Iron chelators in photodynamic therapy revisited: Synergistic effect by novel highly active thiosemicarbazones

Article abstract of DOI:10.1021/ml400422a

In photodynamic therapy (PDT), a noninvasive anticancer treatment, visible light, is used as a magic bullet selectively destroying cancer cells by a photosensitizer that is nontoxic in the dark. Protoporphyrin IX (PpIX) is a natural photosensitizer synthesized in the cell, which is also a chelating agent that if bonded to Fe²⁺ forms heme, a central component of hemoglobin. Therefore, xenobiotic iron chelators can disturb iron homeostasis, increasing the accumulation of PpIX, obstructing the last step of heme biosynthesis, and enhancing PDT efficiency. However, the attempts to use this promising idea have not proved to be hugely successful. Herein, we revisited this issue by analyzing the application of iron chelators highly toxic in the dark, which should have higher Fe²⁺ affinity than the nontoxic chelators used so far. We have designed and prepared thiosemicarbazones (TSC) with the highest dark cellular cytotoxicity among TSCs ever reported. We demonstrate that compound 2 exerts powerful PDT enhancement when used in combination with 5-aminolevulinic acid (ALA), a precursor of PpIX.

Full text of DOI:10.1021/ml400422a

Letter
pubs.acs.org/acsmedchemlett
Iron Chelators in Photodynamic Therapy Revisited: Synergistic Effect
by Novel Highly Active Thiosemicarbazones
Anna Mrozek-Wilczkiewicz,†,‡ Maciej Serda,† Robert Musiol,† Grzegorz Malecki,† Agnieszka Szurko,‡
Angelika Muchowicz,§ Jakub Golab,§ Alicja Ratuszna,‡ and Jaroslaw Polanski*,†
†Institute of Chemistry, University of Silesia, Szkolna 9, PL-40-006 Katowice, Poland
‡Institute of Physics, University of Silesia, Uniwersytecka 4, PL-40-007 Katowice, Poland
§Center of Biostructure Research, Medical University of Warsaw, Banacha 1a, PL-02-097 Warsaw, Poland
S
* Supporting Information
ABSTRACT: In photodynamic therapy (PDT), a noninvasive
anticancer treatment, visible light, is used as a magic bullet
selectively destroying cancer cells by a photosensitizer that is
nontoxic in the dark. Protoporphyrin IX (PpIX) is a natural
photosensitizer synthesized in the cell, which is also a chelating
agent that if bonded to Fe2+ forms heme, a central component
of hemoglobin. Therefore, xenobiotic iron chelators can
disturb iron homeostasis, increasing the accumulation of
PpIX, obstructing the last step of heme biosynthesis, and
enhancing PDT efficiency. However, the attempts to use this
promising idea have not proved to be hugely successful.
Herein, we revisited this issue by analyzing the application of iron chelators highly toxic in the dark, which should have higher
Fe2+ affinity than the nontoxic chelators used so far. We have designed and prepared thiosemicarbazones (TSC) with the highest
dark cellular cytotoxicity among TSCs ever reported. We demonstrate that compound 2 exerts powerful PDT enhancement
when used in combination with 5-aminolevulinic acid (ALA), a precursor of PpIX.
KEYWORDS: Photodynamic therapy, thiosemicarbazones, protoporphyrin IX, triapine, reactive oxygen species
are used commonly.13 Exogenous addition of ALA bypasses the
esferrioxamine (DFO, Figure 1, I) is an iron chelating
Ddrug that has been registered recently for the treatment normal negative feedback mechanisms and causes the
accumulation of PpIX in the tissue.14 The accumulation of
PS in malignant tissue is of clinical importance allowing for
increased treatment selectivity. The difference in accumulation
of PpIX in diseased as compared to healthy tissue results from
abnormal activity of enzymes regulating the formation of PpIX
(porphobilinogen deaminase) and heme (ferrochelatase) in
cancer cells.15 This phenomenon is the basis of the concept of
combination therapy, where iron chelators are used as adjuvants
in ALA-PDT.16 The use of nontoxic iron chelators deprives the
cells of Fe2+ crucial for the last step of heme biosynthesis,
thereby increasing the accumulation of PpIX. Although
successful application of iron chelators in PDT is a promising
idea, the achieved synergy levels in such strategies have been
relatively low so far.17 Minimized dark cytotoxicity of the PS,
including iron chelator enhancement, is considered to be a
substantial prerequisite for the safety of PDT.18
of iron overload diseases.1 Applications of DFO, thiosemicar-
bazones (TSCs), or other iron chelators have been widely
investigated as potential anticancer therapeutic agents;2,3
however, triapine (Figure 1, II) is the first TSC that has
entered phase 2 clinical trials as a drug candidate for these
conditions.4 Several paradigms have been proposed to explain
the molecular mechanisms involved in the activity of these
compounds,5 including triapine itself,6 with the hope of
obtaining FDA approval. Potential cytostatic/cytotoxic mech-
anisms of TSCs include inhibition of cellular iron uptake from
transferrin,7 mobilization of iron from cells, inhibition of
ribonucleotide reductase, the iron-containing enzyme involved
in the rate-limiting step of DNA synthesis,8 and formation of
reactive oxygen species (ROS).9 The latter mechanism is
significant in light of recent reports of the role of ROS
generation in increasing the chelator activity against tumor
cells.5,10
Herein, we present novel thiosemicarbazones (Figure 1) with
the highest dark cellular cytotoxicity among all previously
reported TSCs. We demonstrate that compound 1 exerts
The essence of PDT11 is based on producing singlet oxygen
and free radicals in a reaction triggered by excitation of the
photosensitizer (PS) by light of appropriate wavelength. The
variety among PSs or their prodrugs offers good activity and
selectivity.12 Among them, 5-aminolevulinic acid (ALA) and its
derivatives, which are precursors of protoporphyrin IX (PpIX),
Received: October 24, 2013
Accepted: January 23, 2014
© XXXX American Chemical Society
A
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ACS Medicinal Chemistry Letters
Letter
S1 in the Supporting Information). For characterization data,
see Supporting Information (Figure S2−S4).
Compound 1 was structurally characterized by single-crystal
X-ray. The compound crystallized in the monoclinic P21/c
space group (Table S1 and Figure S5 in the Supporting
Information); CCDC 908257 contains the supplementary
crystallographic data, freely available at http://www.ccdc.cam.
ac.uk/conts/retrieving.html or from the Cambridge Crystallo-
graphic Data Centre, 12 Union Road, Cambridge CB2 1EZ,
UK; fax +44 1223−336−033 or e-mail deposit@ccdc.cam.ac.
uk). In the structure of the compound, the thione sulfur atom
S(1) was cis disposed with respect to the azomethine nitrogen
atom N(2). The C(1)−S(1) bond length of 1.669(2) Å was
adequate to form a carbon−sulfur double bond. The N(1)−
H(1) group was capable of acting as a H-donor, with the N(5)
of the pyridine ring acting as the acceptor in the intramolecular
hydrogen bond formation. Moreover, in the structure of the
compound π-stacking interactions between the aromatic
(phenyl and pyridine) rings were visible but distances of
3.565 and 3.913 Å indicated rather weak interactions.
Dark cytotoxicity of 1−4 and I was studied using HCT 116
colorectal carcinoma cells with wild-type (+/+) or deleted
(−/−) tumor suppressor TP53 gene. The p53 is tumor
suppressor responsible for induction of the apoptosis. It may
regulate the ROS formation in the cells acting as both pro- or
antioxidant.19 Furthermore, after oxidative stress its activity may
increase leading to cell death. Thus, we decided to involve these
p53 positive and negative cells into consideration. Raji and
HeLa cell lines were used for the additional characterization of
cytostatic/cytotoxic effects of investigated compounds, while
normal human-derived fibroblasts (NHDF) were used to
investigate the selectivity of cytostatic/cytotoxic activity among
cancer and normal cells. In an evaluation of IC50 values (Table
1), we observed that compound 1 showed the highest activity
against the tested cells, with IC50 values of 0.81 and 0.76 nM
against HCT 116+/+ and HCT 116−/−, respectively. The Raji
cells were even more susceptible to compound 1, with ∼2.4-
fold lower IC50 value. In contrast to compound 1, which
displayed high toxicity against normal NHDF cells (IC50 = 1.73
nM), compound 2, based on the quinoline moiety, exhibited a
differential effect, inhibiting the HCT 116 cancer cell line with
an IC50 value that was 2770-fold more potent than that for
NHDF cells in the dark cytotoxicity tests.
Figure 1. Structures of the TSCs (1−4), DFO (I), and triapine (II).
powerful photocytotoxic activity when used in combination
with ALA-PDT. Very strong activity was also observed under
these conditions for compound 2, which exerts slightly lower
dark cytotoxicity, but with an IC50 value still in the nanomolar
range. On the contrary, little to no synergy was observed for
low activity TSC compounds 3−4 and for reference
compounds DFO (I) or triapine (II) under similar conditions.
At the same time, the synergy level described by the
combination index (CI) does not correlate closely to the
dark cytotoxicity of the compounds.
TSCs were synthesized by reacting equimolar amounts of the
respective (hetero)aromatic ketones or carbaldehydes and
thiosemicarbazide under microwave irradiation (closed vessel
mode). The reactions were carried out in EtOH with a catalytic
amount of acetic acid (Scheme 1). Products were isolated by
crystallization and purified by column chromatography (Figure
Fluorescence intensity time profile of PpIX assembly in HCT
116+/+ cells growing in medium supplemented with ALA is
presented in Figure S6 (see Supporting Information). The
experiments were performed in medium deprived of phenol red
so as not to coincide with the radiation activating PpIX, under
the reduced light intensity.
Scheme 1
The addition of ALA led to a steady increase in the PpIX
concentration, which reached a plateau after 18−24 h. These
experiments allowed us to determine the optimal incubation
time for the cells cultured with ALA. The maximal
concentration of PpIX was achieved after about 24 h after
which no further PpIX increase was observed. During the whole
test (74 h), viability of the cells was confirmed using the MTS
assay. Since serum suppresses the fluorescence signal due to
PpIX binding to proteins,20 the cells were cultured in serum-
free medium; however, it did not affect their proliferation to a
large extent. ALA concentration, which was optimized in the
other series of tests, was set to 1 mM. The influence of TSCs
on the cellular PpIX formation was tested under different TSC
concentrations for various incubation times. The experiments
B
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ACS Medicinal Chemistry Letters
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Table 1. Dark- and Photocytotoxicity in ALA-PDT-TSC Treatment for Compounds 1−4 and I
a
toxicity IC50 (μM)
1
2
3
4
DFO (I)
HCT116 +/+
HCT116 −/−
Raji
0.00081 0.00013
0.00076 0.00007
0.00032 0.00003
0.57 0.16
0.00384 0.00146
0.01677 0.00558
0.02123 0.00568
0.176 0.007
>10
>20
>20
>10
>20
>20
0.95
>20
>2o
>200
>200
nd
b
nd
HeLa
nd
nd
NHDF
0.00173 0.00071
0.47
>20
6.08
nd
c
CI
0.08
1.01
a
b
IC50 values for triapine (II) used as reference compound: 1.226 0.136 (HCT 116+/+). CI values for triapine: 0.85 (HCT 116+/+). nd: not
c
determined. ALA-TSC-PDT combination therapy; CI, combination index. For detailed calculations, compare Supporting Information.
indicated that the TSC addition did not influence formation of
PpIX in cultured HCT 116+/+ cells (data not shown). For
highly active TSCs, the concentration of each compound did
not exceed a nanomolar scale. Therefore, the potential to form
an iron complex with TSC is extremely low, which suggests that
this level iron−TSC complex is not high enough to produce a
noticeable effect in the synthesis of heme. Similarly, no increase
in PpIX concentration was observed for dark nontoxic TSCs 3,
4, or I at much higher concentrations of ca. 50 μM. In turn,
greater concentrations of toxic TSCs 1 and 2 resulted in cell
death.
favoring heme content. Surprisingly, in our experiment, an
excess of iron did not affect photocytotoxic behavior of the
tested TSCs (see Supporting Information). This result
suggested that TSCs did not affect heme synthesis by iron
chelation as suggested in many publications.23,24 The decrease
of ferrochelatase expression in HCT 116 cells25,26 can be one of
the factors influencing this atypical behavior.
As cytostatic/cytotoxic mechanisms of TSCs can include a
number of effects during ALA-PDT-TSC treatment, we
additionally confirmed the formation of ROS. The positive
results of these experiments indicated generation of ROS in
HCT 116+/+ cells by the compounds 1, 2, and II (Figure S7 in
the Supporting Information). Thus, a synergistic effect can be
additionally enhanced by the coexistence of two direct cytotoxic
effects; the first resulting from a typical PDT triggered by a PS
produced endogenously and, regardless of this event, the
formation of ROS by TSC in the Fenton reaction.
In conclusion, a significantly potentiated photocytotoxicity is
observed when TSC compounds 1 and 2 are used in
combination with ALA-PDT. Compound 2 appears to be the
most effective in the combination therapy studies, revealing the
highest selectivity in terms of relatively low dark cytotoxicity
against normal NHDF cells. The similarity of the studied TSCs
to triapine, the new important drug candidate in phase 2 clinical
trials, makes the observed effects worthy of careful attention.
These studies reveal novel applications for TSCs and offer
better understanding of the intracellular targeting mechanisms
of TSCs. Further studies on the larger series of TSCs as well as
potential applications of triapine are in progress.
Photocytotoxic behavior of TSCs in the combined ALA-
PDT-TSC experiment was determined as presented in Table 1.
Interactions between irradiated PpIX (given as light dose at
constant ALA concentration = 1 mM) and TSC (as
concentration) were measured according to Chou−Thalay
method.21 Increasing the doses in a constant manner we
calculated the combination index (CI) as a ratio of the sum of
the doses in TSC or ALA-PDT to the doses of each therapy
alone, having the same cytotoxic effect22 (see Supporting
Information for the detailed combination of doses, Table S2). A
value of CI below 1 indicates synergy, CI amounting to 1
indicates an additive effect, whereas the CI value greater than 1
implies antagonism. A combination of compound 2 with ALA-
PDT yielded a significant synergy, CI = 0.08, at a nanomolar
concentration of 2. Moreover, CI appeared to be stable for
compound 2 over the whole range of tested light doses (PDT)
and TSC concentrations. This means that the concentration of
TSC 2 can be reduced by almost 60-fold to achieve a result
comparable to its effect if used alone at the light dose of 9.8 J/
cm2, or alternatively, the light dose can be reduced by ca. 4
times in the combination treatment with TSC 2 at a
concentration level of 11 nM. This flexibility offers the
potential to minimize side effects while maintaining the drug
efficacy. These conclusions are based on calculations of DRI
(dose reduction index) according to the Chou−Thalay method
(data not shown).21 For compound 1, a weaker synergy was
observed. Compounds 3 and I seemed to act independently
from PDT, and a value consistent with additivity could be
observed in this case. In contrast, compound 4 displayed an
antagonism with PDT. In case of triapine (II), we detected very
weak synergy (CI value equal to 0.85).
ASSOCIATED CONTENT
* Supporting Information
■
S
Spectroscopic characterization data, figures, tables, graphs, and
detailed description of experimental procedures. This material
is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*(J.P.) E-mail: polanski@us.edu.pl.
■
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
The results of these experiments indicated that TSC can
influence photocytotoxic behavior in the combined ALA-PDT-
TSC regimen. Moreover, subnanomolar concentrations of TSC
do not appear to be high enough to deprive the cellular
mechanism of iron through simple chelation. To test this
proposal, we tested the influence of the addition of exogenous
iron (FeCl3). Hypothetically, the excess of iron competes with
the ability of TSC to chelate heme iron, reducing PpIX and
Funding
J.P. is supported by the NCBiR Warsaw PBS2/A5/40/2014.
A.M.-W. appreciates the support of the NCN grant N405/
068440 and the DoktoRIS studentship. M.S. was supported by
a TWING scholarship and by NCN grant DEC-2011/01/N/
NZ4/01166. J.G. was supported by a grant from the European
C
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ACS Medicinal Chemistry Letters
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Commission seventh Framework Programme: FP7-REGPOT-
2012-CT2012-316254-BASTION. A.M. is a recipient of the
Polish L’Oreal-UNESCO Awards for Women in Science. R.M.
́
acknowledges the NCN grant 2013/09/B/NZ7/00423.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
PDT, photodynamic therapy; PpIX, protoporphyrin IX; TSC,
thiosemicarbazones; ALA, 5-aminolevulinic acid; DFO, desfer-
rioxamine; ROS, reactive oxygen species; PS, photosensitizer;
CI, combination index; DRI, dose reduction index
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