A novel sulfonamide derivative as a strong and selective apototic agent against hematological malignancies

Article abstract of DOI:10.1007/s11696-019-00984-7

Currently available chemotherapeutic drugs against hematological malignancies have several adverse effects and are associated with high mortality rates. Thus, in this study we evaluated the cytotoxic effect of 26 new sulfonamide derivatives on acute leuke

Full text of DOI:10.1007/s11696-019-00984-7

Chemical Papers
https://doi.org/10.1007/s11696-019-00984-7
ORIGINAL PAPER
A novel sulfonamide derivative as a strong and selective apototic
agent against hematological malignancies
Álisson Bigolin2 · Mariana F. Maioral1,2 · Natália M. Stefanes1,2 · Alessandra Mascarello3 ·
Louise D. Chiaradia‑Delatorre3 · Ricardo J. Nunes3 · Rosendo A. Yunes3 · Maria Cláudia Santos‑Silva1,2
Received: 12 July 2019 / Accepted: 29 October 2019
© Institute of Chemistry, Slovak Academy of Sciences 2020
Abstract
Currently available chemotherapeutic drugs against hematological malignancies have several adverse efects and are asso-
ciated with high mortality rates. Thus, in this study we evaluated the cytotoxic efect of 26 new sulfonamide derivatives
on acute leukemia and multiple myeloma cells in order to try to discover a new selective and safe compound that might be
used as a prototype for new chemotherapeutic agents. The most cytotoxic compound, DFS16, reduced the cell viability of
K562, Jurkat and MM.1S cells in a concentration- and time- dependent manner and it was signifcantly less cytotoxic to
non-tumor cells. On acute leukemia cells, sulfonamide DFS16 activated intrinsic and extrinsic apoptosis with Bax/Bcl-2
inversion, increased FasR expression and ΔΨm loss. In K562, DFS16 induced apoptosis by caspase-3 activation, while in
Jurkat, it induced AIF release and caspase-3 independent apoptosis. In multiple myeloma, DFS16 induced cell cycle arrest
at the G2/M phase and apoptosis with ΔΨm loss. Altogether, the results suggest that the new sulfonamide derivative DFS16
induces apoptotic-like cell death in acute leukemia and multiple myeloma cells. DFS16 is a promising new molecule that
could be used as a prototype for the development of chemotherapeutics against hematological malignancies.
Keywords Sulfonamide · Acute leukemia · Multiple myeloma · Cytotoxicity · Cell cycle · Apoptosis
Introduction
characterization, diagnosis and prognosis. In this group of
malignancies are included acute leukemias (AL) and mul-
tiple myeloma (MM). AL are characterized by the halting
of normal hematopoietic diferentiation and by the accu-
mulation of immature cells (blast cells) in the bone marrow
(BM) and/or in the peripheral blood (PB) (Allart-Vorelli
et al. 2015; Swerdlow et al. 2016), while MM is a malignant
clonal B cell neoplasm that usually result in the excessive
production of a monoclonal immunoglobulin (protein M) by
the plasma cells (Choudhry et al. 2018; Touzeau et al. 2018).
Despite the diferent protocols available today against these
diseases, the treatment options for AL and MM are not efec-
tive, especially for MM, which is considered as an incur-
able disease. Besides the strong adverse efects associated
with currently available chemotherapeutics, many patients
develop drug resistance, thus the relapse and mortality rates
remain very high (Kadia et al. 2016; Vasekar et al. 2016).
Considering the poor prognosis of hematological
malignancies and the morbidity associated with chemo-
therapeutic drugs (Choudhry et al. 2018; Vasekar et al.
2016), the search for new synthetic molecules with poten-
tial cytotoxic effect against AL and MM is an urgent
Hematological malignancies are abnormal and uncontrolled
proliferations of neoplastic hematopoietic cells and occupy
the ffth place among new diagnosed cases of cancer. They
are considered as heterogeneous diseases as they have sev-
eral sub-classifcations and diferences regarding the disease
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11696-019-00984-7) contains
supplementary material, which is available to authorized users.
* Maria Cláudia Santos-Silva
maria.claudia.silva@ufsc.br
1
Experimental Oncology and Hemopathies Laboratory,
Clinical Analysis Department, Federal University of Santa
Catarina, Florianópolis, SC CEP 88040-900, Brazil
2
Post-Graduation Program in Pharmacy, Health Science
Center, Federal University of Santa Catarina, Florianópolis,
SC CEP 88040-900, Brazil
3
Structure and Activity Laboratory, Chemistry Department,
Federal University of Santa Catarina, Florianópolis,
SC CEP 88040-900, Brazil
Vol.:(0123456789)
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necessity. The aim is to discover a new compound that
could be used as a prototype for the development of new
and easily administered drugs with higher efficiency and
specificity in inducing tumor cell death and little or negli-
gible side effects to normal cells. In this context, sulfona-
mides are synthetic compounds widely used as antibiotics
since 1935 due to its low cost, low toxicity and excellent
activity against bacterial infections. Over time, the use of
sulfonamides has also been extended as anticancer agents
(Parasca et al. 2013). Although sulfonamides have a com-
mon chemical skeleton, there are a variety of structural
alterations that justify the different mechanisms of action
related to their antitumor activity. Among them, the inhi-
bition of carbonic anhydrase, the induction of microtu-
bule impairment, the cell cycle blockage at G1 phase (by
pRb phosphorylation and by decreasing the expression of
cyclin A and B1 and proteins CDK2 and CDC2) and the
inhibition of angiogenesis (Sabt et al. 2018). Methanesul-
fonamide derivatives, for instance, are capable of binding
to cell DNA and induce cytostatic effects. Amsacrine is
an example of methanesulfonamide that is currently used
as a chemotherapeutic in the treatment of lymphomas and
leukemias (Lee et al. 2017).
Experimental
Chemical synthesis
The 26 new synthetic sulfonamide derivatives included in
this study were kindly provided by Dr. Alessandra Mas-
carello and Dr. Louise Domeneghini Chiaradia-Delatore
under the supersvision of Dr. Ricardo José Nunes and Dr.
Rosendo Augusto Yunes, from the Structure and Activity
Laboratory, Chemistry Department, Federal University of
Santa Catarina. Of them, 10 were derived from the 4-(2-ami-
noethyl) benzenesulfonamide (Gly Series) (Scheme 1) and
16 were derived from the 3,3-diphenylpropylamine (DFS
Series) (Scheme 2). Briefy, the compounds were synthe-
sized by 1 mm of the corresponding methyl ester (a or c),
1 mmol of sulfonyl b1-10 or d1-16, respectively, and 1 mmol
of pyridine in dichloromethane (DCM), at room tempera-
ture and under magnetic stirring. Thereafter, the DCM was
evaporated; the crude product was solubilized in methanol
and poured on crushed ice and, fnally, the fnal products
were vacuum fltered and recrystallized with the appropri-
ate solvent when necessary (Mascarello, 2012). The struc-
tures were characterized by nuclear magnetic resonance
spectroscopy (1H), 13 NMR spectra, infrared spectra and
spectrometry of mass. All new sulfonamides are soluble in
dimethylsulfoxide (DMSO).
Taking into advantage the promising data about sulfon-
amides as anticancer agents, this study aimed to evaluate
the cytotoxic effect of 26 new sulfonamide derivatives on
hematological malignancies and to investigate the main
mechanisms involved in cell death induced by the most
effective compounds on AL and MM cell lines.
Cell culture
K562 acute myeloid leukemia (AML) and Jurkat T cell acute
lymphoblastic leukemia (ALL) cell lines were originally pur-
chased from the American Type Culture Collection (ATCC,
Scheme 1 Synthetic route of
sulfonamides derived from the
4-(2-aminoethyl) benzenesul-
fonamide (Gly Series)
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Scheme 2 Synthetic route of
sulfonamides derived from from
the 3,3-diphenylpropylamine
(DFS Series)
USA). MM cell line (MM.1S) was kindly provided by Dr.
Anamika Dhyani (University of Campinas, Brazil). L929
murine fbroblasts were purchased from the Rio de Janeiro
Cell Bank (BCRJ, Brazil). Cells were cultured in Roswell
Park Memorial Institute Medium (RPMI) (GIBCO®, Brazil)
supplemented with 10% heat inactivated fetal bovine serum
(FBS), 100 U/mL penicillin, 100 µg/mL streptomycin and
10 mM HEPES under 5% CO2 humidifed atmosphere, at
37 °C in 25 cm2 and 75 cm2 culture fasks.
Efect on peripheral blood (PB) cells
PB samples from six non-smoking healthy volunteers were
collected at the Polydoro Ernani de São Thiago Univer-
sity Hospital (Florianópolis, Brazil) after approval by the
University Human Research Ethics Committee–CEPSH
No746.486/2014). Mononuclear cells (MC) were isolated
by Ficoll-Hypaque (density = 1.070 g/mL). PBMC (1
× 106 cells/well) were incubated for 24 h with DMSO,
paclitaxel (positive control) or sulfonamide DFS16 and
cell viability was assessed by the MTT assay as previ-
ously described. For the hemolysis test, the red blood cells
(RBC) were isolated by centrifugation and then incubated
with saline (negative control, 0% lysis), distilled water
(positive control, 100% lysis) and increasing concentra-
tions of DFS16. The % hemolysis was calculated using
the following formula (Boyum, 1968):
Viability assays
For the screening, Jurkat (1 × 105 cells/well), K562 and
MM.1S cells (5 × 104 cells/well) were incubated for 24 h
with 100 µM of sulfonamides Gli 1-10 and DFS 1-16 (dis-
solved in DMSO, Merck Millipore®). The same volume of
DMSO was added to control wells and cell viability was
assessed by the methylthiazoletetrazolium (MTT) assay
(Sigma Chemical Co., USA) (Mosman, 1983). Optical
density of control groups (untreated cells) was considered
as 100% of viable cells. Concentration and time-response
curves were carried out with the most cytotoxic compound,
DFS16. K562, Jurkat, MM.1S and L-929 cell lines were
treated with increasing concentrations (1-100 µM) of DFS16
for 24, 48 and 72 h. The IC50 values (half-maximal inhibi-
tory concentrations) were calculated using GraphPad Prism
6 (GraphPad Software®, USA). The selectivity index (SI)
was calculated by dividing the IC50 obtained on L929 cell
line by the IC50 obtained on AL (K562 and Jurkat) and MM
(MM.1S) cell lines. A high selectivity was considered as
SI≥5, a moderate selectivity as 2≤SI<5 and a low selectiv-
ity as IS<2 (Dahham et al. 2015).
[(sample absorbance − negative control absorbance)
% Hemolysis =
× 100.
(positive control absorbance − negative control absorbance)]
Cell cycle analysis
K562, Jurkat and MM.1S cells (5 × 105 cells/well) were
incubated with DFS16 at their respective 24 h IC50. After
12 h, cells were harvested and cell cycle analysis was
assessed according to the kit protocol (PI/RNAse Solu-
tion Kit, Immunostep®). Analysis was performed by fow
cytometry (FACSCantoIITM, Becton–Dickinson Immuno-
cytometry Systems) using the PE channel (496-578 nm)
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and the data were analyzed using Infnicyt software ver-
complemented by Bonferroni or Tukey post hoc tests. Sta-
tistical analyses were performed using GraphPad Prism 6
software.
sion 1.7 (Cytognos®, Spain).
Apoptosis assays
Apoptotic cell death was frst observed by the ethidium bro-
mide (EB) and acridine orange (AO) method. K562, Jur-
kat and MM.1S cells (1 × 106 cells/well) were incubated
for 12 h with sulfonamide DFS16 (24 h IC50). Then, cells
were resuspended with a solution of EB 1% and AO 1%
(1:1), morphological changes were observed in a fuores-
cence microscope (Olympus BX-FLA, Olympus®, Japan)
at 590 nm (emission) and representative felds were photo-
graphed. The DNA fragmentation assay was performed in
K562, Jurkat and MM.1S cells (6 × 106 cells/well) treated
for 24 h with DFS16 (24 h IC50) or paclitaxel (10 μM, used
as a positive control). DNA extraction was performed using
a commercial kit (QIAGEN®, USA) and DNA samples
were separated by electrophoresis in 2% agarose gel and
observed using a transilluminator. Apoptotic cell death was
also assessed by the Annexin V-FITC Apoptosis Detection
kit (Immunostep®) according to the manufacturer’s instruc-
tions. Analysis was performed by fow cytometry as previ-
ously described.
Results and discussion
New sulfonamide derivatives are cytotoxic
against hematological malignant cells
Current therapeutic strategies against hematologic malig-
nancies include a variety of chemotherapeutic drugs and
hormonal agents that aim to disturb cellular homeostasis
and induce neoplastic cells to apoptosis (Sahu et al. 2017).
Although initial remission is often achieved, most patients
progress to relapse as a result of drugs resistance (Hojjat-
Farsangi, 2015, Foo and Michor, 2014). Thereby, the search
for new therapeutic options, including the synthesis of new
molecules as new bioactive compounds, may improve the
prognosis and the long term survival of cancer patients. In
the present study, the initial screening aimed to identify the
cytotoxic efectiveness of 26 new sulfonamide derivatives
on K562, Jurkat and MM.1S cells (Fig. 1a, b, c). The com-
pounds that signifcantly reduced the cell viability when
compared to the control groups (non-treated cells) were DFS
1-5, DFS 7-16, Gli 1, Gli 3-8 and Gli 10 in K562 cell line,
DFS 1-2, DFS 4-6, DFS 8-16 and Gli 1-10 in Jurkat cell line
and DFS 1-5, DFS 7-13, DFS 15-16 and Gli 5 in MM cell.
Considering these results, the fve compounds that pro-
vided the best cytotoxic effect on these three cell lines
(DFS8, DFS12, DFS15, DFS16 and Gli5) were chosen to
be evaluated under diferent concentrations (1–100 µM)
for 24 h. As shown in Fig. 1d, e, f, all the frst selected
sulfonamides reduced the cell viability in a concentration-
dependent manner. The analysis of their chemical structures
(Schemes 1 and 2) demonstrates that the four sulfonamides
from the DFS series (DFS8, DFS12, DFS15 and DFS16)
have an electronegative group at the 4-position of the aro-
matic ring connected to the sulfur portion of the molecule,
which seems to be an important point for the creation of
a pharmacophore. However, the comparison between the
molecular structures of sulfonamides DFS5 (not selected)
and DFS16 (selected), both having a NO2 group at the
4-position of the aromatic ring, reveals that the presence
of this electronegative group only is not enough to improve
their cytotoxic activity. This diference seems to be associ-
ated with the presence of a NO2 group at the 2-position of
the aromatic ring, which gives more activity to the structure.
According to the literature, compounds with IC50 higher
than 30 μM are considered inactive (Burger and Fiebig,
2014). Therefore, based on this criterion and on the fact that
DFS16 was signifcantly more cytotoxic than the other com-
pounds, this sulfonamide was the only derivative selected
Evaluation of apoptotic factors
K562, Jurkat and MM.1S cells (5×105 cells/well) were incu-
bated for 12 h with sulfonamide DFS16 (24 h IC50). For the
evaluation of the mitochondrial membrane potential (ΔΨm),
cells were incubated with a MitoView 633 ™ (Biotium®,
USA) solution (diluted 1:10.000) and analyzed by fow
cytometry as previously described. For protein expression,
cells were fxed and permeabilized with BD Cytofx/Cytop-
erm (BD Biosciences®, USA) (except cells used in FasR
analysis). Subsequently, cells were stained with anti-Bcl-2-
FITC (no 1F-668-T100, Exbio®, USA), anti-Bax-PerCP (no
SC-7480 PercP, Santa Cruz Biotechnology®, USA), anti-
activated-caspase-3-V450 (no 560627, BD Biosciences®,
USA), anti-AIF-FITC (no SC-13116 FITC, Santa Cruz
Biotechnology®, USA), anti-Ki-67-FITC (no 11-5699-41,
eBioscience®, USA), anti-survivin-PE (no SC-17779 PE,
Santa Cruz Biotechnology®, USA) and anti-FasR-PE (no
SC-8009 PE, Santa Cruz Biotechnology®, USA). After
staining, cells were washed and resuspended in PBS for
cytometer analysis as previously described.
Statistical analysis
The results were expressed as mean standard error of the
mean (SEM) and each experiment was repeated at least
three times independently. Statistical analysis was conducted
using the paired t test or ANOVA one-way and two-way,
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Fig. 1 Efect of new sulfonamide derivatives on hematological tumor
cells and non-tumor cells. a–c Screening of the 26 new synthetic sul-
fonamides on K562 (a), Jurkat (b) and MM.1S (c) cells. d–f Cyto-
toxic efect of frst selected sulfonamides DFS8, DFS12, DFS15,
DFS16 and Gli5 on K562 (d), Jurkat (e) and MM.1S (f) cells. g–i
Concentration- and time-response curves of sulfonamide DFS16 on
K562 (g), Jurkat (h) and MM.1S (i) cells. j, k Cytotoxic efect of sul-
fonamide DFS16 on L-929 fbroblasts (j) and PBMC (k). l Hemolytic
efect of DFS16 on RBC. Each fgure represents the mean SEM of
at least three independent experiments. *Diference when compared
to the control groups. #Statistical similarity with the highest cytotoxic
compound in each series. p<0.05, one-way ANOVA followed by
Bonferroni or Tukey post hoc tests or t-test
Table 1 IC50 values calculated for DFS16. IC50 on AL cells K562 and
Jurkat, on MM cells MM-1.S and on normal fbroblasts L929
K562 cells, and found an IC50 of 6.9 μM after 48 h incuba-
tion, which was higher than DFS16 at the same conditions
(IC50 of 4.3 μM). The divergences in cytotoxicity between
the compounds investigated and those described in the lit-
erature are directly related to their structures, as it is well
established that variations in the aromatic rings size and
modifcations of certain substituents are able to modify the
compounds activity.
IC50 (µM)
K562
Jurkat
MM-1.S
L-929
24 h
48 h
72 h
13.0 0.3
4.3 0.4
2.7 0.4
6.1 0.5
4.6 0.6
1.4 0.9
5.9 0.3
2.9 0.6
0.6 0.2
20.9 1.4
7.2 0.7
4.7 0.6
Sulfonamide DFS16 is not cytotoxic to non‑tumor
for further experiments. DFS16 reduced the cell viability
of K562, Jurkat and MM.1S cells in a concentration and
time-dependent manner (Fig. 1g–i), with IC50 values lower
than 15 μM after 24 h treatment and lower than 3 μM after
72 h (Table 1). The strong cytotoxicity of DFS16 seems
to be associated with the presence of a NO2 group at the
2-position of the aromatic ring, as it was the only compound
with an electronegative group at this position. The cytotoxic
efect of sulfonamides has been extensively investigated by
the scientifc community (Hu, 2006; Doungsoongnuen et al.
2011; Pogorzelska et al. 2018). A study conducted by Liu
et al. (2012) investigated the efect of MPSP-001, a benze-
nesulfonamide capable of destabilizing the microtubules, on
cells
Currently available chemotherapeutics are associated with
low specifcity and high morbidity and mortality rates as
they are also cytotoxic to normal cells (Kadia et al. 2016,
Vasekar et al. 2016). Thus, the search for new bioactive
compounds must include their effect on non-neoplas-
tic cells. Our results revealed that sulfonamide DFS16
reduced the cell viability of L929 fbroblasts at a lower
range when compared to hematological malignancies cells
(Fig. 1j). The choice of this cell line was to mimic the
medullar microenvironment which has fbroblasts in its
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composition. The IC50 values (Table 1) in L929 cells were
signifcantly higher when compared to tumor cells. The SI
comparing non-tumor cells (L929) to hematological tumor
cells showed a moderate selectivity over K562 (SI = 1.61)
cells and a high selectivity over Jurkat and MM.1S cells
(SI = 3.41 and 3.62, respectively).
Sulfonamide DFS16 induced cell cycle arrest in MM
cells
Multiple cell signaling pathways can be influenced by
chemical compounds, such as the induction of DNA dam-
age, the disruption of cell cycle progression and the induc-
tion of apoptosis (Galluzzi et al. 2018). Several studies have
reported the interaction of sulfonamides with the microtu-
bule dynamics by preventing the mitotic spindle formation
and resulting in cell cycle blockage (Liu et al. 2012; Fortin
et al. 2011; Pogorzelska et al. 2018; Sabt et al. 2018). In the
present study, sulfonamide DFS16 increased the proportion
of cells at the sub-G0/G1 phase (dead cells) in K562, Jur-
kat and MM.1S cell lines when compared with the control
groups (Fig. 2a, b, c). This result confrms the previously
discussed data that demonstrates that this new compound
induced potent cell death to hematological neoplastic lin-
eages. In addition, DFS16 blocked the G2/M phase of
MM.1S cells, as refected by a signifcant 10% increase in
the percentage of cells at this phase when compared with the
control group. As the literature reports the capacity of sul-
fonamide derivatives to modulate the microtubules dynamics
and, therefore, prevent chromosome segregation during cell
division, an event related to the G2/M phase (Pogorzelska
In addition, when DFS16 was evaluated in PBMC
(Fig. 1k), the results demonstrated a 41.5% reduction in
cell viability, which was similar to paclitaxel, a chemother-
apeutic drug already used in clinics. Finally, the impact of
DFS16 on healthy RBC was evaluated to verify whether
its systemic use would be compromised. According to
the literature, a chemotherapeutic that induces hemoly-
sis, however potent, has its therapeutic use limited due
to the adverse efects to the patients (Mocan, 2010). The
results showed that only treatment with 39 µM of DFS16,
a concentration three times higher than the higher IC50 cal-
culated in hematological malignancies (13.0 0.3 µM in
K562) was able to induce signifcant hemolysis on normal
RBC (Fig. 1L). Thus, as the selected sulfonamide did not
cause signifcant hemolysis on RBC even at concentrations
much higher than its IC50, this suggests that it could be
safely administered intravenously.
Fig. 2 Efect of sulfonamide DFS16 on the cell cycle of hemato-
logical tumor cells. a–c The cell cycle of K562 (a), Jurkat (b) and
MM.1S cells (c) treated with DFS16 (IC50) was evaluated after PI
staining by fow cytometry. d K562, Jurkat and MM.1S cells were
incubated for 12 h in the absence (control groups) and in the pres-
ence of DFS16 (IC50) and the medium fuorescence intensity (MFI)
was determined by fow cytometry after incubation with anti-KI67.
Histograms (e) illustrate one experiment, grey line represents control
groups and colored lines represent DSF16 groups. Each point and/or
fgure represents the mean SEM of at least three independent exper-
iments. *p<0.05 when compared to the control groups, t-test or one-
way ANOVA followed by Tukey post hoc test
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et al. 2018; Sabt et al. 2018), this might explain MM.1S
cycle blockage by DSF16. Still, further studies should be
carried out to elucidate whether this compound is inducing
disturbances in the cell cytoskeleton or modulating cyclines
and CDKs involved in cell cycle progression. Additionally,
as after only 12 h incubation, no statistical diference was
observed in the other two cell lines, further experiments
should be conducted at diferent incubation periods to clarify
if DSF16 would then be able to block K562 and Jurkat cells
cycles or if the heterogeneity of hematological malignancies
play a role in the diferent responses to this sulfonamide
derivative.
cell death that maintains the morphology of cellular organi-
zations and whose main biochemical characteristics include
phosphatidylserine exposure, caspase activation and mito-
chondrial depolarization. One of the last apoptotic steps is
the cellular DNA fragmentation in multiple fragments of 180
to 200 base pairs by endonucleases, which form a breaking
pattern called “ladder pattern” when separated on agarose
gel. Finally, there is the formation of prolongations in the
plasma membrane called blebs which lead to cell fragments
called apoptotic bodies. These structures are recognized
as phagocytic targets and phagocytosed by immune cells
(Zhang et al. 2015, Goldar et al. 2015, Galluzzi et al. 2018).
As apoptosis is a regulated type of cell death that results in
low infammatory events, it is a very desirable target in the
search for new compounds with chemotherapeutic potential.
In our study, apoptosis was confrmed by the DNA frag-
mentation assay, as the “ladder pattern” was observed in
DFS16-treated K562, Jurkat and MM.1S cells (Fig. 3b).
Finally, the percentage of apoptotic cells was determined
by fow cytometry by detecting phosphatidylserine exposure
after DFS16 treatment. As expected, the results showed an
increase of 53.7%, 79.8% and 121.6% in Annexin V-positive
K562, Jurkat and MM.1S cells, respectively, after treatment
with the sulfonamide, when compared with the respective
controls (Fig. 3c). As we have discussed, the activation of
pathways that result in apoptosis is very advantageous for
new bioactive compounds as apoptosis is a regulated type
of cell death, therefore resulting in fewer adverse efects
to adjacent cells and, consequently, to less side efects in
clinics (Galluzzi et al. 2018). In the present study, we have
demonstrated by three diferent methodologies that DFS16
induced an apoptotic-like cell death on hematological malig-
nancies cells, which corroborates other studies that reported
the induction of apoptosis in diferent neoplastic cells after
treatment with sulfonamides (Fortin et al. 2011; Lee et al.
2017; Liu et al. 2012; Pogorzelska et al. 2018; Sabt et al.
2018).
Interestingly, as shown in Fig. 2d, DFS16 reduced the
expression of the cell proliferation marker KI67 in Jurkat
cells (16%), but not in K562 or MM.1S. Uncontrolled cell
proliferation is one of the main characteristics of neoplas-
tic cells. KI67 nuclear protein is an important marker of
cycling cells, whether they are healthy or tumoral, and it
is used in clinics to evaluate cancer prognosis (Jaafari-
Ashkavandi et al. 2018). To date, no published articles have
been found demonstrating the infuence of sulfonamides on
KI67 expression. However, because this protein is strongly
associated with cell proliferation, tumor progression and
a poor prognosis especially in patients with difuse large
B-cell lymphoma (Kof et al. 2015), its decreased expression
in Jurkat cells after DFS16 treatment which may indicate
that this compound also has some cytostatic activity. Nev-
ertheless, the non-modulation of KI67 observed in K562
and MM.1S cells could be a resistance mechanism against
DFS16 treatment as, according to the literature, malignant
cells try to replicate more in response to cytotoxic agents
(Galluzzi et al. 2018). Especially in MM.1S cells, as DFS16
induced cell cycle arrest, therefore, a decreased KI67 expres-
sion was expected, an attempt to escape cell death induced
by DFS16 is very likely as this is known to be a very resist-
ant lineage and, in clinics, a very aggressive malignancy.
Sulfonamide DFS16 induced apoptotic‑like cell
death in malignant cells
Sulfonamide DFS16 activates intrinsic and extrinsic
apoptosis
As previously mentioned, disruptions in cell proliferation
and cell cycle arrest are mechanisms desired by new chem-
otherapeutic compounds because if the cell fails to repair
these damages, signaling pathways are initiated so that the
cell can be destroyed (Galluzzi et al. 2018). Morphological
changes resulting from cell death induced by sulfonamide
DFS16 were frst observed by fuorescence microscopy. Fig-
ure 3a shows that K562, Jurkat and MM.1S cells exhibited
late apoptosis characteristics (formation of apoptotic bodies
and loss of membrane integrity) after treatment with DFS16.
K562 cells also exhibited characteristics of cells in initial
apoptosis such as chromatin condensation and pycnosis.
According to the literature, apoptosis is a regulated type of
It is well established that the processes responsible for the
regulation of apoptosis involve two main pathways: extrin-
sic apoptosis or the death receptor pathway, and intrinsic
or mitochondrial apoptosis (Zhang et al. 2015). Intrinsic
apoptosis involves mitochondrial disruption in response to
internal and/or external stimuli. When a signal stimulates
mechanisms that interfere with cellular homeostasis, such
as DNA damage, members of the Bcl-2 family are inhib-
ited or activated. The inhibition of antiapoptotic protein
Bcl-2 and the Bax-facilitated opening of the mitochondrial
permeability transition pore result in a disruption of ΔΨm
and in the release of pro-apoptotic proteins to the cytosol
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Fig. 3 Apoptosis induced by sulfonamide DFS16 on hematological
tumor cells. a K562, Jurkat and MM.1S cells treated with DFS16
(C50) for 12 h were stained with EB/AO and observed in a fuores-
cence microscope. Green cells stained with AO indicate intact cell
membranes, while red cells stained with EB indicate loss of mem-
brane integrity. White arrows indicate cells with morphological
changes suggestive of apoptosis. Scale bars (red lines) represent
100 µM. b Cells were incubated with DFS16 (IC50) for 24 h and
DNA fragmentation was evaluated. c Cells were incubated for 12 h
in the absence (control groups) and in the presence of DFS16 (IC50)
and Annexin V-positive cells were quantifed by fow cytometry. His-
tograms (d) illustrate one experiment, grey line represents control
groups and colored lines represent DSF16 groups. Each point and/or
fgure represents the mean SEM of at least three independent exper-
iments. *p<0.05 when compared to the control groups, t-test or one-
way ANOVA followed by Tukey post hoc test
(Galluzzi et al. 2018). Sulfonamide DFS16 decreased
Bcl-2 expression in MM.1S cells by 14% (Fig. 4a) and
increased Bax expression in both K562 (30%) and Jurkat
(25%) cell lines (Fig. 4b). According to the literature, a
decrease of Bcl-2 antiapoptotic protein or an elevation of
Bax pro-apoptotic protein is, by itself, sufcient to invert
the Bax/Bcl-2 ratio, which favors the pro-apoptotic signal
and results in the activation of apoptosis (Galluzzi et al.
2018, Sabt et al. 2018). DFS16 was able to increase the
Bax/Bcl-2 ratio in all three cell lines (Fig. 4c, d), dem-
onstrating the predominance of Bax in relation to Bcl-2
(K562 = 1.16, Jurkat = 1.30, MM1.S = 1.25). Possibly as
a result of the imbalance between Bcl-2 family members,
treatment with DFS16 resulted in the reduction of ΔΨm
in all three hematological malignancies cell lines: 8.4% in
K562, 44.4% in Jurkat and 37.8% in MM.1S (Fig. 4e, f).
The ΔΨm loss by DFS16 resulted in AIF release on Jurkat
cells (24% increase in AIF expression) (Fig. 4g, h). After
being released, AIF migrates directly to the nucleus, where
it induces DNA condensation and fragmentation indepen-
dently of caspase activation (Sabt et al. 2018). AIF expres-
sion was not increased in K562 and MM.1S cells after
DFS16 treatment, which suggests that other mitochondrial
proteins such as cytochrome c might be involved in cell
death induced by this compound on these two cell lines.
Cytochrome c, the main mitochondrial protein involved in
apoptosis, is located in the space between inner and outer
mitochondrial membranes where it acts as an electron
transporter in the transport chain. When released through
the mitochondria it decreases ATP production and is asso-
ciated with oxidative stress. Then, cytochrome c induces
the formation of apoptosome, a multimeric complex
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Fig. 4 Efect of DFS16 on intrinsic apoptosis. K562, Jurkat and
MM.1S cells were incubated for 12 h in the absence or in the pres-
ence of DFS16 (IC50) and the MFI was determined by fow cytom-
etry. Treated and untreated cells were stained with anti-Bcl-2 and
anti-Bax (a–d) or anti-AIF (g, h), while the ΔΨm was determined
using the MitoView 633 ™ kit (e, f). Dotplots and histograms (c, f,
h) illustrate one experiment, grey line represents control groups and
colored lines represent DSF16 groups. Bax/Bcl-2 ratio (d) was deter-
mined by dividing the MFIBax by the MFIBcl-2 and compared with the
control group. Each point and/or fgure represents the mean SEM of
at least three independent experiments. *p<0.05 when compared to
the control groups, t-test or one-way ANOVA followed by Tukey post
hoc test
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Chemical Papers
Fig. 5 Efect of DFS16 on extrinsic and common apoptosis. K562,
Jurkat and MM.1S cells were incubated for 12 h in the absence or in
the presence of DFS16 (IC50) and the MFI was determined by fow
cytometry. Treated and untreated cells were stained with anti-FasR
(a, b), anti-activated caspase-3 (c, d) or anti-survivin (e, f). Histo-
grams (b, d, f) illustrate one experiment, grey line represents control
groups and colored lines represent DSF16 groups. Each point and/or
fgure represents the mean SEM of at least three independent exper-
iments. *p<0.05 when compared to the control groups, t test or one-
way ANOVA followed by Tukey post hoc test
ATP-dependent that results in the activation of caspase 9
and caspase-3 (Goldar et al. 2015).
As previously discussed, the activation of both the intrin-
sic and the extrinsic apoptosis converge to the activation
of efector caspases, such as caspase-3, -6 and -7 (Galluzzi
et al. 2018). We have demonstrated that sulfonamide DFS16
signifcantly increased active-caspase-3 expression in K562
(18%) and MM1.S (7%) cells (Fig. 5c, d), confrming the
execution of apoptosis and suggesting that cytochrome c
might be involved in these cells’ death mechanisms. While
DFS16 seems to induce very classical intrinsic and extrinsic
apoptosis in K562 cells, in MM.1S cell line, this sulfonamide
seems to activate diferent pathways than those observed on
AL cells. In MM.1S cells, DFS16 induced cell cycle arrest at
G2/M phase and an apoptotic-like cell death with ΔΨm loss,
DNA fragmentation and a slight, however signifcant cas-
pase-3 activation. These diferences may be explained by the
Extrinsic apoptosis is activated by receptors present on
the plasma membrane, such as FasR and TRAIL-R. These
receptors are capable of triggering an intracellular signal-
ing cascade through the cleavage of pro-caspase-8 and -10
and the formation of the death-inducing complex (DISC)
in the cytoplasm, consequently activating caspase-3 (Sabt
et al. 2018). DFS16 increased FasR expression in both K562
(12%) and Jurkat (30%) cells Fig. 5a, b), which indicates
that this sulfonamide derivative is also capable of activate
the extrinsic apoptosis in AL cells. In MM.1S cells, though
FasR modulation was not observed, extrinsic apoptosis
may not be excluded, as other membrane receptors might
be involved in cell death.
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fact that AL is originated from immature cells, while MM
originates from a diferentiated disease and, considering the
heterogeneity of hematological malignancies, suggest that
other pathways, including other types of cell death should be
investigated to elucidate the sulfonamide mechanisms of cell
death in MM. In Jurkat cells, even though DFS16 appears to
activate apoptosis, as demonstrated by ΔΨm loss, increased
expression of Fas, Bax and AIF and DNA fragmentation, no
signifcant change in caspase-3 was observed. Nevertheless,
Jurkat was the only investigated cell line in which a signif-
cant AIF release was observed after DFS16 treatment. As
this protein is known to trigger a caspase-independent cell
death, this might explain the non-activation of caspase-3 by
DFS16 in Jurkat cells and might be one of the mechanisms
of sulfonamides in lymphoblastic malignancies. Addition-
ally, several other proteins released by the mitochondria have
pro-apoptotic activities independently of caspases. Endonu-
clease G, for instance, is involved in DNA fragmentation and
ΔΨm loss and, after release into the cytoplasm, translocates
to the nucleus to execute the apoptotic signal. SMAC pro-
tein also promotes apoptosis by inhibiting the endogenous
inhibitors of caspases, such as survivin, which is consid-
ered an antiapoptotic protein because of its ability to inhibit
caspase-9, -3 and -7 (Galluzzi et al. 2018). Interestingly,
survivin was overexpressed in all three cell lines treated with
sulfonamide DFS16 (Fig. 5e, f). According to the literature,
this protein is overexpressed in most human neoplasms and
is related to cell resistance, therefore, it is associated with a
worse prognosis. Our result indicate that K562, Jurkat and
MM1.S cells are resisting against the cytotoxic efects of
DFS16, however, despite the eforts to escape cell death, this
new sulfonamide is still able to induce these hematological
malignancies cells to die, as we have demonstrated in this
paper, which is a very promising and interesting result.
It is important to highlight that several other possible
routes should be investigated in regard of sulfonamide
DFS16 mechanisms on hematological malignancies, such
as the activation of endoplasmic reticulum stress, the partici-
pation of ROS and the activation of MAP kinases and NFkB
pathways (Galluzzi et al. 2018, Sabt et al. 2018).
activated both intrinsic and extrinsic apoptosis with cas-
pase-3 activation, which indicates a more classical mecha-
nism. In MM, DFS16 induced cell cycle arrest at the G2/M
phase and apoptosis with ΔΨm loss and DNA fragmenta-
tion, which might hypostatize that cell cycle mechanisms are
frst involved in cell death induced by this compound on MM
cells. Finally, DFS16 seems to be selective for malignant
cells and might be administered intravenously. The superior-
ity of sulfonamide DFS16 when compared with the other 25
compounds investigated in this paper, as well as its strong
and selective cytotoxic activity against hematological malig-
nancies, might be explained by its interesting chemical struc-
ture, specially by the presence of an electronegative group
at the 4-position of the aromatic ring connected to the sulfur
portion of the molecule, which seems to be an important
point for the creation of a pharmacophore. Additionally, the
presence of a NO2 group at the 2-position of the aromatic
ring seems to give even more activity to DFS16 structure.
Altogether, the results presented in this paper suggest that
DFS16 is a promising molecule that could be used as a pro-
totype for the development of new chemotherapeutics to
treat hematological malignancies and further experiments
should be conducted to elucidate its cell death mechanisms.
Acknowledgements This study was supported by fellowships from
CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Supe-
rior) (Brazil) and CNPq (National Counsel of Technological Scientifc
Development) (Brazil) n°306682/2015-0.
Compliance with ethical standards
Conflict of interest On behalf of all authors, the corresponding author
states that there is no confict of interest.
References
Allart-Vorelli P, Porro B, Baguet F, Michel A, Cousson-Gélie F (2015)
Haematological cancer and quality of life: a systematic litera-
ture review. Blood Cancer J. 5:305–315. https://doi.org/10.1038/
bcj.2015.29
Boyum A (1968) Isolation of mononuclear cells and granulocytes from
human blood. Scand J Clin Lab Invest 97:77–89
Burger AM, Fiebig HH (2014) Preclinical screening for new anticancer
agents. In: Figg WD, Mcleod HL (eds) Handbook of antican-
cer pharmacokinetics and pharmacodynamics, 4th edn. Humana
Press, Totowa, pp 29–44
Conclusions
The set of results presented in this study suggests that the
novel sulfonamide derivative DFS16 induced apoptotic-like
cell death on diferent hematological malignancies, yet by
the activation of diferent pathways. On ALL cells, sulfona-
mide DFS16 decreased the cell proliferation marker Ki67
and activated extrinsic and intrinsic apoptosis by decreasing
FasR and by inverting Bax/Bcl-2 ratio. This led to ΔΨm loss
and AIF release with no caspase-3 modulation, which sug-
gests a caspase-3-independent apoptosis. On AML, DFS16
Choudhry P, Galligan D, Wiita AP (2018) Seeking convergence and
cure with new myeloma therapies. Trends Cancer 4:567–582.
https://doi.org/10.1016/j.trecan.2018.05.005
Dahham SS, Tabana YM, Iqbal MA, Ahamed MB, Ezzat MO, Majid
AS, Majid AM (2015) The anticancer, antioxidant and antimi-
crobial properties of the sesquiterpene β-caryophyllene from the
essential oil of Aquilaria crassna. Molecules 20:11808–11829.
https://doi.org/10.3390/molecules200711808
Doungsoongnuen S, Worachartcheewan A, Pingaew R, Suksrichavalit
T, Prachayasittikul S, Ruchirawat S, Prachayasittikul V (2011)
1 3

Chemical Papers
Mocan T (2010) Hemolysis as expression of nanoparticles-induced
cytotoxicity in red blood cells. Biotech Mol Bio Nanomed. 1:6–11
Mosman T (1983) Rapid colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxicity assays. J
Immunol Methods 65:55–63
Investigation on biological activities of anthranilic acid sulfona-
mide analogs. EXCLI J 10:155–161
Foo J, Michor F (2014) Evolution of acquired resistance to anticancer
therapy. J. Teoretical Biol 355:10–20. https://doi.org/10.1016/j.
jtbi.2014.02.025
Parasca OM, Gheaţă F, Pânzariu A, Geangalău I, Profre L (2013)
Importance of sulfonamide moiety in current and future therapy.
Rev Med Chir Soc Med Nat Iasi 117:558–564
Fortin S, Wei L, Moreau E, Lacroix J, Côté MF, Petitclerc E, Kotra
LP, Gaudreault RC (2011) Substituted phenyl 4-(2-oxoimidazoli-
din-1- yl)benzenesulfonamides as antimitotics. Antiproliferative,
antiangiogenic and antitumoral activity, and quantitative structure-
activity relationships. Eur J Med Chem 46:5327–5342. https://doi.
org/10.1016/j.ejmech.2011.08.034
Pogorzelska A, Żołnowska B, Sławiński J, Kawiak A, Szafrański K,
Belka M, Bączek T (2018) Synthesis of 2-alkylthio-N-(quinazolin-
2-yl) benzenesulfonamide derivatives: anticancer activity, QSAR
studies, and metabolic stability. Monatsh Chem 149:1885–1898.
https://doi.org/10.1007/s00706-018-2251-6
Galluzzi L, Vitale I, Aaronson SA, Abrams JM, Adam D, Agostinis P
et al (2018) Molecular mechanisms of cell death: recommenda-
tions of the Nomenclature Committee on Cell Death. Cell Death
Difer 25:486–541. https://doi.org/10.1038/s41418-017-0012-4
Goldar S, Khaniani MS, Derakhshan SM, Baradaran B (2015) Molecu-
lar Mechanisms of Apoptosis and Roles in Cancer Development
and Treatment. Asian Pac J Cancer Prev 16(6):2129–2144. https
://doi.org/10.7314/apjcp.2015.16.6.2129
Sabt A, Abdelhafez OM, El-Haggar RS, Madkour HMF, Eldehna WM,
El-Khrisy EEAM et al (2018) Novel coumarin-6-sulfonamides as
apoptotic anti-proliferative agents: synthesis, in vitro biologi-
cal evaluation, and QSAR studies. J Enzyme Inhib Med Chem
33:1095–1107. https://doi.org/10.1080/14756366.2018.1477137
Sahu P, Kashaw SK, Jain S, Sau S, Iyer AK (2017) Assessment of pen-
etration potential of pH responsive double walled biodegradable
nanogels coated with eucalyptus oil for the controlled delivery
of 5-fuorouracil: in vitro and ex vivo studies. J Control Release
253:122–136. https://doi.org/10.1016/j.jconrel.2017.03.023
Swerdlow SH, Campo E, Pileri SA, Harris NL, Stein H, Siebert R
et al (2016) WHO Classifcation of tumours of haematopoietic
and lymphoid tissues, 4th edn. IARC, Lyon
Hojjat-Farsangi M (2015) Novel and emerging targeted-based cancer
therapy agents and methods. Tumour Biol 36:543–556. https://
doi.org/10.1007/s13277-015-3184-x
Hu L (2006) Synthesis and structure-activity relationships of carbazole
sulfonamides as a novel class of antimitotic agents 128 against
solid tumors. J Med Chem 49:6273–6282. https://doi.org/10.1021/
jm060546h
Touzeau C, Maciag P, Amiot M, Moreau P (2018) Targeting Bcl-2
for the treatment of multiple myeloma. Leukemia 32:1899–1907.
https://doi.org/10.1038/s41375-018-0223-9
Jaafari-Ashkavandi Z, Mehranmehr F, Roosta
E (2018)
MCM3and Ki67 proliferation markers in odontogenic cysts and
ameloblastoma. J Oral Biol Craniofac Res 9:47–50
Vasekar M, Rizvi S, Liu X, Vrana KE, Zheng H (2016) Novel immu-
notherapies for hematological malignancies. Curr Mol Pharmacol
9:264–271
Kadia TM, Ravandi F, Cortes J, Kantarjian H (2016) New drugs in
acute myeloid leukemia. Ann Oncol 27:770–778. https://doi.
org/10.1093/annonc/mdw015
Zhang D, Tang B, Xie X, Xiao YF, Yang SM, Zhang JW (2015) The
interplay between DNA repair and autophagy in cancer. Can-
cer Biol Ther 16(7):1005–1013. https://doi.org/10.1080/15384
047.2015.1046022
Kof JL, Ramachandiran S, Bernalmizrachi L (2015) A time to kill:
targeting apoptosis in cancer. Int J Mol Sci 16:2942–2955. https
://doi.org/10.3390/ijms16022942
Lee YC, Chen YJ, Huang CH, Chang LS (2017) Amsacrine-induced
apoptosis of human leukemia U937 cells is mediated by the inhi-
bition of AKT- and ERK-induced stabilization of MCL1. Apop-
tosis 22:406–420. https://doi.org/10.1007/s10495-016-1307-5
Liu ZL, Tian W, Wang Y, Kuang S, Luo XM, Yu Q (2012) A novel
sulfonamide agent, MPSP-001, exhibits potent activity against
human cancer cells in vitro through disruption of microtu-
bule. Acta Pharmacol Sin 33:261–270. https://doi.org/10.1038/
aps.2011.156
Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional afliations.
Mascarello, A (2012) Rational search for bioactive molecules against
diabetes, leukemia and tuberculosis. Doctoral dissertation. Federal
University of Santa Catarina, Florianópolis, Brazil
1 3