Novel pyridinecarboxaldehyde thiosemicarbazone conjugated magnetite nanoparticulates (MNPs) promote apoptosis in human lung cancer A549 cells

Article abstract of DOI:10.1007/s00775-019-01728-4

The present study highlights the apoptotic activity of magnetic Fe₃O₄ nanoparticulates functionalized by glutamic acid and 2-pyridinecarboxaldehyde thiosemicarbazone (PTSC) toward human lung epithelial carcinoma A549 cell line. To this aim, the Fe₃O₄ nanoparticulates were prepared using co-precipitation method. Then, the glutamic acid and Fe₃O₄ nanoparticulates were conjugated to each other. The product was further functionalized with bio-reactive PTSC moiety. In addition, the synthesized Fe₃O₄@Glu/PTSC nanoparticulates were characterized by physico-chemical techniques including scanning electron microscope (SEM), energy dispersive X-ray (EDX), X-ray diffraction (XRD), Fourier-transform infrared (FT–IR) spectroscopy and zeta potential analysis. The effects of in vitro cell viability in Fe3O4@Glu/PTSC nanoparticulate indicated the anti-proliferative properties in a dose-dependent manner (IC₅₀ = 135.6?μM/mL). The high selectivity for tumor cells and far below of activity in HEK293 non-tumorigenic cells is considered as an important feature for this complex (SI, 3.48). Based on the results, PTSC failed to reveal any activity against A549 cells alone. However, Fe₃O₄ nanoparticulates had some effects in inhibiting the growth of lung cancer cell. Furthermore, Bax and Bcl-2 gene expressions were quantified by real-time PCR method. The expression of Bax increased 1.62-fold, while the expression of Bcl-2 decreased 0.76-fold at 135.6?μM/mL concentration of Fe₃O₄@Glu/PTSC compared to untreated A549 cells. Furthermore, the Fe₃O₄@Glu/PTSC nanoparticulate-inducing apoptosis properties were evaluated by Hoechst 33258 staining, Caspase-3 activation assay and Annexin V/propidium iodide staining. The results of the present study suggest that Fe₃O₄@Glu/PTSC nanoparticulates exhibit effective anti-cancer activity against lung cancer cells.

Full text of DOI:10.1007/s00775-019-01728-4

JBIC Journal of Biological Inorganic Chemistry
https://doi.org/10.1007/s00775-019-01728-4
ORIGINAL PAPER
Novel pyridinecarboxaldehyde thiosemicarbazone conjugated
magnetite nanoparticulates (MNPs) promote apoptosis in human lung
cancer A549 cells
Alireza Habibi1 · Seyed Ataollah Sadat Shandiz2 · Ali salehzadeh1 · Zeinab Moradi‑Shoeili3
Received: 27 April 2019 / Accepted: 2 October 2019
© Society for Biological Inorganic Chemistry (SBIC) 2019
Abstract
The present study highlights the apoptotic activity of magnetic Fe3O4 nanoparticulates functionalized by glutamic acid and
2-pyridinecarboxaldehyde thiosemicarbazone (PTSC) toward human lung epithelial carcinoma A549 cell line. To this aim,
the Fe3O4 nanoparticulates were prepared using co-precipitation method. Then, the glutamic acid and Fe3O4 nanoparticu-
lates were conjugated to each other. The product was further functionalized with bio-reactive PTSC moiety. In addition, the
synthesized Fe3O4@Glu/PTSC nanoparticulates were characterized by physico-chemical techniques including scanning
electron microscope (SEM), energy dispersive X-ray (EDX), X-ray difraction (XRD), Fourier-transform infrared (FT–IR)
spectroscopy and zeta potential analysis. The efects of in vitro cell viability in Fe3O4@Glu/PTSC nanoparticulate indi-
cated the anti-proliferative properties in a dose-dependent manner (IC50 =135.6 µM/mL). The high selectivity for tumor
cells and far below of activity in HEK293 non-tumorigenic cells is considered as an important feature for this complex (SI,
3.48). Based on the results, PTSC failed to reveal any activity against A549 cells alone. However, Fe3O4 nanoparticulates
had some efects in inhibiting the growth of lung cancer cell. Furthermore, Bax and Bcl-2 gene expressions were quantifed
by real-time PCR method. The expression of Bax increased 1.62-fold, while the expression of Bcl-2 decreased 0.76-fold at
135.6 µM/mL concentration of Fe3O4@Glu/PTSC compared to untreated A549 cells. Furthermore, the Fe3O4@Glu/PTSC
nanoparticulate-inducing apoptosis properties were evaluated by Hoechst 33258 staining, Caspase-3 activation assay and
Annexin V/propidium iodide staining. The results of the present study suggest that Fe3O4@Glu/PTSC nanoparticulates
exhibit efective anti-cancer activity against lung cancer cells.
Keywords Lung cancer · A549 cells · Fe3O4@Glu/PTSC · Apoptosis
Introduction
Non-small cell lung carcinoma (NSCLC) is a heterogeneous
class of tumors which accounts for approximately 85% of all
lung cancer. However, the conventional treatment process
has limited infuence due to the occurence of most diagnostic
Electronic supplementary material The online version of this
cases in advanced stages [1]. Thiosemicarbazones (TSCs)
article (https://doi.org/10.1007/s00775-019-01728-4) contains
are considered as the substantial ligands used as potential
therapeutic agents against lung cancer [2]. Thiosemicarba-
zones have extensive and long-term biomedical applications
due to their potential biological activity including antitumor,
antimicrobial, antifungal, antiviral and antimalarial activity
[3–5]. Thiosemicarbazones induce apoptosis (programmed
cell death) based on caspase activation and BAX/Bcl-2 regu-
lation mechanisms [6, 7]. Because cancer chemotherapy is
always accompanied by some side efects such as fatigue,
nausea/vomiting, diarrhea, febrile neutropenia and rash [8].
supplementary material, which is available to authorized users.
* Seyed Ataollah Sadat Shandiz
Ata.sadatshandiz@iauctb.ac.ir
1
2
3
Department of Biology, Rasht Branch, Islamic Azad
University, Rasht, Iran
Department of Biology, Central Tehran Branch, Islamic Azad
University, Tehran, Iran
Department of Chemistry, Faculty of Sciences, University
of Guilan, P.O. Box 41335–1914, Rasht, Iran
Vol.:(0123456789)
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JBIC Journal of Biological Inorganic Chemistry
Thus, reducing side efects and increasing drug efciency
should be considered for developing tumor treatment [9].
Some recent studies demonstrated the signifcant higher
anti-cancer infuence of thiosemicarbazone-metal complexes
compared to the ligand alone [10, 11]. In another in vivo and
in vitro study, the anti-tumor activity of 2-pyridinecarboxal-
dehyde thiosemicarbazone (PTSC) was confrmed [12]. The
Fe2+ ion can form stable complexes with thiosemicarbazone
chelating agents like other elements such as Cu, Mn, Zn
and Ga [13, 14]. Iron is an essential element for deference
cellular processes including cell proliferation and growth
[15]. Cancer cells are sensitive to iron exclusion due to their
energy generation and DNA synthesis, compared to normal
cells [16]. Magnetic iron oxide (Fe3O4) nanoparticulates
(MIONs) have been widely used as diagnostic felds, bio-
medical and drug delivery agents [17]. MIONs like other
nanoparticulates such as liposomes, polymeric micelles,
dendrimers and colloidal gold have been used in targeted
cancer therapies. The MIONs based on active targeting bind
their ligand to receptors expressed by tumor cells [18]. A
large body of research was conducted to develop new thio-
semicarbazone compounds and their metal complexes as
antitumor agents [19–21]. To the best of our knowledge,
this is the frst report to conduct the simple route for syn-
thesizing novel Fe3O4@Glu/PTSC nanoparticulate as an
efective agent against lung cancer cells. In the present
study, magnetic thiosemicarbazone-Fe3O4 nanoparticulate
(Fe3O4@Glu/PTSC) was synthesized through anchoring
2-pyridinecarboxaldehyde thiosemicarbazone (PTSC) on
the surface of glutamic acid functionalized iron oxide nano-
particulates. The magnetic Fe3O4@Glu/PTSC was character-
ized by several physico-chemical techniques. The inhibitory
concentration (IC50) for A549 cancer cells and cytotoxicity
concentration (CC50) for HEK293 non-tumorigenic cells
were separately determined by methylthiazol tetrazolium
(MTT) assay. Finally, the apoptotic activities of Fe3O4@Glu/
PTSC were analyzed by Hoechst33258 staining, Caspase-3
activation assay, annexin V/propidium iodide staining and
real-time PCR method.
the precipitate was separated by centrifugation and washed
twice with 5–10 mL of diethyl ether.
Fe3O4 and Fe3O4@Glu/PTSC nanoparticulate
synthesis
Fe3O4 nanoparticulates were prepared using the described
co-precipitation method by Zhang [23] and Yazdani et al.
[24]. FeCl3·6H2O (7.57 g) and FeCl2·4H2O (3.17 g) were
mixed in double-distilled water (300 mL) and was stirred
at 75–80 °C for 1 h. Next, NH3 (40 mL) was added to
this mixture and stirred at 75–80 °C for 3 h under nitro-
gen gas. The resulting sediment was separated using the
magnet and washed 2–3 times with distilled water and
dried at 70 °C. To functionalize Fe3O4 nanoparticulates
with l-glutamic acid, IONs were synthesized in the pres-
ence of l-glutamic acid (0.625 g), following the proce-
dure described above. Finally, glutamic acid functional-
ized IONs (1.829 g) and PTSC (0.625 g) were added to
ethanol (20 mL) and the mixture was stirred at 75–80 °C
for 3 h under nitrogen gas to provide Fe3O4@Glu/PTSC
nanoparticulate. After separation and washing procedure,
the resulting precipitate was dried at 70 °C for 8 h.
Determination of Fe3O4@Glu/PTSC nanoparticulates
Fourier-transform infrared (FT–IR) spectra of synthesized
compounds were analyzed by Nicolet IR 100 spectropho-
tometer. The powder X-ray difraction (PXRD) data were
collected with an X-ray difractometer (JEOL JDX-803).
The size, shape and metal amount of modifed Fe3O4@
Glu/PTSC nanoparticulate were determined by scanning
electron microscopy (MIRA3 FEG-SEM) and energy dis-
persive X-ray (EDX) analysis. The physical stability of
Fe3O4 nanoparticulates coated with PTSC was determined
using Zeta potential analyzer.
Cell culture
Materials and methods
The human lung epithelial carcinoma A549 cell line and
HEK293 kidney immortalized cell line were purchased
from Iranian Biological Resource Center. Using estab-
lished protocol by Cree [25], A549 cells were cultured
in RPIM1640 containing 10% fetal bovine serum (FBS)
including 0.05 mg/mL penicillin G and 0.08 mg/mL strep-
tomycin at 37 °C and 5% CO2 in a humidifed incubator. In
addition, according to the protocol reported by Kirschner
et al. [26], the HEK293 cells were grown in Dulbecco’s
modifed Eagle medium and prepared for various tests.
Synthesis of PTSC ligand
PTSC ligand was synthesized using the established method
reported by Kovacevic et al. [22]. In brief, the commer-
cially available thiosemicarbazide (0.91 g) and 2-pyri-
dinecarboxaldehyde (0.95 mL) were added to ethanol
(100 mL). The reaction mixture was stirred at 60–70 °C
for 2 h. Two drops of glacial acetic acid were added to
the mixture and refuxed for 6 h. Then, the solution was
cooled to 5 °C to give a yellow PTSC precipitate. Finally,
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JBIC Journal of Biological Inorganic Chemistry
Hoechst 33258 staining
Cell viability assay
The apoptotic cells were detected by Hoechst 33258 staining
as described previously [33]. The A549 cells were seeded
in six-well plates (2×106 cells/well) and treated with IC50
concentration (135.6 µM/mL) of Fe3O4@Glu/PTSC fol-
lowed by overnight incubation. Then, the cells were col-
lected and washed with PBS. In the next procedure, the cells
were stained with Hoechst 33258 dye at 37 °C for 15 min.
Finally, apoptotic and live cells were expanded on slides and
were distinguished under fuorescence microscope.
MTT assay was conducted to evaluate the cytotoxicity
efects of PTSC, Fe3O4 nanoparticulates and Fe3O4@Glu/
PTSC complex against human lung cancer cell line (A549)
according to the protocol of Zhang et al. [27]. The HEK293
kidney immortalized cell line was used as a control in the
selectivity analysis for non-tumorigenic origin [28]. To cal-
culate the selectivity index (SI) of the Fe3O4@Glu/PTSC,
the equation: CC50 (no cancer cells)/IC50 (cancer cells) was
used [29]. In brief, the cells (2×106 cells/well) were seeded
in 96-well plates. Next, the cells were treated with diferent
concentrations (62.5, 125, 255, 500, 1000 μM/mL) of PTSC,
Fe3O4 nanoparticulates and Fe3O4@Glu/PTSC nanoparticu-
late and incubated at 37 °C with 5% CO2 for 24 h. After-
ward, 0.5 mg/mL of the MTT was added to each well and
the A549 cells were incubated for 4 h. The medium in each
well was replaced by 200 μL of dimethyl sulfoxide (DMSO)
and gentle shaking for 30 min to dissolve the MTT formazan
crystals and the absorbance of the converted dye in living
cells was measured at 590 nm with a microplate reader (Bio-
Rad, Hercules, CA, USA).
Quantitative real‑time PCR
In the present study, mRNA expression levels of Bcl-2 and
Bax genes were evaluated using SYBR Green real-time
quantitative PCR analysis [34–36]. Total cellular RNA
was extracted using TransZol reagent (Transgen Biotech)
according to the manufacturer’s protocol. The cells were
cultured in a 12-well plate. To separate the adherent cells,
washing was done with PBS for three times. The PBS was
discarded and 1 mL TransZol was added into each well and
incubated for a few minutes. After scrapping of, the cells
were transferred to an RNase-free microfuge tube. The cells
were dispersed with pipetting up and down and incubated
for 5 min at room temperature. To provide cellular emul-
sion, 0.1 mL chloroform was added to the microfuge tube
and the cells were shaken for 15 s. Incubation was done
for 3 min at the room temperature of 4 °C and centrifuged
at 10,000 rpm/min, for 10 min and the supernatant were
transferred to a new microfuge tube. Then, isopropyl alcohol
of 0.25 mL was added to microfuge tube and was entirely
mixed. The microfuge tube was incubated for 10 min at the
room temperature of 4 °C and centrifuged at 10,000 rpm/
min for 10 min. After observing some colloidal precipita-
tions at the bottom of the microfuge tube, the supernatant
was discarded and 1 mL 96% ethanol was added into the
tube. After vortexing, the microfuge tube was centrifuged
at 8000 rpm/min, 4 °C for 15 min. The supernatant was
discarded and the ethanol air-dried at the room temperature
for several minutes. The precipitates were dissolved with
1–2 mL of RNA lysate and incubated at the room tempera-
ture for 1 min. Then, the microfuge tube was centrifuged
at 12,000 rpm/min for 1 min. The liquid was divided into
small-volume aliquots and stored at − 80 °C. Finally, the
concentration of extracted RNA was detected at A260/
A230 ratio using a nanodrop spectrophotometer. In addi-
tion, cDNA was synthesized using EasyScript® First-Strand
cDNA Synthesis SuperMix (Transgen Biotech) based on
manufacturer’s instructions and was amplifed within tar-
get and control sequences. In a microtube, 5 µg total RNA,
1 µL Anchored oligo (DT) 18 primer (0.5 µg/µL) and 20 µL
DNase-free water were mixed and incubated at 65 °C for
Cell death assays
Caspase 3 activity assay
The caspase-3 enzyme activity was measured using the
caspase-3 colorimetric Protease Assay Kit (Invitrogen™
Novex™) [30]. In this test, the free chromophore p-nitroan-
ilide (PNA) is generated following the efect of caspase-3
on tetrapeptide substrates DEVD-PNA [31]. The reaction
mixture includes 50 μL of cell extract protein, 50 μL of
2 × reaction bufers (containing 0.5 μL dithiothreitol) and
5 μL of 4 mM DEVD-PNA substrate in a total volume of
105.5 µL. The reaction mixture was incubated at 37 °C for
24 h and the absorbance of the product was measured using
microplate reader (Biotek Epoch, USA) at a wavelength of
405 nm according to manufacturer’s instruction.
Flow cytometry
To distinguish the apoptosis and necrosis of the cancer
cells, a fuorescein isothiocyanate annexin V and propidium
iodide (Annexin V-FITC Apoptosis Detection Kit, Merk)
was assessed according to the manufacturer’s protocol [32].
Briefy, the A549 cells (~106 cells/well) were grown in six-
well plates at 37 °C in a humidifed atmosphere contain-
ing 5% CO2 for 24 h. Afterwards, the cells were exposed to
Fe3O4@Glu/PTSC nanoparticulate with IC50 concentration
with an un-treated A549 cells as a positive control.
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JBIC Journal of Biological Inorganic Chemistry
5 min and put on ice for 2 min. In addition, they were added
to other components including 1 µL EasyScript RT/RI
Enzyme Mix and 10 µL 2×ES Reaction Mix. The resultant
mixture was incubated at 42 °C for 15 min. To inactivate
Enzymes, the mixture was incubated at 85 °C for 5 s and
stored in −80 °C. Primer sequences for the reverse transcrip-
tion reactions were as follows: Bax forward: 5′-TTGCTT
CAGGGTTTCATCCA-3′ and Bax reverse: 5′-AGACAC
TCGCTCAGCTTCTTG-3′, Bcl-2 forward: 5′-TGGCCA
GGGTCAGAGTTAAA-3′ and Bcl-2 reverse: 5′-TGGCCT
CTCTTGCGGAGTA-3′. The glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) was utilized as the housekeeping
gene to normalize within a given sample. Primer sequences
for the reverse transcription reactions were GAPDH forward:
5′-CCCACTCCTCCACCTTTGAC-3′ and GAPDH reverse:
5′-CATACCAGGAAATGAGCTTGACAA-3′. qRT-PCR
reactions were performed using SYBR® Premix Ex Taq™
kit (TaKaRa, Japan) in an ABI 7500 real-time PCR system
(Applied Biosystems, USA) after testing the primer pairs
by Premix Ex Taq (TaKaRa, Japan) and gene length analy-
sis via electrophoresis on a 2.5% agarose gel. The thermal
cycling conditions according to manufacturer’s instructions
were 30 at 95 °C, 60 at 65 °C and 60 s at 72 °C for 40 cycles.
Finally, the reaction was terminated at 4 °C. The reactions
were conducted in triplicate. The relative quantifcations of
Bax and Bcl-2 mRNA expression levels were compared with
the GAPDH mRNA level using the 2−ΔΔCt method.
(C=S) vibration in PTSC structure [39]. The emerged band
at nearby 1700 cm−1 in Fe3O4@Glu/PTSC spectrum refers
to the asymmetric vibrations of glutamate COO-groups [40].
The peaks at 3407 and 3469 cm−1 can be attributed to the
asymmetric (N–H) vibration of the terminal NH2 group [39].
Furthermore, the peaks at 1537 and 1598 cm−1 are related
to the stretching vibration of C=C bonds in the unsaturated
aromatic structure. Therefore, a comparison between FT–IR
peaks in commercial PTSC and synthesized Fe3O4@Glu/
PTSC nanoparticulates revealed the complex successful
formation.
The powder XRD pattern for Fe3O4@Glu/PTSC nano-
particulates is shown in Fig. 1. Based on the XRD analy-
sis, the typical difraction peaks correspond to the refec-
tion planes (111), (220), (311), (222), (400), (422), (511)
and (440) at 2θ degree of 18.63, 29.53, 35.52, 43.04, 52.68,
56.73 and 62.55, respectively, which can be indexed to the
face-centered cubic phase of ferrite. The difraction patterns
of the characteristic peaks are in good agreement with the
data presented previously [41]. The size of the Fe3O4@Glu/
PTSC nanoparticulate was calculated from the most intense
peak (311) by the Scherrer’s equation: D=kλ/βcosθ, where
D indicates the average crystalline size, k shows the Scher-
rer constant (0.89), λ represents the X-ray wavelength used,
β is considered as the angular line width at half maximum
intensity and θ means the Bragg’s angle in degree unit [42].
The average calculated crystallite size was found to be about
8 nm for Fe3O4@Glu/PTSC nanoparticulate.
Statistical analysis
The SEM images of Fe3O4@Glu/PTSC nanoparticulate
(Fig. 2) revealed that the approximately spherical, along
with some moderately agglomerated particles are available
in the sample. In addition, the spherical nanoparticulates
were in the size range of about 20–50 nm. The main reason
for the diference between the measured values in the XRD
and the Scherrer equation and the observed values in the
SEM images can be attributed to the relative accumulation
of synthesized nanoparticulate. As shown in EDX analysis
(Fig. 3), the Fe3O4@Glu/PTSC is composed of Fe, S, O,
ANOVA was used for statistical analysis using Graph Pad
Prism 5.0 software. P<0.05 was considered as the statistical
signifcance. All measurements in the present study were
evaluated in triplicate.
Results
Synthesized complex data analysis
To synthesis magnetic thiosemicarbazone-Fe3O4 nano-
particulate, the glutamic acid and Fe3O4 nanoparticulates
were frst conjugated to each other. The product was further
functionalized with bio-reactive PTSC moiety by covalent
bonding of carboxylic functional groups of the glutamic acid
and NH2-terminuses of PTSC. Figure S1 (supporting infor-
mation) represents the FT-IR spectra of the PTSC and fnal
Fe3O4@Glu/PTSC nanoparticulate, respectively. The signif-
cant absorption at 567 cm−1 and 621 cm−1 is attributed to
stretching frequency of Fe–O [37]. The functionalization of
Fe3O4 nanoparticulates with PTSC ligand can be identifed
by the band of 1641 cm−1, which is related to (C=N) stretch-
ing vibration [38]. The band at 813 cm−1 was assigned to
Fig. 1 XRD patterns of the synthesized Fe3O4@Glu/PTSC nanopar-
ticulates
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JBIC Journal of Biological Inorganic Chemistry
Fig. 2 SEM image of the
synthesized Fe3O4@Glu/PTSC
nanoparticulates
Fig. 3 EDX image of Fe3O4@Glu/PTSC nanoparticulates
C, N atoms, without observing any other impurities. Based
on characterization results, the PTSC ligand was success-
fully conjugated on the surface of IONs. The zeta potential
measurement for the Fe3O4 nanoparticulate-coated PTSC
dissolved in deionized water was about − 23.4 mV which
supplies electrostatic repulsion forces between the nano-
particulates (Fig. S2: Supporting information). In a good
physical colloidal stability, a zeta potential value between
− 30 and + 30 mV is appropriate for providing a sufcient
repulsive force between the particles [43].
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JBIC Journal of Biological Inorganic Chemistry
Fe3O4@Glu/PTSC inhibits the growth of A549 cells
Enhancing Fe3O4@Glu/PTSC through activating
caspase‑3
The growth inhibitory concentration (IC50), cytotoxic
concentration (CC50) of Fe3O4@Glu/PTSC nanopartic-
ulate against A549 cells and human embryonic kidney
293 cells (HEK293) were defined using MTT assay. The
CC50 for HEK293 cells was 472.28 μg/mL, while the IC50
for A549 cells was 135.6 μM/mL. In vitro cell viability
effects of Fe3O4@Glu/PTSC nanoparticulate indicated
the anti-proliferative properties in a dose-dependent man-
ner. The Fe3O4@Glu/PTSC nanoparticulates were sig-
nificantly more toxic than PTSC and Fe3O4 nanoparticle
alone (P < 0.001) (Fig. 4a). Furthermore, an increased
survival rate was observed in HEK293 cells at low con-
centrations after treatment for 24 h (Fig. 4b). The calcu-
lated selectivity index against the A549 cell line was 3.48.
The activation of apoptotic properties of nanoparticulate was
evaluated by colourimetric caspase-3 assay [33, 44]. Based
on the results, the caspase-3 activity of A549 cells increased
1.6 times after 24 h Fe3O4@Glu/PTSC treatment compared
to untreated cells (P<0.05) (Fig. 5).
Fe3O4@Glu/PTSC induce apoptosis in A549 cells
To evaluate the efect of Fe3O4@Glu/PTSC on apoptosis
and necrosis, A549 cells were exposed to Annexin V/PI
staining followed by fow cytometry analysis. In the viable
cells (>58%), both Annexin V and PI were negative (down
left), while Annexin V-positive/PI-negative (down right)
or Annexin V/PI double-positive (upright) occurred in the
apoptotic cells, indicating the cells tolerated early (>33%)
or late (nearly 7%) apoptosis, respectively. Increasing the
Fig. 4 Anti-proliferative activ-
150
A
ity of PTSC, Fe3O4 NPs and
Fe3O4 NPs
PTSC
Fe3O4@Glu/PTSC
Fe3O4@Glu/PTSC cell lines.
Cells were grown for 24 h in the
presence above iron complex
and viability was determined by
MTT assay. ***P<0.001 means
a signifcant diference between
A549 cells treated with Fe3O4@
Glu/PTSC. Fe3O4@Glu/PTSC
nanoparticulates indicate the
anti-proliferative properties
in a dose-dependent manner
(red bars, IC50 =135.6 µM/
mL). However, PTSC indicated
no activity against A549 cells
alone (blue bars), while Fe3O4
nanoparticulates present some
efects in inhibiting lung cancer
cell growth (pale green bars)
(a). Dose–response of HEK293
after 24-h treatment with difer-
ent concentrations of Fe3O4@
Glu/PTSC (CC50=472.28).
Thus, an increased survival rate
can be observed in HEK293
cells at low concentrations.
Finally, the cytotoxic efects
of higher concentrations were
observed in the Fe3O4@Glu/
PTSC (b)
100
50
0
1000
500
250
125
62.5
control
Concentrations (µM/ml)
150
100
50
B
***
***
0
1000
500
250
125
62.5
control
concentraꢀon (µM/ml)
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JBIC Journal of Biological Inorganic Chemistry
of lung cancer cells. However, the treated cells indicated a
signifcant change with diferent concentrations of Fe3O4@
Glu/PTSC nanoparticulate. Furthermore, the morphological
changes in the apoptotic cells were observed with an increas-
ing reduction of cellular volume, staining bright and con-
densed or fragmented nucleus gradually in a dose-dependent
manner (Fig. 7).
Fe3O4@Glu/PTSC upregulate BCL‑2 gene
and downregulate BAX gene
Fig. 5 The activity of caspase-3 was assessed by colorimetric analy-
sis. Data are mean SD (vertical bars), caspase 3 activated 1.61 times
when the A549 cells were treated with Fe3O4@Glu/PTSC compared
to untreated cells
Based on the results, the qRT PCR indicated that the mag-
netic Fe3O4 nanoparticulate and PTSC play a signifcant
synergistic role on Bax over-expression. The Bax mRNA
expression in the treated A549 cells increased to 1.62-fold
in 24 h treatments compared to the untreated control cells
(Fig. 8a). However, the Bcl-2 mRNA expression indicated
a signifcant decrease of 0.76-fold at IC50 concentration of
Fe3O4@Glu/PTSC nanoparticulate compared to untreated
control cells (Fig. 8b).
percentage of early and late apoptotic cells treated with
Fe3O4@Glu/PTSC nanoparticulate was considerably higher
than that of the untreated cells (P<0.05) (Fig. 6). Annexin
V-negative and PI-positive stained cells refer to necrotic
cells (less than 3%) (upleft).
Fe3O4@Glu/PTSC increase morphological changes
in A549 cells
Discussion
In the present study, Hoechst 33258 staining assay was used
to study cell death from another aspect. Based on the result,
the untreated A549 cells stayed with a natural morphology
Some studies reported a strong relationship between thio-
semicarbazones and their metal complexes for inhibiting
the growth of lung cancer cell. The results of another study
Fig. 6 Staining the untreated A549 cells and Fe3O4@Glu/PTSC
treated cells was performed with Annexin V/PI, followed by FACS
analysis. The untreated primary living cells were over 94%. The cell
groups included (down left), dual-negative stained cells indicating
live cell group (down right), Annexin V-positive/PI-negative stained
cells referring to early apoptosis (upright), Annexin V/PI dual-posi-
tive stained cells representing late apoptosis (upleft) and Annexin
V-negative and PI-positive stained cells displaying dead cells
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JBIC Journal of Biological Inorganic Chemistry
Fig. 7 The morphology of apoptotic A549 cells was recognized with
Hoechst 33258 staining and observed using fuorescence microscope
(×400). Based on the results, control cells indicate normal morphol-
ogy and 24-h exposure to 135.6 µM/mL Fe3O4@Glu/PTSC (IC50
dose) shows decreased cell volume, condensed chromatin, frag-
mented nuclei and brightly stained cells (scale bar 50 µm)
Fig. 8 Level of BAX (a) and Bcl-2 (b) mRNA expression in the cells
treated with Fe3O4@Glu/PTSC and untreated cells. qRT PCR analy-
sis of apoptosis-related genes indicated that Bax gene were up-reg-
ulated in Fe3O4@Glu/PTSC-treated cells (1.6-fold), but Bcl-2 was
down-regulated (0.76 less than control)
reported the synergistic activity of the copper complexes
of Dp4e4mT and DpC in combination with gemcitabine or
cisplatin against lung cancer cells [45]. In addition, a series
of novel nickel (II) thiosemicarbazone complexes (1–4)
inhibited the migration and metastasis in lung cancer cells
[46]. The toxicity of 2-acetylpyridine-thiosemicarbazones
Ga (III) complex against human fetal lung fbroblast cells
(MRC-5) was shown via Topo-I inhibition and distinct DNA
cleavage [47]. Some of palladium thiosemicarbazone com-
plexes exhibited better activity than their parent thiosemi-
carbazone analogues on A549 cell line [48]. By considering
these studies, the present research aimed to determine the
efect of Fe3O4 magnetite nanoparticulates (MNPs) Pyri-
dinecarboxaldehyde thiosemicarbazone complex called as
Fe3O4@Glu/PTSC on A549 cell line. The present project
was conducted to identify the anti-proliferative activity of
this compound on these cells and compare the inhibitory
concentration (IC50) of the drug with its cytotoxic concentra-
tion (CC50) on cancer cell line and normal cells. In addition,
the mechanism of inhibiting this complex was evaluated,
along with the changes in gene expression of BAX and BCL-
2 in treated cells with Fe3O4@Glu/PTSC. The results indi-
cated that Fe3O4@Glu/PTSC complex has a proper anti-pro-
liferative activity (IC50, 135.6 µM/mL) against the growth
of A549 lung cancer cells after 24-h incubation (Fig. 4a).
However, less toxic efect on the non-tumorigenic HEK293
cells (CC50, 472.28 µM/mL) was observed in the compound
(Fig. 4b). Furthermore, the drug selectivity index of Fe3O4@
Glu/PTSC exhibited a high degree of cytotoxic selectivity
(SI, 3.48), because the SI values greater than 2 can be rec-
ommended in developing anticancer drugs [49]. The com-
puted SI is higher than that of Matesanz et al. (max, 2.46)
for other metal heterocyclic thiosemicarbazone complex on
the A549 cells [50]. Unexpectedly, the thiosemicarbazone
revealed no activity against A549 cells alone, indicating that
this compound is unable to penetrate cells. The fnding is
consistent with that of Hall et al. [51]. Furthermore, Fe3O4
nanoparticulates could prevent from growing lung cancer
cell, which is in line with that of Zhang et al. [52]. The
results can provide further support for the hypothesis that the
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JBIC Journal of Biological Inorganic Chemistry
thiosemicarbazone is efective against A549 tumor cells and
iron oxide nanoparticulates act as drug delivery compounds.
Some examples were presented to illustrate the applications
of Fe3O4 nanoparticulates as drug-delivery vehicles [42, 52,
53]. The ability of PTSC to inhibit the growth of tumor cells
can be correlated with its efcacy to increase Fe efux and
prevent from uptaking Fe by cancer cells [54]. Accordingly,
in the present study, Fe3O4@Glu/PTSC activated apoptotic
pathways due to the PTSC iron chelator feature and tumor
cell death. Therefore, we performed apoptotic confrmatory
experiments to represent its inhibitory activity on grow-
ing and proliferating cancer cell. To evaluate the mode of
apoptosis, the most efective enzyme activity was observed
for caspase-3, which increased 1.6-fold compared with
the control after 24-h incubation with Fe3O4@Glu/PTSC
(Fig. 5). In line with the results of the present study, some
demonstrated that some similar chelators with PTSC have
proximity activity on caspase-3 in other cell lines. Shandiz
et al. [40] demonstrated 1.7-fold activity of caspase 3 via
thiosemicarbazide on the MCF-7 cell line and Qi et al. [55]
reported an increase of 1.5–2.2-fold of caspase via 2-pyri-
dinecarboxaldehyde thiosemicarbazones Ga(III) complexes.
However, no study had focused on the efect of caspase
assay on this complex. These minor diferences are prob-
ably related to the structural properties of Fe3O4@Glu/PTSC
and thiosemicarbazide. Since caspase activation is associ-
ated with h-cytochrome release from the mitochondrion
[56], the enhanced caspase activity in treated cells compared
to untreated cells, indicated that Fe3O4@Glu/PTSC induced
the mitochondrial pathway of apoptosis. Apoptotic cells can
be further determined using biochemical features including
the expression of surface markers such as phosphatidylserine
(PS), as well as the recognition ligand for phagocytosing
dying cells [57]. PS is located inside the plasma membrane
and is considered as a negatively charged phospholipid.
PS exteriorization is a primary characteristic of the early
apoptosis. The transposition of PS can be traced by Annexin
V protein which has a high afnity [58]. Annexin V (+)/
PI (−) staining in the A549 tumor cells after Fe3O4@Glu/
PTSC treatment indicated induced early apoptosis (>33%)
compared to the control cells (0.23%) (Fig. 6). Thus, this
thiosemicarbazone Fe3O4 nanoparticulate compound con-
tains unique properties interpreting Fe3O4@Glu/PTSC as a
potential anti-proliferative agent against A549 lung cancer
cells. Therefore, both caspase activity and Annexin-V data
refer to the fact that Fe3O4@Glu/PTSC activates intrinsic
mitochondrial-mediated apoptosis in cultured lung cancer
cells. Based on the results, PTSC activity conjugated with
Fe3O4 is more than the case conjugated with metal ions such
as Ga(III),as it was reported in the study of Qi et al. [55]. In
addition, using 2-pyridyl derivatives alone can reduce early
apoptosis and induce late apoptosis [54]. Thus, the function-
alization of Fe3O4 nanoparticulates with thiosemicarbazones
can promote early apoptosis in lung cancer cells, indicating
that iron nanoparticulate enhances early apoptosis. Further-
more, it plays a role in drug delivery, which is consistent
with the results of Namvar et al. [59].
The changes in Bax and BCL-2 gene expression are con-
sidered as another factor which may explain the relatively
good correlation between Fe3O4@Glu/PTSC and apopto-
sis. BCL-2 is markedly localized in the outer membrane of
mitochondria and is divided into pro-apoptotic BAX/BAK
proteins, anti-apoptotic proteins, which are the same pro-
survival BCL-2 family proteins (BCL-2, BCL-XL, BCL-W,
MCL-1, A1/BFL-1) and pro-apoptotic BH3-only proteins
[1, 60]. Some BH3-only proteins such as BIM, tBID and
PUMA are directly connected to BAX/BAK and activate
them, which subsequently inhibit the pro-survival BCL-
2-like proteins. Furthermore, it becomes permeable outer
mitochondrial membrane, which allows to release cytosolic
apoptogenic factors such as cytochrome c Smac/DIABLO,
leading to the activation of caspase cascade [60]. Finally, the
extrinsic and intrinsic pathway meets together via activat-
ing caspase-3 and interacting through pro- and antiapop-
totic proteins [61]. The tumor cells survive under undesir-
able stress conditions and overcome apoptotic pathways
by their increased ratio of anti-apoptotic to pro-apoptotic
Bcl-2 proteins [1]. Thus, regulating can be efcient for the
lung cancer treatment by anti-apoptotic Bcl-2. In the pre-
sent study, the modifcation of expressing BAX and Bcl-2
in A459 cell line was performed by Fe3O4@Glu/PTSC via
qRT-PCR. This Nano-complex could reduce the expression
of Bcl-2 mRNA and induce the expression of BAX mRNA,
which is considered as one of the pro-apoptotic molecular
pathways of Fe3O4@Glu/PTSC for preventing from growing
lung cancer cells. In fact, Fe3O4@Glu/PTSC induces can-
cer cell apoptosis and inhibits the growth of NSCLC cells
through regulating the Bcl-2 family proteins and triggering
the caspase-3 activation. However, Fe3O4@Glu/PTSC efect
on other cell lines and the expression of other genes lines,
as well as its efect in vivo on health cells are suggested for
further research.
Conclusion
Based on the results, the novel Fe3O4@Glu/PTSC indicated
promising an anti-proliferation activity against cancer cells.
In addition, the results suggested that the synergistic efect
of PTSC and Fe3O4 nanoparticulates trigger BAX up-reg-
ulation and Bcl 2 down-regulation, which can lead to the
activation of intrinsic apoptosis pathway.
Acknowledgements We express gratefulness to the biology depart-
ment, Islamic Azad University, Rasht, Iran.
1 3

JBIC Journal of Biological Inorganic Chemistry
34. Pearce MC, Gamble JT, Kopparapu PR, O’Donnell EF, Mueller MJ,
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Compliance with ethical standards
Conflict of interest The authors do not have any confict of interest
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