotoxic activities, 17B significantly triggered cellular apoptosis of MCF7 and U2OS cancer
cells as presented in Figure 3. Although apoptosis is also triggered in A549 cells treated with
17B, this change was not statistically significant. Percentages of cells undergoing apoptosis
were determined to be 9.40±2.23, 78.25±2.34 and 40.84±1.23 in A549, MCF7 and U2OS
cells, respectively. We further wondered whether 17B-induced apoptosis is mediated by the
transcriptional inhibition of anti-apoptotic members and activation pro-apoptotic members. To
test this possibility, expression levels of BCL2, BCLXL, BAX, CASP3 and CASP9 levels
were determined by qPCR. Quantitative analysis of 17B-treated cells demonstrated that 17B-
triggered apoptosis is mediated by the transcriptional activation of pro-apoptotic BAX,
CASP3 and CASP9 and inhibition of anti-apoptotic BCL2 and BCLXL. Particularly, while
the expression levels of BCLXL was significantly downregulated in MCF7 and U2OS cells in
a time-dependent manner, it was not significantly altered in A549 cells treated with 17B. In
contrast, BCL2 remained unchanged in MCF7 and U2OS cells, but significantly altered in
A549 cells treated with 17B in a time-dependent manner. Furthermore, we also tested the
activation of pro-apoptotic BAX and caspase-dependent activation of apoptosis in cells
treated with 17B. Significant transcriptional activation of BAX was observed in all three cell
lines. Consistent with the activation of BAX, expression levels of caspase 3 and 9 was also
found to be markedly increased in MCF7 breast cancer cells treated with 17B. Also,
expression levels of caspase 3 and 9 was found to be slightly increased in A549 and U2OS
cells, yet these changes was statistically insignificant. These findings further supported to
notion that 17B targets members of the apoptotic machinery to trigger apoptosis of cancer
cells.
17B impairs progression through cell cycle and modulates regulators of cell cycle
Our results thus far have suggested that 17B might induce cell cycle arrest. To elucidate this
possibility we further performed cell cycle analysis to observe the effect of 17B in cell cycle
arrest by PI staining with fluorescence-activated cell sorting (FACS) analysis in which cells
can be quantified according to their DNA content which reflects cell cycle status (G0/G1, S,
and G2/M). Consistent with its apoptotic activity, 17B significantly induced accumulation of
cells in the G0/G1 phase in MCF7 and U2OS cells, indicating that 17B-mediated killing of
cancer cells might be result of DNA damage in these cells. Also, as in the case of apoptosis,
17B did no significant effect to cell cycle arrest in A549 lung cancer cells, further suggesting
quite resistance of these cells to 17B-mediated killing. Percentages of cells accumulated in the
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G0/G1 phase of cell cycle was determined to be 66.67±2.21, 73.56±2.20 and 80.61±2.2 in
A549, MCF7 and U2OS cells treated with 17B, respectively. We further wondered whether
17B-induced cell cycle arrest is a result of the modulation of genes involved in the regulation
of cell cycle. To test this possibility, expression levels of CDKN1B, CDKN2B, P53 and
CDK2 levels were determined by the help of qPCR. Confirming the findings obtained in the
cell cycle analysis, CDKN2B expression levels were markedly elevated as a result of 17B
treatments in MCF7 and U2OS cells in a time-dependent manner. P53 and CDKN1B showed
a significant increase only in MCF7 cells in a time-dependent manner, indicating the
activation of DNA damage response and irreversibly DNA damage in these cells. Also,
although differentially expressed in in MCF7 and U2OS cells, expression changes of CDK2
was not statistically significant. In contrast to these findings, consistent with the results of the
apoptosis and cell cycle analysis, we obtained controversial findings in A549 cells. While the
expression levels of CDK2 was significantly elevated, CDK2B expression was significantly
reduced in these cells. These results further suggest that 17B might not be an anti-proliferative
agent specific for lung cancer cells. In contrast, it specifically and efficiently targets to
eliminate breast cancer and osteosarcoma cells through modulating cellular signaling
pathways to induce cell cycle arrest and apoptosis.
17B interferes with the migration of cancer cells
To further reveal the mechanisms of anti-tumorigenic activity of 17B, we wondered whether
17B interferes with the migration of cancer cells and tested migration capacity of cancer cells
treated with 17B by using wound healing assay method in which migration of cells are
monitored. We found that 17B significantly reduced the migration capacities of cancer cells.
In particular, while the wounds in SF and DMSO treated groups were healed at end of the
third day, they were closed at the end of the fourth day in 17B treated group in A549 cells.
Consistently, in U2-OS cells, while the wounds were completely closed at the end of 24 hours
in control groups, they were closed after 72 hours in the 17b group. Similarly, in MCF7 cells,
while the wounds were completely closed at the end of fourth day in control groups, they
were closed after 9 days in the 17b group. These findings have indicated that 17B interferes
with the migratory capacities of cancer cells by restricting their mobility.
DISCUSSION
Because thiosemicarbazones are a well-known class of amine derivatives that have a wide
range of pharmacological effects including antibacterial, antiviral, and antitumor activities,
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they have gained considerable attention in pharmaceutical industry (da Silva et al., 2010). The
mechanisms in which thiosemicarbazone derivatives exert their antitumorigenic properties
have been linked to various biological activities including inhibition of ribonucleotide
reductase, generation of reactive oxygen species (ROS), inhibition of cell cycle, chelation of
metal ions, modulation of cellular signaling pathways regulating cell proliferation and death.
Strikingly, several phase I and phase II clinical trials were initiated for thiosemicarbazone
derivatives. Findings of the clinical trial studies have shown that thiosemicarbazones alone or
in combination with other anti-neoplastic agents such as cisplatin and gemcitabine, interferes
with the development and progression of several types of tumors (Mrozek-Wilczkiewicz et
al., 2019a).
Accordingly here, we demonstrate as yet undetermined anti-cancer properties of a novel
thiosemicarbazone derivative 17B. To explore the mechanism of 17B action on cancer cells,
we analyzed the effects of 17B on cell viability, apoptosis, cell cycle and migration capacities
of cancer cells. Strikingly, 17B restricted viability of A549, MCF7 and U2OS cells and
exerted cytotoxicity in a dose-dependent manner. In line with the anti-proliferative and
cytotoxic activities, 17B was also significantly triggered cellular apoptosis of MCF7 and
U2OS cancer cells but not A549 lung cancer cells. In addition, pro-apoptotic BAX was
activated in all three cell lines and caspases (3 and 9) was only activated in MCF7 breast
cancer cells and anti-apoptotic BCLxl significantly downregulated in MCF7 and U2OS cancer
cells in a time-dependent manner, indicating specific inhibitory function of 17B in breast
cancer and osteosarcoma. Since several anti-cancer drugs such as 5-FU which inhibits DNA
synthesis and induces cell cycle arrest at G1/S phase affect the progression of the cell cycle
and arrest the cell cycle at different phases, we further confirmed apoptosis findings with
cycle analysis and expression analysis of genes involved in the regulation of cell cycle. In
consistent with the apoptotic functions of 17B, 17B impaired progression through cell cycle
and modulated regulators of cell cycle. Particularly, it was induced cell cycle arrest MCF7
and U2OS but not A549 cancer cells by accumulating cells at G0/G1 phase, indicating that
17B interferes with the G1/S transition. Analysis of genes involved in the regulation of cycle
further supported these findings. CDK1B, CDK2B and p53 was shown to be activated in
MCF7 cells in time-dependent manner. P53 and CDK2B was also activated after 24 hours of
post treatments, their levels were later diminished. As one of the therapeutic actions of anti-
cancer drugs is to slow down the migration capacity of cancer cells to prevent the spread of
the tumor cells to make metastasis, we analyzed anti-migratory capacity of 17B in all three
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cell lines. Our findings were further revealed that 17B significantly interfered with the
migration of cancer cells and delayed wound healing. In line with our observations, Rejmund
et al. also showed in their study that a novel TSCs containing piperazine interfered with the
G1/S transition and resulted in differential expression of PARP, caspase-3, 8, 9, p53, cyclin E,
cdc2, p21, and cytochrome c genes in a variety of cancer cell lines (Rejmund et al., 2018).
Also, Malarz et al. showed that p21 and cdc2 but not p53 are activated in response TSCs
(Malarz et al., 2018). Similarly, thiosemicarbazones based on di-2-pyridine ketone and
quinoline
moiety
was
reported
to
alter
expression
of
cyclin
D1,
cdc2, p21, p53 and cytochrome c proteins and induce apoptosis in HCT 116 p53+/+ and MCF-
7 cancer cell lines (Mrozek-Wilczkiewicz et al., 2019b).
In line with our findings, it has been reported that thiosemicarbazone derivatives are potential
therapeutic agents in cancer therapy. In particular, Brockman et al. reported that 2-
formylpyridine thiosemicarbazone has antileukemic activities in murine animal models
(Brockman et al., 1956). Later studies have shown that Triapine, a thiosemicarbazone
derivative, exhibits anti-cancer activity in the L1210 leukemia mouse model (Finch et al.,
2000). Also, Di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT), synthesized
as a novel iron chelator thiosemicarbazones derivative, has been shown to affect tumor
formation and development in various cancers by impairing tumor cell migration and
metastasis (Li et al., 2016). Wangpu et al. (Wangpu et al., 2016) synthesized two novel
thiosemicarbazones derivative, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone and di-
2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone, and showed that these
compounds suppress cell migration and metastasis by increasing the expression of the
NDRG1 (N-myc downstream regulated gene-1) gene in HT29 colon cancer and DU-145
prostate cancer cells. Similarly, pterostilbene carboxaldehyde thiosemicarbazone (PTERC-T)
was shown to inhibit cancer cell migration and metastasis (Nikhil et al., 2016). Taken
together, in accordance with previous observations, our results have proven that this newly
synthesized thiosemicarbazone compound increases the potential for the novel anti-cancer
drug because it stimulates apoptosis in cancer cells, stops cell cycle and slows the capacity of
cells to migrate.
Moreover, in accordance with our findings, Rejmund et al. showed that TSCs containing
piperazinyl fragment have strong anti-cancer properties compared to their previously designed
TSC compounds–DpC and Dp44mT. Particularly, this novel TSCs, piperazinylogs of
Triapine, was reported to inhibit progression through cell cycle, induce apoptosis and related
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gene expression in cancer cells, concluding that Triapine containing piperazine leads to more
potent and selective anticancer activity (Rejmund et al., 2018). In addition, Serda et al. also
evaluated structure-activity relationship of several TSCs containing retro fragments that have
been used in previously designed TSCs. They have demonstrated that use of an N4-based
piperazine or morpholine ring in TSCs resulted in more potent and selective anti-cancer
activity in various cancerous cell lines (Serda et al., 2014). In another study, Serda et al. have
also showed that quinoline-based thiosemicarbazones have greater anti-proliferative activity
than halogenophenyl compounds (Serda et al., 2012). In line with these previous observations,
compound 17b showed promising anti-cancer activities with potent and selective anti-
proliferative and apoptotic activity, which further proposed that the exchange of the piperidine
group including the S form of the chiral thiosemicarbazone improved anticancer activity
cancer cell lines (Tasdemir et al., 2015).
CONCLUSIONS
Here we demonstrated an as yet undetermined anti-cancer property of a novel
thiosemicarbazone derivative 17B which induce cell cycle arrest and trigger apoptosis by
modulating cellular signaling pathways and reduce the migration capacity of cancer cells.
Although there are several limitations such as the lack of in vivo effects of 17B, findings of
the present have strongly indicated that 17B might be a novel anti-cancer drug for the
treatment of breast cancer and osteosarcoma but not for lung cancer. However, there are some
limitations of our study,
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FIGURE LEGENDS
Figure 1. Chemical structure of 17B ((S)-(+)-2-[(4-piperidin-1-il)benzilidin])-N-(1-
feniletil)hidrazinkarbotiyoamid).
Figure 2. 17B restricts cell viability and exerts cytotoxicity in a dose-dependent manner.
Cells were treated with either with controls or 17B and analyzed for cell viability, LDH
activity and morphology. 17b significantly impaired with the viability of cancer cells and
induced cytotoxicity in all cancer cells. Also, significant morphological changes were
observed.
Figure 3. 17B induces apoptosis and activates caspase-mediated killing. Cells were treated
with either with controls or 17B and cell cycle were analyzed to observe induction of
apoptosis and activation of pro-apoptotic members as well as inactivation of anti-apoptotic
members. 17B strongly induced apoptosis of MCF7 and U2OS cells but not A549 cells and
modulated expression levels of pro-apoptotic and anti-apoptotic mediators in MCF7 and
U2OS cells.
Figure 4. 17B impairs progression through cell cycle and modulates regulators of cell cycle.
Cells were treated with either with controls or 17B and cell cycle were analyzed to observe
cell cycle arrest. 17B significantly induced cell cycle arrest in in MCF7 and U2OS cells by
interfering with the G1/S transition and modulating genes responsible from the regulation of
cell cycle progression.
Figure 5. 17B interferes with the migration of cancer cells. Cells were treated with either with
controls or 17B and wound closure were observed in a time-dependent manner. 17B
interfered with the migration of cancer cells and significantly delayed wound healing. The
black arrows were used to indicate the boundary lines of scratch.
Figure 6. Mechanism of action of tumor suppression mediated by novel thiosemicarbazone
derivative 17B. 17B impairs progression through cell cycle by interfering with the G1/S
transition by modulating genes involved in the regulation of cell cycle. Also, it triggers
apoptosis by activating the expression levels of pro-apoptotic (BAX, CASP3 and CASP9) and
inhibiting the expression of anti-apoptotic mediators (BCL2 and BCLxl) and reduces the
migration capacity of cancer cells.
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ATCC
DMSO
FCS
American Type Culture Collection
Dimethyl-Sulfoxide
Fetal Calf Serum
FACS
HBSS
LDH
PI
Fluorescence-Activated Cell Sorting
Hank's Balanced Salt Solution
Lactate Dehydrogenase
Propidium Iodide
TSCs
Thiosemicarbazones
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Authors Contributions: E.B. designed the study and performed experiments. D.T.K. and
A.K.I. designed and synthesized thiosemicarbazone derivative 17B and performed its
chemical characterization. K.A and I.B helped for cell culture experiments. E.B. and I.B.
analyzed the data, performed statistics and drafted manuscript. E.A.C. supervised this project
and edited final draft.
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Highlights
 17B restricts cell viability and exerts cytotoxicity in a dose-dependent manner
 17B induces apoptosis and activates caspase-mediated killing
 17B inhibits progression through cell cycle by interfering G1/S transition
 17B interferes with the migration of cancer cells
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