Copper-dependent cytotoxicity of 8-hydroxyquinoline derivatives correlates with their hydrophobicity and does not require caspase activation

Article abstract of DOI:10.1021/jm301053a

This study reports the structure-activity relationship of a series of 8-hydroxoquinoline derivatives (8-HQs) and focuses on the cytotoxic activity of 5-Cl-7-I-8-HQ (clioquinol, CQ) copper complex (Cu(CQ)). 8-HQs alone cause a dose-dependent loss of viability of the human tumor HeLa and PC3 cells, but the coadministration of copper increases the ligands effects, with extensive cell death occurring in both cell lines. Cytotoxic doses of Cu(CQ) promote intracellular copper accumulation and massive endoplasmic reticulum vacuolization that precede a nonapoptotic (paraptotic) cell death. The cytotoxic effect of Cu(CQ) is reproduced in normal human endothelial cells (HUVEC) at concentrations double those effective in tumor cells, pointing to a potential therapeutic window for Cu(CQ). Finally, the results show that the paraptotic cell death induced by Cu(CQ) does not require nor involve caspases, giving an indication for the current clinical assessment of clioquinol as an antineoplastic agent.

Full text of DOI:10.1021/jm301053a

Article
pubs.acs.org/jmc
Copper-Dependent Cytotoxicity of 8‑Hydroxyquinoline Derivatives
Correlates with Their Hydrophobicity and Does Not Require Caspase
Activation
Saverio Tardito,‡,§,∥ Amelia Barilli,‡,§ Irene Bassanetti,† Matteo Tegoni,† Ovidio Bussolati,‡
Renata Franchi-Gazzola,‡ Claudio Mucchino,† and Luciano Marchio*,†
̀
†Dipartimento di Chimica, Universita
̀
degli Studi di Parma, Viale delle Scienze 17/A, 43123 Parma, Italy
‡Dipartimento di Scienze Biomediche, Biotecnologiche e Traslazionali, Universita
Parma, Italy
̀
degli Studi di Parma, Via Volturno 39, 43100
S
* Supporting Information
ABSTRACT: This study reports the structure−activity
relationship of a series of 8-hydroxoquinoline derivatives (8-
HQs) and focuses on the cytotoxic activity of 5-Cl-7-I-8-HQ
(clioquinol, CQ) copper complex (Cu(CQ)). 8-HQs alone
cause a dose-dependent loss of viability of the human tumor
HeLa and PC3 cells, but the coadministration of copper
increases the ligands effects, with extensive cell death occurring
in both cell lines. Cytotoxic doses of Cu(CQ) promote
intracellular copper accumulation and massive endoplasmic
reticulum vacuolization that precede a nonapoptotic (para-
ptotic) cell death. The cytotoxic effect of Cu(CQ) is
reproduced in normal human endothelial cells (HUVEC) at
concentrations double those effective in tumor cells, pointing
to a potential therapeutic window for Cu(CQ). Finally, the results show that the paraptotic cell death induced by Cu(CQ) does
not require nor involve caspases, giving an indication for the current clinical assessment of clioquinol as an antineoplastic agent.
unfolded protein response (UPR) and inhibit protein synthesis
in an effort to relieve the ER from excessive protein overload. If
the stress persists, the expression of the transcription factor
CHOP (CCAAT-enhancer-binding protein homologous pro-
tein) increases, initiating the apoptotic cascade.7,8
INTRODUCTION
■
Several types of programmed cell death (PCD) have been
reported in the literature.1 Caspase-dependent apoptosis is the
most common type of PCD through which normal and tumor
cells die in an organized manner, both during tissue
development and under conditions of stress, such as chemo-
therapeutic treatment.2 From a morphological point of view,
apoptosis is manifested with cell shrinkage, chromatin
condensation, and the appearance of apoptotic bodies
containing nuclear fragments. The apoptotic process can be
activated either by extracellular ligands through transmembrane
death receptors (extrinsic pathway) or as a consequence of
cellular injury that ultimately leads to mitochondrial membrane
permeabilization and release of cytochrome c (intrinsic
pathway). The complex biochemical machinery activated
during the cell death process usually promotes the activation
of caspase-3, which dismantles cellular architecture and leads to
cell death.3,4 However, caspase-independent apoptosis has also
been documented.1 Caspase-independent apoptotic cell death
usually comprises the translocation of endonucleases
(ENDOG) and/or apoptosis-inducing factor (AIF) from the
mitochondria to the nucleus.5,6 The extensive stress on the
endoplasmic reticulum (ER) caused by the accumulation of
unfolded proteins in its lumen can trigger the apoptotic
pathway. In the presence of this stress, cells activate the
We have recently reported that an elevated intracellular
copper concentration activates the UPR, either by inhibiting the
proteasome, which is responsible for the degradation of
misfolded proteins, or by promoting protein misfolding.9 This
latter process is most likely due to the redox ability of the metal
ion because intracellular copper accumulation is accompanied
by a decrease of the reduced form of glutathione (GSH) and an
increase of its oxidized form (GSSG).10 Interestingly, the
copper-dependent UPR does not lead to apoptosis because of
the inhibition of caspase-3 by the metal, but rather an
alternative type of PCD characterized by extensive cytoplasmic
vacuolization, which is the morphological hallmark of para-
ptosis, is induced.9 Other examples of caspase-3-independent,
paraptosis-like cell death associated with ER stress and UPR
upon treatment of lymphoma cells with cannabinoids were
recently reported.11 In another report, the use of curcumin
induced paraptosis in malignant breast cell lines.12,13 These
Received: July 19, 2012
Published: November 22, 2012
© 2012 American Chemical Society
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findings provide further evidence that paraptosis can be
activated in different cell lines with structurally and functionally
unrelated compounds and may serve as an alternative, drug-
induced death pathway for cancer and normal cells unable to
undergo apoptosis.
was already recognized as an antiparasitic drug, even though its
use was discontinued in some countries because of the
occurrence of subacute myelo-optic neuropathy.22 CQ is
currently used as an antifungal and antibacterial in the
commercial cream Vioform. In mice, CQ proved to be able
to inhibit the accumulation of β amyloid (Aβ) in the brain and
to mobilize the Aβ from existing deposits,23 which are of great
relevance for treatments of Alzheimer disease.24,25 The CQ-
related compound PBT226 recently underwent phase II clinical
trials as an anti-Alzheimer compound.27 Moreover, CQ has
entered phase I clinical trials for the treatment of relapsed or
refractory hematological malignancies (NCT00963495).
In previous research, we have shown that nitrogen donor
ligands behave as copper ionophores in cancer cells, thus
leading to a toxic accumulation of the metal, which eventually
triggers paraptotic cell death.9,10,28 The goal of this project is to
investigate whether nonapoptotic programmed cell death is
activated in tumor cell lines by the combined treatment with
copper and 8-HQ analogues, with a particular focus on the
copper ionophore CQ.29,30
Copper is an essential metal ion involved in several highly
conserved biochemical processes. Its oxidation state shifts from
+2 to +1 upon internalization because of the reducing
intracellular environment. The metal is taken up by the cell
through the copper transporter hCTR1,14,15 and a number of
chaperones deliver it to its intracellular destinations, i.e., Cu−
Zn superoxide dismutase and cytochrome c oxidase. A mild
intracellular excess of copper can normally be balanced by the
cells through an increased expression of metallothioneins or
through the activation of specific pumps, such as ATP7A and
ATP7B,16−18 whereas high copper concentrations cause the
activation of the UPR, as described above. For this reason,
despite its essential role in living cells, copper has been
frequently employed in the preparation of cytotoxic complexes.
In many instances, the choice of Cu(II)-based compounds as
cytotoxic agents originates from the overproduction of reactive
oxygen species (ROS) driven by the metal. Alternatively,
copper-binding agents have been employed for their ability to
direct the toxicity of the metal toward specific intracellular
targets.19,20
In cultured cells, the internalization of copper complexes
does not usually involve the hCTR1 copper-specific transporter
but rather occurs primarily via passive diffusion.9,21 For this
reason, the chemical features of the ligand appear to be the
main determinants of the activity of the complex because they
may (i) modulate its permeability through the cell membranes
by virtue of its lipophilic character, (ii) stabilize a specific redox
state of the metal, (iii) drive the complex to specific cellular
targets, and (iv) exhibit an intrinsic cytotoxic activity when
dissociated from the metal. 8-Hydroxyquinoline (8-HQ)
derivatives are planar, N,O-donating ligands that have a high
affinity for Cu(II) (Figure 1). Particular interest has recently
been dedicated to 5-Cl-7-I-8-HQ (clioquinol, CQ) because CQ
RESULTS
■
Molecular Structures and Stability Constants of Cu(II)
Complexes with 8-HQ Derivatives. The Cu(II) complexes
can be prepared by mixing CuCl2·2H2O and the corresponding
ligands in the 1:1 or 1:2 molar ratio as specified in the
Experimental Section. Complexes of formula [Cu(L)(H2O)2]
are obtained with the 5-SO3-8-HQ and 5-SO3-7-I-8-HQ
ligands, whereas the remaining ligands give rise to complexes
of [Cu(L)2] general formula (Figure 1). Given the different
lipophilic properties of the ligands, different solvents were
employed to prepare solutions of the ligands, whereas
CuCl2·2H2O was dissolved in methanol. The [Cu(L)(H2O)2]
or [Cu(L)2] complexes readily precipitated from the reaction
mixtures as brown or green powders. These compounds are
usually insoluble in aqueous media and in common organic
solvents and are poorly soluble in DMSO. For this reason, the
biological tests were performed by mixing CuCl2·2H2O and the
corresponding ligands directly into the cell culture media at a
1:1 molar ratio. The final concentration of the complex for the
biological tests was in the 1−30 μM range such that it did not
precipitate in the medium. On the basis of our determined
stability constants for 8-HQ and 5-SO3-8-HQ (see below) in
this range of concentrations, we expect that in aqueous media
and with no competitor ligands, 40−50% of the total Cu(II)
forms [Cu(L)] while the remaining 50−60% is present as
[Cu(L)2] and “free” Cu2+ ions (25−30% each) (Supporting
Information, Figure S1).
8-HQ and its derivatives are characterized by the presence of
a N,O-binding system that is suitable for interacting strongly
with Cu(II) ions. Figure 1 depicts the general molecular
structures of the complexes of formula [Cu(5-R-7-R′-8-HQ)2].
−
The presence of the −SO3 group leads to the formation of
complexes in which the metal/ligand ratio is 1:1 because of the
involvement of the sulfonate moiety in metal coordination.31−33
When R and R′ are halogen or alkyl substituents, the metal/
ligand ratio becomes 1:2 and the ligands are arranged in a trans
geometry, with the nitrogen and oxygen atoms on the opposite
side with respect to the metal center. The molecular structures
of [Cu(8-HQ)2]34 and [Cu(CQ)2]35 were previously reported,
whereas that of [Cu(5,7-Me-8-HQ)2] is reported in Figure 1B
and in the Supporting Information, Figure S5.
Figure 1. (A) Molecular structures of Cu(II) complexes with 8-HQ
derivatives. (B) ORTEP diagram of the X-ray molecular structure of
[Cu(5,7-Me-8-HQ)2], symmetry code = −x, 1 − y, −z.
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Figure 2. Dose−response curves for ligands and copper complexes in HeLa and PC3 cells. HeLa and PC3 cells were incubated for 48 h with
different concentrations of ligands (upper panels) or copper complexes (lower panels). Cell viability after treatment was assessed using the resazurin
method. The data presented are the mean SEM of three independent experiments, each performed in triplicate. Error bars are visible when larger
than the symbols.
The stability constants of [Cu(5-SO3-8-HQ)(H2O)2] and
[Cu(8-HQ)2] were determined by potentiometry in a 1:1
DMSO/water (w/w) solution. The Cu(II) complexes with
these ligands were remarkably stable, with log β1 and log β2
values of about 11.5 and 22.5 for both 8-HQ and 5-SO3-8-HQ
(see Supporting Information, Table S1). Previous studies
reported the stability of these ligands in water,36 although most
studies were performed in water/organic mixtures such as
alcohol37 or water/dioxane mixtures38,39 to address solubility
problems. These studies reported formation constants that fall
in the range log β1 of 11−14 and log β2 of 22−25.40,41 In this
study, we used a DMSO/water mixture as an attempt to
increase the solubility of the various species in order to
determine the stability constants in homogeneous conditions.
Unfortunately, the poor solubility of the remaining ligands (or
their complexes) in this mixture made it difficult to measure the
stability constants for the other complexes. Our values fall
within the range determined in different solvents, showing that
the presence of DMSO does not significantly affect the stability
of the complexes. Moreover, the similarity of the values of log β
for 5-SO3-8-HQ and 8-HQ suggests that any interaction
between the [Cu(L)] species and a ligand molecule via the
sulfonate group (as found in the solid state) is negligible in
solution. The influence exerted by the substituents in the 5 and
7 positions on the hydroxyquinoline moiety is also expected to
have minimal steric influence on the geometry of the Cu(II)
complexes and therefore on their stability due to the
positioning of these groups at the exterior of the coordination
site (see Figure 1).
for by their log β values. In fact, the stability constants of the
coordination of Cu(II) with the first ligand (giving [Cu(L)],
log β1) and the second ligand ([Cu(L)2], log K2 = log β2 −
log β1) are approximately the same. As a result, the amount of
[Cu(L)2] in solution for a HL/Cu(II) ratio of 1 is quite high
(∼20% total copper for both ligands), likely leading to the
precipitation of this neutral complex from the solution (see
discussion in the Supporting Information). This finding is in
agreement with the poor solubility of the [Cu(L)2] complex
with 8-HQ (∼10−6 M),36 which precipitates out of solution at a
HL/Cu(II) ratio greater than 1.2, even at pH ≈ 4.5 and CCu
≈
10−4 M.
The stability of the complexes [Cu(8-HQ)2] and [Cu(5-SO3-
8-HQ)(H2O)2] in the presence of human serum albumin
(HSA) was evaluated by means of circular dichroism (Figure
S4).42 The concentration of albumin in the samples was 85 μM,
close to that used in the biological experiments (around 60
μM). The CD spectra in the presence of CuCl2 (control),
[Cu(8-HQ)2], and [Cu(5-SO3-8-HQ)(H2O)2] show a Cotton
effect in the range 450−650 nm related to the Cu(II) d−d
transitions and indicative of the coordination of Cu(II) to the
high affinity binding site at the N-terminus of the protein. By
comparison of the CuCl2/HSA, [Cu(8-HQ)2]/HSA, and
[Cu(5-SO3-8-HQ)(H2O)2]/HSA systems, a lower Cotton
effect is observed in the presence of hydroxyquinoline ligands,
indicating that albumin is not able to completely displace the
metal from the ligands. Overall, these data demonstrate that the
complexes are stable to a significant extent even in the presence
of albumin.
Interestingly, the [Cu(L)2] compounds can precipitate out as
solids when using the 1:1 molar ratio (except those with the 5-
SO3-8-HQ and 5-SO3-7-I-8-HQ ligands that form complexes
with Cu/L 1:1 stoichiometry). This behavior can be accounted
Effects of the Ligands and Complexes on HeLa and
PC3 Cell Viability. The cytotoxic potential of the ligands was
evaluated in two human tumor cell lines derived from cervical
carcinoma, HeLa, and prostatic adenocarcinoma, PC3. As
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shown in Figure 2 (upper panels), all ligands present cytotoxic
activity except for 5-SO3-8-HQ and 5-SO3-7-I-8-HQ, which are
almost ineffective on both cell lines up to the maximum
concentration employed (30 μM). For six out of the eight 8-
HQ derivatives, adding an equimolar amount of CuCl2
dramatically increases the effect of the ligands in both HeLa
and PC3 cells (Figure 2, lower panels), decreasing the IC50
values of the active ligands in both cell models. The addition of
copper shifts the range of ligand IC50 from 13.5−25.2 to 1.9−
12.8 μM and from 8.6−25.7 to 1.3−16.2 μM in HeLa and PC3,
respectively (Table 1 and Table S3, for comparison).
Interestingly, a nonlinear correlation between the lipophilic
character of the ligand, which is modulated by the peripheral
functional groups, and the cytotoxic activity of the correspond-
ing copper complex was found. When the ligands are ranked by
increasing lipophilic character, i.e., with increasing log P values,
the activities of the corresponding complexes describe a bell-
shaped curve (Figure 3). According to this relationship, the
highest cytotoxic activity is expected to be exhibited by ligands
with intermediate log P values, namely, between 1.5 and 3.
Although the complex Cu(CQ) is significantly less active
than Cu(8-HQ) in both cell lines, we decided to further
investigate its biological behavior, given the known and
documented use of clioquinol as a drug43 and the recent
attention paid to its anticancer potential in vitro and in
vivo.30,44,45
a
Table 1. Molecular Structures of the Copper Complexes
b
and the IC50 (μM) Values in HeLa and PC3 Human Tumor
Cell Lines
To evaluate the effect of the drug and its copper complex in
nonmalignant cells, their effects have been tested in normal
human cells (HUVEC, human umbilical vein endothelial cell;
HF, human fibroblast). The vascular endothelium is one of the
first tissues that drugs come in contact with during the
administration of chemotherapy. The study of the effects of the
two compounds on vascular endothelial cells is therefore of
peculiar interest, given their potential application to clinical
practice. Because endothelial cells are routinely grown in the
presence of 20% FBS, for these specific experiments, the serum
concentration was raised from 10% to 20% for all cell types. In
HUVEC, the cytotoxic activity of the ligand remains negligible
at all concentrations tested, whereas the corresponding complex
exhibits an IC50 of 25.2 μM. Notably, the presence of higher
serum levels diminishes the cytotoxic effect of Cu(CQ) in
tumor cell lines, with the IC50 values of Cu(CQ) being 14.8 and
10.9 μM in HeLa and PC3, respectively (Figure 4). This effect
is likely due to the copper binding activity of the serum
components. Indeed, when Cu(CQ) is tested under low serum
conditions (10%), its cytotoxic activity appears to be
considerably increased in all cell models, although with
approximately the same differential efficacy in normal and
tumor cells (Figure S7).
As far as normal human fibroblasts are concerned,
concentrations up to 8.9 μM of the copper complex do not
exert any significant effect, either on cell number or on lactate
dehydrogenase (LDH) release (Figure S8). Beyond this
concentration, LDH release significantly increases and cell
number concomitantly decreases, indicating a cytotoxic effect.
However, a complete cell loss cannot be detected even at the
a
b
Only the CuL fragment is depicted. The cytotoxic activity is
measured for mixtures composed of CuCl2 and hydroxyquinoline
derivatives in a 1:1 molar ratio.
Figure 3. Correlation between cytotoxic activity of copper complexes and ligand log P. The activities of the copper complexes, calculated as the
inverse of IC50 in HeLa and PC3 cells, were matched with corresponding ligand log P values. The curves were extrapolated with the GraphPad Prism
software.
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Figure 4. Comparison of dose−response curves of CQ and Cu(CQ)
in HeLa, PC3, and HUVEC cells. Different concentrations of CQ and
Cu(CQ) were tested for 48 h on the cells in the presence of 20% FBS.
After the treatment, viability was assessed using the resazurin method.
The data presented are the mean
SEM of three independent
experiments, each performed in quadruplicate. Error bars are visible
when larger than the symbols.
maximum concentration tested (30 μM), suggesting that a
fraction of the cell culture is highly resistant to Cu(CQ).
Characterization of Cu(CQ)-Dependent Cytotoxicity.
It has been reported that the cytotoxicity induced by clioquinol
is maximally increased by the presence of copper and is
associated with caspase activation.46 To assess whether the
metal and/or caspases are required for the Cu(CQ) cytotoxic
effect, HeLa and PC3 cells were incubated with increasing
doses of Cu(CQ) in the presence of a high affine extracellular
copper chelator, ammonium tetrathiomolybdate (TM),47,48 or
of a cell-permeable pan-caspase inhibitor, z-VAD-FMK.
As shown in Figure 5A, the addition of TM dramatically
reduces the cytotoxic effect of the complex: the IC50 of
Cu(CQ) moves from 9.0 and 8.9 μM to 24.5 and 21.5 μM in
HeLa and PC3, respectively, and the dose−response profile of
the complex in the presence of TM becomes similar to that of
the CQ ligand alone. Interestingly, higher doses of TM have a
moderately toxic effect on HeLa cells, as shown by the dose−
response curve of Cu(CQ) in the presence of 40 μM TM
(Figure S9). On the contrary, curves obtained upon treatment
of HeLa and PC3 cells with the complex in the absence and
presence of z-VAD-FMK are comparable, suggesting that
copper, not caspases, is essential for the Cu(CQ) mechanism of
action.
Figure 5. Characterization of Cu(CQ)-dependent cytotoxicity. (A)
HeLa and PC3 cells were treated for 48 h with different concentrations
of CQ or Cu(CQ), either in the absence or presence of 20 μM TM or
50 μM z-VAD-FMK or with 14 μM CuCl2. Cell viability was assayed
using the resazurin method. The data presented are the mean SEM
of three independent experiments, each performed in quadruplicate.
Error bars are visible when larger than the symbols. (B) HeLa and PC3
cells were treated for the indicated times with 14 μM Cu(CQ) in the
absence or presence of 20 μM ammonium tetrathiomolybdate (TM)
or with 14 μM CuCl2. The intracellular copper content was measured
by means of ICP-AES, as described in the Experimental Section. The
bars are the mean SEM of two independent measurements in two
different experiments. (C) HeLa and PC3 cells were untreated or
treated with cisplatin (CisPt, 20 and 50 μM, respectively), 50 μM CQ,
or 14 μM Cu(CQ). After 18 h, caspase-3 activity and LDH release
were assayed in cell lysates and in the incubation medium, respectively.
The data are the mean SEM of four independent experiments. (D)
The levels of cleaved PARP-1 and caspase-3 proteins were assessed in
cell lysates of both HeLa and PC3 cells, either untreated or treated for
18 h with 20 and 50 μM CisPt, respectively, 50 μM CQ, or 14 μM
Cu(CQ). The experiment was repeated twice with comparable results.
Representative Western blots are shown.
To directly assess the effect of Cu(CQ) on cellular copper
homeostasis, the intracellular content of the metal has been
measured by means of ICP-AES in cells treated for different
times with 14 μM Cu(CQ). A marked time-dependent
accumulation of copper can be observed in both cell models
(Figure 5B), with the maximum effect being evident after 6 h,
when copper content is 70- and 50-fold increased in HeLa and
PC3 cells, respectively, with respect to control (untreated cells).
As expected, the simultaneous incubation of the cells with
Cu(CQ) and 20 μM TM lowers the intracellular copper
content to values comparable to those of untreated cells
(Figure 5B), thus confirming the ionophoric properties of
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clioquinol. Consistently with the absence of effects on HeLa
and PC3 cells viability (Figure 5A), treatment with 14 μM
CuCl2 only causes a modest increase of intracellular copper
content, as compared to that occurring upon incubation with
the same concentration of Cu(CQ) (Figure 5B).
Data obtained from the dose−response experiment per-
formed in the presence of the caspase inhibitor excluded the
possibility of substantial involvement of caspases in mediating
the cytotoxic effect of Cu(CQ) but not in their eventual
activation. We have therefore addressed the activity of caspase-
3, the final executor in caspase-dependent apoptosis, in
Cu(CQ)-treated cells. Caspase-3 is not activated in HeLa and
PC3 cells treated with the complex at the IC75 (14 μM), a
concentration at which the culture is undergoing substantial cell
death as shown by the marked release of LDH, a marker of
membrane rupture (Figure 5C). Conversely, the activation of
caspase-3 is readily detectable upon treatment of HeLa cells
with CQ at the maximum concentration tested (50 μM).
Consistent with the results obtained by means of the
biochemical assay, Western blot analysis demonstrates the
cleavage of caspase-3 in HeLa cells treated with 50 μM CQ but
not in HeLa and PC3 cells incubated with Cu(CQ) (Figure
5D).
During the apoptotic process, poly ADP-ribose polymerase-1
(PARP-1), a nuclear protein involved in the repair of DNA
breaks, is cleaved by caspases and inactivated to spare energy in
favor of the apoptotic cell death.49,50 In Figure 5D, it is shown
that PARP-1 is extensively cleaved in both HeLa and PC3 cells
upon treatment with the apoptosis inducer cisplatin, moder-
ately cleaved in 50 μM CQ-treated HeLa cells, and not cleaved
at detectable levels under any other conditions. Altogether,
these results exclude the possibility of activation of caspases and
more specifically of caspase-3 during the cell death process
induced by Cu(CQ) in HeLa and PC3 cells. Conversely, a
caspase-3 dependent apoptotic pathway appears to be involved
in clioquinol driven cytotoxicity, since the pan-caspase inhibitor
z-VAD-FMK partially protects HeLa cells from the cytotoxic
effect of CQ on both morphological (Figure 6A) and
biochemical (Figure 6B) grounds.
Finally, to further explore the molecular mechanisms
underlying Cu(CQ) cytotoxicity, the subcellular localization
of apoptosis-inducing factor (AIF) has been addressed in HeLa
cells by means of confocal microscopy. As shown in Figure S10,
a clear-cut nuclear staining of AIF is readily detectable upon
treatment with staurosporin, a toxin known to induce the
nuclear translocation of the factor from mitochondria.51 At
variance, CQ and Cu(CQ)-treated cells do not show any
appreciable nuclear staining, since the signal remains typically
cytoplasmic as in control, untreated cells.
Figure 6. Role of caspases in CQ-dependent cytotoxicity. HeLa cells
were treated with 50 μM CQ, both in the absence and in the presence
of 50 μM z-VAD-FMK for 18 h (control is untreated cells). (A) Phase
contrast images of representative microscopic fields (200×) are shown.
(B) Cell viability was assayed with the resazurin method, and LDH
release was determined in the incubation medium. Data are the mean
SD of three replicates within a representative experiment.
ligands, i.e., amino acids,40 and thus suggest a moderate/high
stability of these complexes with 1:1 and 1:2 Cu/L
stoichiometry. Nevertheless, it is generally accepted that once
a Cu(II) complex enters the cells, the reducing environment
favors the Cu(II) → Cu(I) reduction, causing a decrease in
ligand affinity for the metal. The presence of other highly affine
Cu(I) ligands within the cell most likely results in the
displacement of the ligand from the ion.53,54
The majority of cytotoxic drugs must enter the cell to express
their cytotoxic potential. Entry may occur through active
transport, as proposed for the internalization of cisplatin by the
copper transporter hCTR155,56 or by passive diffusion across
the plasma membrane. In the latter case, the drug must be
endowed with the appropriate lipophilicity so that it can cross
the cell membrane and reach the threshold intracellular
concentration. An overly lipophilic compound would accumu-
late in the membrane, while a large hydrophilicity would
prevent interactions with the lipid bilayer. Consistent with the
results described in this study, the literature reports several
examples of homologous series of species whose cytotoxic
activity correlates with lipophilicity.57−59 Among the 8-HQ
derivatives examined, the most hydrophilic ligands 5-SO3-8-
HQ and 5-SO3-7-I-8-HQ do not display any cytotoxic activity
when complexed with copper, whereas the most active
complexes are those with ligands of intermediate lipophilicity,
namely, 8-HQ, 5,7-Me-8-HQ, and 5-Cl-8-HQ.
DISCUSSION
■
We have recently shown that different classes of nitrogen-donor
ligands behave as copper ionophores and thus cause a toxic
accumulation of the metal in cancer cells, which ultimately
undergo a peculiar type of UPR-dependent, nonapoptotic cell
death.9 In the present study, we address the structure−activity
relationship of copper complexes with 8-HQ derivatives, as well
as their ability to trigger cell death. 8-HQ analogues are ligands
with a high affinity for copper that they bind through a N,O-
donor moiety.30,46,52 The stability constants of the correspond-
ing complexes, which describe the interaction between the
ligands and the Cu(II) ions, are orders of magnitude higher
than those exhibited by endogenous Cu(II)-complexing
Although copper forms the most active complex with 8-HQ,
we have decided to focus on the effects of CQ−copper
complex. CQ is a known antibiotic drug,60 has been extensively
investigated as an anti-Alzheimer compound,24,25,61 and has
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Figure 7. Cu(CQ)-induced cytoplasmic vacuolization. (A) HeLa, PC3, and HUVEC cells were grown with 10% FBS and treated with 50 μM CQ
and 8 μM (HeLa and PC3) or 15 μM (HUVEC) Cu(CQ) for 18 h. Control is untreated cells. Phase contrast images of representative microscopic
fields (400×) are shown. (B) HeLa cells were treated with 8 μM Cu(CQ) for 18 h, and the immunostaining of endoplasmic reticulum was
performed with calnexin, as described in the Experimental Section. The phase contrast (left) and the immunostaining (right) images of the same
representative microscopic field (1000×) are shown.
ionophoric properties that have been previously investigated
with copper and zinc.29,30 It has been demonstrated that the
CQ−copper complex cytotoxicity in breast cancer cells is due
specifically to the metal binding and transport ability of
clioquinol.62 Consistently, we show here that the simultaneous
addition of CuCl2 to CQ-treated HeLa and PC3 cells results in
a time-dependent increase of intracellular copper in both cell
models (Figure 5B). Moreover, the incubation of the cells in
the presence of the cell-impermeable copper chelator
ammonium tetrathiomolybdate (TM)47,48 hinders copper
accumulation (Figure 5A) and protects both HeLa and PC3
cells from the cytotoxic effect of Cu(CQ) (Figure 5B).
Interestingly, when the concentration of TM is raised to 40
μM, it selectively affects the growth of HeLa cells (Figure S9),
which is in agreement with the anticancer mechanism of TM. It
has been proposed that TM, by virtue of its ability to sequester
copper, directly affects malignant cell growth63 and acts as an
antiangiogenic factor impairing vascular endothelial cells
functions.64
fails to rescue cell death (Figure 5A). Moreover, while a
classical proapoptotic stimulus, i.e., cisplatin, activates caspase-3
in HeLa and PC3, cytotoxic concentrations of Cu(CQ) do not
(Figure 5C,D). Intriguingly, the treatment of HeLa cells with
high concentrations of CQ alone moderately activates caspase-3
(Figure 5C) and the addition of the caspase inhibitor z-VAD-
FMK partially rescues HeLa cells from the cytotoxic effect
observed upon treatment with high doses of CQ (Figure 6),
thus indicating that the mechanism of action of the ligand
substantially differs from that of the complex, at least in this
cellular model.
One of the substrates of caspase-3 is poly ADP-ribose
polymerase-1 (PARP-1), an enzyme that signals DNA breaks
and recruits the DNA repair complex. Because PARP-1
activation is energy consuming, cells committed to apoptotic
death cleave and inactivate PARP-1. On the other hand, it was
recently shown that the activation of PARP-1 is one of the key
steps of programmed necrosis.67,68 In our cell models, PARP-1
is cleaved upon cisplatin-induced apoptosis (Figure 5D), but it
is not cleaved upon Cu(CQ) treatment, confirming the
nonapoptotic nature of the death process triggered by the
complex. A further independent evidence confirming that the
Cu(CQ)-dependent cell death is distinct and distinguishable
from apoptosis comes from the lack of translocation of
apoptosis inducing factor (AIF) from mitochondria to the
nucleus upon treatment with the copper complex (Figure S10).
The anticancer activity of CQ alone30 or in combination with
Zn(II) and Cu(II) ions was recently reported to activate a
caspase-3-dependent cell death process65 and more specifically
to activate the apoptotic pathway in breast cancer KCL2 cells.66
On the contrary, our results show that Cu(CQ)-dependent
cytotoxicity does not require the activation of caspases in HeLa
and PC3 cells because the pan-caspase inhibitor z-VAD-FMK
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mg, 1.39 mmol) in 20 mL of methanol, and a brown solid precipitated
immediately. After 30 min of stirring, the solid was filtered and
vacuum-dried (196 mg, yield 68%). IR (cm−1): 428m, 646m, 745s,
772m, 964m, 1064m, 1084m, 1309s, 1364s, 1463m, 1583m, 3063w.
Anal. Calcd for C18H10CuCl2N2O2 (420.74): C, 51.38; H, 2.40; N,
6.66. Found: C, 51.61; H, 2.58; N, 6.29%.
Synthesis of [Cu(5-Me-7-Me-8-HQ)2]. A pale green solution of
CuCl2·2H2O (123 mg, 0.72 mmol) dissolved in methanol (5 mL) was
added to a pale pink solution of 5,7-dimethyl-8-hydroxyquinoline (250
mg, 1.44 mmol) in 20 mL of methanol, and a green solid precipitated
immediately. After 30 min of stirring, the solid was filtered and
vacuum-dried (221 mg, yield 76%). Suitable crystals for X-ray data
collection were grown from agarose gel. IR (cm−1): 443m, 663s, 745m,
789s, 997w, 1134m, 1167m, 1353s, 1435m, 1506m, 1572w, 2915w.
Anal. Calcd for C22H18CuN2O2 (405.93): C, 65.09; H, 4.47; N, 6.90.
Found: C, 64.83; H, 4.64; N, 6.71%.
Synthesis of [Cu(5,7-Cl-8-HQ)2]. A pale green solution of
CuCl2·2H2O (100 mg, 0.58 mmol) dissolved in methanol (5 mL)
was added to a solution of 5,7-dichloro-8-hydroxyquinoline (250 mg,
1.16 mmol) in 10 mL of acetone, and a dark green solid precipitated
immediately. After 30 min of stirring, the solid was filtered and
vacuum-dried (132 mg, yield 46%). IR (cm−1): 482m, 657m, 750s,
980m, 1112m, 1254w, 1369s, 1435s, 1479m, 1539m, 1764w, 2622w,
2811w, 3049w, 3077w. Anal. Calcd for C18H8CuCl4N2O2 (489.63): C,
44.15; H, 1.65; N, 5.72. Found: C, 44.34; H, 1.82; N, 5.56%.
Synthesis of [Cu(CQ)2]. A pale green solution of CuCl2·2H2O (70
mg, 0.41 mmol) dissolved in methanol (5 mL) was added to a solution
of clioquinol (250 mg, 0.82 mmol) in 10 mL of acetone, and a dark
green solid precipitated immediately. After 30 min of stirring, the solid
was filtered and vacuum-dried (207 mg, yield 75%). IR (cm−1): 487m,
586m, 657s, 750s, 975m, 1112m, 1375s, 1435s, 1539m, 1753w, 2626w,
2813w, 3043w, 3069w. Anal. Calcd for C18H10CuCl2I2N2O2 (672.53):
C, 32.15; H, 1.20; N, 4.17. Found: C, 32.33; H, 1.13; N, 4.39%.
Synthesis of [Cu(5,7-I-8-HQ)2]. A pale green solution of
CuCl2·2H2O (53 mg, 0.31 mmol) dissolved in methanol (5 mL)
was added to a solution of 5,7-diiodo-8-hydroxyquinoline (250 mg,
0.62 mmol) in 20 mL of acetone/chloroform (1/1), and a green solid
precipitated immediately. After 30 min of stirring, the solid was filtered
and vacuum-dried (238 mg, yield 90%). IR (cm−1): 482w, 657m, 750s,
980m, 1112m, 1249w, 1364s, 1435s, 1545s, 1753w, 2626w, 2813w,
3069w, 3073w. Anal. Calcd for C18H8CuI4N2O2 (855.43): C, 25.27; H,
0.94; N, 3.27. Found: C, 25.09; H, 1.08; N, 3.17
Cell Cultures and Experimental Treatments. The HeLa cell
line, derived from human cervical carcinoma, was obtained from
ATCC, while human prostatic carcinoma PC3 cells were kindly
provided by Prof. S. Bettuzzi (Department of Experimental Medicine,
University of Parma, Italy). Cells were routinely grown in high glucose
Dulbecco’s modified Eagle’s medium (DMEM) (4.5 g/L glucose)
supplemented with 10% fetal bovine serum (FBS), 4 mM glutamine,
100 U/mL penicillin, and 100 μg/mL streptomycin.
Human umbilical vein endothelial cells (HUVECs), obtained as
previously described,71 were cultured in medium M199 supplemented
with 20% fetal bovine serum (FBS), endothelial cell growth
supplement (ECGS, 37.5 μg/mL), heparin (75 U/mL), and glutamine
(2 mM).
Human foreskin fibroblasts (HFs) were obtained as previously
described72 and routinely grown in low glucose Dulbecco’s modified
Eagle’s medium (DMEM) (1 g/L glucose) supplemented with 10%
fetal bovine serum (FBS), 4 mM glutamine, 100 U/mL penicillin, and
100 μg/mL streptomycin.
All cells were maintained at 37 °C in a humidified atmosphere of 5%
CO2 in air, pH 7.4.
Although the enzymes responsible for the paraptosis
execution remain to be identified, the morphology of dying
cells can nonetheless provide useful hints to the identification
of the mechanisms involved in this type of cell death. An
increasing number of reports refer to paraptosis as non-
apoptotic cell death characterized by a massive cytoplasmic
vacuolization due to endoplasmic reticulum swelling. In
particular, these features have been frequently observed upon
treatment with copper-based compounds.9,28,69,70 Consistently,
HeLa, PC3, and HUVEC cells treated with cytotoxic doses of
Cu(CQ) display the prototypical traits of paraptosis (Figure 7).
These results confirm the ionophoric nature of 8-HQ
derivatives for copper, pinpoint the ligand hydrophobicity as
a determinant of the activity of the complex, and demonstrate
that the ligand dependent copper accumulation triggers a
nonapoptotic cell death, morphologically defined as paraptosis,
that does not activate or depend on caspase activity.
Altogether these findings may support the combined use of
CQ and copper for the treatment of tumors characterized by
innate or acquired apoptosis resistant phenotype. On the
contrary the combination of clioquinol, copper, and proapop-
totic agent may be evaluated carefully in order to avoid
conflicting effects on caspase activation. The differential
cytotoxicity of Cu(CQ) in tumor and normal cells also
supports its potential chemotherapeutic application in clinical
practice, with reasonably minor adverse side effects.
EXPERIMENTAL SECTION
■
All reagents and solvents were commercially available. Infrared spectra
were recorded from 4000 to 700 cm−1 on a Perkin-Elmer FT-IR Nexus
spectrometer equipped with a Thermo-Nicolet microscope. Com-
pound purity was assessed by means of combustion analysis (Carlo
Erba EA 1108 automated analyzer) with a confirming purity of ≥95%
for all copper complexes.
Synthesis of [Cu(5-SO3-8-HQ)(H2O)2]. A pale green solution of
CuCl2·2H2O (94 mg, 0.55 mmol) dissolved in methanol (5 mL) was
added to a solution of 8-hydroxyquinoline-5-sulfonic acid (125 mg,
0.56 mmol) in 10 mL of methanol/water (1/1), and then the mixture
was stirred for 30 min. A green solid precipitated. The solvent was
concentrated under vacuum, and the solid was filtered and vacuum-
dried (156 mg, yield 88%). IR (cm−1): 454s, 547m, 624m, 706m,
778w, 1024m, 1139s, 1320w, 1369w, 1490m, 1572w, 2847w broad,
3091w sharp, 3165m broad. Anal. Calcd for C9H9CuNO6S (322.78):
C, 33.49; H, 2.81; N, 4.34. Found: C, 33.17; H, 2.59; N, 4.16%.
Synthesis of [Cu(5-SO3-7-I-8-HQ)(H2O)2]. A pale green solution
of CuCl2·2H2O (61 mg, 0.35 mmol) dissolved in methanol (5 mL)
was added to a pale yellow solution of 8-hydroxy-7-iodo-5-
quinolinesulfonic acid (123 mg, 0.35 mmol) in 30 mL of methanol/
water (8/2), and the mixture was stirred for 30 min. A green solid
precipitated. The solvent was concentrated under vacuum, and the
solid was filtered and vacuum-dried (131 mg, yield 83%). IR (cm−1):
591s, 728m, 816m, 1035s, 1167s, 1380m, 1484m, 1556w, 1626w,
3073w, 3195w, 3343m broad, 3526m sharp. Anal. Calcd for
C9H8CuINO6S (448.68): C, 24.09; H, 1.80; N, 3.12. Found: C,
23.81; H, 1.97; N, 3.33%.
Synthesis of [Cu(8-HQ)2]. A pale green solution of CuCl2·2H2O
(147 mg, 0.86 mmol) dissolved in methanol (5 mL) was added to a
yellow solution of 8-hydroxyquinoline (250 mg, 1.72 mmol) in 20 mL
of methanol, and a brown solid precipitated immediately. After 30 min
of stirring, the solid was filtered and vacuum-dried (233 mg, yield
77%). IR (cm−1): 504m, 635w, 739s, 821s, 1106s, 1265m, 1315s,
1375s, 1495s, 1578m, 3057w. Anal. Calcd for C18H14CuN2O2
(351.85): C, 61.44; H, 3.44; N, 7.96. Found: C, 61.27; H, 3.57; N,
7.71%.
Ligands or copper complexes (obtained by mixing ligands and
CuCl2, 1:1 vol/vol, in the culture medium) were added to complete
growth medium from 35 mM stock solutions in DMSO to obtain the
concentrations required. Ammonium tetrathiomolybdate (TM, 10
mM in H2O) was diluted to the final concentrations of 20 or 40 μM in
complete growth medium. The caspase inhibitor z-VAD-FMK (10
mM in DMSO) was added to complete growth medium and used at
50 μM.
Synthesis of [Cu(5-Cl-8-HQ)2]. A pale green solution of
CuCl2·2H2O (119 mg, 0.69 mmol) dissolved in methanol (5 mL)
was added to a yellow solution of 5-chloro-8-hydroxyquinoline (250
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Cell Count and Viability Assay. Cells were seeded in complete
growth medium in 96-well plates (7.5 × 103 cells/well) or in 24-well
cell culture plates (5 × 104 cells/well) for viability assay or cell count,
respectively. After 24 h, the growth medium was renewed and
supplemented with the compounds as indicated by the experimental
design. After 48 h of incubation under the selected conditions, cells
were counted with a cell counter ZM (Coulter Electronics Ltd., Luton,
U.K.) after culture trypsinization while cell viability was tested by
replacing the culture medium with a solution of resazurin (110 μg/mL
resazurin in PBS) in serum-free medium (1:10). Fluorescence at 572
nm was measured in each well with a fluorometer (Wallac 1420
Victor2 multilabel counter, Perkin-Elmer), and cell viability was
calculated with the equation viability = [(Vt − B)/(Vu − B)] × 100,
where Vt and Vu are the fluorescence values obtained in treated and
untreated cells, respectively, and B is the background value. Dose−
response curves were fitted with nonlinear regression analysis, and IC50
values were calculated with GraphPad Prism 5.0.
Calculation of log P Values. The log P values of the ligands,
calculated with the software ALOPGPS (VCCLAB virtual computa-
tional chemistry laboratory73), are in good agreement with the
retention time exhibited in the reverse phase RPLC column. 5-SO3-8-
HQ was the first to elute (0.58 min) because of its marked hydrophilic
character, whereas the most lipophilic ligand 5,7-I-8-HQ had the
longest retention time (5.61 min) (Figure S6).
caspase-3 substrate Ac-DEVD-pNA (200 μM, Alexis Biochemicals).
The absorbance at 405 nM was read with a microplate reader (Wallac
1420 Victor2 multilabel counter, Perkin-Elmer) after 2 h at 37 °C.
Caspase-3 activity under each condition is expressed as the percent of
the value obtained for the untreated control cells after subtraction of
the blank value.
LDH Release. Cells were grown to subconfluence on 24-well plates
and treated as required. Lactate dehydrogenase (LDH), released from
necrotic cells into the medium, was assessed with a CytoTox 96
nonradioactive cytotoxicity assay (Promega, Milano, Italy). The
amount of LDH released was expressed as percent of LDH released
from untreated cells (percent of control).
Immunofluorescence. HeLa cells (8 × 104 cells) were seeded on
chamber slides and grown to subconfluence, then treated as indicated
in Figures 7 and S10. Cells were then fixed in 3.7% paraformaldehyde
and permeabilized with ice-cold methanol for calnexin staining or fixed
and permeabilized in methanol for AIF detection. After blocking with
5% anti-goat serum, fixed cells were incubated at 4 °C overnight with
rabbit polyclonal antibodies (1:50 anti-calnexin, Cell Signaling, and
1:100 anti-AIF, Santa Cruz Biotechnology). After being washed, cells
were incubated with 488 Alexa Fluor goat anti-rabbit IgG antibody
(1:800) and then visualized by means of a Nikon Eclipse 300 inverted
fluorescence microscope (for calnexin) or a LSM 510 META Zeiss
confocal microscope (for AIF).
Copper Uptake. For intracellular copper determination, 1 × 106
HeLa or 1.5 × 106 PC3 cells were seeded in 100 mm dishes. After 24
h, growth medium was renewed, and the monolayers were treated as
indicated in Figure 5. After the incubation, three culture plates/
condition were used for analytical copper determination. Cells were
rapidly washed twice with ice-cold PBS and collected in 1.5 mL of
trypsin solution. The samples were diluted to 5 mL with concentrated
HNO3, mineralized by a microwave procedure, and analyzed for
copper content using ICP-AES (inductively coupled plasma atomic
emission spectroscopy). One culture plate/condition was used for
protein quantification according to a modified Lowry method.72 The
copper content under each condition is expressed as nmol/mg protein.
Western Blot Analysis. Cells, grown to subconfluence on 10 cm
diameter dishes, were treated as required by the experimental plan. At
the end of the treatment, both adherent and floating cells were
collected, rinsed twice in PBS, and lysed in lysis buffer containing 20
mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA,
1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophos-
phate, 1 mM Na3VO4, 1 mM NaF, 2 mM imidazole, and a cocktail of
protease inhibitors (cOmplete, Mini, EDTA-free, Roche). Lysates were
then sonicated for 5 s and centrifuged at 12000g for 10 min at 4 °C.
After protein quantification of the supernatant with the Bio-Rad
protein assay, 30 μg of protein was mixed with Laemmli buffer 4×,
warmed at 95 °C for 5 min, and loaded on a gel for SDS−PAGE.
Resolved proteins were then transferred to polyvinylidene difluoride
membranes (Millipore, Immobilon-P). After a 2 h incubation at room
temperature in 10% blocking solution (Roche Diagnostics SpA, Milan,
Italy), blots were exposed overnight at 4 °C to anti-caspase-3 (full-
length and cleaved, rabbit polyclonal 1:1000, Cell Signaling) or anti-
PARP1 (full-length and cleaved, mouse monoclonal 1:500, Santa Cruz
Biotechnology) primary antibodies and diluted in a 5% bovine serum
albumin solution in TBS Tween 0.1%. The blots were then washed
and exposed for 1 h at room temperature to HRP-conjugated anti-
mouse or anti-rabbit antibodies (ExactaCrutz, Santa Cruz Biotechnol-
ogy), diluted 1:10000 in blocking solution. Immunoreactivity was
visualized with the Immobilon Western chemiluminescent HRP
substrate (Millipore).
Materials. Serum was obtained from Lonza, Basel, Switzerland.
DMEM and M199 were provided by Euroclone, Italy. Z-VAD-FMK
was provided by Enzo Life Sciences. Staurosporine was purchased
from Sigma-Aldrich. Unless otherwise indicated, Sigma Aldrich was the
source of all other chemicals, including ligands.
ASSOCIATED CONTENT
* Supporting Information
■
S
Stability constants determination, competition studies with
human serum albumin, X-ray structure of [Cu(5,7-Me-8-HQ)2]
(CCDC 891695) and crystallographic data in CIF format, cell
viability assays (HeLa, PC3, HUVEC, and HF), confocal
microscope images of AIF translocation. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: 0039 0521 905419. E-mail: marchio@unipr.it.
■
Present Address
∥The Beatson Institute for Cancer Research, Switchback Road,
Glasgow G61 1BD, U.K.
Author Contributions
§These authors contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
University of Parma, Italy, is acknowledged for financial
support. The authors gratefully acknowledge the Consorzio
Interuniversitario di Ricerca in Chimica dei Metalli nei Sistemi
Biologici (CIRCMSB) for helpful discussions and Dr.
Massimiliano Bianchi (Dipartimento di Scienze Biomediche,
̀
Biotecnologiche e Traslazionali, Universita degli Studi di
Caspase-3 Activity. Cells were grown to subconfluence on 10 cm
diameter dishes and treated as required. At the end of the treatment,
both adherent and floating cells were collected and rinsed once in PBS.
Pellets were resuspended in 500 μL of 50 mM Hepes, 0.1% CHAPS,
10 mM EDTA, 5% glycerol, and 10 mM DTT and vigorously
vortexed. After centrifugation at 12000g for 10 min at 4 °C, the protein
content in the supernatant was determined with the Bio-Rad protein
assay and adjusted to the same concentration. Aliquots of 80 μg of
protein were distributed in each well of a 96-well plate, along with the
Parma, Italy) for his contribution to the acquisition of confocal
microscope images. A.B. is supported by a research scholarship
of the University of Parma Medical School, Italy.
ABBREVIATIONS USED
■
AIF, apoptosis inducing factor; ATP7A, copper transporting
ATPase A; ATP7B, copper transporting ATPase B; CHOP,
CCAAT-enhancer-binding protein homologous protein; CQ,
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(15) Haas, K. L.; Putterman, A. B.; White, D. R.; Thiele, D. J.; Franz,
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clioquinol; DMSO, dimethyl sulfoxide; ENDOG, endonuclease
G; ER, endoplasmic reticulum; GSH, glutathione; GSSG,
oxidized glutathione; hCTR1, human copper transporter 1; 8-
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