Novel Mechanism of Cytotoxicity for the Selective Selenosemicarbazone, 2-Acetylpyridine 4,4-Dimethyl-3-selenosemicarbazone (Ap44mSe): Lysosomal Membrane Permeabilization

Article abstract of DOI:10.1021/acs.jmedchem.5b01399

Selenosemicarbazones show marked antitumor activity. However, their mechanism of action remains unknown. We examined the medicinal chemistry of the selenosemicarbazone, 2-acetylpyridine 4,4-dimethyl-3-selenosemicarbazone (Ap44mSe), and its iron and copper complexes to elucidate its mechanisms of action. Ap44mSe demonstrated a pronounced improvement in selectivity toward neoplastic relative to normal cells compared to its parent thiosemicarbazone. It also effectively depleted cellular Fe, resulting in transferrin receptor-1 up-regulation, ferritin down-regulation, and increased expression of the potent metastasis suppressor, N-myc downstream regulated gene-1. Significantly, Ap44mSe limited deleterious methemoglobin formation, highlighting its usefulness in overcoming toxicities of clinically relevant thiosemicarbazones. Furthermore, Cu-Ap44mSe mediated intracellular reactive oxygen species generation, which was attenuated by the antioxidant, N-acetyl-l-cysteine, or Cu sequestration. Notably, Ap44mSe forms redox active Cu complexes that target the lysosome to induce lysosomal membrane permeabilization. This investigation highlights novel structure-activity relationships for future chemotherapeutic design and underlines the potential of Ap44mSe as a selective anticancer/antimetastatic agent.

Full text of DOI:10.1021/acs.jmedchem.5b01399

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Article
Novel Mechanism of Cytotoxicity for the Selective Selenosemicarbazone,
-Acetylpyridine 4,4-Dimethyl-3-Selenosemicarbazone
Ap44mSe): Lysosomal Membrane Permeabilization
2
(
Zaynab Al-Eisawi, Christian Stefani, Patric Jansson, Akanksha Arvind, Philip C Sharpe,
Maram Basha, George M Iskander, Naresh Kumar, Zaklina Kovacevic, Darius J.R. Lane,
Sumit Sahni, Paul Vincent Bernhardt, Des R Richardson, and Danuta Sandra Kalinowski
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01399 • Publication Date (Web): 08 Dec 2015
Downloaded from http://pubs.acs.org on December 15, 2015
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Journal of Medicinal Chemistry is published by the American Chemical Society.
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Novel Mechanism of Cytotoxicity for the Selective Selenosemicarbazone, 2-Acetylpyridine 4,4-
Dimethyl-3-Selenosemicarbazone (Ap44mSe): Lysosomal Membrane Permeabilization
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†
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Zaynab Al-Eisawi , Christian Stefani , Patric J. Jansson , Akanksha Arvind , Philip C. Sharpe ,
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Maram T. Basha , George M. Iskander^, Naresh Kumar^, Zaklina Kovacevic , Darius J.R. Lane ,
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†*
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Sumit Sahni , Paul V. Bernhardt , Des R. Richardson and Danuta S. Kalinowski
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Molecular Pharmacology and Pathology Program, Department of Pathology, The University of
Sydney, Sydney, NSW, 2006, Australia
‡
School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, QLD, 4072,
Australia
^School of Chemistry, University of New South Wales, Sydney, NSW, 2052, Australia
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Abstract
Selenosemicarbazones show marked anti-tumor activity. However, their mechanism of action
remains unknown. We examined the medicinal chemistry of the selenosemicarbazone, 2-
acetylpyridine 4,4-dimethyl-3-selenosemicarbazone (Ap44mSe), and its iron and copper complexes
to elucidate its mechanisms of action. Ap44mSe demonstrated a pronounced improvement in
selectivity towards neoplastic relative to normal cells compared to its parent thiosemicarbazone. It
also effectively depleted cellular Fe, resulting in transferrin receptor-1 up-regulation, ferritin down-
regulation and increased expression of the potent metastasis suppressor, N-myc downstream
regulated gene-1. Significantly, Ap44mSe limited deleterious methemoglobin formation,
highlighting its usefulness in overcoming toxicities of clinically-relevant thiosemicarbazones.
Furthermore, Cu-Ap44mSe mediated intracellular reactive oxygen species generation, which was
attenuated by the anti-oxidant, N-acetyl-L-cysteine, or Cu sequestration. Notably, Ap44mSe forms
redox active Cu complexes that target the lysosome to induce lysosomal membrane
permeabilization. This investigation highlights novel structure-activity relationships for future
chemotherapeutic design, and underlines the potential of Ap44mSe as a selective anti-cancer/anti-
metastatic agent.
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Introduction
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Trace metals, including iron (Fe) and copper (Cu), are elements that are vital for normal cellular
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-3
function. The participation of Fe and Cu in a wide range of cellular processes is due to their
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innate ability to redox cycle and act as both electron donors and acceptors. For example, Fe has
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crucial roles in oxygen transport, electron transfer, energy metabolism, DNA synthesis and cell
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cycle progression. Moreover, Fe acts as a cofactor for the key enzyme, ribonucleotide reductase
(
RR), that catalyzes the reduction of ribonucleotides to deoxyribonucleotides and is the rate-limiting
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step in DNA synthesis. Due to their rapid cell growth and proliferation, it is not entirely surprising
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that Fe is crucial for the proliferation of cancer cells.
It is also well recognized that Cu is vital for the activity of many enzymes essential for cellular
respiration, defense against reactive oxygen species (ROS), melanin synthesis, formation of
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connective tissue and Fe metabolism. It is also an essential co-factor for tumor angiogenesis and
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metastasis. In fact, Fe and Cu metabolism is profoundly altered in many human cancers.
Considering the crucial roles of these metals, the development of novel agents that target Fe and Cu
have become a promising anti-cancer strategy.
Chelators, such as desferrioxamine (DFO, Fig. 1A), have been traditionally used for the treatment
of Fe overload disease. Early studies investigating the anti-tumor efficacy of DFO demonstrated
2
,10
only modest efficacy in vitro and in vivo. The limited membrane permeability of DFO resulted in
11,12
poor anti-cancer activity and prevented its application in cancer therapy.
Recently, a number of
studies have demonstrated that thiosemicarbazone chelators are an evolving class of agents, with di-
-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT; Fig. 1B) showing effective and
selective anti-tumor activity in vitro and in vivo that can overcome resistance to conventional
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chemotherapy.
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The anti-proliferative activity of more potent chelators, such as Dp44mT and those of the 2-
acetylpyridine thiosemicarbazone series, such as 2-acetylpyridine 4,4-dimethyl-3-
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thiosemicarbazone (Ap44mT; Fig. 1C), is likely mediated by their effects on multiple molecular
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targets. In addition to binding to Fe, thiosemicarbazones possess the ability to bind Cu.
The
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resultant Fe and Cu complexes are capable of engaging in redox cycling to generate ROS, such as
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hydroxyl radicals, via the Fenton reaction.
These ROS mediate cellular injury to induce
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apoptosis.
Other modes of anti-cancer activity mediated by thiosemicarbazones include the up-regulation of
21-23
the metastasis suppressor protein, N-myc downstream regulated gene-1 (NDRG1)
and the
inhibition of cell cycle progression (G /S phase) through the modulation of the expression of the
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cyclin family of proteins (e.g., cyclin D1), GADD family of proteins (e.g., GADD45), and
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cyclin-dependent kinase 2. Furthermore, the accumulation of Cu thiosemicarbazone complexes in
the lysosome activates the lysosomal apoptotic pathway through lysosomal membrane
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permeabilization (LMP). The induction of LMP causes the proteolytic activation of Bid, which is
cleaved by the lysosomal cathepsins, B and D, to subsequently induce mitochondrial outer
membrane permeabilization, resulting in cytochrome c release and apoptosome-dependent caspase
27-29
activation, and ultimately, apoptosis.
Studies have highlighted the potential of chalcogensemicarbazones, such as selenosemicarbazones
30-34
as anti-cancer agents.
The substitution of chalcogen atoms with selenium in
chalcogensemicarbazones resulted in increased activity as anti-bacterial, anti-parasitic and anti-viral
35-37
agents.
Selenosemicarbazones and their Pt(II) and Pd(II) complexes were also found to
31,33,34
demonstrate marked anti-tumor activity in vitro.
These results demonstrated the potential
application of selenosemicarbazones as novel compounds in cancer therapy.
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We investigated the impact of substituting the thiocarbonyl sulfur in Ap44mT (Fig. 1C) with
selenium to generate, 2-acetylpyridine 4,4-dimethyl-3-selenosemicarbazone (Ap44mSe; Fig. 1D),
in an effort to develop highly potent, yet well tolerated, anti-cancer agents and gain further insight
into the structure-activity relationships of selenosemicarbazones. In this study, we examined the
electrochemical and intracellular behavior of Ap44mSe (Fig. 1D) and its Fe and Cu complexes in
SK-N-MC neuroepithelioma cells. Herein, it is demonstrated that the anti-proliferative activity of
Ap44mSe is linked with the ability of its resultant Cu complex to redox cycle, leading to
intracellular ROS generation and LMP. Our findings underline for the first time the importance of
ROS generation and LMP in the anti-cancer activity of Ap44mSe and demonstrate lysosomal
targeting as a novel mechanism of selenosemicarbazone anti-proliferative activity. Moreover,
substitution of thiocarbonyl S for Se in Ap44mSe, resulted in markedly higher anti-tumor
selectivity and lower metHb generation relative to its parent thiosemicarbazone and a lead di-2-
pyridylketone thiosemicarbazone, Dp44mT. The novel structure-activity relationships highlighted
herein are important for future chemotherapeutic design.
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Results and Discussion
Synthesis and Structural Characterization
2-Acetylpyridine 4,4-dimethyl-3-selenosemicarbazone (Ap44mSe) was prepared by S-methylation
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of Ap44mT with methyl iodide in the presence of a base. This was followed by substitution of the
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S-methyl group by selenium using sodium hydrogen selenide under argon. The Cu complexes
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were prepared following methods employed previously for Ap44mT and its complexes. Using
cupric acetate provides both a coordinating co-ligand and a base to deprotonate the NH group of the
selenosemicarbazone, which coordinates in its imino-selenolate anionic form. The IR spectrum of
-1
this complex reveals the presence of coordinated acetate (1607 cm ) in addition to several other
II
strong IR bands from the coordinated ligand. The ligand and Cu complexes exhibit good solubility
in MeCN and DMF. Further development of the hydrochloride salt of Ap44mSe would enhance
aqueous solubility for future assessment in preclinical studies.
As an appropriate example, the crystal structure of [Cu(Ap44mSe)(OAc)] was determined. A view
of the molecule is shown in Fig. 2A and selected bond lengths are in Table 1. The Cu atom is in a
distorted square planar coordination geometry comprising the coordinated N, N, Se- donor set of the
deprotonated selenosemicarbazone and a monodentate coordinated acetato ligand. The second
acetate O-atom (O2) is more than 2.8 Å from the Cu ion and there are no other closer contacts with
the metal (Fig. 2A).
There are some interesting parallels with the structure of the homologous thiosemicarbazone Cu
1
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complex of Ap44mT, namely [Cu(Ap44mT)(OAc)]. The larger covalent radius of Se compared to
S lengthens both the Cu-X (X= Se, S) and C8-X bond lengths by around 0.13 Å. This has an
influence on the coordinate angles as well. The trans coordinate angle N1-Cu-X1 is effectively set
by the rigid ligand and decreases further away from linearity in [Cu(Ap44mSe)(OAc)] due to the
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longer Cu-Se and C8-Se bonds. The C8-Se-Cu bond is close to 90° (ca. 3° smaller than the S-donor
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in its analogous thiosemicarbazone), which is expected for the heavier chalcogen donor atom.
When cupric perchlorate was used as a precursor in the presence of a 1:2 Cu:ligand ratio, the
expected bis-selenosemicarbazone complex was not obtained, but instead an unusual dinuclear Se-
bridged complex was isolated. Its crystal structure was determined and a view of the complex
cation appears in Fig. 2B. The structure comprises two Cu ions and three selenosemicarbazone
ligands; two of them connecting the Cu ions via Cu-Se-Cu bridges. The environment of Cu1
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resembles bis-thiosemicarbazone complexes such as [Cu(Ap44mT) ], where a tetragonally
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elongated (Jahn-Teller distorted) 6-coordinate geometry is present. The elongated axis is defined
by the trans donor atoms, N1B and Se1B, from ligand ‘B’, whose bonds to Cu1 are elongated by
more than 0.2 Å compared to the corresponding N1A and Se1A donor atoms of ligand ‘A’ (Table
2
). The environment of Cu2 is different and the metal is in a pseudo-square planar environment,
comprising an unusual N Se donor set with an additional bridging Se atom (Se1A) at the apex.
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However, Se1A is approximately 0.5 Å further from the Cu than the equatorially coordinated Se
donors (Se1B and Se1C) and formally within the coordination sphere (Fig. 2B). The environment of
Cu2 resembles the geometry found in [Cu(Ap44mSe)(OAc)], where Se1B replaces the equatorial
acetate.
Electrochemistry
The electrochemical properties of [Cu(Ap44mSe)(OAc)] and [Cu
investigated in MeCN:H O (70:30 v/v). These complexes exhibit good solubility in MeCN and
DMF, but are not soluble in water alone at millimolar concentrations. The solvent mixture of
MeCN:H O (70:30 v/v) provided a good compromise. Indeed, it enabled the electrochemical
2 3 4
(Ap44mSe) ](ClO ) were
2
2
properties to be studied under conditions where a high concentration of water was present, and thus,
these conditions are more biologically relevant than investigations in pure organic solvent.
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Furthermore, this solvent combination has been utilized in our previous studies on other
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thiosemicarbazone systems
enabling a useful comparison.
II/I
The mononuclear complex [Cu(Ap44mSe)(OAc)] yielded a quasi-reversible Cu redox couple at -
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20 mV vs. the NHE (Fig. 3A). This Cu redox potential is similar to that found in the complex
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[Cu(Ap44mT)(OAc)] (-204 mV vs. the NHE). On the initial sweep (data not shown), a higher
potential pre-wave was seen which disappears on subsequent cycles. The data shown in Fig. 3A
correspond to the second cycle. Under these conditions (30% water), hydrolysis of the acetate
ligand most likely occurs.
The dinuclear complex [Cu (Ap44mSe) ](ClO ) was also examined under the same conditions. The
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two Cu centers bear very different coordination environments (Fig. 2B). In the solid state, one Cu
ion is chelated by two Ap44mSe ligands, while the other is only coordinated by one
selenosemicarbazone and resembles [Cu(Ap44mSe)(OAc)]. The cyclic voltammetry of this
II/I
dinuclear complex demonstrates two well-separated, quasi-reversible Cu couples at -217 and -461
mV vs. the NHE (Fig. 3B). By analogy with the cyclic voltammetry of [Cu(Ap44mSe)(OAc)] (Fig.
3A), the higher potential couple is assigned to the 1:1 Cu:L component, while the lower potential
couple is attributed to the CuL
2
moiety. Interestingly, there is no significant difference between the
(Ap44mSe) ](ClO ) and that found for [Cu(Ap44mSe)(OAc)].
higher redox potential of [Cu
2
3
4
Normally, the proximity of another Cu ion bridged by two Se atoms would be expected to raise the
II/I
Cu redox potential, but it is unchanged. This suggests that the dinuclear complex dissociates in
+
solution to generate [Cu(Ap44mSe)
2
] and [Cu(Ap44mSe)(OH
2
)] in the presence of water.
II
We have previously found that 1:2 Cu :thiosemicarbazone complexes from this family partially
dissociate to give a mixture of 1:1 and 1:2 Cu:ligand complexes due to a Jahn-Teller distortion that
II
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weakens a pair of trans axial coordinate bonds in the 6-coordinate Cu state. In the present case,
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the initial cathodic sweep produced two Cu peaks of equal magnitude (Fig. 3C), which illustrates
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that the putative separated [Cu (Ap44mSe)(OH
2 2
)] and [Cu (Ap44mSe) ] complexes are stable. In
+
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the second voltammetric cycle, the magnitude of the [Cu(Ap44mSe)(OH )] couple increases
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/-
relative to the lower potential [Cu(Ap44mSe)
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]
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I
-
I
dissociation of [Cu (Ap44mSe)
2
] to liberate free ligand and more [Cu (Ap44mSe)(OH
2
)] at the
I
electrode surface; only reduction to Cu triggers partial ligand dissociation. This chemical reaction
coupled to electron transfer proceeds relatively slowly and the rate of the reaction may be followed
-1
at different sweep rates (Fig. 3C). At rapid sweep rates (100 mV s and above), the first and second
cycles are not significantly different and no dissociation can occur on this timescale. However, at 25
-1
and 50 mV s the higher potential cathodic wave is significantly enhanced on the second cycle due
I
to the ligand dissociation from Cu proceeding to a greater extent (Fig. 3C).
EPR Spectroscopy
Electron paramagnetic spectroscopy is a particularly useful technique for interrogating the
II
coordination geometry of Cu complexes. The frozen (DMF) solution X-band EPR spectrum of
[
Cu(Ap44mSe)(OAc)] is shown in Figure 4. The spectrum is rich in fine structure. Hyperfine
coupling between the single electron spin and both the Cu nucleus (I = 3/2) and the cis N-donors (I
1) is apparent. The spin Hamiltonian parameters were obtained by spectral simulation (see
Supporting Information Fig. S1). For comparison, the EPR spectrum of [Cu(Ap44mT)(OAc)]
=
18
(published previously) is shown and clearly the two spectra are quite similar. Both are
II
characteristic of a mononuclear Cu complex in a pseudo-square planar N
2
OS(e) coordination
environment. The similarity of the g values and hyperfine coupling constants indicates that
substitution of Se for S has only a small effect on the EPR properties. Also, the only Se isotope with
77
41
non-zero spin is Se (I = ½), but it is only 7.6% abundant, and thus, no additional hyperfine
coupling was resolved.
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2 3 4
Interestingly, the dinuclear complex [Cu (Ap44mSe) ](ClO ) is EPR silent. It is important to note
that EPR was carried out in DMF, so the dinuclear complex is evidently persistent in the absence of
9
water, in contrast to the electrochemistry in mixed aqueous solvent. The fact that the two d metal
centers are bridged by the two Se-donors leads to strong anti-ferromagnetic coupling and no EPR
spectrum.
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Biological Studies
Ap44mSe Demonstrates Anti-Proliferative Activity Against Tumor Cells that is Much Greater
than DFO, but Similar to Ap44mT and Dp44mT
The ability of the selenosemicarbazone, Ap44mSe, to inhibit cellular proliferation was assessed
using SK-N-MC neuroepithelioma cells, as the effect of other chelators and thiosemicarbazones on
1
5,40,42
its growth has been well characterized.
The activity of Ap44mSe was compared to the parent
1
5
compound, Ap44mT, and two positive control chelators (Table 2), which included: (1) DFO, that
2
is used for Fe overload therapy; and (2) Dp44mT, a thiosemicarbazone with marked anti-
1
3,40,43
proliferative activity (Fig. 1).
The selenosemicarbazone, Ap44mSe, showed potent anti-proliferative efficacy against SK-N-MC
cells, with an IC50 value of 0.011 μM (Table 2). In comparison, the parent ligand, Ap44mT, also
demonstrated marked anti-cancer activity (0.009 μM; Table 2). Dp44mT showed the greatest anti-
proliferative efficacy of all the ligands examined, with an IC50 value of 0.007 µM that was
significantly (p < 0.001) lower than that of DFO, and slightly less than that for Ap44mT and
Ap44mSe (Table 2). The clinically used agent, DFO, demonstrated poor anti-proliferative effects,
15,39
with an IC50 of 19.31 µM (Table 2), which was similar to previously observed results.
This
study highlights the potent anti-proliferative activity of the selenosemicarbazone, Ap44mSe.
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The Cu Complexes of Ap44mSe, But Not its Fe Complex, Demonstrate Similar Activity to the
Ligand Alone
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Previous studies have illustrated that complexation of chelators with metals can result in significant
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changes in their biological activity.
To determine the effect of complexation on the anti-
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proliferative behavior of Ap44mSe, the 1:2 Fe:ligand complex and the 1:1 and 1:2 Cu:ligand
complexes were prepared and their anti-proliferative activity was examined using SK-N-MC
neuroepithelioma cells relative to the Fe and Cu complexes of the ligands, DFO, Dp44mT and
Ap44mT (Table 2).
In comparison with their free ligands, the Fe complexes of all the chelators demonstrated poor anti-
proliferative activity and resulted in IC values of >25 μM for the 1:1 DFO-Fe complex and >0.8
50
μM for 2:1 ligand to Fe complexes of Dp44mT, Ap44mT and Ap44mSe (Table 2). The Cu
complexes of DFO demonstrated IC50 values that were slightly, but significantly (p < 0.01-0.05)
less than the ligand alone, namely 11.44-11.81 μM relative to 19.31 μM (Table 2). On the other
hand, the anti-proliferative activity of the Cu complexes of Dp44mT, Ap44mT and Ap44mSe were
either similar to (p > 0.05), or significantly (p < 0.001-0.01) less than, their respective ligands
(Table 2). Notably, unlike the Fe complex, the Cu complexes of Ap44mSe (i.e., 1:1 and 2:1
ligand:metal complex) retained similar (p > 0.05) anti-proliferative activity as the ligand (Table 2),
suggesting Cu complexation may play a role in the anti-proliferative efficacy of Ap44mSe.
Ap44mSe Demonstrates Markedly Greater Selective Anti-Proliferative Activity (Therapeutic
Index) Against Tumor Cells Versus Normal Mortal Cells Relative to Ap44mT or Dp44mT
The selective anti-proliferative efficacy of Ap44mSe relative to Dp44mT and Ap44mT towards
neoplastic cells was assessed by comparing SK-N-MC neuroepithelioma cells to mortal MRC-5
fibroblasts (Table 3). Their selectivity was assessed by calculating an in vitro therapeutic index
(
Table 3). This parameter represents the ratio of the IC50 values of normal to neoplastic cells (i.e.,
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IC50 (MRC-5)/IC50 (SK-N-MC); Table 3), with higher values representing greater selectivity
towards cancer cells.
Neoplastic cells were found to be markedly and significantly (p < 0.01-0.05) more sensitive than
normal MRC-5 cells to the anti-proliferative activity of Ap44mT, Dp44mT and Ap44mSe (Table
3). Of the ligands examined herein, Ap44mSe was found to be the most selective, based on an IC50
value of 2.10 μM against MRC-5 cells and 0.011 μM against SK-N-MC cells (Table 3). Both
Dp44mT and Ap44mT demonstrated similar anti-proliferative activity towards MRC-5 cells,
resulting in IC values of 0.54 and 0.45 μM, respectively, and therapeutic indices of 77 and 50,
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respectively (Table 3). Importantly, although Ap44mSe demonstrated slightly decreased anti-
proliferative effects against SK-N-MC cells relative to Dp44mT and Ap44mT, it showed the
greatest selectivity of all the ligands examined (Table 3), and this property highlights its potential as
a selective anti-cancer agent.
5
9
Ap44mSe Demonstrates Fe Mobilization Efficacy from Cells that is Markedly Greater than
DFO and Similar to that of Ap44mT and Dp44mT
As the ability of chelators to bind Fe from critical cellular Fe pools plays a role in their anti-tumor
42
59
activity, the ability of Ap44mSe to mobilize intracellular Fe from prelabeled SK-N-MC
59
neuroepithelioma cells was examined (Fig. 5A). The efflux of cellular Fe mediated by Ap44mSe
was compared to that of DFO, Dp44mT and Ap44mT, which have been characterized in this cell-
15,39,40
type
and did not induce apoptosis during the short incubation period utilized.
1
5,40
59
As shown previously,
control medium alone led to very little Fe efflux from cells, namely
59
3
.7% of intracellular Fe (Fig. 5A). Similarly, DFO at 2.5 and 25 µM also demonstrated limited
59
59
15,40
ability to mobilize Fe, resulting in the release of 3.8% and 6.2% of cellular Fe, respectively.
In contrast, incubation of SK-N-MC cells with either Dp44mT and Ap44mT at 2.5 or 25 µM led to
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a significant (p < 0.001) increase in Fe mobilization efficacy relative to the control, resulting in
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the efflux of 32-34% of cellular Fe (Fig. 5A). Similarly, Ap44mSe also mediated significantly (p
5
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< 0.001) increased levels of cellular Fe release at both 2.5 and 25 µM relative to the control,
5
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resulting in the mobilization of 29.4% and 29.9% of cellular Fe, respectively (Fig. 5A). Notably,
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Ap44mSe was significantly (p < 0.001) more effective than DFO at releasing cellular Fe at both
2.5 and 25 µM (Fig. 5A). At a concentration of 2.5 µM, Ap44mSe was slightly, but significantly (p
59
<
0.01-0.05), less effective than Dp44mT or Ap44mT in terms of Fe mobilization efficacy, but
was comparable (p > 0.05) to Dp44mT and Ap44mT at 25 µM (Fig. 5A). These results demonstrate
5
9
the similar activity of Ap44mSe relative to Dp44mT and Ap44mT in mediating Fe mobilization
from prelabeled cells, which could play a role in its high anti-proliferative efficacy.
5
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59
Ap44mSe Demonstrates Marked Efficacy at Inhibiting Cellular Fe Uptake from Fe-
Transferrin that is Much Greater than DFO and Similar to Dp44mT and Ap44mT
5
9
The ability of Ap44mSe to inhibit the cellular uptake of Fe from the physiological Fe transport
protein, transferrin (Tf), by SK-N-MC neuroepithelioma cells was also assessed and compared to
the control ligands, DFO, Dp44mT and Ap44mT (Fig. 5B). This activity was examined as
inhibiting cancer cell proliferation by targeting Fe by ligands involves not only increasing cellular
42
Fe mobilization form cells, but also the prevention of cellular Fe uptake from Tf.
1
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59
As previously observed,
DFO demonstrated poor ability to inhibit the cellular uptake of Fe
59
59
from Fe
2
-Tf, decreasing internalized Fe uptake to 97.7% and 95.1% of the control at
concentrations of 2.5 and 25 µM, respectively (Fig. 5B). At 2.5 µM, DFO showed comparable
5
9
ability to inhibit cellular Fe uptake relative to the control medium, but demonstrated significantly
(
p < 0.05) greater efficacy at 25 µM (Fig. 5B). In contrast, Dp44mT was able to markedly and
59
59
significantly (p < 0.001) inhibit cellular Fe uptake from Fe
2
-Tf, decreasing it to 20.0% and 6.0%
of the control at 2.5 and 25 µM, respectively (Fig. 5B). Similarly, Ap44mT significantly (p < 0.001)
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inhibited Fe uptake from Fe
2
-Tf, to 14.5% and 6.6% of the control at 2.5 and 25 µM,
respectively (Fig. 5B). Both Dp44mT and Ap44mT were significantly (p < 0.001) more effective at
59
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15,40
inhibiting cellular Fe uptake from Fe -Tf relative to DFO at 2.5 and 25 µM.
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The selenosemicarbazone, Ap44mSe, significantly (p < 0.001) reduced Fe uptake from Fe -Tf to
28.5% of the control at 2.5 µM, but was significantly (p < 0.01) less effective than Ap44mT at this
59
concentration (Fig. 5B). Additionally, at 25 µM, Ap44mSe markedly reduced Fe uptake to 8.7%
of the control and was comparable in efficacy to its thiosemicarbazone counterparts, Dp44mT and
Ap44mT (Fig. 5B). On the other hand, Ap44mSe was significantly (p < 0.001) more effective than
59
DFO at inhibiting Fe uptake at both 2.5 and 25 µM. Although Ap44mSe was not as effective at
59
inhibiting cellular Fe uptake relative to Ap44mT, these results indicate that its ability to inhibit Fe
uptake from Tf could still play a significant role in the anti-proliferative activity of Ap44mSe.
Ap44mSe Alters the Expression of the Classical Iron-Regulated Genes, Transferrin Receptor-1
and Ferritin, and Up-Regulates the Metastasis Suppressor Protein, NDRG1
5
9
To determine whether the ability of Ap44mSe to chelate Fe, as observed through the Fe efflux
5
9
(Fig. 5A) and Fe uptake (Fig. 5B) studies above, was involved in its anti-cancer activity, we
further investigated if Ap44mSe could alter the expression of crucial proteins that are regulated by
cellular Fe levels. The ability of Ap44mSe (10 µM) to alter the expression of the Fe uptake protein,
4
4
transferrin receptor-1 (TfR1), and the Fe storage protein, ferritin, was examined in comparison
with DFO (100 µM), Dp44mT (10 µM) and Ap44mT (10 µM) after a 24 h/37°C incubation using
SK-N-MC neuroepithelioma and SK-Mel-28 melanoma cells (Fig. 6). Notably, these ligand
concentrations were utilized considering the low Fe chelation efficacy of DFO observed
45
43
15
previously and herein (Fig. 5A, B) relative to the marked activity of Dp44mT, Ap44mT and
Ap44mSe (Fig. 5A, B). Furthermore, these cell-types were used as their Fe metabolism is well
4
2,46,47
characterized and their response to chelators, such as DFO etc., is well known.
The expression
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of the metastasis suppressor, NDRG1, was also assessed upon incubation with Ap44mSe relative to
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DFO, Dp44mT and Ap44mT, due its pronounced regulation by cellular Fe levels via hypoxia-
2
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inducible factor-1α-dependent and independent mechanisms
and its potential as a promising
2
1,22
therapeutic target against cancer.
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As previously demonstrated,
incubation with either DFO (100 µM) or Dp44mT (10 µM)
significantly (p < 0.01-0.05) increased the expression of TfR1 in both SK-N-MC and SK-Mel-28
cells relative to the control (Fig. 6A,B), further indicating that these agents were able to sequester
2
,49
cellular Fe.
Similarly, Ap44mT and Ap44mSe also significantly (p < 0.01-0.05) increased the
levels of TfR1 expression relative to the control in both cell-types, resulting in a 2.9- to 3.7-fold
increase in TfR1 expression (Fig. 6A,B). The Ap44mSe-mediated increase in TfR1 expression was
comparable to that observed with DFO, Dp44mT and Ap44mT in both SK-N-MC and SK-Mel-28
cells. The up-regulation of TfR1 expression by all chelators examined herein demonstrates the well
4
4
known response of cells to Fe depletion.
In contrast to TfR1 up-regulation, ferritin expression was down-regulated following incubation with
the chelators (Fig. 6A, B). In these studies, ferritin in SK-N-MC cell lysates was resolved into its
component H- and L- chains (Fig. 6A), while this was not apparent in SK-Mel-28 cells where
ferritin migrated as a single band (Fig. 6B). The ligands, DFO, Dp44mT and Ap44mT mediated a
significant (p < 0.001-0.01) decrease in ferritin expression in both SK-N-MC and SK-Mel-28 cells
(
Fig. 6A,B), resulting in a 1.7- to 8.3-fold decrease in ferritin expression. Incubation with Ap44mSe
also induced a significant (p < 0.001-0.01) decrease in ferritin expression relative to the control,
namely a 2.5- to 4.3-fold decrease in ferritin levels in SK-N-MC and SK-Mel-28 cells, respectively
(
Fig. 6A, B). This decrease in ferritin expression observed for all ligands examined herein indicates
the classical response to cellular Fe depletion, where less ferritin is expressed due to the decreased
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need for Fe storage.
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Assessing NDRG1, multiple bands were observed in both cell-types (Fig. 6A, B), as reported
45,50,51
previously in a variety of tumor cells.
These bands are thought to represent different cleavage
21,52
forms or phosphorylation states of this protein.
Thus, the densitometry represents the total sum
of all bands. The expression of NDRG1 was significantly (p < 0.05) increased upon DFO
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incubation in SK-N-MC cells and represented a 3-fold increase in NDRG1 expression (Fig. 6A).
Levels of NDRG1 were also slightly, but not significantly (p > 0.05), increased upon incubation
21,53
with DFO in SK-Mel-28 cells (Fig. 6B). As previously observed,
Dp44mT, markedly and
significantly (p < 0.001-0.01) increased NDRG1 levels in both cell-types, resulting in a 11.6- to
33.9-fold increase in expression (Fig. 6A, B). In fact, Dp44mT was the most effective of the
chelators examined at inducing NDRG1 expression. These results are in agreement with the ability
of Dp44mT to increase the expression of Fe-regulated genes, such as NDRG1 and TfR1, in tumor
1
3
tissue upon in vivo administration. Additionally, Ap44mT also significantly (p < 0.001-0.01)
increased NDRG1 expression by 4.5- and 16.8-fold in SK-N-MC and SK-Mel-28 cells, respectively
(Fig. 6A, B). Similarly, the selenosemicarbazone, Ap44mSe, also significantly (p < 0.01) increased
NDRG1 levels in both cell-types relative to the control, representing a 5.5- to 12.7-fold increase in
NDRG1 expression that was comparable to its thiosemicarbazone counterpart, Ap44mT (Fig. 6). In
fact, the increase in NDRG1 levels mediated by Ap44mSe was significantly (p < 0.01-0.05) greater
than that of DFO in both cell-types.
5
4-56
Considering that NDRG1 potently inhibits metastasis in multiple tumor-types,
and that
57
metastasis kills 90% of cancer patients, this property of Ap44mSe is important in terms of its
potential efficacy as an anti-metastasis/tumor agent.
Ascorbate Oxidation Studies: Ap44mSe-Fe Complex Does Not Oxidize Ascorbate
In order to determine whether redox cycling played a role in the anti-proliferative activity of
Ap44mSe, the ability of its Fe complex to mediate the oxidation of a physiological substrate,
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ascorbate, was assessed (Fig. 7A). Notably, in these studies the Ap44mSe Cu complex was not
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assessed due to its low Cu redox potential (Fig. 3) that does not facilitate the oxidation of
ascorbate and this was confirmed by preliminary studies in this investigation. However, the redox
activity of both the Cu and Fe complexes of Ap44mSe relative to Dp44mT and Ap44mT were
assessed using an alternative method below (Fig. 7B).
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The oxidation of ascorbate catalyzed by the Ap44mSe Fe complex was assessed in comparison to
its parent Ap44mT counterpart (Fig. 7A). Both Dp44mT and EDTA were included as positive
1
5,39,40
controls to accelerate ascorbate oxidation as shown previously,
while DFO acted as a negative
15,40
control as its Fe complex inhibits ascorbate oxidation.
The results in the ascorbate oxidation
assays are represented as Fe-binding equivalents (IBE) due to the different denticity of the chelators
examined (i.e., EDTA and DFO form 1:1 Fe/ligand complexes and are hexadentate, while
Ap44mSe, Ap44mT and Dp44mT form 1:2 Fe/ligand complexes and bind in a tridentate manner).
Three IBEs were examined, namely 0.1, 1, and 3, to assess the redox activity of their Fe complexes.
An IBE of 0.1 corresponds to an excess of Fe to chelator, while an IBE of 1 represents a completely
filled coordination sphere (i.e., the formation of a 1:1 EDTA Fe complex or a 2:1 tridentate Fe
complex). On the other hand, an IBE of 3 represents an excess of chelator relative to Fe.
15,39,40
In agreement with previous studies,
the Fe complex of EDTA demonstrated the greatest
ability to mediate ascorbate oxidation, increasing it to 476% and 487% of the control at IBEs of 1
and 3, respectively (Fig. 7A). In contrast, the DFO-Fe complex acted in a protective manner,
markedly inhibiting ascorbate oxidation to 43% and 21% of the control at IBEs of 1 and 3,
respectively (Fig. 7A). The Fe complex of Dp44mT increased ascorbate oxidation to 251% and
15,39,40
1
77% of the control at IBEs of 1 and 3, respectively (Fig. 7A), as shown in previous studies.
Although the Fe complex of Dp44mT demonstrated significantly (p < 0.01) decreased levels of
ascorbate oxidation relative to the Fe-EDTA complex, it showed significantly (p < 0.001) greater
ability to mediate ascorbate oxidation compared to Fe-DFO at IBEs of 1 and 3.
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In contrast, the Fe complex of Ap44mT did not effectively catalyze the oxidation of ascorbate due
III/II
15
to its low Fe
redox potential (+49 vs. NHE) that lies within a similar region to the two-electron
5
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ascorbate/dehydroascorbate couple (+60 mV vs. NHE). Thus, there is little electrochemical
driving force to enable the reaction. In fact, the Ap44mT-Fe complex resulted in 102% and 88% of
ascorbate oxidation at IBEs of 1 and 3, respectively, and thus, was comparable to the control (Fig.
7A).
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Additionally, the Fe complex of Ap44mSe mediated similar levels of ascorbate oxidation to that
observed with the Fe complex of Ap44mT, catalyzing 109% and 92% of ascorbate oxidation
relative to the control at IBEs of 1 and 3, respectively (Fig. 7A). The levels of ascorbate oxidation
catalyzed by Fe complex of Ap44mSe were similar to both the control and the Fe complex of
III/II
Ap44mT. This suggests that, like the Fe complex of Ap44mT, the Fe
redox potential of the Fe
complex of Ap44mSe may be too low to effectively mediate ascorbate oxidation.
Cu Complexation by Ap44mSe, but not Fe, Induces Cellular ROS Generation
To further investigate the ability of Ap44mSe to form redox active Fe and Cu complexes, we
examined their ability to catalyze the oxidation of the non-fluorescent molecule, 2',7'-
dichlorodihydrofluorescein
diacetate
(H
2
DCF-DA),
to
its
fluorescent
counterpart,
dichlorofluorescein (DCF), in SK-N-MC cells (Fig. 7B). Notably, DCF is a widely used probe to
4
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examine levels of intracellular redox stress.
2 2
In these studies, hydrogen peroxide (H O ; 50
µM), Ap44mT and Dp44mT were used as positive controls, as their ability to increase cellular ROS
1
8,43
has been previously documented.
To examine the ability of Ap44mSe to generate ROS, SK-N-MC cells were incubated with the
o
chelators (2.5 µM alone or in the presence of Fe or Cu) for 30 min/37 C, with or without a 15
o
min/37 C pre-incubation with FeCl
3
(1.25 µM; 2:1) or CuCl
2
(2.5 µM; 1:1) and/or the non-toxic Cu
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chelator, tetrathiomolybdate (TM; 5 µM). In these studies, TM was used as it is known to remove
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Cu from thiosemicarbazone-Cu complexes and prevent their redox activity.
The results were
quantified by flow cytometry and are expressed as a percentage of the control (Fig. 7B).
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Incubation of cells with the positive control, H O , led to a significant (p < 0.001) increase in
2
2
H DCF oxidation to 133.1 ± 5.9% of control (Fig. 7B). In contrast, incubation of cells with FeCl ,
2
3
CuCl
2
, or TM alone did not increase H
2
DCF oxidation relative to control cells (Fig. 7B). The
incubation of cells with Dp44mT, Ap44mT, or Ap44mSe alone did not significantly (p > 0.05)
increase H DCF oxidation relative to control cells. The pre-incubation of cells with FeCl followed
2
3
by the addition of Dp44mT, Ap44mT, or Ap44mSe also had no significant (p > 0.05) effect on
H DCF oxidation relative to control cells (Fig. 7B).
2
In contrast, the pre-incubation of cells with CuCl
2
followed by the addition of Dp44mT, Ap44mT,
DCF oxidation to 218%,
70% and 164% of the control, respectively (Fig. 7B). In the presence of CuCl , Ap44mSe
DCF oxidation relative to Dp44mT, but it was
or Ap44mSe mediated a significant (p < 0.001) increase in intracellular H
2
1
2
mediated significantly (p < 0.05) decreased H
2
comparable to the thiosemicarbazone counterpart, Ap44mT (Fig. 7B). Pre-incubation of cells with
TM and CuCl followed by the addition of Dp44mT, Ap44mT, or Ap44mSe resulted in a
significant (p < 0.001-0.01) decrease in H DCF oxidation to 103%, 98% and 98%, respectively,
relative to cells incubated with CuCl and Dp44mT, Ap44mT, or Ap44mSe (Fig. 7B).
2
2
2
Collectively, these results demonstrate the ability of the Cu complex of Ap44mSe to catalyze the
formation of intracellular ROS. Significantly, the Cu chelator, TM, was able to attenuate this effect
and highlights the importance of Cu chelation in the ability of Ap44mSe to catalyze ROS
formation.
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Ap44mSe Prevents Methemoglobin Formation
In the design of novel anti-cancer agents, the development of drugs that specifically target cancer
cells with minimal side effects to normal cells is essential. Notably, methemoglobinemia and
hypoxia are problematic side effects associated with the administration of 3-aminopyridine-2-
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®
carboxaldehyde thiosemicarbazone (3-AP; Triapine ), that has entered >20 phase I and II clinical
61-65
trials.
Thus, it is critical to avoid these detrimental effects when designing novel
thiosemicarbazone anti-cancer agents.
We previously demonstrated that the ability of 3-AP and Dp44mT to form redox active Fe
complexes is vital in the formation of methemoglobin (metHb), an oxidation product of
6
2
oxyhemoglobin (oxyHb) that cannot bind oxygen. Thus, it was crucial to assess the ability of
Ap44mSe to mediate metHb formation as this may provide an insight into its tolerability in vivo. In
this study, the ability of Ap44mSe and its Cu and Fe complexes (25 μM) to catalyze metHb
generation was examined in intact red blood cells (RBCs) after a 3 h incubation at 37°C and metHb
62
formation was assessed by standard methods (Fig. 8). This experiment was carried out in
comparison to the control chelators, DFO, Dp44mT and Ap44mT and their Cu and Fe complexes
6
2
(25 μM), as their ability to catalyze the formation of metHb has been previously characterized.
As expected, metHb levels present in control RBCs and RBCs incubated with FeCl
low, representing 0.25% and 0.20% of total Hb, respectively (Fig. 8). Additionally, RBCs incubated
with CuCl alone demonstrated a small, but significant (p < 0.05), increase in metHb levels
3
alone were
2
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2
representing 1.1% of total Hb (Fig. 8). As previously observed, DFO and its Fe and Cu complexes
did not significantly (p > 0.05) increase oxyHb oxidation relative to the control (Fig. 8).
In contrast, Dp44mT and its Fe and Cu complexes were able to significantly (p < 0.01-0.001)
increase oxyHb oxidation relative to the control, resulting in a 71-, 191- and 78-fold increase in
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metHb levels, respectively (Fig. 8). Importantly, the Fe complex significantly (p < 0.001) increased
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the generation of metHb relative to the ligand alone, as previously demonstrated. On the other
hand, the Cu complex of Dp44mT did not potentiate metHb formation and was comparable to the
ligand alone (Fig. 8). As the Cu complexes of these ligands do not have the appropriate redox
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potential to oxidize oxyHb,
these results could suggest that transmetallation occurs, whereby the
Cu complex donates Cu to the cell, releasing the ligand which then binds intracellular Fe which
then acts to oxidize oxyHb. Notably, the mechanism of transmetallation by this general class of
1
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ligands is known.
Ap44mT and its Fe and Cu complexes also significantly (p < 0.001-0.05) increased the levels of
oxyHb oxidation compared to the control to 2.0%, 11.2% and 2.1%, respectively. As observed for
Dp44mT, the addition of Fe significantly (p < 0.01) increased the ability of Ap44mT to catalyze
oxyHb oxidation, while the addition of Cu did not promote metHb generation and was comparable
to Ap44mT alone (Fig. 8). Importantly, the ability of Ap44mT and its Fe and Cu complexes to
mediate oxyHb oxidation was significantly (p < 0.001-0.01) decreased relative to the corresponding
Dp44mT ligand or its Fe and Cu complexes (Fig. 8).
Interestingly, the levels of metHb catalyzed by Ap44mSe or its Cu complex were found to be
comparable to the control and were significantly (p < 0.01) less than Dp44mT or its Cu complex,
respectively (Fig. 8). Although the Ap44mSe Fe complex was significantly (p < 0.01) more active
at oxidizing oxyHb than the control, it was significantly (p < 0.001) less potent than the Dp44mT
Fe complex (Fig. 8), and less effective (p > 0.05) than the Ap44mT-Fe complex. The differences in
the ability of Dp44mT and Ap44mSe to catalyze metHb formation may be due to differences in (1)
their lipophilicity, which would affect their ability to gain access to intracellular oxyHb; or (2) their
ability to interact directly with and oxidize oxyHb.
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In summary, unlike Dp44mT and Ap44mT, the ligand, Ap44mSe mediated decreased levels of
oxyHb oxidation that was comparable to the control. This property highlights the potential
tolerability of Ap44mSe in vivo and its clinical usefulness relative to either Dp44mT or Ap44mT.
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The Ap44mSe-Cu Complex Targets Lysosomal Integrity
Previous work in our laboratory revealed that the lysosome is an important cellular target in the
II
19
anti-cancer activity of the Cu thiosemicarbazone complex, Cu (Dp44mT). The redox active
II
Cu (Dp44mT) complex was found to accumulate in lysosomes, inducing cellular apoptosis by
1
9
disruption of lysosomal integrity. Due to the structural similarities between Ap44mSe, Ap44mT
and Dp44mT (Fig. 1) and their ability to form redox active Cu complexes (Fig. 7B), we examined
whether Ap44mSe also targets the lysosome using the lysosomotropic fluorophore, acridine orange
(
AO; Fig. 9A). The lysosomotropic agent, AO, is well known to accumulate in intact lysosomes to
give a red punctate fluorescence, but it exhibits a diffuse green fluorescence upon redistribution to
1
9,66,67
the cytosol or nucleus after lysosomal membrane permeabilization (LMP).
To examine the ability of Ap44mSe, relative to its thiosemicarbazone counterparts, Ap44mT and
Dp44mT, to affect lysosomal membrane integrity and AO localization, SK-N-MC cells were
incubated with the chelators (2.5 µM) for 30 min/37˚C, with or without a 15 min/37˚C pre-
3 2
incubation with FeCl (1.25 µM; 1:2 complex) or CuCl (2.5 µM; 1:1 complex). These cells were
additionally pre-incubated with either: (1) control medium alone, or medium containing the
glutathione (GSH) synthesis inhibitor, buthionine sulfoximine (BSO; 100 µM), for 16 h/37˚C; or
(
2) control medium alone or medium containing the anti-oxidant, N-acetyl-L-cysteine (NAC; 5
mM), for 15 min/37˚C; or (3) control medium alone, or medium containing the non-toxic Cu
chelator, TM (5 µM), for 15 min/37˚C. Notably, BSO is known to enhance oxidative stress induced
by Dp44mT by decreasing cellular levels of GSH through the inhibition of γ-glutamylcysteine
19,68
69
synthetase.
In contrast, the anti-oxidant and GSH precursor, NAC, has been demonstrated to
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prevent the redox stress-mediated cytotoxicity of Dp44mT and its Cu complex. The red
fluorescence intensity was quantified with the image processing and analysis software, ImageJ
v1.48 (National Institutes of Health, MD, USA; Fig. 9B).
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Upon examining control cells by fluorescence microscopy, a granular red fluorescence consistent
with AO accumulation in intact lysosomes was observed (Fig. 9A(a)). No significant difference in
red fluorescence was observed upon pre-incubation with either: FeCl
3 2
, CuCl , BSO, NAC or TM
(
Fig. 9A(b-f), 9B) relative to the control (Fig. 9A(a)), suggesting that these pre-incubation
conditions did not significantly (p > 0.05) affect lysosomal integrity. Upon incubation of the cells
with Dp44mT, Ap44mT, or Ap44mSe alone, no significant (p > 0.05) effect was observed on red
fluorescence (Fig. 9A(g),(o),(w), 9B) relative to the control. Similarly, the pre-incubation of cells
with FeCl followed by incubation with Dp44mT, Ap44mT or Ap44mSe had no significant (p >
3
0
.05) effect on red fluorescence relative to the control (Fig. 9A(h),(p),(x), 9B). Furthermore, the
pre-incubation of cells with BSO or NAC with FeCl followed by incubation with Dp44mT,
3
Ap44mT or Ap44mSe also had no significant (p > 0.05) effect on red fluorescence compared to the
control (Fig. 9A(i-j),(q-r),(y-z), 9B).
2
In contrast, pre-incubation of cells with CuCl (2.5 μM) followed by incubation with Dp44mT,
Ap44mT or Ap44mSe resulted in a marked and significant (p < 0.001) decrease in red fluorescence
to 14%, 27% and 34%, respectively, of the control (Fig. 9B). This was accompanied by
disappearance of lysosomal red vesicles consistent and the simultaneous increase in cytosolic green
19,66,67
fluorescence consistent with the redistribution of AO to the cytosol,
damage (Fig. 9A(k),(s),(aa)). Notably, Dp44mT mediated the greatest reduction in red fluorescence
in the presence of CuCl and was significantly (p < 0.001-0.01) more effective in decreasing red AO
indicating lysosomal
2
2
fluorescence than either Ap44mT or Ap44mSe in the presence of CuCl (Fig. 9B).
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Furthermore, pre-incubation with BSO prior to the addition of CuCl and the chelators, Dp44mT,
Ap44mT, or Ap44mSe, led to a significant (p < 0.001) decrease of visible red fluorescence to 26%,
17% and 28%, respectively, relative to the control (Fig. 9A(l),(t),(ab), 9B). In fact, BSO
significantly (p < 0.01-0.05) potentiated the ability of Ap44mT or Ap44mSe co-incubated with
CuCl to decrease red AO fluorescence relative to Ap44mT or Ap44mSe and CuCl co-incubation
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alone (Fig. 9B). This observation suggested that the BSO-mediated depletion of GSH sensitized
these cells to LMP by Ap44mT or Ap44mSe in the presence of CuCl2.
Importantly, pre-incubation of cells with NAC or TM prior to the addition of CuCl and either
2
Dp44mT, Ap44mT, or Ap44mSe, successfully inhibited the relocation of AO from the lysosome to
the cytosol (Fig. 9A(m-n),(u -v),(ac-ad)). Indeed, this NAC or TM pre-incubation significantly (p <
0
.001) increased red fluorescence relative to cells incubated with CuCl
2
and the chelators alone
and either
(
Fig. 9B). Furthermore, pre-incubation with TM or NAC prior to the addition of CuCl
2
Dp44mT, Ap44mT, or Ap44mSe resulted in levels of red fluorescence that were comparable to the
control (Fig. 9B). Hence, both the anti-oxidant, NAC, and the Cu chelator, TM, decreased the
2
pronounced ability of the chelators in the presence of CuCl to mediate lysosomal damage,
demonstrating the importance of both ROS generation and Cu chelation, respectively, in their
ability to induce LMP.
Collectively, these results in Fig. 9 demonstrate that like Dp44mT and Ap44mT, the
selenosemicarbazone, Ap44mSe, can mediate LMP in SK-N-MC cells via its Cu complex. This
effect was potentiated upon pre-incubation with BSO that mediates the depletion of cellular
1
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69
GSH.
Moreover, both the anti-oxidant and GSH precursor, NAC, and the Cu chelator, TM,
could prevent Cu-Ap44mSe induced LMP. This AO study (Fig. 9), together with the DCF
fluorescence data (Fig. 7B), suggests that Ap44mSe can form redox active Cu complexes that can
mediate LMP.
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The Copper Complex of Ap44mSe Mediates LMP and the Redistribution of Cathepsin D to the
Cytosol
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To further investigate the effect of Ap44mSe on the lysosome, we examined the ability of
Ap44mSe and its Cu complex to alter the intracellular distribution and co-localization of the
lysosomal membrane glycoprotein, lysosome-associated membrane protein 2 (Lamp2), with the
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soluble lysosomal enzyme, cathepsin D,
in SK-N-MC cells (Fig. 10). Upon damage to the
lysosomal membrane, soluble enzymes present in the lysosomal lumen, such as cathepsin D, are
2
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released into the cytosol to trigger apoptotic signaling pathways. Thus, the release of cathepsin D
into the cytosol upon LMP leads to its decreased co-localization with the lysosomal membrane
14
protein, Lamp2, and is a suitable marker to examine LMP.
In these studies, SK-N-MC cells were successively incubated in 3 steps: (1) for 16 h/37˚C with
either medium alone (control), or medium containing BSO (100 µM); or alternatively, for 15
min/37˚C with either medium alone (control) or medium containing NAC (5 mM) or TM (5 µM);
(
2) for 15 min/37˚C with either medium alone or medium containing CuCl
medium alone or medium containing Ap44mSe (2.5 µM) for 30 min/37˚C. The effect of FeCl
the presence of Ap44mSe on LMP was not further investigated in this study as no change in
lysosomal integrity was observed upon co-incubation of FeCl with Ap44mSe in the AO
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(2.5 µM); or (3) with
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in
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experiments (Fig. 9). The cells were then examined by immunofluorescence microscopy to assess
the co-localization of cathepsin D (green; Fig. 10A) and Lamp2 (red; Fig. 10A). Nuclei were
stained blue with 4',6-diamidino-2-phenylindole (DAPI; Fig. 10A(a,e,i,m,q,u,y)). The levels of
green fluorescence due to cathepsin D were quantified with the image processing and analysis
software, ImageJ v1.48 (Fig. 10B).
Control SK-N-MC cells stained for Lamp2 and cathepsin D demonstrated a vesicular pattern
consistent with that of lysosomes (Fig. 10A(b-c)). Significantly, upon overlaying the Lamp2 (Fig.
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0A(b)) and cathepsin D (Fig. 10A(c)) images, a yellow granular pattern was observed,
demonstrating their co-localization in intact lysosomes (Fig. 10A(d)). Similarly, pre-incubation with
CuCl alone did not significantly (p > 0.05) alter cathepsin D and Lamp2 localization, with a yellow
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punctate vesicular pattern being observed ((Fig. 10A(h)), suggesting intact lysosomes. Additionally,
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there was no significant (p > 0.05) change in cathepsin D green fluorescence upon CuCl incubation
relative to the control (Fig. 10B). In agreement with the AO results (Fig. 9), Ap44mSe alone did not
significantly (p > 0.05) affect the integrity of the lysosomal membrane, resulting in a similar yellow
punctate pattern (Fig. 10A(l)) and comparable levels of green fluorescence relative to the control
(Fig. 10A(d), 10B).
Conversely, the pre-incubation of cells with CuCl followed by Ap44mSe resulted in a marked
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alteration in the morphology of Lamp2 (Fig. 10A(n)) and cathepsin D (Fig. 10A(o)) staining with a
distinct loss of yellow punctate structures in the overlay (Fig. 10A(p)) relative to the control (Fig.
10A(d)). In fact, under the latter condition, a significant (p < 0.001) loss of green cathepsin D
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fluorescence (Fig. 10A(o)) was observed upon CuCl and Ap44mSe incubation, resulting in a
decrease to 24% of the control value (Fig. 10B). This collectively suggested the redistribution of
cathepsin D from the lysosome into the cytosol after LMP. These observations are consistent with
our AO studies above (Fig. 9) and suggest that co-incubation of Ap44mSe and CuCl
lysosomal integrity.
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impairs
Additionally, pre-incubation of cells with the GSH synthesis inhibitor, BSO, prior to CuCl
Ap44mSe incubation, also demonstrated a marked decrease in the punctate staining of both Lamp2
Fig. 10A(r)) and especially cathepsin D (Fig. 10A(s)) relative to the respective Controls (Fig.
0A(b), (c)). This effect resulted in a pronounced reduction in the co-localization of these proteins,
evident by reduced yellow fluorescence in the overlay (Fig. 10A(t)) compared to the control (Fig.
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(
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0A(d)). Moreover, a significant (p < 0.001) decrease in green cathepsin D fluorescence was
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observed (Fig. 10B).
In contrast, pre-incubation of cells with the anti-oxidant, NAC, or the Cu chelator, TM, prior to
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CuCl and Ap44mSe incubation, restored the punctate staining of Lamp2 (Fig. 10A(v)(z)) and
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cathepsin D (Fig. 10A(w)(aa)) relative to the CuCl and Ap44mSe incubation alone (Fig.
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0A(n),(o)). Moreover, in the overlay of Lamp2 and cathepsin D, the yellow vesicular pattern
consistent with intact lysosomes was maintained upon pre-incubation with NAC (Fig. 10A(x)) or
TM (Fig. 10A(ab)) prior to CuCl and Ap44mSe incubation. In fact, the pre-incubation with NAC
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or TM prior to treatment with CuCl
fluorescence that were significantly (p < 0.001) increased to 89% or 90% of the control,
respectively, relative to when the incubation was performed with CuCl and Ap44mSe alone (24%;
Fig. 10B). These results were in agreement with the AO experiments (Fig. 9) that demonstrated the
ability of both NAC and TM to prevent CuCl - and Ap44mSe-mediated damage to lysosomes.
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and Ap44mSe, resulted in levels of green cathepsin D
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In conclusion, these results in Figs 7B, 9 and 10 demonstrate that Ap44mSe can form redox active
Cu complexes which generate ROS and target lysosomal membrane integrity to induce LMP. This
effect was prevented by the chelation of Cu by TM, or the supplementation of cellular GSH via
NAC, suggesting that the formation of a redox active Cu-Ap44mSe complex was vital in the
induction of LMP.
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Conclusions
Selenosemicarbazones have gained considerable attention due to their activity as anti-bacterial,
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anti-parasitic, anti-viral and anti-cancer agents.
Significantly, selenosemicarbazones and their
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metal complexes have been observed to exhibit potent anti-cancer activity both in vitro
and in
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vivo with high tolerability.
Despite extensive investigations on the biological activity of
selenosemicarbazones, further studies were required to elucidate their cellular targets and
mechanisms of action. In this investigation, we compared the selectivity and mechanisms of action
of the selenosemicarbazone, Ap44mSe, in comparison to its well characterized thiosemicarbazone
counterparts, Ap44mT and Dp44mT.
Notably, an important structure-activity relationship derived from this study is that the substitution
of chalcogen S atom in the thiocarbonyl moiety with selenium did not markedly affect anti-
proliferative activity, but led to a marked 3-4-fold increase in the selective anti-tumor activity (i.e.,
therapeutic index) against cancer cells relative to normal mortal cells. This represents a marked
improvement in efficacy, as a major problem with current anti-tumor chemotherapy is that it suffers
from non-specific side effects against normal tissue. Hence, future chemotherapeutic design of
thiosemicarbazones, particularly the highly potent second generation of di-2-pyridylketone
21,70
thiosemicarbazones,
could implement selenium substitution of the thiocarbonyl group.
Another significant structure-activity relationship was that Ap44mSe relative to the parent ligand,
Ap44mT, exhibited less metHb generation activity as the ligand, Fe complex and also Cu complex
(
Fig. 8). This is important, as metHb is a side effect observed upon the clinical administration of the
6
1,63,71,72
thiosemicarbazone, 3-AP,
and is also observed after the in vivo administration of
62
Dp44mT. In fact, the metHb generation by the Ap44mSe was markedly and significantly less
pronounced than that mediated by Dp44mT in erythrocytes (Fig. 8). As an additional appropriate
clinically relevant property, Ap44mSe induced the up-regulation of the metastasis suppressor
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protein, NDRG1, which has been shown to be important in suppressing the metastasis of a variety
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of aggressive tumors.
This property is vital, as metastasis results in the death of 90% of cancer
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patients, and hence, agents that can effectively up-regulate NDRG1 as an important therapeutic
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target are essential to develop. Of note, Ap44mSe was not as effective as Dp44mT in up-
regulating NDRG1. However, its ability to achieve markedly higher anti-tumor selectivity and
lower metHb generation than either Dp44mT or Ap44mT, means collectively, it has greater
potential as an anti-cancer/anti-metastatic chemotherapeutic.
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For the first time, the current study identified that Ap44mSe could target lysosomal integrity via the
formation of redox active Cu complexes. Intracellular DCF studies demonstrated that Cu, but not
Fe, complexes of Ap44mSe mediated significant levels of ROS generation that could be attenuated
by the non-toxic Cu chelator, TM (Fig. 7B). Significantly, the Cu complex, but not the Fe complex,
of Ap44mSe targeted the lysosome to induce LMP, as observed through both AO and cathepsin D
fluorescence studies. Clearly, the formation of ROS and Cu complexation was critical for this
effect, as both the anti-oxidant, NAC, and Cu chelator, TM, could prevent the Cu-Ap44mSe-
mediated LMP. Importantly, the redistribution of cathepsin D from the lysosome to the cytosol
following Cu-Ap44mSe-mediated LMP is a critical step in the initiation of apoptosis, and is an
28,29
early event in the apoptotic cascade prior to mitochondrial destabilization.
In conclusion, for the first time, we demonstrate the ability of Ap44mSe to form redox active Cu
complexes that mediate ROS formation and target the lysosome to induce LMP. Hence, the ability
of selenosemicarbazones to induce cytotoxicity via the lysosome represents an important
therapeutic approach worthy of further examination. Moreover, the current study highlights the
importance of the thiocarbonyl S to Se substitution in generating advantageous pharmacological
properties and the therapeutic potential of Ap44mSe as an anti-cancer/anti-metastatic agent that
limits metHb formation and displays a very high therapeutic index.
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