A thiosemicarbazone derivative induces triple negative breast cancer cell apoptosis: possible role of miRNA-125a-5p and miRNA-181a-5p

Article abstract of DOI:10.1007/s13258-019-00866-y

Background: Breast cancer, the most commonly diagnosed malignancy in women, accounts for the highest cancer-related deaths worldwide. Triple negative breast cancer (TNBC), lacking the expression of estrogen, progesterone and HER2 receptors, has an aggressive clinical phenotype and is susceptible to chemotherapy but not to hormonal or targeted immunotherapy. In an attempt to identify potent and selective anti-TNBC agents, a set of thiosemicarbazone derivatives were screened for their cytotoxic activity against MDA-MB 231 breast cancer cell line. Methods: MTT assay was used to examine cell viability. P53 phosphorylation status, poly (ADP-ribose) polymerase (PARP) cleavage as well as Bcl2 and Bax protein levels were assessed by Western blot. Quantitative Real Time-PCR was carried out to characterize miRNAs expression levels. Results: Combining Cisplatin + thiosemicarbazone compound 4 showed potent anti-TNBC potential. Cisplatin + compound 4 significantly enhanced p53 phosphorylation, induced Bax amount, reduced Bcl2 protein levels, enhanced PARP cleavage and modulated miRNAs expression profile in TNBCs, with a particular overexpression of miR-125a-5p and miR-181a-5p. Intriguingly, miR-125a-5p and miR-181a-5p could significantly downregulate BCL2 expression by binding to their target sites in the 3′UTR. Conclusions: Collectively, our results demonstrate an anti-TNBC activity of Cisplatin + thiosemicarbazone compound 4 combination mediated via induction of apoptosis.

Full text of DOI:10.1007/s13258-019-00866-y

Genes & Genomics
https://doi.org/10.1007/s13258-019-00866-y
Online ISSN 2092-9293
Print ISSN 1976-9571
RESEARCH ARTICLE
A thiosemicarbazone derivative induces triple negative breast cancer
cell apoptosis: possible role of miRNA‑125a‑5p and miRNA‑181a‑5p
Rania El Majzoub1 · Mohammad Fayyad‑kazan1 · Assaad Nasr El Dine2 · Rawan Makki1 · Eva Hamade1 ·
René Grée3 · Ali Hachem2 · Rabih Talhouk4 · Hussein Fayyad‑Kazan1 · Bassam Badran1
Received: 14 May 2019 / Accepted: 29 August 2019
© The Genetics Society of Korea 2019
Abstract
Background Breast cancer, the most commonly diagnosed malignancy in women, accounts for the highest cancer-related
deaths worldwide. Triple negative breast cancer (TNBC), lacking the expression of estrogen, progesterone and HER2 recep-
tors, has an aggressive clinical phenotype and is susceptible to chemotherapy but not to hormonal or targeted immunotherapy.
In an attempt to identify potent and selective anti-TNBC agents, a set of thiosemicarbazone derivatives were screened for
their cytotoxic activity against MDA-MB 231 breast cancer cell line.
Methods MTT assay was used to examine cell viability. P53 phosphorylation status, poly (ADP-ribose) polymerase (PARP)
cleavage as well as Bcl2 and Bax protein levels were assessed by Western blot. Quantitative Real Time-PCR was carried out
to characterize miRNAs expression levels.
Results Combining Cisplatin+thiosemicarbazone compound 4 showed potent anti-TNBC potential. Cisplatin+compound
4 signifcantly enhanced p53 phosphorylation, induced Bax amount, reduced Bcl2 protein levels, enhanced PARP cleavage
and modulated miRNAs expression profle in TNBCs, with a particular overexpression of miR-125a-5p and miR-181a-5p.
Intriguingly, miR-125a-5p and miR-181a-5p could signifcantly downregulate BCL2 expression by binding to their target
sites in the 3′UTR.
Conclusions Collectively, our results demonstrate an anti-TNBC activity of Cisplatin+ thiosemicarbazone compound 4
combination mediated via induction of apoptosis.
Keywords Thiosemicarbazone derivatives · Breast cancer · Apoptosis · miRNAs
Introduction
subgroups can be partly related to the expression of dif-
ferent breast-cancer-related genes. Among these are genes
encoding estrogen receptor (ER) (Bartlett et al. 2011), pro-
gesterone receptor (PR) (Purdie et al. 2014) and human
epidermal growth factor receptor 2 (HER2) (Rimawi et al.
2013). Patients with ER and/or PR positive status, which
accounts for approximately 80% of hormone-sensitive
breast cancers, have a better prognosis than those with
negative status (Carey et al. 2006). Patients with upreg-
ulated HER2 expression are treated by trastuzumab, a
monoclonal antibody which blocks HER2 growth factor
receptor (Baselga et al. 1996; Hudis 2007). Triple negative
breast cancer (TNBC), being characterized by the absence
of ER, PR and HER2, represent about 10–20% of all breast
cancers diagnosed and is considered a highly aggressive
breast cancer subtype with poor prognosis (Podo et al.
2010; Carey et al. 2010; Boyle 2012). Due to the lack of
known specifc molecular therapeutic targets, the partially
Breast cancer, representing the second leading cause of
cancer death in women, is the most common cancer type
that develops in women worldwide, and its incidence rate
continues to rise (Kamangar et al. 2006). Breast cancer is
a heterogeneous disease that includes diferent subtypes
showing diverse clinical behaviors and sensitivities to
treatment (Parker et al. 2009). Identifying breast cancer
Rania El Majzoub and Mohammad Fayyad-kazan contributed
equally to this work.
Hussein Fayyad-Kazan and Bassam Badran Joint senior co-
authors.
* Bassam Badran
bassam.badran@ul.edu.lb
Extended author information available on the last page of the article
Vol.:(0123456789)
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Genes & Genomics
successful conventional chemotherapy, including doxo-
rubicin and taxane, is basically used for treating TNBC
patients (Liedtke et al. 2008; Oakman et al. 2011; Joen-
suu and Gligorov 2012). TNBC is being, nowadays, inten-
sively investigated for identifying predictive biomarkers
or designing novel efcient therapeutic agents. Although
a large number of chemical compounds, with anti-cancer
potential, have been identifed, so far, their clinical use
is limited by their high toxicity and adverse impact on
normal cells (Buolamwini 1999). Thiosemicarbazones,
belonging to a wide group of thiourea derivatives, exhibit
various valuable biological properties and have therefore
gained special pharmaceutical and medicinal interest
(Pelosi 2010). Over the past 50 years, thiosemicarbazones
have been well characterized for their antiviral, antibacte-
rial, antifungal, and antimalarial activities (Pelosi 2010).
In addition, thiosemicarbazones exhibit anticancer activity
where diferent thiosemicarbazone derivatives and syn-
thetic analogues, such as Triapine, are being used, today,
for cancer treatment (Kalinowski and Richardson 2005;
Hu et al. 2006; Yu et al. 2006; Vandresen et al. 2014).
Thiosemicarbazones derivatives confer anticancer efects
in free and in metal-complexed forms. In free forms, their
biological activity is related to their ability to inhibit
topoisomerase II α and ribonucleotide reductase enzymes
(Bisceglie et al. 2018). The alternative anticancer mode
of action is based on their coordination chemistry with
functional proteins containing metal ions in their struc-
ture. This coordination complex is preferred to occur with
transition metals, particularly Zinc (Zn), Copper (Cu), Iron
(Fe), Cobalt (Co), Nickel (Ni), Platinum (Pt), and Pal-
ladium (Pd). It is note worthy that the metal-complexed
forms confer better anticancer activity than their free
counterparts (Bisceglie et al. 2018).
humid environment in an incubator with 5% CO2. When
reaching 85% confuence, cells were harvested using 0.25%
trypsin and then seeded in 96- or 6-well plates, depending
on the performed experiment. Cells were at 70% confuent
prior to treatment.
Peripheral blood mononuclear cells (PBMC) were
obtained from the peripheral blood of healthy donors, and
were immediately purifed using gradient centrifugation in
Ficoll-PaqueTM Plus (Amersham Biosciences, USA).
The mixture was centrifuged for 40 min at 18–20 °C
(400×g) without a break. Bufy coat was aspirated and washed
twice with balanced salt solution (PBS) and then resuspended
in RPMI 1640 medium provided with 10% fetal bovine serum,
HEPES (20 nM), penicillin (100 U/mL), and streptomycin
(100 µg/mL). Cells were activated upon being treated with
Mitogen phytohemagglutinin-P (PHA-P) (5 µg/ml; sigma) and
human interleukin (IL-2) (10 ng/ml; R&D Systems).
MTT assay
PBMC and MDA-MB-231 cells were frst seeded in 96-well
plates (8×103 cells/well). Cells were treated with the difer-
ent indicated products at concentrations ranging from 100 to
1 μM for 48 h. Cell viability was examined using Cell Prolif-
eration Assay, MTT (Thiozolyl Blue Tetrazoluim Bromide;
Sigma; USA; M-5655-1G).
Western blotting
Protein samples were charged into stacking gel wells and
then run until bromophenol blue reached the bottom of
the gel. Gels were then allowed to transfer on nitrocellu-
lose membranes (1 h at 4 °C; 80 V). The membranes were
then incubated (2 h at room temperature) in the presence of
blocking solution: 3% BSA (Bovine Serum Albumin, Sigma
A2153), prepared in Tween PBS. The used primary anti-
bodies were: monoclonal antibodies for p53 (Santa Cruz
Biotechnology Inc., Texas, USA; sc-126), Bcl2 (Santa Cruz
Biotechnology Inc., Texas, USA; sc-7382), Bax (Cell signal-
ing; USA; D2E11), PARP (abcam; ab191217) and GAPDH
(abcam; USA; ab9485)—the latter was used to ensure equal
loading of samples.
In this study, we screened, in vitro, the antitumor activity of
diferent thiosemicarbazone derivatives against a TNBC cell
line. Our results identifed one thiosemicarbazone derivative,
designated compound 4, to intensify cisplatin-mediated cyto-
toxicity against TNBCs probably via upregulating the expres-
sion of certain tumor suppressor miRNAs, miR-125a-5p and
miR-181a-5p, as well as enhancing cisplatin-dependent phos-
phorylation of p53 and activation of apoptosis.
MiRNA profling and data analysis
Materials and methods
Trizol total RNA isolation reagent (Roche Diagnostics,
vilvoorde, Belgium) was used to exact total RNA from
cells. NanoDrop Spectrophotometer was used to quantify
the concentration. In a frst step, and in order to synthesize
cDNA from miRNAs, a TaqMan miRNA Reverse Transcrip-
tion Kit (#4366596; Applied Biosystems, Gent, Belgium)
and Megaplex RT primers (Human Pool A, #4399966;
Applied Biosystems, Gent, Belgium) were used following
Cell culture
MDA-MB-231 cells were obtained from American Type
Culture Collection (ATCC) (Rockville, USA). They were
cultivated in DMEM and provided with 10% heat inactivated
FBS, 2 mM l-glutamine, and 1% of penicillin and strepto-
mycin. Cells were cultivated in 75 cm2 fasks at 37 °C within
1 3

Genes & Genomics
the manufacturer’s protocol. Using 500 ng of total template
RNA, this enabled simultaneous reverse transcription of 380
mature human miRNAs. RT was carried out using Mastercy-
cler Epgradient thermocycler (VWR International, Leuven,
Belgium) with the following parameters: 40 cycles of 16 °C
for 2 min, 42 °C for 1 min, 50 °C for 1 s, and a fnal step of
80 °C for 5 min to inactivate the reverse transcriptase. The
products were then diluted with RNase-free water, mixed
with TaqMan gene expression Master Mix and then charged
into TaqMan Human miRNA Array A (#4398965; Applied
Biosystems, Gent, Belgium), corresponding to a 384-well
formatted plate and real-time PCR-based microfuidic card
containing embedded TaqMan primers and probes in each
well for the 380 diferent mature human miRNAs. RNU48
transcript was used as a normalization control. Quantita-
tive PCR (qPCR) was carried out following the manufac-
turer’s instructions. Real-time PCR was carried out using
ABI PRISM 7900HT sequence detection system (Applied
Biosystems, Gent, Belgium) with the following parameters:
50 °C for 2 min, 94.5 °C for 10 min, followed by 40 cycles
of 95 °C for 30 s and 59.7 °C for 1 min. RNU48 embed-
ded in the TaqMan Human miRNA Arrays was considered
as an endogenous control. The relative expression levels of
miRNAs were calculated following the comparative ΔΔCt
method as described before (Livak and Schmittgen 2001;
Schmittgen and Livak 2008). The fold changes in miRNAs
Plasmid constructions
A 710-bp fragment of BCL2 3′UTR containing the miR-
125a-5p and miR-181a-5p potential target sites was cloned
downstream of the Renilla luciferase gene (EcoRI/XhoI
sites) in the psiCHECK-1 plasmid (Promega, Mannheim,
Germany) and designated as psiCHECK 3′UTR WT. PCR
primers used for amplifcation of the BCL2 3′ UTR were:
BCL2:
Forward primer: 5′-TCTGTATTAACTTTGGAATGT
ACTCTG-3′
Reverse primer: 5′-CAAAGGTCTGATCATTCT
GTTCC-3′.
The constructs were verifed by sequencing.
Site directed mutagenesis
QuikChange site-directed mutagenesis mutation/deletion
of the miR-125a-5p and miR-181a-5p potential target sites
respectively was carried out following the manufacturer’s
guidelines (Stratagene, La Jolla, CA, USA) and designated
as psiCHECK-UTRdel. QuikChange site-directed mutagen-
esis was carried out using the following primers:
The following primers were used to carry out the deletion:
BCL2 (miR-125a-5p deleted):
Forward: 5′-CCTCCCCGGCGGGCAACAGAATG-3′
Reverse: 5′-CATTCTGTTGCCCGCCGGGGAGG-3′.
BCL2 (miR-181a-5p deleted):
expression levels were calculated by the equation 2−ΔΔCt
.
Taqman miRNA assay for individual miRNAs
Forward primer: 5′-GTATTAACTTTGACTCTGTTCAAT
G-3′
For each miRNA, reverse transcription was carried out using
10 ng of purifed total RNA, 100 mM dNTPs, 50 U MutliS-
cribe reverse transcriptase, 20 U RNase inhibitor, and 50 nM
of RT primer samples using the TaqMan MicroRNA Reverse
Transcription kit (Applied Biosystems, Gent, Belgium).
Reactions (15 μl) were incubated: 30 min at 16 °C, 30 min
at 42 °C, and 5 min at 85 °C. Real Time-PCR reactions
(5 μl of RT product, 10 μl TaqMan 2×Universal PCR master
Mix (Applied Biosystems, Gent, Belgium), and 1 μl TaqMan
MicroRNA Assay Mix containing PCR primers and TaqMan
probes) were performed using ABI Prism 7900HT Sequence
Detection System (Applied Biosystems, Gent, Belgium) at
the following parameters: 95 °C for 10 min followed by 40
cycles at 95 °C for 15 s and 60 °C for 1 min. The expres-
sion levels 2−ΔΔCt of miRNAs were calculated as previously
described (Schmittgen and Livak, 2008).
Reverse primer: 5′-CATTGAACAGAGTCAAAGTTAATA
C-3′.
The constructs were verifed by sequencing.
Cell culture
Hela cells were cultivated in Dulbecco’s Modifed Eagle’s
Medium (DMEM, Lonza, Verviers, Belgium) provided with
10% heat inactivated fetal bovine serum (Invitrogen Europe,
Paisley, UK), 2 mM l-glutamine, 50 IU/ml Penicillin, and
50 μg/ml Streptomycin (all from Lonza, Verviers, Belgium).
Luciferase assays
Assays were performed in a 24-well format. Hela cells were
con-transfected, using Lipofectamine 2000 (Invitrogen, Merel-
beke, Belgium) according to the manufacturer’s guidelines,
with reporter plasmids (psiCHECK/psiCHECK 3′UTR WT/
psiCHECK 3′UTR deleted) (100 ng) along with miR-125a-5p
and miR-181a-5p-mimic/miR-negative control-mimic at fnal
concentration of 10 μM (miRIDIAN mimic, Dharmacon, Geel,
Belgium) and control frefy plasmid pGL3-CMV (100 ng).
The cells were frst assessed for their expression levels of
Bioinformatics
MicroRNA Data Integration Portal (mirDIP) (http://ophid
.utoronto.ca/mirDIP/) was searched for potential miRNA
target sites. BCL2 was identifed as having putative miR-
125a-5p and miR-181a-5p target sites.
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Genes & Genomics
miRNAs of interest using quantitative RT-PCR, as described
below. After 40 h post transfection, cells were collected and
luciferase levels were determined using the Dual luciferase
reporter assay system (Promega, Mannheim, Germany) fol-
lowing the manufacturer’s guidelines. Relative protein levels
corresponded to Renilla/Firefy luciferase ratios.
(ER−, PR− and HER2−), using MTT assay. The inhibitory
activities (IC50) are summarized in Table 2. Compounds 2, 3,
4, 5, and 6 showed a cytotoxic efect against MDA-MB 231
cells at concentrations close to those exhibited by Cisplatin
(known anticancer agent) (Table 2), and lower than other
compounds.
Interestingly, combining compound 4 with Cisplatin fur-
ther enhanced the anticancer activity of Cisplatin (Table 2).
On the other hand, no signifcant improvement of Cisplatin
cytotoxic efect was detected upon combining it with either
compound 2, 3, 5, or 6 (data not shown).
Quantitative PCR for BCL2 expression
Quantitative mRNA expression was determined by real-time
PCR, with PRISM 7900 sequence detection system (Applied
Biosystems, Gent, Belgium), and the TaqMan Master mix kit
with EF1- α mRNA was used as an internal control. Human
Taqman gene expression assays for BCL2 (Hs04986394_s1)
and EF1-α (Hs00951278_m1) were purchased from Applied
Biosystems.
For a chemical compound to be benefcial as an antican-
cer drug, it must exhibit preferential anti-proliferative activ-
ity against tumor cells over normal and mortal cell-types.
Hence, the ability of 4 versus that of cisplatin, to selectively
target cancer cells, was evaluated by examining its cytotoxic
efect against a mortal cell-type, namely peripheral blood
mononuclear cells (PBMC). Noteworthy, PBMCs have been
widely used as control cells to assess the specifcity of many
potential anticancer agents (Varamini et al. 2007; Pinto et al.
2011; Bendale et al. 2017). Interestingly, none of the indicated
compounds showed signifcant cytotoxicity towards PBMCs
(Table 2). Moreover, the combination of Cisplatin+compound
4 exerted no cytototoxic efect against PBMCs (Table 2).
Chemistry
The thirteen thiosemicarbazone derivatives (Table 1) were
prepared starting from the suitable isothiocyanate (commer-
cially available or prepared from the corresponding amine)
(Wong and Dolman 2007). Treatment of isothiocyanate with
hydrazine hydrate gave the thiosemicarbazide derivative
which was condensed with the appropriate aldehyde to give
the corresponding thiosemicarbazone.
H
N
H
N
H
N
H
N
R1 NCS
+
H2N
NH2 . H2O
R1
N
R2
R1
NH2
S
S
Statistical analysis
Efect of Cisplatin + compound 4 combination
on apoptotic components
Presented data corresponds to mean SEM of at least three
independent experiments and was analyzed using Student’s
t-test. P-values < 0.05 (*) and < 0.01(**) were considered
signifcant.
Since our above results revealed that the combination of Cis-
platin+compound 4 has a striking cytotoxic efect against
TNBCs and in order to get further insight into the molecular
mechanisms underlying the observed cell death, we assessed
the efect of this treatment on diferent components regulat-
ing cellular apoptosis. The p53 protein is a tumor suppressor
that becomes active once phosphorylated. In order to check
whether the observed potent anticancer potential of Cispl-
atin+compound 4 was associated with p53 activation, we
assessed the phosphorylation level of p53 in untreated cells
versus Cisplatin, compound 4 and Cisplatin+compound 4
treated cells. A slight increase in p53 phosphorylation was
observed in Cisplatin or compound 4 treated cells. Interest-
ingly, p53 phosphorylation level was more pronounced in
Cisplatin+compound 4 treated cells (Fig. 1a, b).
Results
Antiproliferative activity of diferent
thiosemicarbazone derivatives against MDA‑MB‑
231 breast cancer cell line
All the synthesized thiosemicarbazone compounds (1–13)
were screened for their in vitro anticancer activity against
the triple negative breast cancer cell line MDA-MB-231
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Genes & Genomics
Table 1 The 13
Compound
1
R1
R2
thiosemicarbazone derivatives
used in this study
2
3
OH
HO
HO
OH
OH
OH
HO
HO
Br
4
5
6
Br
Cl
Cl
H3CO
7
8
H3CO
H3C
OH
9
NC
OH
Br
10
11
12
13
H3C
Br
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Genes & Genomics
Table 2 In vitro antiproliferative activity of thiosemicarbazone deriv-
Efect of Cisplatin + compound 4 combination
on MDA‑MB‑231 cells’ miRNA expression profle
atives
Compounds (C)
IC50 (µM)
Abnormal miRNA expression has been associated with
the development of breast cancer and other tumors (Iorio
et al. 2005; Kumar et al. 2007) and miRNAs are consid-
ered important molecular biomarkers and therapeutic targets
in many diseases especially cancer (Raisch et al. 2013). In
order to check whether miRNAs are involved in regulating
the molecular mechanisms involved in triggering the cyto-
toxic efect of Cisplatin+compound 4 combination against
TNBCs, we assessed the diference in miRNA expression
profle in Cisplatin+compound 4 treated versus untreated
MDA-MB-231 cells. RNA was frst prepared from those
cells and TaqMan Low Density Array (TLDA) was then
applied to determine the miRNA expression profles. 15
miRNAs appeared to be diferentially expressed in treated
versus untreated control cells (Table 3). In a second step,
quantitative Real Time-PCR (qRT-PCR) was carried out to
validate the diferential expression of these miRNAs. The
efect of Cisplatin + compound 4 on MDA-MB-231 cells
miRNA expression profle was confrmed for 10 miRNAs
exhibiting signifcantly altered expression levels where miR-
23a, miR-125a-5p, miR-181a-5p, and miR-330 were upreg-
ulated whilst miR-133b, miR-324-5p, miR-521, miR-372,
miR-509-5p, and miR-636 were downregulated in treated
versus control cells (Fig. 3).
MDA-MB 231
PBMC
C1
C2
C3
C4
C5
C6
C7
C8
> 100
–
70 7.8
60 5.2
60 1.5
60 1.7
70 2.4
> 100
> 100
> 100
> 100
> 100
> 100
> 100
44.8 3.1
16 2.9
> 100
> 100
> 100
> 100
> 100
–
–
–
–
–
–
–
C9
C10
C11
C12
C13
Cisplatin
Cisplatin+C4
> 100
> 100
PBMC (Peripheral Blood Mononuclear Cells) and MDA-MB-231
cells were treated with diferent concentrations (5, 25, 50, 75 and
100 µM) of indicated compounds for 48 h. IC50 value corresponds to
the concentration of compound required to inhibit cell growth by 50%
in comparison to untreated cells. Each value represents a mean SD
(n=3)
Once activated, p53 can activate the expression of BAX, a
BCL2 family member that triggers apoptosis. In parallel with
p53 phosphorylation status, Bax protein levels increased
in Cisplatin + compound 4 treated cells versus untreated
cells (Fig. 1a, c). Moreover, an increase in Bax levels was
observed after treating cells with either Cisplatin or com-
pound 4 alone.
miR‑125a‑5p and miR‑181a‑5p negatively regulate
BCL2 expression upon targeting the 3′UTR
Using the computational microRNA Data Integration Portal
(mirDIP), BCL2 was identifed as a potential target for miR-
125a-5p and miR-181a-5p. We, therefore, checked whether
BCL2 expression could be directly regulated by miR-
125a-5p and/or miR-181a-5p. A Renilla luciferase reporter
gene-based vector was designed in which 200 bp fragment
of the 3′-UTR of BCL2 containing the miR-125a-5p and the
miR-181a-5p target sequences was cloned downstream of
the reporter gene. This vector was referred to as psiCHECK-
UTRwt. In parallel, the same procedure was applied to clone
this BCL2 3′-UTR fragment with a deleted miR-125a-5p
(psiCHECK-UTRdel1) or miR-181a-5p (psiCHECK-
UTRdel2) target site. Transient transfection of psiCHECK-
UTRwt in Hela cells led to~20 to 30% decrease in reporter
luciferase activity, when compared with the psiCHECK
control vector (Fig. 4a, b). However, no decrease in reporter
luciferase activity was observed in cells transfected with
either psiCHECK-UTRdel1 or psiCHECK-UTRdel2
(Fig. 4a, b). Notably, a more substantial reduction in reporter
luciferase activity was observed in Hela cells being co-trans-
fected with either miR-125a-5p and psiCHECK-UTRwt
(~ 65%) or miR-181a-5p and psiCHECK-UTRwt (~ 55%)
Further, we assessed the efect these products on the pro-
tein levels of Bcl2, an anti-apoptotic component. Diferently
from Cisplatin that caused only a slight decrease in Bcl2
levels, compound 4 triggered a more pronounced reduction
in Bcl2 protein amount (Fig. 1a, d). Bcl2 amount was strik-
ingly lowered in Cisplatin+compound 4 treated cells.
Cleavage of poly(ADP-ribose) polymerase (PARP) by
caspases is a key event taking place during apoptosis. Here,
we checked the ability of our products to trigger PARP
cleavage. In contrast to untreated conditions where PARP
was present in its full length uncleaved form, the cleaved
form appeared in response to Cisplatin. Interestigly, the
amount of cleaved PARP was further enhanced in cells
treated with Cisplatin+compound 4 (Fig. 2a, b).
Altogether, these observations indicated that Cispl-
atin+compound 4 combination can interfere with the sig-
nalling pathways regulating MDA-MB-231 cells apopotosis.
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Genes & Genomics
Fig. 1 Efect of thiosemicarbazone derivative compound 4 on Bax
and BCL2 expression and p53 phosphorylation. a MDA-MB-231
cells were treated, for 48 h, with Cisplatin (45 µM), C4 (60 µM) or
Cisplatin+compound 4 (16 µM). Total protein extracts were prepared
and p53, Bax, BCL2 and GAPDH were detected as described in
materials and methods. Representative western blot was shown. Lev-
els of phosphorylated p53 (b), Bax protein (c) and BCL2 protein (d)
were quantifed by densitometer and normalized to GAPDH
Fig. 2 Efect of thiosemicarbazone derivative compound 4 and cispl-
atin on PARP cleavage. a MDA-MB-231 cells were treated, for 48 h,
with Cisplatin (45 µM) or Cisplatin+compound 4 (16 µM). Total
protein extracts were prepared and PARP and GAPDH levels were
detected. Representative western blot was shown. Levels of cleaved
PARP form (b) were quantifed by densitometer and normalized to
full PARP form and GAPDH
in comparison to cells being transfected with psiCHECK-
UTRwt alone (Fig. 4a, b). Further, the negative efect of
either miR-125a-5p or miR-181a-5p was bypassed in cells
co-transfected with either miR-125a-5p and psiCHECK-
UTRdel1 or miR-181a-5p and psiCHECK-UTRdel2
(Fig. 4a, b). Remarkably, co-transfection of Hela cells with
psiCHECK-UTRwt and both miR-125a-5p and miR-181a-5p
resulted in more striking negative efect (~75%) in com-
parison to cells co-transfected with psiCHECK-UTRwt and
either miR-125a-5p or miR-181a-5p (Fig. 4c). Altogether,
these data indicate that BCL2 expression could be negatively
regulated by miR-125a-5p and miR-181a-5p.
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Genes & Genomics
Table 3 miRNA signature identifed by TLDA technique
Lentiviral transduction of MDA‑MB‑231
cells and the negative efect of miR‑125a‑5p
and miR‑181a‑5p on BCL2 mRNA levels
MicroRNA
Cisplatin
Cisplatin+C4
P value
hsa-miR-23a
hsa-miR-330
hsa-miR-125a-5p
hsa-miR-181a-5p
hsa-miR-489
miR-133b
miR-324-5p
miR-372
miR-509-5p
miR-521
miR-636
miR-616
miR-655
miR-708
2.5
3.8
5.5
6.1
5.5
7.2
0.021
0.015
0.001
0.003
0.03
0.013
0.015
0.023
0.014
0.023
0.011
0.041
0.044
0.039
0.04
Lentiviral based systems have been developed to generate
replication incompetent lentiviral vectors that were ef-
ciently used in the transduction of both dividing and non-
dividing mammalian cells and provided stable, long term
expression of RNA of interest. To investigate the efects
of miR-125a-5p and miR-181a-5p on MDA-MB-231 cells
and more specifcally on BCL2 expression, lentiviral vec-
tors (lenti-miR-125a-5p, lenti-miR-181a-5p) containing
copGFP as a reporter gene were produced and transduced
into MDA-MB-231 cells. A scrambled lenti-miR-ctrl was
used as a negative control. After optimization of trans-
duction conditions, we defned an MOI (Multiplicity of
infection) of six as the one providing the highest trans-
duction rate. This MOI was then chosen to transduce
MDA-MB-231 cells. The efcacy of transduction was
measured 1 week after transduction. Measurement of
GFP expression by fow cytometry showed 87–92% trans-
duction efciency. Subsequent fow cytometry sorting of
11.5
13.5
7.1
0.12
0.02
0.1
0.012
0.01
0.018
0.28
0.21
0.27
0.29
3.3
0.45
0.144
0.32
0.33
0.14
0.31
0.41
0.43
0.48
0.51
miR-758
TLDA analysis unravelled 15 miRNAs to be diferentially expressed
in treated versus non-treated cells with a p value<0.05
Fig. 3 Ten miRNAs are diferentially expressed after Cispl-
atin+compound 4 treatment. MDA-MB-231 cells were cultivated in
the absence or presence of Cisplatin+compound 4. RNU48-normal-
ized miRNA levels were quantifed by qRT-PCR. The statistical sig-
nifcance was determined using unpaired Student’s t test (*p < 0.05,
**p < 0.01, ***p < 0.001 v.s. untreated control cells)
1 3

Genes & Genomics
Fig. 4 A repressive efect of miR-125a-5p and miR-181a-5p on BCL2
expression via targeting BCL2 3′-UTR. Hela cells were transiently
co-transfected with psiCHECK-UTRwt (bearing 200 bp of the BCL2
3′-UTR), psiCHECK-UTRdel1 or del2 (miR-125a-5p or miR-181a-5p
target site was deleted, respectively) with miRNA control mimic,
miR-125a-5p or mR-181a-5p mimics. Transfection with empty
psiCHECK was used to assess the background promoter activity of
the vector. Data represent mean SEM of three independent experi-
ments, each performed in triplicate. a The efect of miR-125a-5p.
b The efect of miR-181a-5p. c The synergistic efect of both miR-
125a-5p and miR-181a-5p. *p<0.05; **p<0.01 vs. psiCHECK
transfected cells; Student’s t-test
the GFP-positive cells resulted in a 99% pure population.
Quantitative real-time RT-PCR revealed an approximate
twofold decrease of the mRNA level of BCL2 in the lenti-
miR-125a-5p, and lenti-miR-181a-5p transduced MDA-
MB-231 cells compared to the Lenti-miR-ctrl transduced
cells (Fig. 5) indicating that BCL2 expression is negatively
regulated by miR-125a-5p and miR-181a-5p.
against various cancer cell lines, including pancreas, lung,
ovary, kidney, and prostate (Murren et al. 2003). In this
study, we examined the anti-cancer potential of 13 thiosemi-
carbazone derivatives against TNBC cells. Among these,
diferent compounds showed moderate cytotoxic potential
against TNBC cells. Intriguingly, combining compound 4
with Cisplatin resulted in a striking cytotoxic efect. Notably,
this cytotoxicity was selective against cancer cells without
harming the normal PBMCs. Such observations highlight an
advantageous efect of compound 4 by reinforcing the selec-
tive cytotoxic potential of Cisplatin against TNBC cells.
The tumor suppressor p53 plays an essential role in control-
ling genomic integrity, cell cycle, and apoptosis in response
to various stress signals (Hu et al. 2012). P53 is activated fol-
lowing phosphorylation in its N-terminal domain by protein
kinases of the MAPK family (Eliaš et al. 2014). Activated
p53 can then translocate to the nucleus, bind to its target
sequences, and activate the transcription of genes involved
in cell cycle arrest and apoptosis induction (such as BAX,
BAD and BIM) (Yao et al. 2014; Mollereau and Ma 2014).
On the other hand, accumulation of phosphorylated p53 in
the mitochondria leads to apoptosis induction upon interacting
with pro- and anti-apoptotic BCL2 family members. In fact,
binding of phosphorylated p53 to the pro-apoptotic proteins
Bax and Bak leads to conformational changes alleviating the
antagonism mediated by anti-apoptotic components (Westphal
et al. 2011). Another major event taking place during apop-
tosis is the cleavage of PARP by various caspases, especially
caspase-3 (Kaufmann et al. 1993). Interestingly, our results
Discussion
TNBCs, being characterized by absence of ER, PR and
HER2, are among the most aggressive subtypes of epithe-
lial breast malignancies. For patients with TNBC, endocrine
therapies including Tamoxifen and aromatase inhibitors as
well as HER2 targeted therapies including Trastazumab and
Lapatinib are not efcient. On the other hand, TNBCs are
sensitive to chemotherapy where anthracyclines and taxa-
nes are commonly used for high risk patients (Chacón and
Costanzo 2010). Despite this chemo-susceptibility, patients
with TNBCs are at higher relapse risk than those presenting
hormone positive breast cancer (Hudis and Gianni 2011).
Designing an optimal chemotherapy which will ensure a
longer overall survival of TNBC patients remains a chal-
lenge. Thiosemicarbazones correspond to a group of mol-
ecules with promising therapeutic potential, particularly
cancer treatment (Pelosi 2010). For instance, Triapine
(3-aminopyridine-2-carboxaldehyde-thiosemicarbazone)
is now used in clinical trials due to its potent cytotoxicity
1 3

Genes & Genomics
Fig. 5 Lentiviral-mediated miR-125a-5p and miR-181a-5p expression
and their efect on BCL2 expression in MDA-MB-231 cells. miR-
125a-5p, miR-181a-5p, and BCL2 expression were determined by
qRT-PCR in MDA-MB-231 cells, miR-125a-5p, miR-181a-5p, and
miR-Ctrl transduced MDA-MB-231cells. Mean of three independ-
ent experiments are shown. *p<0.05 (lenti-miR-125a-5p, lenti-miR-
181a-5p vs. lenti-miR-Ctrl transduced MDA-MB-231cells; Student’s
t-test)
showed that Cisplatin+compound 4 combination induced p53
phosphorylation, and this was accompanied with elavated Bax
levels, reduced Bcl2 amount and enhanced PARP cleavage,
thus indicating that Cisplatin+compound 4 anticancer efect
could be attributed, at least in part, to their ability to trigger
cell apoptosis. So far, the exact mechanism underlying the
ability of compound 4, specifcally, to enhance the anticancer
potential of cisplatin is still unclear. The biological activities
of thiosemicarbazone derivatives are often related to chela-
tion of metal ions. They can coordinate to the metal center
in N,S-bidentate mode, but when additional coordinating
groups exist, more binding modes become possible. Particu-
larly, the presence of three hydroxyl groups on compound 4
might provide a more favorable simultaneous coordination for
one or more metal ions (such as Fe2+ or Cu2+) which in turn
could lead to downregulation of key enzymatic and regula-
tory functions regulating cell survival such as Ribonucleo-
tide Reductase (iron-dependent enzyme) activity. Moreover,
it could also be possible that the three hydroxyl groups present
in compound 4 might render it soluble in aqueous medium
and therefore can afect its ionization and lipid solubility.
Epigenetic modifcations, including miRNAs expression,
have been associated with cancer initiation and progression
(Peña-Chilet et al. 2014). MiRNAs are regulatory elements
capable of modulating a wide array of physiological pro-
cesses, including proliferation, diferentiation and apoptosis,
upon modulating gene expression via binding the 3′UTR
in target mRNA. Numerous miRNAs have been identifed
to be deregulated between normal and tumor breast tissues
(Iorio et al. 2009; Leivonen et al. 2014). Besides, miRNAs
can be involved in the tumorgenesis process via modulat-
ing the expression of certain oncogenes or tumor suppressor
genes (Zhang et al. 2007; Cho 2007; Hummel et al. 2010).
Intriguingly, drug-sensitive and drug resistant cancer cell
lines exhibit distinct miRNA signatures (Chen et al. 2010;
Zhou et al. 2010; Pogribny et al. 2010) and thus can serve
not only as biomarkers but also as anticancer therapeutic
targets. In this study, we examined changes in miRNA signa-
ture after the treatment of TNBC cells with Cisplatin+com-
pound 4 combination. Importantly, this treatment afected
the expression of 10 miRNAs, among which 4 were upregu-
lated and 6 were downregulated. Upon screening for targets
of the upregulated miRNAs, we identifed the antiapoptotic
BCL2 gene as a putative target for miR-125a-5p and miR-
181a-5p. The direct interaction between miR-125a-5p, miR-
181a-5p, and BCL2 was confrmed using dual-luciferase
reporter assay. Indeed, we showed that miR-125a-5p and
miR-181a-5p, via targeting their potential binding sites in
BCL2 gene, can negatively regulate gene expression. In fact,
a repressive efect of miR-125a-5p on BCL2 expression has
already been established. For instance, miR-125a-5p was
reported to be involved in repressing BCL2 expression in
response to CD40 ligand in human Leukemic B-cells (Willi-
mott and Wagner 2012). Moreover, miR-125a-5p was shown
to inhibit cell proliferation and trigger apoptosis in colon
cancer upon targeting BCL2 (Tong et al. 2015). Further,
miR-125a-5p was described to enhance chemotherapy sen-
sitivity to Cisplatin via downregulating BCL2 expression in
gallbladder cancer (Yang et al. 2017). Mir-181a-5p has also
been described to enhance drug sensitivity in lymphocytic
1 3

Genes & Genomics
Buolamwini JK (1999) Novel anticancer drug discovery. Curr Opin
Chem Biol 3:500–509
leukemia cells upon targeting multiple anti-apoptotic genes
including BCL2 (Zhu et al. 2012).
Carey LA, Perou CM, Livasy CA, Dressler LG, Cowan D, Conway
K, Karaca G, Troester MA, Tse CK, Edmiston S et al (2006)
Race, breast cancer subtypes, and survival in the Carolina Breast
Cancer Study. JAMA 295:2492–2502
Carey L, Winer E, Viale G, Cameron D, Gianni L (2010) Triple-nega-
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Clin Oncol 7:683–692
Conclusion
In conclusion, we report here a potent anticancer efect for a
new drug combination, Cisplatin+thiosemicarbazone com-
pound 4 against TNBC cells upon modulating the activity
and expression of diferent cellular elements (p53, Bax,
Bcl2, PARP and miRNAs) and triggering cellular apoptosis.
Chacón RD, Costanzo MV (2010) Triple-negative breast cancer.
Breast Cancer Res 12:S3
Chen G-Q, Zhao Z-W, Zhou H-Y, Liu Y-J, Yang H-J (2010) Systematic
analysis of microRNA involved in resistance of the MCF-7 human
breast cancer cell to doxorubicin. Med Oncol 27:406–415
Cho WCS (2007) OncomiRs: the discovery and progress of microR-
NAs in cancers. Mol Cancer 6:60
Eliaš J, Dimitrio L, Clairambault J, Natalini R (2014) The p53 protein
and its molecular network: modelling a missing link between
DNA damage and cell fate. Biochim Biophys Acta Proteins Pro-
teom 1844:232–247
Acknowledgements All participants are acknowledged in the
authorship.
Funding This work is supported by the Lebanese University and the
Lebanese National Council for Scientifc Research (CNRS-L). Financial
support by the “Ligue contre le Cancer, Conseil Interrégional Grand
Ouest” is also gratefully acknowledged.
Hu W, Zhou W, Xia C, Wen X (2006) Synthesis and anticancer activ-
ity of thiosemicarbazones. Bioorg Med Chem Lett 16:2213–2218
Hu W, Feng Z, Levine AJ (2012) The regulation of multiple p53 stress
responses is mediated through MDM2. Genes Cancer 3:199–208
Hudis CA (2007) Trastuzumab—mechanism of action and use in clini-
cal practice. N Engl J Med 357:39–51
Compliance with ethical standards
Hudis CA, Gianni L (2011) Triple-negative breast cancer: an unmet
medical need. Oncologist 16(Suppl 1):1–11
Conflict of interest Rania El Majzoub, Mohammad Fayyad-kazan,
Assaad Nasr El Dine, Rawan Makki, Eva Hamade, René Grée, Ali
Hachem, Rabih Talhouk, Hussein Fayyad-Kazan and Bassam Badran
declare that they have no confict of interest.
Hummel R, Hussey DJ, Haier J (2010) MicroRNAs: predictors and
modifers of chemo- and radiotherapy in diferent tumour types.
Eur J Cancer 46:298–311
Iorio MV, Ferracin M, Liu C-G, Veronese A, Spizzo R, Sabbioni S,
Magri E, Pedriali M, Fabbri M, Campiglio M et al (2005) Micro-
RNA gene expression deregulation in human breast cancer. Can-
cer Res 65:7065–7070
Ethical approval All procedures performed in studies involving human
participants were in accordance with the 1964 Helsinki declaration and
its later amendments.
Iorio MV, Casalini P, Piovan C, Di Leva G, Merlo A, Triulzi T,
Ménard S, Croce CM, Tagliabue E (2009) microRNA-205 regu-
lates HER3 in human breast cancer. Cancer Res 69:2195–2200
Joensuu H, Gligorov J (2012) Adjuvant treatments for triple-negative
breast cancers. Ann Oncol 23(Suppl 6):vi40–vi45
Informed consent Informed consent was obtained from all individual
participants included in the study.
Kalinowski DS, Richardson DR (2005) The evolution of iron chelators
for the treatment of iron overload disease and cancer. Pharmacol
Rev 57:547–583
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Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional afliations.
Afliations
Rania El Majzoub1 · Mohammad Fayyad‑kazan1 · Assaad Nasr El Dine2 · Rawan Makki1 · Eva Hamade1 ·
René Grée3 · Ali Hachem2 · Rabih Talhouk4 · Hussein Fayyad‑Kazan1 · Bassam Badran1
Rania El Majzoub
Eva Hamade
rania.elmajzoub@gmail.com
eva.hamade@ul.edu.lb
Mohammad Fayyad-kazan
mfayyadk@gmail.com
René Grée
rene.gree@univ-rennes1.fr
Assaad Nasr El Dine
Ali Hachem
nasreldine_assaad@hotmail.com
ahachem@ul.edu.lb
Rawan Makki
Rabih Talhouk
rawan_makki@hotmail.fr
rtalhouk@aub.edu.lb
1 3

Genes & Genomics
3
4
Hussein Fayyad-Kazan
hfayyadk@gmail.com
University Rennes, CNRS, ISCR (Institut des Sciences
Chimiques de Rennes), UMR 6226, 35000 Rennes, France
Department of Biology, Faculty of Arts and Sciences,
American University of Beirut, Beirut, Lebanon
1
Laboratory of Cancer Biology and Molecular Immunology,
Faculty of Sciences-I, Lebanese University, Hadath, Beirut,
Lebanon
2
Laboratory for Medicinal Chemistry and Natural Products,
Faculty of Sciences-I and PRASE-EDST, Lebanese
University, Hadath, Beirut, Lebanon
1 3