A new chalcone derivative with promising antiproliferative and anti-invasion activities in glioblastoma cells

Article abstract of DOI:10.3390/molecules26113383

Glioblastoma (GBM) is the most common and most deadly primary malignant brain tumor. Current therapies are not effective, the average survival of GBM patients after diagnosis being limited to few months. Therefore, the discovery of new treatments for this highly aggressive brain cancer is urgently needed. Chalcones are synthetic and naturally occurring compounds that have been widely investigated as anticancer agents. In this work, three chalcone derivatives were tested regarding their inhibitory activity and selectivity towards GBM cell lines (human and mouse) and a non-cancerous mouse brain cell line. The chalcone 1 showed the most potent and selective cytotoxic effects in the GBM cell lines, being further investigated regarding its ability to reduce critical hallmark features of GBM and to induce apoptosis and cell cycle arrest. This derivative showed to successfully reduce the invasion and proliferation capacity of tumor cells, both key targets for cancer treatment. Moreover, to overcome potential systemic side effects and its poor water solubility, this compound was encapsulated into liposomes. Therapeutic concentrations were incorporated retaining the potent in vitro growth inhibitory effect of the selected compound. In conclusion, our results demonstrated that this new formulation can be a promising starting point for the discovery of new and more effective drug treatments for GBM.

Full text of DOI:10.3390/molecules26113383

molecules
Article
A New Chalcone Derivative with Promising Antiproliferative
and Anti-Invasion Activities in Glioblastoma Cells
Daniel Mendanha 1,2 , Joana Vieira de Castro 1,2, Joana Moreira 3,4, Bruno M. Costa 2,5 , Honorina Cidade 3,4
,
Madalena Pinto 3,4 , Helena Ferreira 1,2,
*
and Nuno M. Neves 1,2,
*
1
3B’s Research Group, I3Bs—Research Institute on Biomaterials, Biodegradables and Biomimetics,
University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and
Regenerative Medicine, AvePark, Parque de Ciência e Tecnologia, Zona Industrial da Gandra, Barco,
4805-017 Guimarães, Portugal; daniel.mendanha@i3bs.uminho.pt (D.M.);
joana.castro@i3bs.uminho.pt (J.V.d.C.)
2
3
ICVS/3B’s-PT Government Associate Laboratory, 4805-017 Guimarães, Portugal; bfmcosta@med.uminho.pt
Laboratory of Organic and Pharmaceutical Chemistry, Department of Chemical Sciences,
Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal;
up201302558@edu.ff.up.pt (J.M.); hcidade@ff.up.pt (H.C.); madalena@ff.up.pt (M.P.)
Interdisciplinary Centre of Marine and Environmental Research (CIIMAR), University of Porto,
Edifício do Terminal de Cruzeiros do Porto de Leixões, Avenida General Norton de Matos s/n,
4450-208 Matosinhos, Portugal
4
5
Life and Health Sciences Research Institute (ICVS), School of Medicine, Campus Gualtar, University of Minho,
4710-057 Braga, Portugal
*
Correspondence: helenaferreira@i3bs.uminho.pt (H.F.); nuno@i3bs.uminho.pt (N.M.N.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Glioblastoma (GBM) is the most common and most deadly primary malignant brain tumor.
Current therapies are not effective, the average survival of GBM patients after diagnosis being
limited to few months. Therefore, the discovery of new treatments for this highly aggressive brain
cancer is urgently needed. Chalcones are synthetic and naturally occurring compounds that have
been widely investigated as anticancer agents. In this work, three chalcone derivatives were tested
regarding their inhibitory activity and selectivity towards GBM cell lines (human and mouse) and a
non-cancerous mouse brain cell line. The chalcone 1 showed the most potent and selective cytotoxic
effects in the GBM cell lines, being further investigated regarding its ability to reduce critical hallmark
features of GBM and to induce apoptosis and cell cycle arrest. This derivative showed to successfully
reduce the invasion and proliferation capacity of tumor cells, both key targets for cancer treatment.
Moreover, to overcome potential systemic side effects and its poor water solubility, this compound
was encapsulated into liposomes. Therapeutic concentrations were incorporated retaining the potent
in vitro growth inhibitory effect of the selected compound. In conclusion, our results demonstrated
that this new formulation can be a promising starting point for the discovery of new and more
effective drug treatments for GBM.
Citation: Mendanha, D.; Vieira de
Castro, J.; Moreira, J.; Costa, B.M.;
Cidade, H.; Pinto, M.; Ferreira, H.;
Neves, N.M. A New Chalcone
Derivative with Promising
Antiproliferative and Anti-Invasion
Activities in Glioblastoma Cells.
Molecules 2021, 26, 3383. https://
doi.org/10.3390/molecules26113383
Academic Editor:
Diego Muñoz-Torrero
Received: 5 May 2021
Accepted: 31 May 2021
Published: 3 June 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Keywords: glioblastoma; chalcone; cell death; drug delivery; liposomes
1. Introduction
Glioblastoma (GBM), the most common malignant type of primary brain tumor
in the central nervous system (CNS), presents specific pathophysiology characteristics,
such as nuclear atypia, increased mitotic activity, microvascular proliferation, and tissue
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
necrosis [
grade IV, the highest into its malignant scale of CNS tumors [
annual incidence rate of 0.59 to 5 per 100,000 people, with studies indicating a rise in
incidence in the last few years [ ]. The mean age of primary GBM patients is 62 years old,
1
,2]. The World Health Organization (WHO) classifies this type of tumor with a
1]. GBM has an approximate
3
and the median overall survival is approximately 14.6 months, revealing the lack of efficacy
of current treatments [4].
Molecules 2021, 26, 3383. https://doi.org/10.3390/molecules26113383
https://www.mdpi.com/journal/molecules

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The currently available treatment consists of the maximal surgical resection of the
tumor, followed by a radiotherapy treatment schedule conjugated with the use of the alky-
lating agent temozolomide (TMZ) [4–6]. The high invasiveness capacity of GBM, together
with its intra- and intertumoral heterogeneity, leads to very different patient responses to
the treatment and makes difficult the development of alternative and effective therapeutic
regimens [7,8]. As recently reviewed, some promising drugs have been investigated in
clinical trials, although with variable efficacy [9]. Consequently, a better alternative to the
current treatment is yet to be established.
The development of new drug-based therapies for GBM is highly dependent on the
ability of the drugs to cross the blood–brain barrier (BBB) and reach the tumor site in
therapeutic concentrations. The BBB is a tight structure responsible for maintaining a
homeostatic environment in the CNS [10
in conjugation with the presence of efflux pumps at the luminal side of the BBB prevents
the crossing of lipophilic molecules, xenobiotics, toxic metabolites, and drugs [12 13].
,11]. The high transendothelial electrical resistance
,
The inability to efficiently cross the biological barriers and the systemic side effects are
two of the main reasons for the limited availability of new chemotherapeutic agents for
GBM treatment.
The use of new strategies to improve the pharmacokinetics and safety of newly devel-
oped as well as established drugs has been intensively investigated in the last decades. One
of the most promising approaches includes the use of nanocarriers to protect and deliver
the therapeutic agents at the tumor site. Among the different nanocarriers developed,
liposomes present distinctive advantages as drug delivery devices, including increased
targeting capacity, long-term efficacy, enhanced drug stability, reduced drug toxicity, and
increased circulation time [14–16].
Natural and synthetic chalcones were intensively explored for a wide range of bio-
logical activities, such as antimicrobial [17], anti-inflammatory [18], antioxidant [19], and
antitumor activity [20]. Considering their simple chemistry, ease of synthesis, and the
large number of replaceable hydrogens facilitating chemical modifications, this class of
compounds was intensively explored for cancer treatment [20]. Regarding their antitumor
effects, several studies have shown that chalcone derivatives can present antiproliferative
activity on cancer cells, and several molecular targets have already been identified and
studied [20–22].
The ability of some types of chalcones and derivatives to induce cell death in cancer
cells can bring a new class of therapeutic agents to GBM treatment. Recently, different
studies using natural and synthetic chalcones against GBM have shown the potential
therapeutic activity of those compounds. Different cell death mechanisms induced by these
molecules have already been described, such as apoptosis mediated by caspases [21,23],
autophagy [21], methuosis [24], and unfolded protein response mediated cell death [25].
We already reported several chalcones with promising growth inhibitory activity in human
cancer cell lines [26
activity in A375-C5 (melanoma), MCF-7 (breast adenocarcinoma), and NCI-H460 (non-
small cell lung cancer) cells [33]. Moreover, the effect of chalcones in NCI-H460
–32]. Among these, chalcones 1–3 (Figure 1) revealed antiproliferative
1–2
demonstrated a pronounced anti-mitotic effect, blocking mitotic progression by affecting
spindle assembly and, therefore, arrested cells at metaphase, culminating in cell death [33].
Considering the potential antitumor activity of these compounds in other cancer cell lines,
we hypothesized that these chalcone derivatives can have a therapeutic effect on GBM
cells, by inducing selective and controlled cell death. Moreover, the ability of the most
promising compound to be encapsulated in liposomes while maintaining their biological
effect was also accessed.

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Figure 1. Chemical structure of chalcones 1–3.
2. Results
2.1. In Vitro Cytotoxic Activity of Chalcones 1–3 on GBM
Chalcones 1–3 were evaluated regarding their potential cytotoxic effect on GBM cells
as well as on a healthy brain cell line. A range of concentrations from 2.5 M to 100 M of
µ
µ
these compounds was tested to verify their effectiveness towards two different GBM cell
cultures, namely human glioblastoma astrocytoma (U87) and murine glioma (GL261) cells,
as well as a non-tumor cell line (bEnd.3), at different time points (Figure 2).
Figure 2. Cytotoxic effect of chalcone derivatives—Cell metabolic activity after treatment (24, 48, and 72 h) with chalcones
), ), and ) on GBM cell lines (U87 and GL261) and a brain endothelial cell line (bEnd.3) by MTS assay. Results are
normalized to control (1% DMSO) and presented as mean ± SEM of three independent experiments.
1
(a
2
(b
3 (c
Compound
in both GBM cell lines at different time points, which was not observed for compound
(Figure 2b). Regarding the chalcone derivative , it was possible to observe a decrease of cell
metabolic activity of GBM cell lines, with a reduction superior to 50% detected only with
the maximal tested concentration of 100 M (Figure 2c). The non-tumor endothelial cell
line bEnd.3, commonly used to recreate the BBB in vitro, showed more resistance towards
chalcone and with a decrease in metabolic activity only at 100 M concentration.
These results evidenced a potential selectivity of these compounds towards GBM cell lines.
1 (Figure 2a) demonstrated the ability to inhibit cell metabolic activity
2
3
µ
1
3
µ

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Moreover, it was possible to observe a dose-response effect on GL261 (p < 0.0001) and
U87 (p < 0.0001) after chalcone
observed in the GL261 cell line (p = 0.0003). Based on these results, the half-maximal
inhibitory concentration (IC50) of chalcone was calculated for the GL261 cell line at 24 h
(25.54 M), 48 h (10.02 M), and 72 h (7.34 M), and the U87 cell line at the same time
points (19.50 M, 16.51 M, and 18.07 M for 24, 48 and 72 h, respectively). Considering
1 treatment (Figure 2a), being a time-dependent effect only
1
µ
µ
µ
µ
µ
µ
the selective and cytotoxic effect observed, the IC50 of chalcone
possible mechanisms of action.
1 was used to explore its
2.2. Impact of Chalcone 1 on GBM Hallmarks
Among the wide range of pharmacologic anticancer therapies that are currently being
investigated or in clinical use, the hallmark targets are the dysregulated proliferation and
highly invasive profiles. The inhibitory effect of chalcone
1 on GBM cell proliferation was
evaluated by assessing the BrdU incorporation during DNA synthesis. Additionally, the
effect on cell viability was assessed by analyzing the membrane integrity and, consequently,
the ability to exclude the dye trypan blue after treatment with the respective IC50 values,
for 24, 48, and 72 h (Figure 3a,b). The invasive profile of U87 treated with chalcone 1 was
assessed by the ability of cells to invade through a Matrigel membrane in response to
chemoattractants (Figure 3c).
Figure 3. Effect of chalcone
1
on GBM hallmarks. Analyses of U87 (
a
) and GL261 (
b) cell proliferation
(left graph) and cell viability (right graph) by BrdU and Trypan Blue assay, respectively. (
c) Analyses
of U87 invasion by Matrigel invasion assay, with representative images of control (CTR; I; 0.25%
DMSO) and chalcone derivative treated (II) with cell nucleus stained with DAPI. Results are presented
as mean
±
SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to CTR.

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A statistical impairment in cell proliferation compared to non-treated cells (control)
was noticeable in both GBM cell lines, after treatment with chalcone 1, at all time points
tested, with a reduction of about 40% in U87 cells and 25% in GL261 cell lines, although
no time-dependent response was observable (p = 0.2404 for U87; p = 0.3277 for GL261)
(
Figure 3a,b: graphs on the left). Considering the total number of viable cells after treatment,
it is possible to observe a significant decrease after 48 h (40
105) and 72 h (35 105)
in U87 cells (Figure 3a: right graph). Similar results after treatment, with a reduction of
24
105 and 88 105 viable cells at 48 and 72 h, respectively, in GL261 cells was observed
(Figure 3b: right graph). After the treatment of U87 cells (a highly invasive cell model of
GBM) with chalcone , a significant reduction (around 50%) of the cells’ invasion capacity
×
×
×
×
1
was observed (p = 0.0005) compared to negative control cells treated with 0.25% DMSO
(chalcone 1 vehicle) (Figure 3c).
2.3. Evaluation of Apoptosis and Cell Cycle Arrest of GBM Cells after Chalcone 1 Treatment
To evaluate the presence of cell death after compound
1 treatment, cells were ex-
posed with a concentration of chalcone corresponding to the 24 h IC50 for 24, 48, and
1
72 h. The Annexin V-FITC/ PI assay was used since it allows to determine if cells are
viable, apoptotic, or necrotic based on the differences in plasma membrane integrity and
permeability (Figure 4).
After chalcone 1 treatment, a clear difference in the morphology and density of cells
was observed by phase-contrast microscopy at all time points. The U87 cells, which in 2D
culture conditions have a star-like morphology, presented a different shape and a noticeable
increase in size after treatment with the tested chalcone derivative (Figure 4a). The GL261
cells respond to treatment by detaching from the flask surface, an indicator of cell death
(data not shown). After the chalcone
1 treatment of the U87 cells, it was possible to observe
an increase in early apoptotic cells after 24 h (p = 0.0267) and 48 h (p = 0.0015) relative
to the control, and an increase in late apoptotic/necrotic cells after 48 h (p = 0.0028) and
72 h (p < 0.0001). Moreover, it was noticeable the decrease in the number of viable cells
at 24 (p = 0.0075), 48 (p < 0.0001), and 72 h (p < 0.0001; Figure 4b,c). Regarding the GL261
cell line, similar results were observed after treatment with chalcone 1, with an increase of
early apoptosis cells at 24 (p = 0.03008), 48 (p = 0.0002), and 72 h (p < 0.0001). After 72 h
of treatment, an increase of around 20% of late apoptotic/necrotic cells was also detected
(p < 0.0001) compared to the control condition. The number of viable cells was significantly
lower at 24 (p = 0.0085), 48 (p < 0.0001), and 72 h (p < 0.0001) when compared to control
cells (Figure 4d,e).
Considering the percentage of late apoptotic/necrotic cells in the U87 cell line after
treatment, we explored the possible mechanisms that could be responsible for this process.
The U87 cells were treated with chalcone 1 at a concentration of the 24 h chalcone 1 IC50
and the cell cycle was analyzed by propidium iodide (PI) staining using flow cytometry,
while the expression of caspases was analyzed by the western blot assay (Figure 5).
The DNA content, correlated with the brightness of the stoichiometric dye (PI) de-
tected in each cell, was quantified by flow cytometry after treatment. The increase in PI
staining (Figure 5a,b) showed an arrest of chalcone
after 48 (p = 0.0362) and 72 h (p = 0.0013). The expression of caspases 3, 8, and 9 were
analyzed to access possible apoptotic pathways triggered by chalcone treatment. No
1 treated cells in the G2/M checkpoint
1
significant differences in the expression of early precursor caspases 8 and 9 were detected.
Moreover, only pro-caspase 3 expression was observed in the treated and non-treated
cells, with no cleavage and, consequently, no caspase 3 expression being detected in the
conditions (Figure 5c).

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Figure 4. Cell death analyses after treatment with chalcone
and after treatment with chalcone with the concentrations of the 24 h IC50 for 24, 48, and 72 h (
of U87 ( ) and GL261 ( ) cell viability at different treatment times with chalcone 24 h IC50 concentrations. Graphical
representation of the percentage of U87 ( ) and GL261 ( ) cells in each quadrant of the dot plot. Results are presented as
mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared to CTR.
1. Morphological analyses of U87 cells without (control—CTR)
1
a). Flow cytometry analyses
b
d
1
c
e

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Figure 5. Cell death mechanisms induced by chalcone
24, 48, and 72 h with chalcone 24 h IC50 concentration. (
treatment. ( ) Representative immunoblot of U87 cells treated with chalcone
independent experiments. * p < 0.05, ** p < 0.01, **** p < 0.0001 compared to CTR.
1
. (
b
a
) Cell cycle profile of U87 cells treated or not (control—CTR) for
) Quantification of cells in different phases of the cell cycle after
. Results are presented as mean SD of three
1
c
1
±
2.4. Characterization and Biological Activity of Liposomes Incorporating Chalcone 1
Considering that one of the main limitations of chalcones is their poor water solubility,
the encapsulation of chalcone
1 in a nanocarrier was performed and the biological activity
of the resulting liposomal formulation was accessed (Figure 6).

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Figure 6. Characterization and biological activity of liposomes incorporating chalcone
1
. Analyses of the size distribution (
a),
stability (
chalcone
b
1
), and zeta potential (
c) of liposomes. Morphological analyses of empty liposomes (I) and liposomes incorporating
(II) by STEM ( ). U87 (
d
e) and GL261 (
f
) GBM cells metabolic activity was quantified by MTS assay in the
1
absence (control—CTR) and presence of liposomes incorporating chalcone
1
at concentrations of IC50, IC50, and 2x IC50
2
at the different time points. Results are presented as mean
**** p < 0.0001 compared to CTR.
±
SD of three independent experiments. * p < 0.05, ** p < 0.01,
Regarding the encapsulation efficiency, we were able to load 49.60
into liposomes. The size of liposomes incorporating (107.6 5.4 nm) or not incorporating
(111.6 3.8 nm) chalcone did not present significant differences (p = 0.1095; Figure 6a).
±
6.29% of chalcone
1
±
±
1
Moreover, the two suspensions presented a homogeneous size (polydispersity index—PDI
inferior to 0.1) and a high degree of stability (no significant variation of size) for 21 days
(Figure 6b). Similar (p = 0.0618) and significant negative charges were also presented by both
liposomes without (
−
26.39
±
1.22 mV) and incorporating chalcone
1
(
−
27.75
±
1.38 mV;

Molecules 2021, 26, 3383
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Figure 6c). Moreover, liposomes with this chalcone derivative maintained a spherical
morphology (Figure 6d).
Different concentrations of chalcone
1 encapsulated into liposomes, based on the IC50
previously calculated, were added to the two GBM cell cultures to evaluate if its cytotoxic
potential is maintained. For this evaluation, the cell metabolic activity was analyzed by
MTS assay. After treatment, the U87 cells’ metabolic activity was significantly reduced
in a dose-response manner at the different time points tested (p < 0.0001). The empty
liposomes showed reduced cytotoxicity towards GBM cells. Indeed, despite a maximal of
20% inhibition after 24 h was observed, cells were able to recover their normal metabolic
activity after 48 and 72 h of culture (Figure 6e). Similar results were observed with the
GL261 cell line, being a dose-dependent response observed at 48 (p = 0.0002) and 72 h
(p < 0.0001), although no differences were found at 24 h (p = 0.0877; Figure 6f).
3. Discussion
Despite the resources and the aggressive treatment regimen, GBM patients still present
a very poor prognosis. Between the wide range of pharmacologic anticancer therapies
that are currently being investigated or in use in the clinic, the selectivity and the ability to
induce controlled cell death of carcinogenic cells are crucial steps in the development of
new, safe, and effective pharmacological strategies. From the three chalcone derivatives
tested, chalcone
selectivity observed, chalcone
1
showed the most promising effects (Figure 2). Indeed, considering the
presented higher cytotoxicity in GBM cells comparing to
1
brain endothelial cells, at the same concentrations of the other two derivatives. Moreover,
considering the importance of a thorough assessment of the compounds in different cells
that can model the heterogeneity found in this type of tumor, we observed different time-
dependent responses between the two GBM cell lines considered. Importantly, the IC50
values calculated for chalcone
of compounds against GBM [25
new candidate treatments for cancer, and particularly for GBM, are closely associated with
the ability to inhibit one or more of the cancer hallmarks [36 37]. Therefore, we evaluated
the ability of chalcone to inhibit GBM cell proliferation and invasion (Figure 3). Indeed,
1
are in the range proposed by different studies for this class
,
34] and other cancer cells [33 35]. The identification of
,
,
1
this chalcone derivative was effective in decreasing the deregulated proliferation, and
the high invasion capacity of GBM cells was compromised when treated with chalcone
1
(
Figure 3), showing its potential to inhibit distinct mechanisms of cancer progression.
Although several studies showed the ability of different chalcones and their derivatives to
inhibit the proliferation of glioma cells [34 38], studies covering its impact on invasion are
,
more limited. However, as described by us (Figure 3c), other studies reveal the potential of
chalcone impact on cancer cell invasion. For instance, the regulation of the tropomyosin
1 gene was suggested as a possible mechanism for decreasing the invasion of glioma cells
when exposed to chalcone derivatives [39], but more studies are needed to establish this
link. The use of chalcones for the treatment of different cancers has also shown that they
can reduce cell migration and invasion by reducing MMP-2 and MMP-9 activity through
the downregulation of JNK signaling pathways [40]. Finally, the modulation of NF-κB and
Akt signaling pathways by a chalcone derivative was also proposed as a mechanism of
action for the decrease of colon cancer cell invasion and migration [41].
The evasion of programmed cell death is a key hallmark of cancer that contributes
to tumor formation and progression [36]. The blockage of cell death in cancer cells is
closely associated with chemotherapeutic resistance, and the inability to maintain an intact
signal transduction pathway leading to cell death [42]. Therefore, the ability of chalcone
1
to induce GBM cell death was evaluated, demonstrating its effectiveness in inducing
GBM cell death (Figure 4). Moreover, the detection of a large percentage of apoptotic
cells instead of necrotic cells after treatment with chalcone , suggests that this chalcone
1
derivative is promoting a programmed cell death via apoptotic pathways. We observed
an increase of late apoptotic cells over time which demonstrates a continuous response
to treatment. Although different cell death mechanisms have been previously reported

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in GBM in response to chalcone derivatives [24
reported [21
of GBM cells with chalcone
,
25], apoptosis is the most commonly
,
23], as observed in this study (Figure 4). We demonstrated that the treatment
1
led to visible cell morphological alterations (Figure 4a).
Indeed, it is possible to detect an increase in cell debris and morphological changes in the
membrane that are visual indications of cells under apoptosis, as previously reported [43].
The current chemotherapeutic agent used in the clinic, TMZ, presents a mechanism of
action by adding methyl groups at N7 and O6 sites on guanines and O3 position on adenines
leading to deficient DNA replication, a G2/M checkpoint arrest, and, consequently, cell
death [44
,45]. We also observed an arrest of cells in G2/M after treatment (Figure 5a,b),
explaining the decrease of proliferation on chalcone
1
treated cells and showing a possible
mechanism responsible for the activation of apoptotic pathways. Indeed, the arrest in this
checkpoint was already reported for different chalcone derivatives in GBM treatment, being
a commonly reported feature when developing pharmacological anticancer drugs [46].
During apoptosis, two different molecular pathways, extrinsic and intrinsic, can be
activated to induce cell death. In the extrinsic pathway, proapoptotic ligands activate cell
surface receptors associated with cell death that lead to the formation of a death-signaling
inducing complex. The intrinsic pathway is activated inside the cell in response to cell
stress, such as DNA damage or oxidative stress [42]. In both pathways exist a proteolytic
activation of caspases leading to cell changes, such as chromatin condensation, DNA frag-
mentation, or membrane blebbing [42]. Therefore, in this work, after chalcone
1 treatment
the expression of caspases 9 and 8, associated with intrinsic and extrinsic pathways, re-
spectively, and the expression of the effector caspase 3 were analyzed. Interestingly, we
did not observe differences in the caspase expression after treatment, observing similar
levels of expression of caspases 8 and 9 in both the control and treated cells (Figure 5c). The
results can be explained by deregulated apoptosis pathways associated with GBM [42]. The
absence of expression of caspase 3, can suggest that apoptosis is executed by other effector
caspases, such as caspases 6 or 7, or by caspase-independent cell death [47]. However,
more studies are needed to clarify the apoptotic pathways activated after treatment with
chalcone derivatives.
The poor water solubility, the reticulum endoplasmic system, and the inability to
cross the BBB are only some of the reasons that justify the increased emphasis on the
development of drug delivery systems for GBM treatment. Particularly, liposomes have
been used to increase the efficacy of chemotherapeutic agents in GBM for over a decade [48].
Therefore, in this work, a liposomal system able to carry chalcone
1 was developed, using
commonly available phospholipids (Figure 6). This liposome suspension presented long-
term stability with a homogeneous size (PDI < 0.2) around 100 nm. Consequently, they
present key characteristics of nanocarriers used to target GBM (Figure 6a,b) [49,50]. Indeed,
drug delivery devices should present a size <150 nm to enter and exit fenestrated capillaries
in the tumor microenvironment and reach the carcinogenic cells [51]. After incorporation
in liposomes, chalcone
1 was able to maintain the cytotoxic effect. However, the results
suggest that the liposomal formulation can take more time to present similar cytotoxic
effects to free chalcone, which can be related to liposome internalization. Indeed, we
observed an increased cytotoxic effect after 48 and 72 h (Figure 6e,f).
Gathering all the results, the proposed mechanisms of action for chalcone
presented in Figure 7.
1 are

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Figure 7. Impact and mechanism of action of chalcone 1 in GBM cell lines.
4. Materials and Methods
4.1. Reagents
Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), and trypLE Ex-
press were purchased from Life Technologies (Carlsbad, CA, USA). CellTiter 96® AQueous
One Solution was purchased from Promega (Madison, WI, USA). ELISA BrdU (colorimetric)
was acquired from Roche (Basel, Switzerland). BD BioCoatTM MatrigelTM Invasion Cham-
bers were acquired from Corning® (Corning, New York, NY, USA). The dead cell apoptosis
kit with Annexin V-FITC/ PI was obtained from BD Bioscience (Franklin Lakes, NJ, USA).
RNAse A and PI were purchased from Invitrogen (Carlsbad, CA, USA). Anti-caspase 8 and
anti-caspase 9 antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).
IRDye 800CW Goat anti-mouse IgG and IRDye 800CW Goat anti-rabbit IgG were acquired
from LI-COR (Lincoln, NE, USA). Anti-caspase 3 was purchased from Abcam (Cambridge,
UK). L-α-phosphatidylcholine (PC) and anti-vinculin antibody were obtained from Sigma-
Aldrich (St. Louis, MI, USA). 1,2-Dioleoyl-sn-glycerol-3-phosphoserine sodium salt (Lipoid
PS 18:1/18:1) was obtained from Lipoid GmbH (Ludwigshafen, Germany) and LabAssay
Phospholipid was obtained from Wako (Osaka, Japan).
4.2. Chalcone Derivatives
Details concerning the synthesis of chalcones 1–3 (Figure 1) were previously re-
ported [33]. Briefly, to a solution of 3,4-dimethoxyacetophenone (1.01–7.93 mmol) in
methanol (25 mL), an aqueous solution of 40% sodium hydroxide (methanol/water) was
added until pH 13–14, under stirring and on ice. Then, a solution of appropriately sub-
stituted benzaldehyde (2.03–15.86 mmol) in methanol was slowly added to the reaction
mixture. The reaction was submitted to 2 h of microwave irradiation of 180 W of power,
with the final temperature of 69 ◦C. Upon completion, the reaction mixture was poured
into ice and the pH was adjusted to approximately 7 with diluted hydrochloride acid.
For chalcone
1, the resulting residue was filtered, washed with water, and purified by
crystallization with methanol (yield: 81%). For chalcones
2
and , the resulting residue
3

Molecules 2021, 26, 3383
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was taken in chloroform and further rinsed with water, dried over anhydrous sodium
sulfate, evaporated under reduced pressure, and the obtained residue was purified by flash
column chromatography (n-hexane: ethyl acetate, 6:4; yield: 67 and 62%, respectively). The
structure elucidation of compounds was established by 1H and 13C NMR techniques and
data were in accordance with the data previously reported [52
dissolved in DMSO at a stock concentration of 10 mM and kept at
freshly prepared to achieve the desired concentrations before each experiment.
,
53]. Compounds
1–3 were
−
20 ◦C. Dilutions were
4.3. Cell Lines and Culture Conditions
Two different established GBM cell line models, U87 (human) and GL261 (murine),
and a brain endothelial cell line, bEnd.3 (mouse) were used during the experiments. The
cell lines were maintained in DMEM supplemented with 10% FBS and 1% penicillin-
streptomycin and incubated at 37 ◦C in a humidified 5% (v/v) CO2 atmosphere.
4.4. Cell Metabolic Activity
The two GBM cell lines (U87 and GL261) and the brain endothelial cell line (bEnd.3)
were seeded in 96-well plates at 3000 cells/100
(U87) per well. The cells were allowed to adhere for 24 h. Then, they were exposed to
different concentrations of chalcones , and for 24, 48, and 72 h. The control was
µL (GL261 and bEnd.3) and 2000 cells/100 µL
1
,
2
3
performed with 1.0% DMSO (compound vehicle), matching the maximum amount of this
organic solvent used with the tested compounds. The cytotoxicity and the IC50 of chalcone
derivatives were assessed using the MTS assay (CellTiter 96® AQueous One Solution).
Briefly, DMEM without FBS and phenol red was mixed with MTS reagent (5:1 volume
ratio) and added to each condition. Cells were incubated for 3 h at culture conditions and
then the absorbance was read in triplicate at 490 nm using a microplate reader (Synergy
HT, Bio-Tek, Winooski, USA). GraphPad Prism 8 software was used to calculate the IC50
values from three independent experiments, each one in triplicate, applying sigmoidal
dose-response (variable slope) non-linear regression after logarithmic transformation.
The same experimental conditions were used to evaluate the cytotoxicity of liposomes
with chalcone
tion necessary to carry the tested chalcone
1
towards the GBM cell lines. Empty liposomes at the same lipidic concentra-
concentrations were used as controls. Results
1
were normalized to the condition cultured without liposomes (only with medium).
4.5. Cell Viability
The U87 and GL261 GBM cells were plated at an initial density of 1.5
well in 6-well plates and grown in culture conditions. Cells were treated with chalcone
at the respective 24 h IC50 values for 24, 48, and 72 h of culture. At each time point, cells
×
105/2 mL per
1
were washed, trypsinized, and the number of viable cells was counted using Countess II
(Invitrogen, Carlsbad, USA) according to the manufacturer’s protocol.
4.6. Cell Proliferation
The U87 and GL261 cells were plated in 96-well plates at a density of 2000 cells/100 µL
and 3000 cells/100 µL, respectively. Then, they were incubated for 24 h in culture condi-
tions to adhere and grown. After this period, cells were treated with the respective 24 h
IC50 of each cell line or 0.25% DMSO (control) for 24, 48, and 72 h. After incubation, a cell
proliferation ELISA BrdU (colorimetric) was performed according to the manufacturer in-
structions (Roche, Basel, Switzerland). Briefly, cells were labeled by addition of 10
µL/well
of BrdU (final concentration of 10 M) and incubated for two additional hours in culture
µ
conditions, allowing for its incorporation into the DNA. After labeling, the medium was
removed by suction and cells were fixed with FixDenat solution for 30 min. The solution
was removed, and the Anti-BrdU-POD solution was incubated for 90 min at room temper-
ature. The antibody conjugate was removed, and the samples were washed three times
with washing solution. The samples were incubated with substrate solution for 30 min at
room temperature, and the absorbance of the samples was subsequently read at 370 nm

Molecules 2021, 26, 3383
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(reference wavelength of 492 nm) in a microplate reader (Synergy HT, Bio-TEK, Winooski,
USA). The results presented represent three independent experiments (in triplicate), after
removing blank values.
4.7. Cell Invasion
The U87 cell invasion was evaluated using BD BioCoatTM MatrigelTM Invasion Cham-
bers (Corning®, Corning, USA). Briefly, inserts with Matrigel were rehydrated for two
hours at cell culture conditions, using DMEM without FBS. The U87 cells were seeded at
2.5
×
104 cells at the top of each insert, in DMEM supplemented with 1% FBS. DMEM
supplemented with 10% FBS was used in the lower chamber to work as a chemoattractant
to the cells. Cells were incubated for 24 h in cell culture conditions with a concentration of
chalcone 1 corresponding to the 24 h IC50 or 0.25% DMSO (control). After incubation, the
non-invasive cells were removed from the upper part of the membrane by gently swabbing
twice. The invasive cells (placed in the bottom part of the membrane) were fixed with cold
methanol for 2 min and washed two times with PBS. The inserts were left to air dry, and
the membrane was stained with DAPI for 5 min. The membranes were cut from the inserts
and analyzed using an inverted fluorescent microscope (Zeiss, Oberkochen, Germany)
with a coupled camera (Axio Imager Z1m, Zeiss, Oberkochen, Germany). Eight photos
per sample covering a broad area of the membrane at 5x magnification were taken for
nucleus counting. Results are presented as the mean number of cells counted per field,
corresponding to three independent experiments in triplicates.
4.8. Cell Cycle Analyses
The U87 cells were plated at an initial density of 1.5
plates and grown overnight at culture conditions. Cells were treated with 24 h chalcone
respective IC50 for 24, 48, and 72 h. Cells were collected, pelleted, washed, and fixed in
cold ethanol (70%, v/v) for one hour at 4 ◦C. After that, cells were washed twice with PBS
containing 0.1% triton and after centrifugation and elimination of the supernatant, 50
of RNAse A (100 g/mL) was added to the pellet and incubated at room temperature for
15 min. After the incubation period, 200 L of PI (50 g/mL) was added, followed by
×
105/2 mL per well in 6-well
1
µL
µ
µ
µ
flow cytometry analyses. A total of 20,000 events were acquired by a flow cytometer (BD
FACSCalibur, BD Biosciences, New Jersey, USA). The percentage of cells in each phase was
analyzed by FlowJo software (version 7.6). At least three independent biological replicates
were performed.
4.9. Cell Death
Cell death was evaluated after treatment with chalcone
according to the manufacturer’s instructions (BD Biosciences, New Jersey, USA). Briefly,
the U87 and GL261 GBM cells were plated at an initial density of 1.5
105/2 mL per well
in 6-well plates and grown overnight at culture conditions. Cells were treated for 24 h
with chalcone at the respective IC50 for 24, 48, and 72 h. Floating and adherent cells were
collected and centrifugated (300 g, 5 min). After supernatant elimination, the pellet was
resuspended in 100 L of 1x annexin-binding buffer and, then, 5 L of FITC annexin V
and 1 L of PI were added. Samples were incubated for 15 min at room temperature in
the dark. After the incubation period, 400 L of 1x annexin-binding buffer was added to
1 by Annexin V-FITC/ PI,
×
1
×
µ
µ
µ
µ
each sample, followed by flow cytometry analysis. A total of 20,000 events were acquired
by a flow cytometer. The percentage of cells in each phase was analyzed using the FlowJo
7.6 software.
4.10. Western Blot Analyses
U87 cells were seeded in 6 well plates at a cell density of 1
adhere for 24 h. Cells were then treated with chalcone at a concentration corresponding to
the 24 h IC50, for 24, 48, and 72 h. After treatment, cells were washed with PBS and 200
of RIPA buffer (Sigma-Aldrich, St.Louis, USA) was added to each well. Cells were scraped
×
105 per well and left to
1
µL

Molecules 2021, 26, 3383
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and collected from the wells and incubated in ice for 30 min while being vortexed several
times during this period. Lysates were centrifuged at 18,000
the supernatant was collected for protein quantification using the PierceTM BCA Protein
Assay Kit (Thermo Scientific, Waltham, USA). Fifteen g of total protein of each sample
×
g for 16 min at 4 ◦C and
µ
were separated in a 12.5% polyacrylamide gel (120 V) and transferred to a nitrocellulose
membrane using a semi-dry protocol. After protein transference, membranes were blocked
◦
one hour with 5% BSA in 1x TBS-T and incubated overnight at 4 C with primary antibodies
of anti-caspase 3 (1:5000), anti-caspase 8 (1:200), anti-caspase 9 (1:200), and anti-vinculin
(1:200) prepared in the blocking solution. Then, the membranes were washed three times
with TBS-T, and the secondary antibodies, namely IRDye 800CW Goat anti-mouse IgG
(1:15,000) and IRDye 800CW Goat anti-rabbit IgG (1:15,000), were added to the membrane
for one hour at room temperature in the dark. After incubation, the membranes were
washed three times for 5 min with TBS-T, and the protein bands fluorescence analyzed
using the Odyssey Fc imaging system (LI-COR, Lincoln, USA).
4.11. Liposomes Production and Characterization
4.11.1. Liposomes Preparation
Liposomes were prepared by the thin-film hydration method followed by extru-
sion [54]. Briefly, a lipid film of phosphatidylcholine/phosphatidylserine at a ratio of 7:3
(n/n) was obtained after ethanol and dichloromethane complete evaporation in a rotatory
evaporator. Then, the film was hydrated with PBS (pH 7.4) and vigorously vortexed to
obtain multilaminar vesicles (MLVs). The MLVs were subsequently extruded 23 times
through polycarbonate filters of 0.1
µm pore diameter, using an Avanti-Mini Extruder
(London, UK). To obtain liposomes incorporating chalcone
1, this compound was solubi-
lized in a solution of ethanol/chloroform (3:1, v/v) and mixed with the lipid solution at
1:25 (n/n). To remove the not encapsulated chalcone, PD-10 desalting columns containing
Sephadex G-25 were used according to the manufactures’ instructions (GE Healthcare,
Chicago, USA).
The determination of phosphatidylcholine concentration in the liposome samples was
performed using an enzymatic method, namely LabAssay Phospolipid (FUJIFILM Wako,
◦
Japan). Briefly, samples were incubated for 5 min at 37 C with Chromogen Substrate, and
the absorbance acquired at 600 nm using a microplate reader.
4.11.2. Encapsulation Efficiency Determination
The determination of chalcone
1 encapsulated in the liposomes was performed by the
total disruption of the liposomes with ethanol followed by measuring the absorbance of
the samples at 347 nm using a microplate reader. The encapsulation efficiency (EE%) was
determined according to the following equation:
EE% = (Wt/Wi) ∗ 100
where Wt is the weight of chalcone
1 present in the liposomes after non-encapsulated
compound separation and Wi corresponds to the initial weight of chalcone
1 added to the
liposome formulation.
4.11.3. Size Distribution and Zeta Potential of the Liposomes
The size, PDI, and zeta potential of liposome suspensions (500 µM) were analyzed by
Dynamic Light Scattering using disposable cuvettes and laser Doppler micro-electrophoresis
using a dip cell, at 25
±
0.1 ◦C, in a Malvern Zetasizer NS (Malvern Instruments, Malvern,
◦
UK) equipment. For storage stability assessment, liposomes were kept at 4 C under static
conditions for 21 days, and at defined time points the previous analyses were performed.
4.11.4. STEM Analyses
Scanning-transmission electron microscopy (STEM) was used to assess the morphol-
ogy of the liposomes. The liposome samples were diluted at a final lipidic concentration

Molecules 2021, 26, 3383
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of 20
µM in HEPES solution to avoid the presence of PBS salts after samples drying.
The samples were placed in carbon film grids, left to dry overnight, and analyzed by
High-Resolution Field Emission Scanning Electron Microscopy (Auriga Compact, ZEISS,
Oberkochen, Germany).
4.12. Statistical Analyses
GraphPad Prism 8 software (GraphPad Software Inc., San Diego, USA) was used
to perform statistical analyses. Parametric tests were applied, and an independent t-test
was used when comparing two groups. To compare two or more groups at different time
points and with different conditions, a two-way ANOVA was applied, followed by Sidak
´s
multiple comparison tests. Results are presented as normalized mean SD or mean SEM,
±
±
and statistical significance was defined as p < 0.05 for a 95% confidence interval.
5. Conclusions
In this work, we report evidence suggesting a potential new chalcone derivative (
with cytotoxicity as well as antiproliferative and anti-invasion activities towards GBM
cells. Moreover, at therapeutic concentrations, chalcone presented lower cytotoxicity
1)
1
towards the brain endothelial cell line. The induction of apoptosis by the selected chalcone
derivative in GBM cells was triggered by the cell cycle arrest in the G2/M checkpoint.
Chalcone 1 was successfully encapsulated in liposomes while maintaining its therapeutic
activity. Therefore, the development of liposomes incorporating chalcone derivatives has
the potential to provide a new treatment alternative for GBM.
Author Contributions: Conceptualization, D.M., H.C., and H.F.; investigation, D.M., J.V.d.C., and
J.M.; methodology, D.M.; resources, B.M.C. and M.P.; supervision, H.C., M.P., H.F., and N.M.N.;
writing—original draft, D.M.; writing—review & editing, J.V.d.C., H.C., H.F., and N.M.N. All authors
have read and agreed to the published version of the manuscript.
Funding: This research was funded by FCT to the PhD grant of DM fellowship (PD/BD/143038/2018)
and the projects PATH (PD/00169/2013), FROnTHERA (NORTE-01-0145-FEDER-000023), Cells4_IDs
(PTDC/BTM-SAL/28882/2017) and the NORTE 2020 Structured Project, co-funded by Norte2020
(NORTE-01-0145-FEDER-000021). This research was also supported by the Strategic Funding
UIDB/04423/2020 and UIDP/04423/2020 (Group of Natural Products and Medicinal Chemistry-
CIIMAR) through national funds provided by the FCT and ERDF, within the framework of the
program PT2020. Joana Moreira acknowledges her grant (SFRH/BD/135852/2018).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: We gratefully acknowledge the technicians responsible for the Scanning-transmission
electron microscopy at I3Bs. Figure 7 was created with Biorender.com.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are available from the corresponding author.
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