Synthesis of PJOV56, a new quinoxalinyl-hydrazone derivative able to induce autophagy and apoptosis in colorectal cancer cells, and related compounds

Article abstract of DOI:10.1016/j.bmcl.2019.126851

Quinoxaline derivatives are reported as antineoplastic agents against a variety of human cancer cell lines, with some compounds being submitted to clinical trials. In this work, we report the synthesis, characterization and cytotoxicity potential of a new series of quinoxalinyl-hydrazones. The most cytotoxic compound was (E)-2-[2-(2-pyridin-2-ylmethylene)hydrazinyl]quinoxaline (PJOV56) that presented a time-dependent effect against HCT-116 cells. After 48 h of incubation, PJOV56 was able to induce autophagy and apoptosis of HCT-116 cells, mediated by upregulation of Beclin 1, upregulation of LC3A/B II and activation of caspase 7. Apoptosis was induced along with G0/G1 cell cycle arrest at the highest concentration of PJOV56 (6.0 μM). Thus, PJOV56 showed a dose-dependent mode of action related to induction of autophagy and apoptosis in HCT-116 cells.

Full text of DOI:10.1016/j.bmcl.2019.126851

Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of PJOV56, a new quinoxalinyl-hydrazone derivative able to
induce autophagy and apoptosis in colorectal cancer cells, and related
compounds
Sarah Sant'Anna Maranhãoa, Andrea Felinto Mouraa, Augusto César Aragão Oliveiraa,
Daisy Jereissati Barbosa Limaa, Francisco Washington Araújo Barros-Nepomucenoa,b
Carlos Roberto Koscky Paiera, Alessandra Campbell Pinheiroc,
,
⁎
Thais Cristina Mendonça Nogueirac, Marcus Vinícius Nora de Souzac,d, Claudia Pessoaa,
a Laboratory of Experimental Oncology, Center for Research and Drug Development, Federal University of Ceará, Rua Coronel Nunes de Melo 1000, CEP 60430-275
Fortaleza, CE, Brazil
b Institute of Health Sciences, University for International Integration of the Afro-Brazilian Lusophony, CE 060, Km 51, CEP 62785-000 Acarape, CE, Brazil
c Oswaldo Cruz Foundation, Institute of Technology in Drugs - Farmanguinhos, 21041-250 Rio de Janeiro, RJ, Brazil
d Federal University of Rio de Janeiro, Institute of Chemistry, Department of Organic Chemistry, CP 68563, 21945-970 Rio de Janeiro, RJ, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords:
Quinoxaline derivatives are reported as antineoplastic agents against a variety of human cancer cell lines, with
some compounds being submitted to clinical trials. In this work, we report the synthesis, characterization and
cytotoxicity potential of a new series of quinoxalinyl-hydrazones. The most cytotoxic compound was (E)-2-[2-(2-
pyridin-2-ylmethylene)hydrazinyl]quinoxaline (PJOV56) that presented a time-dependent effect against HCT-
116 cells. After 48 h of incubation, PJOV56 was able to induce autophagy and apoptosis of HCT-116 cells,
mediated by upregulation of Beclin 1, upregulation of LC3A/B II and activation of caspase 7. Apoptosis was
induced along with G0/G1 cell cycle arrest at the highest concentration of PJOV56 (6.0 µM). Thus, PJOV56
showed a dose-dependent mode of action related to induction of autophagy and apoptosis in HCT-116 cells.
Quinoxalines
Cytotoxicity
HCT-116 cells
Cell cycle arrest
Quinoxaline is a low molecular weight heterocyclic compound
comprised of fused benzene and pyridine aromatic rings.1 Quinoxaline
derivatives are rarely found in nature, except in fungal Streptomyces
strains. The quinoxaline antibiotic echinomycin (isolated from S. echi-
natus) is composed of two quinoxaline rings attached to a dimeric cyclic
peptide structure. This natural product acts as a sequence-specific DNA
bis-intercalator, which inserts two planar quinoxaline cores between
DNA base pairs and impairs RNA polymerase function.2 The quinoxa-
line cores also prevent Hypoxia Inducible Factor-1 (HIF-1) isoforms
from binding to DNA. HIF-1 is a critical transcription factor for tumor
progression and aggressiveness under hypoxia.3 Not surprisingly,
echinomycin is also an antineoplastic agent.4 Triostins are antibiotics
isolated from S. triostinicus, and show an echinomycin-like structure,
mechanism of action and antitumor activity.5 Along with echinomycin
and triostins, quinoxapeptins are quinoxaline derivatives linked to bi-
cyclic peptides, but produced by the actinomycete fungus Betula pa-
pyrifera. Quinoxapeptins A and B are strong inhibitors of HIV reverse
transcriptase2. All these cyclic peptides are biologically synthesized by
nonribosomal polypetide synthetases (NRPS), whose cDNAs were
cloned and expressed in E. coli for heterologous syntheses of these
natural quinoxaline derivatives.6
Most described quinoxalines are synthetic. There are many rela-
tively simple methods for their syntheses, and this has driven research
on and industrial interest in quinoxaline-derived chemicals. A great
variety of compounds with several structural modifications have been
generated.1 Quinoxaline derivatives encompass a broad range of uses
because of the diversity of quinoxaline ring substituents. Their biolo-
gical activities highlight the great therapeutic potential exhibited by
this family of compounds.7 Different quinoxalines are antimicrobial
agents against bacteria (Mycobacterium tuberculosis, Staphylococcus
aureus, Bacillus subtilis, Proteus vulgaris, Klebsiella pneumoniae), viruses
(Herpes simplex virus, human immunodeficiency virus, cytomegalo-
virus, varicella-zoster virus, influenza virus), fungi (Candida albicans)
and protozoa (Entamoeba histolytica, Leishmania amazonensis, Plasmo-
dium falciparum, Trypanosoma cruzi). Quinoxaline derivatives also show
pharmacological properties in the treatment of diabetes, chronic
⁎ Corresponding author.
E-mail address: cpessoa@ufc.br (C. Pessoa).
https://doi.org/10.1016/j.bmcl.2019.126851
Received 8 July 2019; Received in revised form 7 November 2019; Accepted 20 November 2019
0960-894X/©2019ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:SarahSant'AnnaMaranhão,etal.,Bioorganic&MedicinalChemistryLetters,
https://doi.org/10.1016/j.bmcl.2019.126851

S.S. Maranhão, et al.
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inflammation, glaucoma, atherosclerosis and neurological dis-
orders.1,8,9
7.98–8.39 ppm, while the NeH and N]C stretching vibrations in the IR
spectra were at 3107–3396 and 1560–1594 cm−1, respectively.
The nine synthesized quinoxaline derivatives were first tested
against three human cancer cell lines using an MTT assay.22 In this
round of cytotoxicity evaluation, cell growth inhibition was estimated
for all cell lines treated with the different compounds at a single con-
centration (5.0 µg.mL−1). The percentage growth inhibition (GI%) of
all cells treated with each compound, and its molar concentration are
described in Table 2. Only PJOV54, PJOV55 and PJOV56 exhibited cell
growth inhibition against HL-60 (leukemia), HCT-116 (colorectal
cancer) and OVCAR-8 (ovarian cancer), with GI% ranging from 52.32%
to 98.80%. The remaining compounds showed a GI% below 34.0%.
Only PJOV56 induced a growth inhibition over 80% against all cell
lines tested. Therefore, PJOV56 was selected for further investigation of
Like their natural counterparts, synthetic quinoxalines may also
exert antineoplastic activity on tumor cells. More than sixty tumor cell
lines (including breast, lung and central nervous system cancer cells)
had their growth significantly inhibited by quinoxaline derivatives.1
The antitumor quinoxalines XK469 and SH80 reached Phase I clinical
trials against refractory hematologic cancer,10 advanced neuro-
blastoma11 and advanced solid tumors,12 suggesting their potential as
new chemotherapeutics. However, low efficacy and/or toxicity hin-
dered their development as anticancer drugs for clinical use, reinforcing
the need to discover new compounds.
According to the World Health Organization (WHO), nearly 1 in 6
deaths in 2015 were due to cancer, which accounts for 8.8 million
victims. The global economic cost of cancer in 2010 was estimated at
US$ 1.16 trillion. Cancer is presently the second leading cause of death
globally, and a 70% increase in new cases is predicted over the next two
decades. More than 100 types of cancer have been described, each one
with a specific diagnosis and treatment.13 The most prevalent among
them are; lung (13%), colorectal (10%), prostate (8%) and female
breast cancer (12%). These four types of cancer are responsible for 4
out of every 10 new cases diagnosed worldwide.14,15
its antineoplastic activity. These results suggest
a probable SAR
(structure–activity relationship). For example, in the pyridine ring of
PJOV56, PJOV57 and PJOV58, the position of the nitrogen seems to be
crucial for the biological activity. In the furan and thiophene rings of
PJOV50 and PJOV53, the nitro group is apparently critical for the ac-
tivity, and the change of oxygen for sulfur does not change the biolo-
gical response. Between the pyrrole ring of PJOV55 and imidazole ring
of PJOV54, the replacement of one carbon for nitrogen doesn’t interfere
in the biological activity. Furthermore, this series complies with Li-
pinski’s rules, which comprise a useful parameter in drug discovery.21
In a second round of MTT assays with PJOV56-treated cells, the half
maximal inhibitory concentration (IC50) was estimated in comparison
to untreated cells. Six tumor cell lines and one non-tumor cell line were
tested at 72 h of incubation (Table 3). PJOV56 showed a high cytotoxic
effect against all tumor cell lines, having IC50 values ranging from 0.82
to 7.35 µM. Among the tumor cell lines used, HL-60 and HCT-116 cells
were more sensitive to PJOV56 treatment, showing IC50 values of 0.82
and 1.83 µM, respectively. In L-929 non-tumor cells, PJOV56 also
showed a strong cytotoxic effect (IC50 = 2.27 µM). These data suggest
that PJOV56 possesses a similar, strong antitumor effect on cells in vitro
as the quinoxalinyl-hydrazones previously studied by our group,17 and
poor selectivity against neoplastic cells. PJOV56 selectivity against
tumor cells may be improved by using special drug delivery systems,
such as liposomes, nanotubes or antibody-drug conjugates.23,24
Considering the cytotoxicity data presented here, and the high in-
cidence and aggressiveness of colorectal cancer, the HCT-116 cell line
was chosen to further investigate the cell death mechanism induced by
PJOV56. HCT-116 cells were treated with PJOV56 at different con-
centrations, aiming to estimate IC50 at 24 h and 48 h using the MTT
assay (Fig. 2). At 24 h, PJOV56 has no effect (IC50 > 20 µM) against
HCT-116 cells, while IC50 was 3.04 µM at 48 h treatment. As previously
stated, the IC50 value of PJOV56 was 1.83 µM at 72 h of incubation.
These results suggest that the cytotoxicity of PJOV56 against HCT-116
cells is time-dependent, since an increase in IC50 values was observed
with reduced treatment time. Several studies corroborate these find-
ings, showing that quinoxaline derivatives present anticancer potential
against human breast, lung, central nervous system,1,17 colorectal,25–28
osteosarcoma,29 prostate30 and leukemia cancer cell lines in vitro.31,32
Indeed, some antineoplastic quinoxalines have reached clinical
trials.33–35
Colorectal cancer (CRC) alone caused nearly 774,000 deaths in
2015.13 Current treatment depends on the disease stage, and generally
includes chemotherapy combined with radiotherapy and surgical re-
section. Despite therapeutic advances, CRC patients still have a reduced
life expectancy. The selection of proper treatment strategies according
to new genetic biomarker-based CRC profiling is challenging. Single
nucleotide and microsatellite variations in RAS and BRAF genes must be
evaluated to determine the most appropriate therapeutic intervention.
Other genetic biomarkers such as CD274 (B7-H/ B7H1/B7-H1/PD-L1/
PDL1) and ERBB2 (CD340/HER-2/HER2/NEU) may also be considered.
Indeed, new drugs and protocols are required to target newly dis-
covered colorectal cancer molecular biomarkers.16
Aiming to contribute to the drug discovery of more potent and less
toxic anticancer quinoxalines, our group previously evaluated the cy-
totoxic properties of quinoxalinyl-hydrazones with remarkable in vitro
activity against a range of human tumor cell lines. These derivatives
were synthesized from substituted aromatic benzaldehydes.17 Interest-
ingly, previous studies had revealed that quinoxalines synthesized from
heteroaromatic aldehydes displayed excellent activity in other types of
diseases such as tuberculosis and Chagas disease.18–20 In this study, we
describe the synthesis route, structural characterization and anti-
neoplastic potential of a series of nine novel quinoxalinyl-hydrazones
synthesized from heteroaromatic aldehydes, planned according to Li-
pinski's rule of five.21. Owing to the epidemiologic data and therapeutic
challenges regarding CRC, as well as the cytotoxicity data shown here,
the colonic neoplasm cell line HCT-116 was employed as a model for
deeper investigations on the cell death mechanisms induced by the
most potent derivative synthesized, (E)-2-[2-(2-pyridin-2-ylmethylene)
hydrazinyl]quinoxaline (PJOV56).
The quinoxalinyl-hydrazones (2) were synthesized from reactions of
2-hydrazinylquinoxalines (1) and heteroaromatic aldehydes, in
30–73% yields, as shown in Fig. 1 and Table 1. Characterization of
compound 2 was achieved from NMR, IR and MS spectral data. Briefly,
in the 1H NMR spectra, the signal for the N]CH proton is found at
Subsequent experiments were performed on HCT-116 cells after
48 h of incubation with PJOV56 at three different concentrations (1.5,
3.0 and 6.0 μM), chosen based on the IC50 values obtained in the MTT
assay. The qualitative morphological analyses of HCT-116 cells after
48 h of incubation with different concentrations of PJOV56 found
phenotypic changes in comparison to the negative and positive (dox-
orubicin) controls. The main changes were increased cell size, de-
creased number of mitotic cells and increased number and size of in-
tracellular vesicles. These morphological features were intensified as
PJOV56 concentration increased (Fig. 3A). The finding of larger cells
after PJOV56 treatment was corroborated by flow cytometry counting,
in which the displacement of the treated cell populations (1.5, 3.0 and
Fig. 1. Synthesis route of quinoxalynil-hydrazones derivatives. Reagents and
conditions: (a) N2H4·H2O (80%), EtOH, rt, 48 h, 85%; (b) RCHO, EtOH, rt, 24 h,
30–73%.
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Table 1
Structure, melting point, synthesis yield, partition coefficient predictions and biological activity predictions of quinoxalinyl-hydrazone (2a-i) derivatives.
Compound
R
Melting Point (°C)
Yield (%)
clog P Predictiona
Mutagenic/Tumorigenic Predictiona
2a PJOV51
219–220
53
4.4965
None/None
2b PJOV50
2c PJOV52
290–292
190–193
68
67
3.7645
3.8186
None/None
None/None
2d PJOV53
2e PJOV54
262–263
252–254
67
30
3.2484
2.8441
High/High
None/None
2f PJOV55
225–226
39
3.4064
None/None
2g PJOV56
2h PJOV57
247–248
253–255
37
73
3.6830
3.6290
Low/None
None/None
2i PJOV58
247–249
64
3.6290
None/None
a
Calculated using Data Warrior program (http://www.openmolecules.org/datawarrior).
Table 2
Table 3
Cytotoxic effect of synthetic quinoxaline derivatives (2a-i) at single con-
centration (5.0 µg.mL−1) against three human tumor cell lines (HL60; HCT116
and OVCAR8) after 72 h of incubation using MTT assay.
Cytotoxic effect of PJOV56 at different concentrations (0.08–10 µM) against a
panel of cell lines (HL60; HCT116; SF295; K562; MCF7; HEPG2 and L929) after
72 h of incubation using MTT assay.
Compound (Concentration)
HL60
HCT116
OVCAR-8
Cell Line
PJOV56
Doxorubicin
GI%a
IC50 (µM)a
2a PJOV50 (19.68 µM)
2b PJOV51 (16.72 µM)
2c PJOV52 (20.99 µM)
2d PJOV53 (17.65 µM)
2e PJOV54 (21.00 µM)
2f PJOV55 (21.00 µM)
2g PJOV56 (20.07 µM)
2h PJOV57 (20.07 µM)
2i PJOV58 (20.07 µM)
19.73
0.00
4.01
20.98
0.03
6.98
0.01
5.75
0.00
0.00
HL60
0.82
2.62
3.88
1.83
7.00
7.35
2.27
0.06
0.02
0.46
0.25
0.11
0.17
0.21
0.66
0.00
K562
0.17
0.13
0.03
0.85
1.72
0.29
0.01
0.03
0.03
0.01
0.04
0.17
0.00
9.01
3.56
1.72
5.51
SF295
HCT116
MCF7
HEPG2
L929
12.20
98.80
96.29
94.76
0.00
1.10
0.15
2.16
0.77
27.10
77.17
54.26
80.44
18.84
33.71
1.26
3.95
0.32
1.14
0.13
1.33
0.61
57.44
52.32
97.76
10.15
14.87
2.35
0.54
0.45
0.18
1.56
0.00
a
Data are presented as half maximal inhibitory concentration
a
(IC50)
SEM from three independent experiments performed in triplicate
calculated by Prism 6.0 (GraphPad / Intuitive Software for Science, San Diego,
CA). The IC50 of the compounds was obtained from a non-linear regression of
the cytotoxicity curves. Viability data were normalized to control group (DMSO
0.5% in media). Doxorubicin (0.002–0.2 or 0.06–8 µM) dissolved in DMSO was
used as positive control of the experiment.
Data are presented as percentage of growth inhibition (GI%)
SEM from
three experiments performed in triplicate calculated by Prism 6.0 (GraphPad/
Intuitive Software for Science, San Diego, CA). Viability data were normalized
to control group (DMSO 0.5% in media).
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Fig. 2. Cytotoxic effect of PJOV56 at different con-
centrations (0.08–10 µM) against HCT116 cells after
24, 48 and 72 h of incubation using MTT assay. A,
graphical representation between cell viability and
molar concentration. B, half maximal inhibitory
concentration (IC50)
SEM from three in-
dependent experiments performed in triplicate cal-
culated by Prism 6.0 (GraphPad/Intuitive Software
for Science, San Diego, CA). Viability data were
normalized to control group (DMSO 0.5% in media).
Doxorubicin (0.002–0.2 or 0.06–8 µM) dissolved in
DMSO was used as positive control of the experi-
ment.
Fig. 3. Morphological changes (A), DNA fragmentation (B), membrane integrity (C) and cell viability (D) of HCT116 cells treated with PJOV56 (1.5, 3.0 and
6.0 μmol.L−1) after 48 h of incubation. A, light microscopy (200x) analyses of stained cells with quick panoptic kit (black arrows show mitotic cells, gray arrows
shows apoptotic cells and red arrows shows cytoplasmic vesicle formation). B, C and D, cell viability, membrane integrity and DNA fragmentation were measured by
flow cytometry using propidium iodide. Control group received the same amount of vehicle whose concentration was kept constant (DMSO 0.5% in media).
Doxorubicin (0.2 µM) dissolved in DMSO was used as positive control of the experiment. Data were presented as mean
SEM from three independent experiments
performed in triplicate calculated by Prism 6.0 (GraphPad/Intuitive Software for Science, San Diego, CA). Statistical analyses for multiple comparisons were
performed using One-way ANOVA followed by the Turkey‘s Test. *p < 0.05 was considered statistically significant. (For interpretation of the references to colour in
this figure legend, the reader is referred to the web version of this article.)
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Fig. 4. Autophagy induction analysis of HCT116 cells treated with PJOV56 (3.0 and 6.0 μmol.L−1) after 48 h of incubation. Control group (Vehicle or V) received the
same amount of vehicle whose concentration was kept constant (DMSO 0.5% in media). Rapamycin (RAPA; 1.0 µM) dissolved in DMSO was used as positive control
of the experiment. A, acidic vesicular organelles (AVO) were observed by confocal microscopy using acridine orange. B, autophagic vacuoles were observed by
confocal microscopy using Monodansylcadaverine. C, expression of autophagy proteins (Beclin 1, LC3A/B I and II) by western blot. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of this article.)
6.0 µM) in the FSC and SSC dot plot axes was observed (data not
shown). Doxorubicin-treated cells (0.2 µM) presented morphological
changes indicative of apoptosis, like bleb formation and pyknosis
(Fig. 3A). In PJOV56-treated cells, discrete apoptotic features were only
observed at the highest PJOV56 concentration (6.0 µM) (Fig. 3A).
Based on the morphological data, it’s also appropriate to consider
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that the phenotypic alterations induced by PJOV56 resemble autop-
hagic events characterized by the presence of numerous vesicles in the
cytoplasm.36 Autophagy is a cellular homeostatic process that can im-
pair tumors by limiting cell growth and, when overstimulated, can lead
to inhibition or even regression of tumor growth. Autophagic de-
gradation may lead to cell death associated with apoptosis.37 In fact,
PJOV56 was able to induce autophagy in HCT-116 cells, by promoting
the formation of acidic vesicular organelles (stained with acridine or-
ange), and autophagic vacuoles (stained with monodansylcadaverine),
as found at both tested concentrations (3.0 and 6.0 µM) after 48 h of
incubation (Fig. 4A and B). The induction of autophagy was confirmed
by western blot analysis which showed increased levels of Beclin 1 (at
3.0 µM) and microtubule-associated protein 1B/chain 3 (LC3A/B II; at
3.0 and 6.0 µM) (Fig. 4C). Curiously, decreased levels of Beclin 1 were
observed at the higher concentration of PJOV56 (6.0 μM). It is known
that Beclin 1 is essential for promoting stress-induced autophagy in vivo
and in vitro.38 However, Beclin 1 may be cleaved by caspases through
the mitochondrial release of pro-apoptotic factors. Zhou et al. (2017)
showed a correlation between the under-expression of Beclin 1 and the
induction of apoptosis in colorectal cancer cells by icaritine. Another
study, in which HCT-116 cells were treated with a steroidal saponin,
reported the transition from autophagy to apoptosis after cleavage of
Beclin 1.38–40
induce both apoptotic and autophagic cell death processes in tumor cell
lines. Reiners and coworkers showed that compounds XK469 and SH80
were cytostatic in low concentrations (below 5.0 μM) and cytolytic at
higher concentrations.42
Cell viability and membrane integrity were investigated by flow
cytometry after propidium iodide staining of HCT-116 cells treated with
PJOV56 for 48 h. Total cell counting diminished with increased
PJOV56 concentration when compared to untreated cells (p < 0.05)
(Fig. 5B). Despite the reduction in total cell population, membrane
integrity analysis demonstrated that 1.5 and 3.0 µM treatments did not
reduce the viable cell fraction, nor increase the fraction of non-viable
cells when compared to the control (Fig. 5C). A significant decrease of
viable cells was observed only in the 6.0 µM treatment, and this was
accompanied by an increase in the non-viable cell subpopulation. The
fraction of propidium iodide permeable (non-viable) cells augmented
from 9.46% at 3.0 µM to 31.36% at 6.0 µM, while viable cells decreased
from 90.45% at 3.0 µM to 68.31% at 6.0 µM. These data suggest that
the membrane integrity of the cells is preserved, despite the cytotoxic
effect of PJOV56, a finding corroborated by the morphological analyses
(presented above), that indicate the participation of apoptosis in the
PJOV56 induced mechanism of cell death (at least at the higher con-
centration tested).
PJOV56-induced apoptosis was also investigated by flow cytometry
using the Annexin V binding assay. At the highest concentration
(6.0 µM), PJOV56 was able to significantly induce apoptotic events
(early and late apoptosis; 25.60%), when compared to the untreated
control (0.34%) (Fig. 5A-B). Reinforcing this finding, flow cytometry
also showed significant DNA fragmentation of HCT-116 cells (3.89%) at
6.0 µM PJOV56. As expected, the doxorubicin positive control (0.2 µM)
also induced significant DNA fragmentation (6.05%) (Fig. 3B).
Finally, flow cytometry cell cycle analyses were performed because
Seeking to understand the role of apoptosis in the mechanism of
action of PJOV56, we determined the expression of caspase 7 by wes-
tern blot. Caspase 7 is a key protein in apoptosis, functioning as an
effector enzyme that cleaves target proteins, eventually leading to cell
death. As seen in Fig. 5C, caspase 7 activation was induced by PJOV56
at 6.0 µM, corroborating the previous hypothesis about the link be-
tween autophagy and apoptosis.41
Similarly, quinoxaline derivatives XK469 and SH80 were able to
Fig. 5. Apoptosis induction analysis of HCT116 cells treated with PJOV56 (1.5, 3.0 and 6.0 μmol.L−1) after 48 h of incubation. Control group (Vehicle or V) received
the same amount of vehicle whose concentration was kept constant (DMSO 0.5% in media). A, Graphical representation of phosphatidylserine externalization using
Annexin V-PE/7-AAD apoptosis detection kit by flow cytometry. 7-AAD-positive and annexin V-negative cells were considered necrotic. Annexin V-positive cells
(both 7-AAD positive and negative cells) were considered apoptotic cells. 7-AAD-negative and Annexin V-negative cells were considered viable. B, apoptotic cells’
data were presented as mean
SEM from three independent experiments performed in triplicate calculated by Prism 6.0 (GraphPad/Intuitive Software for Science,
San Diego, CA). Statistical analyses for multiple comparisons were performed using One-way ANOVA followed by the Turkey’s Test. *p < 0.05 was considered
statistically significant. C, expression of caspase 7 and cleaved caspase 7 by western blot.
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Fig. 6. Cell cycle analysis of HCT116 cells treated with PJOV56 (1.5, 3.0 and 6.0 μmol.L−1) after 48 h of incubation by flow cytometry. Cells were stained with
propidium iodide and control group received the same amount of vehicle whose concentration was kept constant (DMSO 0.5% in media). Doxorubicin (0.2 µM) and
paclitaxel (0.1 µM) dissolved in DMSO were used as positive control of the experiment. A, graphical representation of DNA content. B, DNA content’ data were
presented as mean
SEM from three independent experiments performed in triplicate calculated by Prism 6.0 (GraphPad/Intuitive Software for Science, San Diego,
CA). Statistical analyses for multiple comparisons were performed using One-way ANOVA followed by the Turkey’s Test. *p < 0.05 was considered statistically
significant.
previous results suggested proliferation arrest in PJOV56-treated HCT-
116 cells. In this context, the literature has also shown that quinox-
alines are able to induce strong G2/M cell cycle arrest associated with
their apoptotic effect.43,44 Once more, cell subpopulations behaved
differently according to PJOV56 concentration. The histogram of
1.5 µM PJOV56-treated cells was similar to that of vehicle-treated cells
(Fig. 6A). Treatment with 3.0 µM PJOV56 increased the cell sub-
population at S-phase by 22%, while the 6.0 µM treatment caused a
23% increase of cells at G0/G1-phase, in comparison to vehicle-treated
cells (Fig. 6). Regarding cell cycle arrest, PJOV56 presented a dose-
dependent effect, which was different from any effect of either re-
ference drug on HCT-116 cells. These contrasts suggest a different
mechanism of action for PJOV56 (at any concentration) to paclitaxel
(0.1 µM). Other quinoxalines also stopped cell growth, acting as anti-
proliferative agents.25,26,45 For instance, Harakeh and coworkers
showed that high dichloroquinoxaline (5.0 and 10.0 μg.mL−1) con-
centrations were considered toxic to leukemic lineages, whereas lower
concentrations produced antiproliferative effects after incubation for
48 h.30 In addition, we suggest that apoptosis observed after PJOV56
treatment is an event linked with HCT-116 cell cycle arrest.46 Corro-
borating this hypothesis, Zhao, Zhou and Wang showed a correlation
between G0/G1 cell cycle arrest and apoptosis in HCT-116 cells after
treatment with an isoprenoid-substituted flavonoid.47
In summary, we report the cytotoxicity of
a new series of
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quinoxaline derivatives against human cancer cell lines, and that the
compound (E)-2-[2-(2-pyridin-2-ylmethylene) hydrazinyl]quinoxaline
(PJOV56) was the best cytotoxic derivative synthesized. In HCT-116
colorectal cancer cells, high concentrations of PJOV56 induced autop-
hagy and apoptosis correlated with G0/G1 cell cycle arrest.
Interestingly, this quinoxalinyl-hydrazone showed a dose-dependent
mode of action, therefore having great potential as a therapeutic tool in
cancer treatment. Further investigations are necessary to confirm the
antitumor effect of PJOV56 in vivo.
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Declaration of Competing Interest
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The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
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Acknowledgments
We thank federal and state Brazilian research agencies CAPES,
CNPq, FAPERJ and FUNCAP for funding. We also thank Fiocruz for
funding and provisioning the necessary equipment and infrastructure
for chemical synthesis and structural characterization of quinoxalinyl-
hydrazones. The authors also thank the National Cancer Institute
(Bethesda, MD, USA) for the donation of the tumor cell lines used in this
study.
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