Triarylpyridine compounds and chloroquine act in concert to trigger lysosomal membrane permeabilization and cell death in cancer cells

Article abstract of DOI:10.3390/cancers12061621

Lysosomes play a key role in regulating cell death in response to cancer therapies, yet little is known on the possible role of lysosomes in the therapeutic efficacy of G-quadruplex DNA ligands (G4L) in cancer cells. Here, we investigate the relationship

Full text of DOI:10.3390/cancers12061621

cancers
Article
Triarylpyridine Compounds and Chloroquine Act in
Concert to Trigger Lysosomal Membrane
Permeabilization and Cell Death in Cancer Cells
Jennifer Beauvarlet 1, Rabindra Nath Das 2 , Karla Alvarez-Valadez 3,4, Isabelle Martins 5,
Alexandra Muller 3,6, Elodie Darbo 1, Elodie Richard 1 , Pierre Soubeyran 1,
Guido Kroemer 3,4,6,7,8 , Jean Guillon 2 , Jean-Louis Mergny 2 and
Mojgan Djavaheri-Mergny 1,3,4,
*
1
Institut Bergonié, INSERM U1218, Université de Bordeaux, 33000 Bordeaux, France;
beauvarletj@gmail.com (J.B.); elodie.darbo@gmail.com (E.D.); robertelo@yahoo.fr (E.R.);
P.Soubeyran@bordeaux.unicancer.fr (P.S.)
2
3
4
ARNA Laboratory, Université de Bordeaux, INSERM U1212, CNRS UMR 5320, 33000 Bordeaux, France;
rabindrachem@gmail.com (R.N.D.); jean.guillon@u-bordeaux.fr (J.G.); jean-louis.mergny@inserm.fr (J.-L.M.)
Metabolomics and Cell Biology Platforms, Institut Gustave Roussy, 94805 Villejuif, France;
alvarezvaladez4@gmail.com (K.A.-V.); alexandra.muller12@gmail.com (A.M.); kroemer@orange.fr (G.K.)
Centre de Recherche des Cordeliers, INSERM UMRS 1138, Sorbonne Université, Université de Paris,
Equipe 11 labellisée par la Ligue contre le Cancer, 75006 Paris, France
5
6
7
8
Gustave Roussy Comprehensive Cancer Institute, 94805 Villejuif, France; isabellemart@gmail.com
Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, 75015 Paris, France
Suzhou Institute for Systems Medicine, Chinese Academy of Sciences, Suzhou 215123, China
Department of Women’s and Children’s Health, Karolinska University Hospital, 17176 Stockholm, Sweden
Correspondence: mojgan.mergny@inserm.fr
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 16 May 2020; Accepted: 12 June 2020; Published: 18 June 2020
Abstract: Lysosomes play a key role in regulating cell death in response to cancer therapies, yet little is
known on the possible role of lysosomes in the therapeutic efficacy of G-quadruplex DNA ligands (G4L)
in cancer cells. Here, we investigate the relationship between the modulation of lysosomal membrane
damage and the degree to which cancer cells respond to the cytotoxic effects of G-quadruplex ligands
belonging to the triarylpyridine family. Our results reveal that the lead compound of this family, 20A
promotes the enlargement of the lysosome compartment as well as the induction of lysosome-relevant
mRNAs. Interestingly, the combination of 20A and chloroquine (an inhibitor of lysosomal functions)
led to a significant induction of lysosomal membrane permeabilization coupled to massive cell death.
Similar effects were observed when chloroquine was added to three new triarylpyridine derivatives.
Our findings thus uncover the lysosomal effects of triarylpyridines compounds and delineate a
rationale for combining these compounds with chloroquine to increase their anticancer effects.
Keywords: Lysosome; lysosomal membrane permeabilization; cell death; cancer; G-quadruplex
ligand; triarylpyridine compounds; resistance to therapy
1. Introduction
Lysosomes are the end points of several vesicular pathways that mediate degradation of intra- and
extra-cellular material in cells [
processes including nutrient sensing, signaling to the nucleus, and the ignition of cell death pathways [
1
]. As a result, lysosomes ensure the regulation of numerous biological
].
2
In particular, lysosomes are essential for the proper function of autophagy, a cytoprotective process
that provides necessary nutrients and energy through promoting the degradation of intracellular
Cancers 2020, 12, 1621; doi:10.3390/cancers12061621
www.mdpi.com/journal/cancers

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material [3,4]. Hallmarks of lysosomal dysfunction include changes in lysosomal size, morphology and
localization, changes in the expression of lysosomal enzymes and alterations of lysosomal membrane
permeability [5].
Lysosomes play major roles in cancer, as they undergo multiple alterations during the
transformation process that contribute to tumor progression and metastasis [6]. Mounting evidence
indicates that lysosomes are involved in the resistance to anticancer compounds with weak base
properties. These drugs may be sequestered within the lysosome, resulting in a loss of accessibility of
the drug to its cellular targets and thereby a reduction of its effectiveness [
lysosomal sequestration can be passive, through diffusion into the acidic lumen of the lysosome where
the drug is protonated and retained [10 11]. However, an active mechanism has also been described
7–10]. The mechanism of
,
that relies on multidrug efflux transporters [12]. Accordingly, inhibition of lysosomal sequestration
(by increasing lysosomal pH, or inhibiting active transport) may increase the sensitivity of cancer cells
to several antineoplastic therapies [13]. Conversely, the modifications of the lysosomal compartment
that occur during cellular transformation can paradoxically render cancer cells more susceptible to
lysosomal membrane permeabilization (LMP) [14
,15], and ultimately lead to lysosomal-dependent cell
death (LDCD) induced by anticancer agents [13,16].
LDCD is characterized as a process that involves LMP and the subsequent release of lysosomal
content such as cathepsins into the cytosol [17]. Extensive lysosomal leakage can cause necrotic cell
death while partial lysosomal membrane destabilization can result in lysosomal cell death through
apoptotic and non-apoptotic dependent mechanisms. LMP is triggered by stimuli that affect lysosomal
membrane stability, including lysosomotropic agents, ROS-generating molecules, or agents that affect
the lipid composition of the lysosomal membrane [17]. Of note, the induction of lysosomal-dependent
non-apoptotic cell death is of tremendous interest for cancer therapy because cancer cells often display
a defective apoptotic machinery rendering them resistant to conventional chemotherapies [18].
Hence, the identification of molecules (or combination of molecules) that promote lysosomal
cell death is an attractive strategy for the treatment of apoptosis- or therapy-resistant cancers.
Quadruplexes (G4) are unusual nucleic acid structures formed by G-rich DNA and RNA [19] that
can be selectively recognized by ligands. A number of G4 ligands have been described and over
a thousand different compounds are now listed in the G4 ligand database (http://www.g4ldb.org).
These compounds can be neutral or bear one or more positive or negative charges thanks to differences
in side chains [20–22]; several of these molecules exhibit exquisite selectivity towards G-quadruplexes
in vitro. The effect of G4 ligands on genomic stability, replication, transcription and splicing and
their roles as anticancer agents is starting to emerge [23], but a detailed analysis of their effects on
lysosome is still lacking. We have previously shown that 20A, a G-quadruplex ligand belonging to the
triarylpyridine family, exerts potent cytotoxic and cytostatic effects on several cancer cells. We found
that one of the gene classes most significantly upregulated by 20A corresponds to the autophagy
lysosomal pathway [24].
In this work, we speculated that 20A may interfere with the lysosomal pathway as autophagy
and lysosomal function are intertwined processes. A more detailed analysis of the lysosomal KEGG
database indeed revealed that 20A led to an increased expression of numerous genes involved in the
lysosomal pathway (i.e., genes coding for lysosomal hydrolases and membrane proteins, as well as
for proteins involved in the synthesis and traffic of lysosomal enzymes) (Figure S1). Based on these
observations, we hypothesized that the enrichment of the lysosomal pathway by 20A would render cells
less vulnerable to the cytotoxic effect of this compound. We thus investigated the effect of chloroquine,
an inhibitor of lysosomal functions, on the cytotoxic effect of G4 ligands from the triarylpyridine
family. The chemical structures of triarylpyridine compounds used in this study are shown in Figure 1.
In addition to the parent compound 20A, three new 20A derivatives were synthesized (Figure S2a)
and then tested for their ability to stabilize G4 structures. These three new 20A derivatives stabilize
the human telomeric quadruplex in a concentration-dependent manner (Figure S2b), as previously
reported for 20A [25].

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Figure 1. Formula of triarylpyridine compounds used in this study.
2. Results
2.1. 20A Accumulates Within the Lysosomal Compartment and Causes Enlargement of the Lysosomes
To investigate the effect of 20A on lysosomal homeostasis, we first stained cells with the lysosomal
marker, LysoTracker Red. As shown in Figure 2a, the size of the lysosomes is significantly increased
following exposure of HeLa cells to 20A. Incidentally, we observed the presence of faint blue fluorescence
puncta in the cytosol of cells subjected to 20A treatment (Figure 2b). In vitro fluorescence experiments
confirmed that 20A emits a weak blue fluorescence following excitation in the UV region (Figure S3).
Co-localization experiments with LysoTracker and MitoTracker (a specific marker of mitochondria)
revealed that the blue fluorescence puncta mostly co-localize with the lysosomes, not with mitochondria
(Figure 2b,c). Altogether, these results indicate that 20A accumulates within the lysosome as early as
6 h after treatment and elicits an enlargement of this compartment.
LysoTracker Red
a)
0.8
*
0.6
0.4
0.2
10µm
10µm
0.0
Control
20A
Figure 2. Cont.

Cancers 2020, 12, 1621
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b)
LysoTracker
Red
20A
fluorescence
MitoTracker
Green
Merge
LysoTracker Red
MitoTracker Green
20A fluorescence
25µm
25µm
c)
Fluorescence profile
Figure 2. 20A accumulates within the lysosomes and causes lysosomal enlargement. (
were treated with or without 5 20A for 24 h and then stained with LysoTracker Red (red signal) to
visualize lysosomes. Left panel, representative confocal fluorescence images are presented for each
condition. Right panel, the average of lysosome size was scored for the experiment in ( ) using Image J
tools (n > 30 cells/condition, p value * < 0.05 using Mann-Whitney test). Scale bars: 10 m. ( ) HeLa cells
were treated with or without 5 20A for 6 h and then stained with LysoTracker Red and MitoTracker
a) HeLa cells
µM
a
µ
b
µM
Green to visualize lysosomes and mitochondria, respectively. Representative confocal images are
presented for LysoTracker Red (red signal), 20A fluorescence (blue signal) and MitoTracker Green
(green signal). Magnified views of boxes are presented for individual or merged signals. Scale bars:
25 µm. (c) The fluorescence intensities of red, blue and green signals plotted along the yellow bar are
shown in the “20A-treated” cells panel.
2.2. 20A and Chloroquine Act in Concert to Trigger Cell Death
The presence of 20A within lysosomes is reminiscent of a mechanism referred to as lysosomal drug
sequestration, which is observed in response to some anticancer compounds [18]. Growing evidence
reveals that lysosomotropic agents such as chloroquine can prevent lysosomal drug sequestration,
thus sensitizing cells to cancer therapies [10,11,26]. To test this hypothesis, we investigated the effect
of a combination of 20A and chloroquine on cell death in both HeLa cervical carcinoma and A549
non-small cell lung cancer cell lines. In order to properly evaluate the effect of the combination of the
two drugs (20A and chloroquine), we decided to use 20A doses that trigger a relatively low level of cell
death (<10%) when used alone. Similarly, a sublethal dose (<10% cell death) of chloroquine (25 µM)
was used.

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As shown in Figure 3a, the combination of chloroquine (25
µM) and 20A (5 or 6
µM) promoted a
robust activation of cell death (50% or more) in HeLa cells as compared to 20A or chloroquine alone.
A similar effect was observed when A549 cells were treated with the combination of 20A (3.5 or 4 M)
and chloroquine (25 M), indicating that chloroquine greatly potentiated 20A-induced cell death.
µ
µ
Similar results were found with another lysosomal inhibitor, Lys05, which is an analogue of chloroquine
(data not shown). As found for HeLa and A549 cells, the combination of 20A and chloroquine promoted
a significant cell death induction in U20S osteosarcoma cells (Figure 3b, left panel). We also evaluated
loss of mitochondrial transmembrane potential (a feature of early apoptosis) and increased cell
membrane permeability (a feature of late apoptosis) by co-staining U2OS cells with TMRM and DAPI
dyes. As shown in Figure 3b (right panel) and Figure S4, the combination of 20A and chloroquine led
to early (TMRMlow/DAPILow) and late cell death (TMRMLow/DAPI High) as earlier as 8 h of treatment.
A significant increase of both features of apoptosis were observed after 16 h of treatment (Figure 3b,
right panel).
As chloroquine is a well-known inhibitor of autophagy and 20A has been shown to promote
autophagy [24], we next explored whether the inhibition of autophagy would be responsible for the
robust induction of cell death by the combination of 20A and chloroquine. To this purpose, we compared
cell death induced by 20A plus chloroquine in autophagy-proficient cells and autophagy-deficient cells
(the latter were generated by disruption of two key autophagy genes, ATG5 and ATG7); (Figure 3c,
left panel and Figure S5 for the whole blots). As shown in Figure 3c (right panel), autophagy-deficient
cells exhibited a similar rate of cell death in response to 20A plus chloroquine as did autophagy-proficient
cells, suggesting that chloroquine (at least at 25
autophagy-independent mechanism.
µM) sensitizes cells to 20A treatment through an
HeLa cells
A549 cells
a)
U20S cells
TMRMLow/ DAPIHigh
b)
U20S cells
TMRMLow/ DAPILow
80
60
80
60
40
20
40
20
0
0
10µm
-
+
-
+
-
+
- +
CQ
20A µM
20A µM
0
3
4
0
3.5
3
4
Figure 3. Cont.

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c)
20A
-
+
-
+ -
+
ATG5-ATG12
(55KDa)
ATG5 (33kDa)
ratio ATG5-ATG12/Actin β:
1
1.3 0.12 0.12 0.13 0.15
ATG7 (78 KDa)
ratio ATG7 /Actin β:
1
1.2
1
1.1 0.01 0.01
Actin β (42 KDa)
Figure 3. 20A and chloroquine act in concert to trigger cell death. (
(right) were treated with the indicated concentration of 20A with or without 25
for 24 h. Cell death was evaluated by scoring the percentage of propidium iodide (PI) positive cells
after flow cytometer analysis. The data represents the mean SD of 6 values obtained from three
independent experiments each performed in duplicate (** p value < 0.01 using Kruskal-Wallis test,
with post-hoc Dunn’s analysis). ( ) U20S cells were treated with the indicated concentration of 20A
with or without 25 M of chloroquine for 16 h. Left panel, cell death was evaluated by scoring the
percentage of propidium iodide positive cells after flow cytometer analysis. The data represents the
mean SD of 9 values obtained from three independent experiments each performed in triplicate
a) HeLa cells (left) and A549 cells
µM of chloroquine
±
b
µ
±
(p value **** < 0.0001, using Mann-Whitney test). Right panel, Cell death was evaluated after 16 h
treatment with 20A ± chloroquine by scoring the percentage of TMRMLow/ DAPILow (early apoptosis)
and TMRMLow/ DAPIHigh (late apoptosis) cells following flow cytometer analysis. The data represents
the mean
±
SD of 9 values obtained from three independent experiments each performed in triplicate
(p value **** < 0.0001, using Mann-Whitney test). (
c) Autophagy-proficient (sgNTC) and -deficient
(sgATG5 and sgATG7) HeLa cells were treated with 5
µM
20A either in the presence (25 M) or absence
µ
of chloroquine for 24 h. Left panel, silencing of key autophagy proteins, ATG5 and ATG7 was checked
by western blotting. The western blots were quantified and given as ratio of ATG5-ATG12/Actin
β
or ATG7/Actin
β. Right panel, cell death was evaluated by scoring the percentage of propidium
iodide positive cells after flow cytometer analysis. The data represents the mean
obtained from three independent experiments each performed in triplicate (p value * < 0.05 and p value
*** < 0.001, ns: not significant using Mann-Whitney test).
±
SD of nine values
The induction of lysosomal membrane permeabilization (LMP) represents one mechanism through
which chloroquine can affect lysosomes [27]. Recently, the detection of Galectin 3 puncta at damaged
endo-lysosome membranes was shown to be a highly sensitive method to evaluate LMP [28]. To explore
this possibility, we used the U20S cell line expressing mCherry-tagged Galectin 3 and counted cells
displaying one or more Galectin 3 puncta. As shown in Figure 4 (a1 and a2), the percentage of cells
with Galectin 3 puncta was nearly equal in cells subjected to 20A and in untreated cells (3% and 2%,
respectively) but increased to 8 % when cells were exposed to chloroquine. However, the percentage
of cells with Galectin 3 puncta massively increased (to 39%) when chloroquine was associated with
20A treatment. It is worth noting that, under the latter condition, 26% of cells with Gal3 puncta
harbored one or two Gal3 puncta and the rest (13%) displayed either 3 or more Gal3 puncta (Figure 4a3),
suggesting several instances of endo-lysosomal membrane damage per cell. We next analyzed whether
the Gal3 puncta were located at the lysosomal membrane by co-staining cells with LAMP1, a specific
marker of the lysosomal membrane.

Cancers 2020, 12, 1621
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b)
a)
Gal3-
mCherry
LAMP1
Hoechst
Merge
a1)
Control
20A
25µm
25µm
25µm
a2)
****
60
40
20
0
25µm
mCherry Galectin 3 Lamp1 Hoescht
***
20A
Control
20A
-
+
-
+
+ CQ
More than 3 puncta
1 or 2 puncta
0
a3)
100
80
60
40
20
0
10µm
20A
-
+
-
+
Zoom Lamp1 staining
+ CQ
Figure 4. 20A and chloroquine act in concert to trigger LMP in cancer cells. (
expressing U2OS cells were treated or not with 3 20A in either the presence or absence of 25
chloroquine for 24 h. (a1) representative fluorescence images of Galectin 3 staining (red signal) and
nuclei stained with Hoechst (blue signal) are shown; scale bar represents a distance of 25 m. (a2) The
percentage of cells displaying at least one Galectin 3 punctae is scored. Data are presented as mean SD
a) mCherry-Galectin3
µM
µM
µ
±

Cancers 2020, 12, 1621
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of 15 values obtained from 5 randomly chosen fields in each of the three independent experiments.
Data are presented as the mean percentage of at least 1000 cells analyzed from five randomly chosen
fields in each of the three independent experiments (*** p value < 0.001 **** and p value < 0.0001
using Kruskal-Wallis test, with post-hoc Dunn’s analysis). (a3) The percentage of cells with >3, 1 and
2, 0 Galectin 3 puncta are scored. (b) mCherry-Galectin3 U2OS cells were treated or not with 3
µM
20A either in the presence or absence of 25
µM chloroquine for 24 h and then immunostained with
an antibody that recognizes LAMP1, a lysosomal marker. Nuclei were counterstained with Hoechst.
Representative of (z-projected) confocal images of Galectin 3 (red), nuclei (blue) and LAMP1 (green)
are shown. Scale bar represents a distance of 25 µm. Zoom image of 20A-treated cells shows that
most Galectin 3 puncta co-localize with LAMP1 (representative co-localization signals are depicted by
white arrowheads). Zoom Lamp1 staining images of cells treated with 20A ± chloroquine are shown.
Scale bar: 10 µm.
As shown in Figure 4b, a significant colocalization was observed when cells were exposed to 20A
plus chloroquine, confirming that Gal3 puncta match to the membrane damage sites at lysosomes.
We also examined the size of the lysosomes in cells treated with 20A plus chloroquine by looking at
the pattern of LAMP1 staining. As shown in Figure 4b (zoom LAMP1 staining), 20A and chloroquine
both caused a dramatic lysosomal enlargement as evidenced by LAMP1 staining. More importantly,
this phenotype was markedly increased when the two compounds were combined.
This observation, combined with the increased size of the lysosomes in cells treated with 20A
plus chloroquine, suggests that the lysosomal swelling process could be a cause of LMP. Altogether,
these results demonstrate that the combination of 20A and chloroquine causes significant lysosomal
membrane permeabilization along cell death induction.
2.3. Apoptosis Is Involved in Cell Death Induced by 20A Plus Chloroquine
We next investigated whether apoptosis is implicated in the massive cell death induced by
20A plus chloroquine. To this purpose, we measured apoptosis-associated parameters such as the
appearance of the cleaved form of caspase-3 and its substrate PARP1. As presented in Figure 5a (whole
blots are shown in Figure S6), the combination of chloroquine and 20A treatment promoted a robust
cleavage of PARP-1 and Caspase 3 in both HeLa and A549 cells, while no significant cleavage was
observed when cells were treated solely with one compound.
To further evaluate the role of caspases in the occurrence of cell death, we used a pan-inhibitor of
caspase activity (Q-VD-Oph) abbreviated as QVD (Figure 5b) and evaluated cell death induction in
response to 20A plus chloroquine. As shown in Figure 5b, the induction of cell death was significantly
reduced when both HeLa and A549 cells were treated with QV-D-Oph prior to the addition of 20A
plus chloroquine, suggesting the contribution of caspases to cellular demise.
Using a pharmacological approach, we also investigated the possible implication of ferroptosis
and necroptosis in cell death induced by 20A plus chloroquine. As shown in Figure S7A, ferrostatin
1, an inhibitor of ferroptosis, markedly inhibited cell death induced by erastin (a known inducer of
ferroptosis) but had no significant effect on cellular demise triggered by the combination of 20A and
chloroquine. Similarly, inhibition of necroptosis by necrostatin 1 did not impact cell death induced
by the combination of 20A and chloroquine (Figure S7B). Together, these data reveal that neither
ferroptosis nor necroptosis are involved in the cytotoxicity effect triggered by 20A plus chloroquine.
To gain further insight into the mechanism linking LMP and cell death, we evaluated the
involvement of two families of cathepsins by using E64d, an inhibitor of the cysteine cathepsin family
and pepstatin A, an inhibitor of the aspartic cathepsin family. While E64d and pepstatin A efficiently
compromised cell death induced by LLOMe, a well-known inducer of cathepsin-dependent cell death,
they failed to inhibit cell death induced by 20A plus chloroquine (Figure S7C). These results suggest
that the cysteine and aspartic cathepsins are unlikely to be involved in the execution of cell death
induced by 20A plus chloroquine.

Cancers 2020, 12, 1621
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a)
HeLa cells
A549 cells
20A
CQ
-
-
-
+
+
-
+
+
-
-
-
+
+
-
+
+
PARP 1 (116kDa)
Cleaved PARP1 (89kDa)
Quantification:
0.07 0.07 0.12 1
0.1 0.1 0.25 1
0.6 0.8 0.7
0.1 0.2 0.2
1
Cleaved forms
of caspase 3
(39, 19,17 kDa)
Quantification:
1
Actin b (42 kDa)
b)
HeLa cells
A549 cells
*
**
80
100
80
60
40
20
0
+QVD
+QVD
60
40
20
0
**
**
20A
CQ
QVD
-
-
-
-
+
+
-
+
+
+
-
+
+
+
20A
CQ
QVD
-
-
-
-
+
-
-
+
-
+
+
+
+
+
+
-
-
-
+
-
+
-
Figure 5. Apoptosis is involved in cell death induced by 20A plus chloroquine. (
and A549 (right panel) cells were treated with 5 M and 3.5 20A, respectively, in the presence or
absence of 25 M chloroquine for 24 h. Apoptotic cell death was assessed by immunoblot analysis of
cleaved forms of either Caspase 3 or PARP1. Actin was detected as a loading control. The Western
blots were quantified and given as ratio of cleaved caspase3/Actin or cleaved Parp1/Actin
HeLa cells (left panel) and A549 (right panel) cells were treated with or without 20 M QV-D-Oph
two hours prior to the addition of 20A (5 M and 3.5 M for HeLa cells and A549 cells, respectively).
Where indicated, cells are also exposed to 25 M chloroquine (CQ) for 24 h. Cell death was assessed by
scoring propidium iodide positive cells. The data represents the mean SD of six values obtained
a) HeLa cells (left panel)
µ
µM
µ
β
β
β (b)
µ
µ
µ
µ
±
from three independent experiments each performed in duplicate (* p value < 0.05 and ** p value < 0.01
using Mann-Whitney test).

Cancers 2020, 12, 1621
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2.4. Chloroquine Significantly Activates LMP Triggered by 20A Derivatives
Next, we investigated whether the cooperation between chloroquine and 20A in induction of
cell death can be extended to other triarylpyridine compounds. For this, we tested three new 20A
derivatives (1a, 1b and 1c) (Figure 1) that were synthesized according to the scheme presented in
Figure S2A. The synthesis and characterization of 1a-c are presented in the experimental section.
We then determined their IC50 in HeLa cells before performing a drug combination assay. As shown
in Figure S8, these compounds significantly inhibited HeLa cell viability (with IC50 values even lower
than the one found for 20A). We then selected sublethal doses (mortality < 10%, as for 20A) for each
compound to analyze LMP and cell death in the presence or absence of chloroquine.
We first analyzed LMP by determining the percentage of cells with Gal3 puncta. As shown in
Figure 6a,b, unlike 20A, all three compounds 1a–c have the ability to induce LMP alone (compared to
untreated cells) as evidenced by the percentage of with Gal3 puncta which amounts to 26%, 31% and
25%, respectively. More importantly, the percentage of Gal3 puncta positive cells was further increased
when chloroquine was added to each ligand (57%, 56% and 45% for 1a, 1b and 1c, respectively, Figure 6b,
left panel). This response was accompanied by an increase in the percentage of cells harboring more
than three Gal3 puncta (Figure 6c, right panel), suggesting that a significant population of cells had
undergone intensive lysosomal membrane damage. Thus, the combination of chloroquine and 20A
derivatives triggered a massive increase in LMP induction.
Hoechst
Control
mCherry Galectin 3
20A
a)
1a
1b
1c
B
25µm
b)
More than 3 puncta
1 or 2 puncta
0
- CQ
+ CQ
100
80
60
40
20
0
100
80
60
40
20
****
****
****
****
0
CQ
-
+
-
+
-
+
-
+
1b
-
+
control 20A 1a
1b
1c
control 20A
1a
1c
Figure 6. Chloroquine enhances LMP triggered by 20A derivatives. (a) mCherry-Galectin3 expressing
U2OS cells were treated with or without 3 20A, 2 1a, 1 1b or 1 µM
µ
M
µ
M
µ
M
1c in either the presence

Cancers 2020, 12, 1621
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or absence of 25
µ
M chloroquine for 24 h. Representative fluorescence merge images are presented
m. ( ) Left Panel, quantification
of the percentage of cells displaying at least one Galectin3 puncta. Data are presented as mean SD
with Galectin 3 (red signal) and Hoechst (blue signal), scale bar 25
µ
b
±
of 15 values obtained from five randomly chosen areas in each of the three independent experiments.
Right panel, the percentage of cells with 0, 1 or 2, or 3 or more Galectin 3 puncta are scored. Data are
presented as the mean percentage of at least 1000 cells analyzed from 5 randomly chosen field in each
of the three independent experiments (**** p value < 0.0001 using Mann-Whitney test).
2.5. Combined Treatment with Chloroquine and 20A Derivatives Significantly Activates Cell Death in Lung
Adenocarcinoma Cells
The induction of LMP by 20A-derivative compounds 1a, 1b and 1c prompted us to investigate if
the combination of chloroquine with 20A derivatives can exacerbate cell death modalities, as seen for
20A. We thus examined the induction of cell death in response to 20A and 20A derivatives in either the
presence or absence of chloroquine in A549 lung adenocarcinoma cells.
As shown in Figure 7a, cell death was markedly enhanced when chloroquine was combined with
1a. Similar results were also observed with 1b and 1c, supporting the idea that the triarylpyridine
compounds cooperate with chloroquine to trigger a robust cell death. To validate our results in a
clinically relevant setting, we also examined the effect of combination of triarylpyridine compounds
with chloroquine in patient–derived xenograft cell lines from lung adenocarcinomas (referred as PDX2
and PDX3 in the original paper [29]). As shown in Figure 7b, both PDX cell lines were highly sensitive
to the combined treatment.
a)
A549 cells
+CQ
- CQ
100
80
60
40
20
0
****
****
***
****
Control 20A
1a
1b
1c
Figure 7. Cont.

Cancers 2020, 12, 1621
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b)
PDX2 cells
PDX3 cells
- CQ
+ CQ
- CQ
+ CQ
100
80
60
40
20
0
100
80
60
40
20
0
****
****
****
****
****
****
****
****
Control 20A
1a
1b
1c
Control 20A
1a
1b
1c
Figure 7. Combination of chloroquine with 20A derivatives significantly enhances cell death in both
A549 lung cancer cells and patient–derived xenograft cell lines from lung cancer. A549 lung cancer
cell lines (a) and two PDX from lung cancer PDX2 and PDX3 (b) were treated with either 3.5 µM 20A,
2.5
was evaluated by scoring the percentage of propidium iodide positive cells by flow cytometer analysis.
The data represents the mean SD of 9 values obtained from three independent experiments each
performed in triplicate (p-value *** < 0.001 and **** < 0.0001 using Mann-Whitney test).
µM 1a, 1.5 µM 1b or 2 µM 1c in the presence and absence of 25 µM chloroquine for 24 h. Cell death
±
3. Discussion
Targeting lysosome in tumors emerges as an attractive approach for overcoming resistance to
therapy. Here, we showed that the triarylpyridine compound 20A accumulates in the lysosomal
compartment and causes lysosome enlargement. The addition of chloroquine to 20A markedly
enhanced the accumulation of enlarged lysosome as compared to a single drug treatment (20A or
chloroquine alone), supporting the idea that lysosome fission/fusion is affected under these conditions.
Calcium fluxes on lysosomal membranes play a major role in fission and fusion processes affecting
these organelles. In fact, the lysosomal Ca2+ channels P2X4 and Mucolipin 1 (TRPML1) were shown to
be involved in lysosomal fusion and fission, respectively [30,
31]. The possible contribution of Na+
and other cations to lysosomal enlargement has also been suggested in the literature. Whether or not
Ca2+ and other cations are involved in the lysosomal phenotype observed in response to 20A and
chloroquine/20A remains to be clarified.
Another factor that regulates the lysosomal size and number is the TFEB transcription factor,
which is responsible for the induction of genes encoding lysosomal proteins through a mechanism
that depends on mTOR inhibition [32,33]. The fact that 20A causes the upregulation of several genes
encoding for lysosomal proteins and inhibition of mTOR (as shown in our previous works [24
,
34]
and Figure S1) argues in favor of the activation of the TFEB protein in response to 20A. It would be
interesting to determine if and how TFEB regulates lysosome functions and cell death in cells exposed to
20A alone or with chloroquine. Further experiments will be necessary to better understand the features
of lysosome enlargement induced by 20A either as a single agent or in combination with chloroquine.
Most importantly, we found that the combination of 20A and chloroquine promotes a significant
induction of lysosomal membrane permeability. Lysosomal membrane permeability can be initiated by
several agents namely; (i) lysosomotropic agents, such as chloroquine, which destabilize the lysosomal
membrane through either hydrophobic or electrostatic interactions [26
,35]; (ii) agents that affect the
lipid composition (i.e., cholesterol or sphingolipid) of the lysosomal membrane; (iii) ROS-generating

Cancers 2020, 12, 1621
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agents that cause lipid peroxidation of this membrane. We found that the addition of antioxidants
did not significantly affect LMP triggered by 20A plus chloroquine, suggesting the implication of
ROS-independent mechanisms in this process (Figure S9A). Whether or not the accumulation of
enlarged lysosomes and/or changes in lysosomal lipid composition are responsible for the induction of
LMP warrants further investigation.
Interestingly, we observed that the cooperative effect of chloroquine and 20A on LMP can be
extended to three other triarylpyridine derivatives, suggesting a general mechanism triggered by the
triarylpyridine family of compounds. As the induction of LMP is considered an attractive strategy for
cancer therapy [14], the characterization of the chemical properties of these molecules and their precise
mode of action in regulating LMP deserves further investigation.
Of particular interest are the results showing that, in addition to the robust activation of LMP,
the combination of chloroquine and triarylpyridine compounds triggers massive cell death in a variety
of cancer cells, including HeLa, U2OS and A549 cell lines, as well as PDX cells from lung cancer patients.
Moreover, we found that apoptosis, but neither necroptosis, ferroptosis nor cathepsin-dependent cell
death are involved in the execution of cell death, as evidenced by specific pharmacological inhibitors
of each pathway. Strikingly, a pan caspase inhibitor only partially recovered cell death triggered by
20A plus chloroquine, suggesting that the induction of cell death required caspase-dependent and
-independent mechanisms. However, the nature of this caspase inhibitor-insensitive component of
cell death remains yet unknown. As mitochondrial outer membrane permeabilization (MOMP) is
a critical step of lysosome-induced apoptosis [36], it would be interesting to explore if BCl-2 family
members (which control MOMP) are implicated in the regulation of the cell death process induced
by 20A plus chloroquine. Of note, caspases seem to operate downstream of LMP (Figure S9B) as a
pan inhibitor of caspases had no effect on LMP induction. Clearly, further research will be necessary
to fully understand the network involved in cell death induced by triarylpyridine compounds either
alone or in combination with chloroquine.
By screening of a series of triarylpyridine coumpounds, we first characterized 20A as a potent
G-quadruplex stabilizing DNA Ligand (G4L) [25]. Here, we showed that three new 20A- derivatives
that induce LMP are also able to stabilize an intramolecular G4 structure. However, it remains
unclear if the effects of these compounds on lysosomes are related to their interaction with G4 or
rather linked to their ability to be sequestrated within the lysosomes. The accumulation of G4Ls in
lysosomes has been described only for two other ligands, BMVC and DAOTA [37,38]. Of note, many
G4Ls, whose localization could be examined by virtue of their fluorescent property, have shown to
display perinuclear cytoplasmic localization. This cytoplasmic localization of G4L was suggested
to be attributed to their ability to target mitochondria DNA or cytoplasmic RNAs [39,40]. However,
we cannot rule out that some G4Ls are sequestered in lysosomes and that this might affect their
anti-proliferative efficacy. In this line, BMVC was shown to be enriched in the lysosomes of cancer cells
resistant to chemotherapy and the induction of LMP with LLOMe promoted its nuclear relocation [37].
Overall, it would be interesting to investigate whether different G4 ligand classes affect the lysosome
compartment; results may depend on a variety of factors, such as net charge (not all G4L are cationic),
hydrophobicity, or potential accumulation in other organelles such as mitochondria.
4. Materials and Methods
4.1. Cell Culture
The human cervical cancer cell lines HeLa and U20S were purchased from the American Type
Culture Collection (ATCC, Manassas, VA, USA) and the human lung carcinoma A549 cell line is a
generous gift of Prof. Jean-François Riou (CNRS UMR 7196, Paris, France). The autophagy-deficient
(ATG5 and ATG7 depleted) HeLa cell lines were generated as previously described [24]. U2OS expressing
mCherry-Galectin3 cell line is a generous gift from Dr. A H. Woldrich (CNRS UMR5, Bordeaux,
France) [41]. The lung adenocarcinoma PDX cell lines (PDX2) and (PDX3) are generous gifts from

Cancers 2020, 12, 1621
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Dr. David Santamaria, INSERM U1218, Bordeaux, France [29]. HeLa cells were grown in RPMI 1640
culture media supplemented with 2 mM glutamine (Invitrogen ,Waltham, MA, USA), 10% fetal bovine
serum, 100 units/mL penicillin, and 100
grown in DMEM supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100
streptomycin. For mCherry-Galectin3 expressing U2OS cells, 200 g/mL hygromycin B (#10687010,
µg/mL streptomycin, while A549, U2OS and PDX cell lines were
µg/mL
µ
Invitrogen, Waltham, MA, USA) was added in cell culture media but removed from media before each
experiment. All cell lines used in this study were cultivated at 37 ◦C in a humidified atmosphere with
5% CO2.
4.2. Reagents and Antibodies
Antibodies against the following proteins were used: ATG5 (#0262-100/ATG5-7C6) from Nanotools
(Teningen, Baden-Württemberg, DE), ATG7 (#8558) ), Cleaved-Caspase3 (#9664), LAMP-1 (#9091)
from Cell Signaling Technology (Danvers, MA, USA), PARP-1 (C-2-10) (#BML-SA249-0050) from Enzo
Life Sciences (Farmingdale, NY, USA), Actin
β (#NB600-501) from Novus Biologicals (Centennial,
CO, USA), HRP conjugated rabbit (#111-035-003) and HRP conjugated mouse (#115-035-174) from
Jackson ImmunoResearch (Ely, Cambridgeshire, UK), Alexa488 conjugated anti-rabbit (#A11008)
from Invitrogen (Waltham, MA, USA). Hoechst 33258 (#14530), E64d (#E8640), Propidium iodide
(#P4864), Chloroquine diphosphate salt (#C6628), QV-D-Oph (#SML0063), butylhydroxyanisole (BHA,
#B1212000), N-acetyl-L-cysteine (NAC, #A8199) were purchased from Sigma-Aldrich (St. Louis,
MO, USA).). Tetramethylrhodamine methyl-ester, TMRM (T-668) was purchased from Molecular
Probes (Eugene, OR, USA). Fluoromount G (#00-4958-02) was purchased from Invitrogen. 20A was
synthesized as previously described [42,43]. Ferrostatin-1 (#341494) was purchased from Calbiochem
(San Diego, CA, USA)), while LLOME (#16008) and Erastin (#17754) were purchased from Cayman
Chemical (Ann Arbor, MI, USA).
4.3. Synthesis of Bis-Triazole Triarylpyridines
A series of novel bis-triazole substituted 2,4,6-triarylpyridine derivatives 1a–c have been synthesized
through a three steps procedure (Figure S2A). The synthetic route involves the base-catalysed
condensation of commercially available p-aminoacetophenone with 4-(methylthio)-benzaldehyde in
PEG300 resulting in the formation of the 2,6-bis(4-aminophenyl)-4-[4-(methylthio)phenyl]pyridine (
2)
using the modified Chichibabin pyridine synthesis [25 43 44]. This 2,6-bis(4-aminophenyl)pyridine 2
,
,
was then converted into the intermediate corresponding diazido derivative 3 which was then subjected
to “click reaction”, a copper-catalyzed azide-alkyne 1,3-dipolar cycloaddition reaction, with three
amine-terminal alkynes to afford the substituted 1,2,3-triazole derivatives 1a, 1b and 1c.
4.3.1. Chemicals and Instrumentation for Chemical Synthesis
Commercially available reagents were used as received without additional purification.
Melting points were determined with an SM-LUX-POL Leitz hot-stage microscope (Leitz GMBH,
Midland, ON, USA) and are uncorrected. IR spectra were recorded on a 380FT-IR spectrophotometer
(Nicolet- Thermo Electron Scientific Instruments LLC, Madison, WI, USA). NMR spectra were recorded
with tetramethylsilane as an internal standard using an AVANCE 300 spectrometer (Bruker BioSpin,
Wissembourg, France). Splitting patterns have been designated as follows: s = singlet; d = doublet;
t = triplet; m = multiplet. Analytical TLC were carried out on 0.25 precoated silica gel plates
(POLYGRAM SIL G/UV254) and visualization of compounds after UV light irradiation. Silica gel
60 (70–230 mesh) was used for column chromatography. High resolution mass spectra (electrospray
in positive mode, ESI+ or MALDI-TOF MS) were recorded on a Q-TOF Ultima apparatus (Bruker
Daltonics, Bremen, Germany). Elemental analyses were found within 0.4% of the theoretical values.
±

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4.3.2. Synthesis of 2,6-bis(4-aminophenyl)-4-[4-(methylthio)phenyl]pyridine (2) (Figure S2A)
p-Aminoacetophenone (5 g, 37 mMol) was added to a suspension of crushed NaOH (1.5 g,
37 mmol) in PEG300 (20 mL) at room temperature. The suspension was stirred and heated to 80 ◦C to
form a clear solution. Then, p-methylthiobenzaldehyde (2.8 g, 18.5 mmol) was added to the reaction
mixture and heated at 110 ◦C for 4 h, after which NH4OAc (42.7 g, 555 mmol) was added and the
heating was reduced to 100 ◦C and stirred for 4 h. After cooling to room temperature, 400 mL cold water
was added to the mixture to get a precipitate which was filtered and further purified through silica
gel chromatography using DCM:MeOH (0–5%) as eluent affording compound
2
as a brown-yellow
powder (52%); 1H-NMR
δ
(300 MHz, CDCl3) 8.37 (d, 4H, J = 8.70 Hz, 2H-20 and 2H-60), 7.95 (s, 2H,
H-3 and H-5), 7.92 (d, 4H, J = 8.70 Hz, 2H-30 and 2H-50), 7.73 (d, 2H, J = 8.70 Hz, H-200 and H-600),
7.44 (d, 2H, J = 8.70 Hz, H-300 and H-500), 5.89 (br, 4H, 2NH2), 2.59 (s, 3H, CH3S). MALDI-TOF MS m/z
[M + H]+ Calcd C24H21N3S: 384.1529, Found: 384.1523.
4.3.3. Synthesis of 2,6-bis(4-azidophenyl)-4-[4-(methylthio)phenyl]pyridine (3) (Figure S2A)
Compound 2 (200 mg, 0.5 mmol) was added to 15 mL 6N HCl and the reaction mixture was cooled
to 0 ◦C and NaNO2 (138 mg, 2 mmol) was added and was stirred for 30 min under cold condition.
Sodium azide (195 mg, 3 mmol) was added to the reaction mixture portion-wise under cold condition;
and the reaction mixture was allowed to stir at room temperature for 2 h. The mixture was neutralized
with sodium bicarbonate and extracted with ethyl acetate (3
×
30 mL). The organic part was evaporated
under vacuum at 30 ◦C to afford compound
3 as yellow crystals (98%); as a yellow solid which was
used for next reaction. IR (KBr) 2105 cm-1 (N3). MALDI-TOF MS m/z [M+H]+ Calcd for C24H18N7S;
436.1339, Found: 436.1326.
4.3.4. General Procedure For The Synthesis of 2,6-bis{4-[4-(substituted-aminoalkyl)-1H-1,2,3-triazol-1-
yl]phenyl}-4-[4-(methylthio)phenyl]pyridines 1a–c
Compound
3 (50 mg, 0.1 mmol) was added to a mixture of 4 mL H2O and THF (1:1), then
CuSO4 5H2O (0.002 mmol) and sodium ascorbate (0.1 mmol) were added to the mixture and stirred.
·
After 5 min, the appropriate substituted alkyne (0.3 mmol) was added and stirred for 12 h at room
temperature. The solvent was evaporated under reduced pressure, and the residue was washed with
water followed by diethyl ether to afford the desired bis-triazolylarylpyridine compound 1a–c.
4-[4-(Methylthio)phenyl]-2,6-bis{4-[4-(3-pyrrolidin-1-ylpropyl)-1H-1,2,3-triazol-1 yl]phenyl}pyridine (1a).
Yellow crystals (84%); 1H-NMR (300 MHz, CDCl3) 8.39 (d, 4H, J = 8.80 Hz, 2H-20 and 2H-60),
δ
7.96 (s, 2H, H-3 and H-5), 7.92 (d, 4H, J = 8.80 Hz, 2H-30 and 2H-50), 7.86 (s, 2H, 2H triazol.), 7.74 (d, 2H,
J = 8.40 Hz, H-200 and H-600), 7.43 (s, 2H, J = 8.40 Hz, H-300 and H-500), 2.91 (t, 4H, J = 7.50 Hz, 2NCH2),
2.63-2.53 (m, 12H, 6 NCH2), 2.59 (s, 3H, CH3S), 2.06-1.96 (m, 4H, 2CH2 pyrrol.), 1.84-1.77 (m, 4H,
2CH2 pyrrol.). 13C-NMR (75 MHz, CDCl3)
δ 157.5 (C-2 and C-6), 151.4 (C-4), 150.2 (C-4 triazol.), 142.1
(C-400), 140.6 (C-10), 138.9 (C-40), 135.9 (C-100), 129.8 (C-30 and C-50), 128.7 (C-200 and C-600), 127.9 (C-300
and C-500), 121.8 (C-20 and C-60), 120.2 (C-5 triazol.), 118.5 (C-3 and C-5), 58.3 (NCH2), 56.8 (NCH2),
28.4 (CH2), 27.4 (CH2), 24.9 (CH2), 16.8 (CH3S). MALDI-TOF MS m/z [M+H]+ Calcd for C42H48N9S;
710.3748, Found: 710.3740.
2,6-Bis{4-[4-(3-(4-methylpiperazin-1-yl)propyl)-1H-1,2,3-triazol-1-yl]phenyl}-4-[4-(methylthio)phenyl]-
pyridine (1b)
Orange crystals (68 %); 1H-NMR (300 MHz, CDCl3) 8.38 (d, 4H, J = 8.70 Hz, 2H-20 and 2H-60),
δ
7.96 (s, 2H, H-3 and H-5), 7.92 (d, 4H, J = 8.70 Hz, 2H-30 and 2H-50), 7.86 (s, 2H, 2H triazol.), 7.73 (d, 2H,
J = 8.50 Hz, H-200 and H-600), 7.43 (d, 2H, J = 8.50 Hz, H-300 and H-500), 2.89 (t, 4H, J = 7.50 Hz, 2NCH2),
2.66-2.42 (m, 20 H, 2NCH2 and 8NCH2 piperaz.), 2.59 (s, 3H, CH3S), 2.32 (s, 6H, 2NCH3), 2.02-1.92 (m,
4H, 2CH2). 13C-NMR (75 MHz, CDCl3)
δ
157.5 (C-2 and C-6), 151.3 (C-4), 150.1 (C-4 triazol.), 142.1
(C-400), 140.6 (C-10), 139.0 (C-40), 136.1 (C-100), 129.7 (C-30 and C-50), 128.8 (C-200 and C-600), 128.0 (C-300

Cancers 2020, 12, 1621
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and C-500), 121.7 (C-20 and C-60), 120.2 (C-5 triazol.), 118.4 (C-3 and C-5), 59.1 (NCH2), 56.5 (NCH2),
54.5 (NCH2), 47.4 (NCH3), 28.0 (CH2), 25.0 (CH2), 16.8 (CH3S). MALDI-TOF MS m/z [M+H]+ Calcd for
C44H54N11S; 768.4279, Found: 768.4269.
2,6-Bis{4-[4-(2-(4-methylpiperazin-1-yl)ethyl)-1H-1,2,3-triazol-1-yl]phenyl}-4-[4-(methylthio)phenyl]-
pyridine (1c).
Yellow crystals (64%); 1H NMR (300 MHz, CDCl3) 8.39 (d, 4H, J = 8.80 Hz, 2H-20 and 2H-60),
δ
7.96 (s, 2H, H-3 and H-5), 7.94 (s, 2H, 2H triazol.), 7.91 (d, 4H, J = 8.80 Hz, 2H-30 and 2H-50), 7.73
(d, 2H, J = 8.40 Hz, H-200 and H-600), 7.43 (d, 2H, J = 8.40 Hz, H-300 and H-500), 3.07 (t, 4H, J = 7.80 Hz,
2NCH2), 2.81 (t, 4H, J = 7.80 Hz, 2NCH2), 2.63-2.43 (m, 16 H, 8NCH2 piperaz.), 2.59 (s, 3H, CH3S),
2.34 (s, 6H, 2NCH3). 13C-NMR (75 MHz, CDCl3)
δ 157.5 (C-2 and C-6), 151.5 (C-4), 148.0 (C-4 triazol.),
142.2 (C-400), 140.8 (C-10), 139.0 (C-40), 136.2 (C-100), 129.8 (C-30 and C-50), 128.8 (C-200 and C-600), 128.0
(C-300 and C-500), 121.9 (C-20 and C-60), 120.8 (C-5 triazol.), 118.5 (C-3 and C-5), 58.6 (NCH2), 55.8
(NCH2), 53.1 (NCH2), 46.5 (NCH3), 24.6 (CH2), 16.8 (CH3S). MALDI-TOF MS m/z [M+H]+ Calcd for
C42H50N11S; 740.3966, Found: 740.3924.
4.4. Quadruplex Stabilization
Binding of 1a
previously described [45]. Compounds were tested at 1–10
the F21T oligonucleotide (FAM-50-GGGTTAGGGTTAGGGTTAGGG-30-TAMRA) at 0.2
,
1b and 1c to G-quadruplexes was investigated by FRET melting analysis as
M concentration in the presence of
M strand
µ
µ
concentration in a 10 mM lithium cacodylate pH 7.2 buffer supplemented with 10 mM KCl and
90 mM LiCl.
4.5. Mitochondrial and Lysosomal Staining and Evaluation of 20A Localization
Cells were grown on glass coverslips on a 6-well plate (6
5 µM 20A for 6h. Cells were stained with 50 nM MitoTracker Green FM (#M7514, Molecular Probes,
×
104 cells/well), treated with or without
Eugene, OR, USA) and 50 nM LysoTrackerTM Red DND-99 (#L7528, Molecular Probes) 30 min before
the end of the incubation time of 20A. Cells were then placed in a perfusion chamber with DMEM
without red Phenol containing 15 nM Lysotracker Red DN-99. Image acquisition was made on an LSM
510 META confocal microscope Zeiss (Oberkochen, Ostalbkreis, DE) equipped with an Apoplan x63
objective. Identical exposures were used for each channel throughout individual experiments, and no
images were altered after capture. Co-localization of blue signal (20A) with red signal (lysosome)
and green signal (mitochondria) was analyzed by superposing the fluorescence profile of each signal.
For some experiments, lysosome size was scored using “the analysis particles” function of Image J
after applying the autoThreshold “Minimum”. For the analysis, we took into consideration particles
that display a size between 0.1 and 1.6 µm2 and circularity between 0.5 and 1 µm.
4.6. Lysosomal Membrane Permeabilization (LMP) Analysis
4.6.1. Quantification of Galectin 3 Puncta
Lysosomal membrane damage was assessed thanks to U2OS expressing mCherry-Galectin3 cells.
Cells were plated on 12-well plates (7.5
×
104 cells/well) treated with or without 20A in either the
presence or absence of 25 M chloroquine for 24 h. Cells were then washed with PBS, fixed with
µ
4% (v/v) paraformaldehyde for 10 min at room temperature and nuclei were then counterstained for
15 min with Hoechst 33258. Image acquisition was made with a DMI8 epifluorescence microscope
(Leica Microsystems, Wetzlar, Hessen, DE) with a x20 objective (NA 0.40) equipped with a digital
CMOS camera (Hamamatsu Photonics, Hamamatsu, Shizuoka, JP) and filter bloc for detection of
phase contrast and blue (exc: 325–375 nm/em: 435–485 nm) and red (exc: 541–551 nm/em: 565–605 nm)
fluorescence. Each image was collected with identical exposure times and scaled equally. Five images
were acquired for each condition and then analysed thanks to Image J software to ImageJ software

Cancers 2020, 12, 1621
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(NIH-National Institutes of Health, Bethesda, MD, USA).The number of Galectin3 puncta within each
cell was scored using the “Find Maxima” tool.
4.6.2. Co-Localization of Galectin 3 with LAMP1
To assess lysosomal membrane permeabilization (LMP), co-localization study of Galectin3 puncta
and LAMP1 was carried out by immunofluorescence analysis. Cells were grown on glass coverslips,
fixed in ice-cold methanol at
20◦C for 15 min and then blocked/permeabilized with 3% BSA containing
−
0.3% Triton X-100 for 1 h. Cells were then incubated with the primary antibody overnight at 4◦C.
After washing, cells were incubated with the secondary antibody (anti-rabbit Alexa Fluor 488 antibody)
for 1 h at room temperature in a dark chamber and stained with 2 µg/mL of Hoechst 33258 (H3569,
Promega, Madison, WI, USA) for 10 min. After two PBS wash, cells were mounted with Fluoromount
(F4680, Sigma-Aldrich, St. Louis, MO, USA) and images were acquired using a Zeiss LSM510 Meta
confocal microscope Zeiss, (Oberkochen, Ostalbkreis, DE) equipped with an oil immersion Apoplan
63
×
objective (NA 0.75). Z-stack series were made with 0.6 µm interval and the corresponding
images represent a z-projection of maximal intensity. Identical exposures were used for each channel
throughout individual experiment. No images were altered after capture. Processing of images was
performed with the ImageJ software.
4.7. Evaluation of Cell Death
Cell death was determined by measuring the plasma membrane permeability using propidium
iodide dye (P4864, Sigma). Briefly, supernatant and attached cells were collected, pelleted at 350 g for
5 min and loaded with 1 µg/mL Propidum iodide for 15 min. Cells were then analyzed by flow cytometry
(BD FACSCalibur, Becton Dickinson, Franklin Lakes, NJ, USA) in the FL2 channel (exc: 488 nm/em:
564–606 nm). For some experiments, cells were stained with 150 nM tetramethylrhodamine methyl ester
(TMRM, from Molecular Probes-Life Technologies) and 5 µ
g/mL 40,6-diaminidino-2-phenylindole (DAPI,
from Molecular Probes-Life Technologies) for 30 min at 37 ◦C. Cytofluorometric determinations of early
and late apoptotis were carried out on a MACSQuant cytometer (Miltenyi Biotec, Bergisch Gladbach,
Germany) upon gating on events displaying normal forward scatter and side scatter parameters.
Apoptosis was also evaluated by western blotting analyses of cleaved forms of either PARP 1 or
caspase 3 [24]. Where indicated, the caspase inhibitor QV-D-Oph (20
the possible implication of caspases in cell demise.
µM) was added to cells to evaluate
4.8. Immunoblot Assay
Cell extracts were prepared in 10 mM Tris, pH 7.4, 1% SDS, 1 mM sodium vanadate, 2 mM
PMSF (93482, Sigma-Aldrich), 1% Protease Inhibitor Cocktail (P8849, Sigma-Aldrich), and 1% Halt
Phosphatase Inhibitor Cocktail (1862495, Thermo Fisher Scientific, Waltham, MA, USA) and treated
with benzonase endonuclease (71205, Merck Millipore) for 5 min at room temperature, and boiled for
5 min. Fractions (30–50 µg) of cellular extract proteins were subjected to SDS-PAGE as previously
described [21]. The densitometry quantification was performed using the ImageJ software.
4.9. Cell viability Assay
MTT assay was used to monitor cell viability in cells subjected to different treatments. Briefly, cells
were seeded in a 96-well plate (4
added to each well to a final concentration of 0.5 mg/mL during 3 h at 37 ◦C. The supernatant of cells was
then removed, and 100 L of DMSO was added per well. The absorbance in each well was measured at
×
103 cells/well). After 24 h of treatment of cells with drug, MTT was
µ
570 nm and 630 nm using a Flexstation 3 microplate reader FlexStation 3 Microplate Reader (Molecular
Devices, San Jose, CA, USA). To determine the IC50 of 20A derivative compounds, a non-linear
regression curve was fit using GraphPad Prism software (GraphPad Software Inc., San Diego, CA, USA).

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4.10. Statistical Analysis
Unless otherwise stated, each experiment was performed three times. Results were expressed as the
mean value standard deviation (SD). Statistical analysis was performed by Mann-Withney unpaired
±
statistical test. p < 0.05 was considered statistically significant. Where indicated, Kruskal-Wallis test,
with post-hoc Dunn’s analysis was used for statistical analysis. The software used was GraphPad
Prism 5.
5. Conclusions
In conclusion, our study uncovers the lysosomal effects of G4 ligands belonging to the
triarylpyridine family and proposes a rationale for combining these compounds with chloroquine
to increase their effectiveness against cancer. It would be interesting to carry out a comprehensive
study on multiple G4 ligands to classify them according to their lysosomal sequestration and their
capacity to cooperated with chloroquine-like agents. In fact, the chemical properties of the molecules
including pKa value(s) and lipophilicity are critical factors that determine the ability of the molecule to
be trapped within the lysosomes and provoke LMP [46]. This information should then be taken into
account for the development of new G-quadruplex ligands designed for optimal cancer therapy.
Supplementary Materials: The following figures are available online at http://www.mdpi.com/2072-6694/12/6/
1621/s1, Figure S1: KEGG database depicting the lysosomal network alterations by 20A, Figure S2: Synthesis of
20A derivatives and stabilization of G4 structure by 20A derivatives, Figure S3: Fluorescence emission spectra of
20A, Figure S4: Evaluation of early and late apoptosis in U2OS cells treated by 20A plus chloroquine, Figures S5 &
S6: Full Western blot images for Western blotting experiments presented in the main MS, Figure S7: Evaluation of
involvement of ferroptosis, necroptosis and cathepsin activity in cell treated 20A plus chloroquine. Figure S8: Cell
viability assay for 20A derivative compounds, Figure S9: Role of caspases and ROS in LMP induced by 20A plus
chloroquine, Figure S10: Detailed information about Figure 2a, Figure S11: Detailed information about Figure 4c.
Author Contributions: Conceptualization of the study was performed by J.B. and M.D.-M.; methodology was
conducted by J.B., R.N.D., K.A.-V., I.M., J.G.; validation was performed by J.B., K.A.-V., I.M., E.R., A.M.; Formal
analysis was performed by E.D., R.N.D., J.G.; investigation was performed by J.B., R.N.D., K.A.-V., I.M., E.R., A.M.;
resources, J.B., E.D., J.G., M.D.-M.; data curation, E.D.; writing—original draft preparation was written by J.B.
and M.D.-M. writing—review and editing were performed by J.B., M.D.-M., P.S., G.K., J.G., J.-L.M.; visualization
was done by J.B., J.-L.M., M.D.-M.; supervision, M.D.-M.; project administration was carried out by J.-L.M. and
M.D.-M.; funding acquisition was obtained by J.-L.M., G.K., M.D.-M.; All authors have read and agreed to the
published version of the manuscript
Funding: This research was funded by Ligue R
SIRIC BRIO (to M.D.M), FR Transbiomed (to M.D-M.), Ligue R
supported by the Mexican National Council of Science and Technology (CONACYT, funding #757821). G.K. is
é
gionale d’Aquitaine, comit
é de Dordogne (to J.L.M and M.D.M.),
é
gionale d’Aquitaine, (to M.D-M). K.A-V is
supported by the Ligue contre le Cancer (équipe labellisée); Agence National de la Recherche (ANR) – Projets
blancs; ANR under the frame of E-Rare-2; ANR “Flash-covid-19” LYSOMOTROP; the ERA-Net for Research on
Rare Diseases; AMMICa US23/CNRS UMS3655; Association pour la recherche sur le cancer (ARC); Association
“Le Cancer du Sein, Parlons-en!”; Canc
Fondation pour la Recherche M
éropôle Ile-de-France; Chancelerie des universités de Paris (Legs Poix),
é
dicale (FRM); a donation by Elior; European Research Area Network on
Cardiovascular Diseases (ERA-CVD, MINOTAUR); Gustave Roussy Odyssea, the European Union Horizon
2020 Project Oncobiome; Fondation Carrefour; High-end Foreign Expert Program in China (GDW20171100085);
Institut National du Cancer (INCa); Inserm (HTE); Institut Universitaire de France; LeDucq Foundation; the LabEx
Immuno-Oncology (ANR-18-IDEX-0001); the RHU Torino Lumière; the Seerave Foundation; the SIRIC Stratified
Oncology Cell DNA Repair and Tumor Immune Elimination (SOCRATE); and the SIRIC Cancer Research and
Personalized Medicine (CARPEM).
Acknowledgments: The authors gratefully acknowledge the BIC platform in Bordeaux. The graphical abstract
was created by BioRender assets.
Conflicts of Interest: The authors declare no conflict of interest.
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