Highly selective suppression of melanoma cells by inducible DNA cross-linking agents: Bis(catechol) derivatives

Article abstract of DOI:10.1021/ja106637e

A series of bis(catechol) quaternary ammonium derivatives were designed and synthesized. We investigated their ability to cross-link DNA induced by tyrosinase and found that the o-quinone is key intermediate in the process by using the nucleophile 3-methy

Full text of DOI:10.1021/ja106637e

Published on Web 10/12/2010
Highly Selective Suppression of Melanoma Cells by Inducible
DNA Cross-Linking Agents: Bis(catechol) Derivatives
Minghui Bai,† Jing Huang,† Xiaolong Zheng,† Zhibin Song,† Miru Tang,†
Wuxiang Mao,† Libo Yuan,† Jun Wu,† Xiaocheng Weng,† and Xiang Zhou*,†,‡
College of Chemistry and Molecular Sciences, State Key Laboratory of Virology, Wuhan
UniVersity, Hubei, Wuhan 430072, P. R. China, and State Key Laboratory of Natural and
Biomimetic Drugs, Peking UniVersity, Beijing 100083, P. R. China
Received July 26, 2010; E-mail: xzhou@whu.edu.cn
Abstract: A series of bis(catechol) quaternary ammonium derivatives were designed and synthesized.
We investigated their ability to cross-link DNA induced by tyrosinase and found that the o-quinone is key
intermediate in the process by using the nucleophile 3-methyl-2-benzothiazolinone hydrazone (MBTH) in
the tyrosinase assay. Their cytotoxicities to B16F1, Hela, and CHO cells were tested by MTT assays. The
specific and potent abilities to kill the tyrosinase-efficient melanoma cells kindled our interest in exploring
the relationship between their abilities of cross-linking DNA and their selective cytotoxicities to cells. Through
an integrated approach including intracellular imaging for detection of the dihydroxyphenyl groups, alkaline
comet assays, and γ-H2AX immunofluorescence assays, the speculation was confirmed. The bis(catechol)
quaternary ammonium derivatives showed notable cell selectivity because they displayed cytotoxicities
after being oxidized by tyrosinase, and they were able to target the DNA efficiently in the tyrosinase-
efficient melanoma cells, forming both alkylated and cross-linked species.
Introduction
Therefore, these strategies targeting tyrosinase could induce
tyrosinase expression in melanocytes.
Traditional chemotherapy and radiation are effective thera-
pies; however, the lack of selective cytotoxicity often results
in intolerable side effects. Thus, there is an urgent need for
improved treatment.1 Melanoma is a malignant tumor that
originates in melanocytes, and these cells produce the pigment
melanin.2 Tyrosinase, an enzyme that is highly expressed in
malignant tumors, mediates the biosynthesis of melanin. Many
tyrosinase inhibitors from artificial or natural sources selectively
block melanogenic pathways.3 Natural tyrosinase inhibitors such
as flavonoids4 are generally free of harmful side effects and
induce significant inhibition of tyrosinase activity. Many groups
have reported the selective treatment of malignant melanoma
using melanocyte-directed enzyme prodrug therapy (MDEPT).5
The prodrug has a catechol or phenolic moiety that permits
tyrosinase oxidation before the release of the active agent.
In our previous study,6 we reported that bis(catechol)
quaternary ammonium derivatives may be used as potent, water-
soluble, and tyrosinase-dependent DNA cross-linking agents
(Scheme 1). DNA cross-linking is a common chemical process
that is a useful chemical tool. DNA cross-linking agents
covalently bind the complementary strands of the DNA double
helix, preventing DNA replication and inducing cytotoxic DNA
double-strand breaks (DSBs).7 Endogenous enzyme-inducible
cross-linking agents have promising therapeutic potential be-
cause of their moderate inducing conditions and low side
effects.8 If a selective therapeutic strategy is developed, the
bis(catechol) quaternary ammonium derivatives may specifically
target malignant cells. These agents may be highly toxic to
tumor cells as a result of the formation of intra- and/or
interstrand adducts. Our approach is similar to two previous
strategies that depend on the activation of tyrosinase, which is
highly expressed in melanoma cells. In this paper, we present
a series of bis(catechol) quaternary ammonium derivatives that
effectively target the nuclei of melanoma cells. These derivatives
have two catechol units that are linked through various aliphatic
and aromatic chains and have the capacity to cross-link DNA.
† Wuhan University.
‡ Peking University.
(1) Kyrgidis, A.; Tzellos, T.; Triaridis, S. J. Carcinog. 2010, 9, 3–18.
(2) Ye, Y.; Chou, G. X.; Mu, D. D.; Wang, H.; Chu, J. H.; Leung,
A. K. M.; Fong, W. F.; Yu, Z. L. J. Ethnopharmacol. 2010, 129, 387–
390.
(3) (a) Yamaoka, Y.; Ohguchi, K.; Itoh, T.; Nozawa, Y.; Akao, Y. Biosci.,
Biotechnol., Biochem. 2009, 73, 1429–1431. (b) Ghani, U.; Ullah, N.
Bioorg. Med. Chem. 2010, 18, 4042–4048.
(4) (a) Seo, Y. K.; Jung, S. H.; Song, K. Y.; Park, J. K.; Park, C. S. Eur.
Food. Res. Technol. 2010, 231, 163–169. (b) Lu, Y. H.; Chen, J.;
Wei, D. Z.; Wang, Z. T.; Tao, X. Y. J. Enzyme Inhib. Med. Chem.
2009, 24, 1154–1160.
(6) Song, Z. B.; Weng, X. C.; Weng, L. W.; Huang, J.; Wang, X. L.;
Bai, M. H.; Zhou, Y. Y.; Yang, G. F.; Zhou, X. Chem.sEur. J. 2008,
14, 5751–5754.
(5) (a) Perry, M. J.; Mendes, E.; Simpl´ıcio, A. L.; Coelho, A.; Soares,
R. V.; Iley, J.; Moreira, R.; Francisco, A. P. Eur. J. Med. Chem. 2009,
44, 3228–3234. (b) Jordan, A. M.; Khan, T. H.; Malkin, H.; Osborn,
H. M. I.; Photiou, A.; Riley, P. A. Bioorg. Med. Chem. 2001, 9, 1549–
1558.
(7) (a) Wang, P.; Song, Y.; Zhang, L. X.; He, H. P.; Zhou, X. Curr. Med.
Chem. 2005, 12, 2893–2913. (b) Noll, D. M.; Mason, T. M.; Miller,
P. S. Chem. ReV. 2006, 106, 277–301. (c) Skladanowski, A.; Koba,
M.; Konopa, J. Biochem. Pharmacol. 2001, 61, 67–72.
(8) Celli, C. M.; Jaiswal, A. K. Cancer Res. 2003, 63, 6016–6025.
9
10.1021/ja106637e  2010 American Chemical Society
J. AM. CHEM. SOC. 2010, 132, 15321–15327 15321

A R T I C L E S
Bai et al.
Scheme 1. Structures of Bis(catechol) Derivatives
Scheme 2. Synthesis of Bis(catechol) Derivatives
A series of cellular assays were also used to investigate the
relationship between DNA cross-linking activity and selective
cytotoxicity.
The cross-linking percentages measured for different com-
pounds are shown in Figure 1B, where they are expressed as
mean values of three individual experiments conducted in
triplicate. The results show that the 3,4-dihydroxy derivatives
9a-f have higher DNA cross-linking abilities than the 2,3-
dihydroxy derivatives 6a-f. On the other hand, the compounds
with biphenyl linkers have higher DNA cross-linking abilities
than those with aliphatic linkers. This probably results from the
fact that the biphenyl core preferably binds and reacts with the
DNA helix. In these cases, the 3,4-dihydroxy derivative with
biphenyl core, 9f, is the most efficient cross-linking agent.
Results and Discussion
Synthesis of Bis(catechol) Quaternary Ammonium Deriva-
tives. The symmetrical bis(catechol) quaternary ammonium
derivatives 6a-f and 9a-f were prepared as shown in Scheme
2. All of the new compounds were fully characterized by high-
1
resolution mass spectrometry (HRMS) and H and 13C NMR
spectroscopy.
Proposed Mechanism of Tyrosinase Oxidation between
DNA and Catechol. These DNA cross-linking agents can form
an o-quinone intermediate induced by tyrosinase. In order to
determine the formation of o-quinone methide, we performed
the 3-methyl-2-benzothiazolinone hydrazone (MBTH) color test
to measure the formation of the intermediate o-quinone in our
current study.9 The dihydroxyphenyl groups were oxidized by
tyrosinase into o-quinones, which reacted with MBTH by a
Michael reaction to form a dark-pink pigment (shown in Figure
2). The formation of the quinone intermediate was monitored
by UV-vis spectroscopy. The addition of MBTH to the reaction
resulted in the formation of MBTH-o-quinone adducts, which
are intensely colored (Figure 2A). As shown in Figure 2B, the
maximum absorption of compound 9f and MBTH is at 260 nm.
After reaction with tyrosinase for 1 h, a peak at 360 nm
appeared, indicating the structural change of the dihydroxyphe-
DNA Cross-Linking. The DNA-DNA cross-linking abilities
of the 12 bis(catechol) quaternary ammonium derivatives were
investigated using a negatively supercoiled plasmid DNA
(pBR322) by denatured alkaline agarose gel electrophoresis. The
nonreacted plasmid was present in its single-stranded DNA
(ssDNA) form. The interstrand cross-linking (ISC) activities of
the compounds were evident as a DNA cross-linking bands,
which run slightly slower than the ssDNA form. As shown in
Figure 1A, ISC was more efficacious for the 3,4-dihydroxy
derivatives 9a-f than for the 2,3-dihydroxy derivatives 6a-f.
This is consistent with our previously published results that 3,4-
dihydroxy substitutions in the benzene ring may be preferable
for tyrosinase-induced DNA cross-linking.6 Compound 9f
produced nearly 100% DNA cross-linking at a concentration
of 15 µM after incubation for 30 min with tyrosinase. Relative
to our earlier published results,6 compound 2 (Scheme 1)
produced only 30% DNA cross-linking at a concentration of
20 µM. The biphenyl-linker-containing compound 9f had the
most potent ISC properties. It was about 4-fold more efficient
than compound 2 as a DNA cross-linking agent.
(9) (a) McMahon, A. M.; Doyle, E. M.; O’Connor, K. E. Enzyme Microb.
Technol. 2005, 36, 808–812. (b) Winder, A. J.; Harris, H. Eur.
J. Biochem. 1991, 198, 317–326.
9
15322 J. AM. CHEM. SOC. VOL. 132, NO. 43, 2010

Suppression of Melanoma Cells by DNA Cross-Linking Agents
A R T I C L E S
Figure 1. Agarose gel electrophoresis of 12 bis(catechol) quaternary ammonium salt derivatives (tyrosinase oxidation). (A) The concentrations of drug and
tyrosinase were fixed at 20 µM and 40 units, respectively. Lanes 1-12: 0.7 µg of pBR322 + 20 µM 6a-6f, 9a-9f + tyrosinase. Lane 13: 0.7 µg of pBR322
+ tyrosinase. (B) Histogram of cross-linking percentages for different compounds. The standard deviations of the cross-linking percentages were obtained
from three individual experiments. (C) Concentration dependence of compounds 9f. Lanes 1-4: concentrations of 9f are 15, 10, 5, 1 µM. Lane 5: 0.7 µg
of pBR322 + 15 µM 9f. Lane 6: 0.7 µg of pBR322 + tyrosinase. Lane 7: 0.7 µg of pBR322.
nyl groups in compound 9f to o-quinones (Figure S1 in the
Supporting Information).
site.10 The resulting intermediate has the advantage of easy
translation of electrons, resulting in the formation of the deoxy
form of tyrosinase and the bis(quinone). The bis(quinone) then
has the corresponding position for the nucleophilic reaction by
DNA, which finally results in DNA cross-links (Figure 3).
Evaluation of the Cytotoxicities of the Bis(catechol) Quaternary
Ammonium Derivatives in Three Cell Lines. To characterize the
activity of these cross-linking agents in biological systems, we
determined their toxicities in the B16F1 murine melanoma cell
line (which highly expresses tyrosinase) and in Chinese hamster
ovary (CHO) and Hela cells (tyrosinase-deficient cell lines).12
The cell damage was assessed using MTT assays, and the results
for each compound are shown in Table 1. Each compound
induced a high level of toxicity in B16F1 cells but exhibited
relatively low toxicities in the tyrosinase-deficient CHO and
Hela cell lines. Compound 9f potently induced cytoxicity in
B16F1 cells with an IC50 of ∼7 µM. These experiments
demonstrate that the bis(catechol) quaternary ammonium deriva-
tives selectively target high-tyrosinase-expressing B16F1 cells.
This mechanism is probably due to DNA alkylation or cross-
linking, which requires tyrosinase as an oxidative agent.
Intracellular Imaging for Detection of the Dihydroxyphenyl
Groups. Boron dipyrromethene (BODIPY) dyes are commonly
used as visible fluorescence probes in intracellular imaging
applications.13 To test whether the dihydroxyphenyl groups were
oxidized by tyrosinase in B16F1 cells, we used BODIPY-
modified catechol 13 (whose synthesis is shown in Scheme S1
The mechanism of catechol oxidation by tyrosinase has been
clearly researched.10 The key point is that catalysis of the
reaction by tyrosinase depends on the ability of the substrate to
bind to the tyrosinase active site. If the substrate can bind well
with the tyosinase active site, tryosinase will oxidize the
substrate efficiently. For our results, 9a-f have structures very
similar to that of 3,4-dihydroxyphenylalanine (DOPA), which
is the natural oxidation substrate of tryosinase.11 Therefore, this
suggested that 9a-f should have a similar ability as DOPA to
bind to the active site of tyrosinase and thus that tyrosinase
should oxidize substrates 9a-f efficiently. In contrast, com-
pounds 6a-f could not be oxidized efficiently by tyrosinase
because of their weak binding abilities to tyrosinase. On the
basis of the tyrosinase-specific catalytic mechanism, tyrosinase
catalyzes the conversion of the bis(catechol) to the corresponding
bis(quinone) in two steps. First, two protons are abstracted from
each of the catechol groups, and the deprotonated catechol
oxygen atoms bind to two Cu(II) ions of tyrosinase in the active
(10) (a) Mun˜oz-Mun˜oz, J. L.; Garc´ıa-Molina, F.; Garc´ıa-Ruiz, P. A.;
Molina-Alarco´n, M.; Tudela, J.; Garc´ıa-Ca´novas, F.; Rodr´ıguez-Lo´pez,
J. N. Biochem. J. 2008, 416, 431–440. (b) Cooksey, C. J.; Garratt,
P. J.; Land, E. J.; Ramsden, C. A.; Riley, P. A. Biochem. J. 1998,
333, 685–691.
(11) (a) Decker, H.; Schweikardt, T.; Tuzek, F. Angew. Chem., Int. Ed.
2006, 45, 4546–4550. (b) Garaud, M.; Pei, D. J. Am. Chem. Soc. 2007,
129, 5366–5367. (c) Mirica, L. M.; Vance, M.; Rudd, D. J.; Hedman,
B.; Hodgson, K. O.; Solomon, E. I.; Stack, T. D. Science 2005, 308,
1890–1892. (d) Solomon, E. I.; Sundaram, U. M.; Machonkin, T. E.
Chem. ReV. 1996, 96, 2563–2605.
(12) Jordan, A. M.; Khan, T. H.; Osborn, H. M. I.; Photiou, A.; Riley,
P. A. Bioorg. Med. Chem. 1999, 7, 1775–1780.
9
J. AM. CHEM. SOC. VOL. 132, NO. 43, 2010 15323

A R T I C L E S
Bai et al.
Table 1. Cytotoxicities (IC50 Values in µM) of the Bis(catechol)
Quaternary Ammonium Salt Derivatives in the Three Cell Lines
IC50 (µM)
compound
B16 F1
Hela
CHO
6a
9a
6b
9b
6c
9c
6d
9d
6e
9e
6f
15.09 ( 1.49
13.65 ( 1.13
20.98 ( 1.76
18.68 ( 0.98
27.98 ( 2.04
21.12 ( 1.33
18.09 ( 1.52
17.98 ( 1.28
10.07 ( 1.12
9.67 ( 1.08
7.88 ( 1.65
7.29 ( 0.75
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
9f
We also found that the fluorescence of BODIPY-modified
catechol 13 in the nucleus of the B16F1 cell was very weak.
The fluorescence quenching of 13 in the nucleus of the B16F1
cell may have resulted from DNA alkylation or cross-linking
with 13 after it was oxidized by the tyrosinase. To confirm our
hypothesis, the fluorescence of 13 in a genomic DNA extraction
mixture was tested in vitro. We found that with increasing
concentration of genomic DNA extract from B16F1 cells, the
fluorescence intensity of 13 at the characteristic peak near 508
nm dramatically decreased (Figure S3 in the Supporting
Information). This may explain that the faintness of the
fluorescence of 13 in the nucleus of the B16F1 cell is the result
of the interaction between the cross-link agent and nuclear DNA.
Figure 2. (A) MBTH color test based on trapping of the intermediate
o-quinones formed from compound 9f. The concentrations of 9f, MBTH, and
tyrosinase were fixed at 500 µM, 1 mM, and 40 units, respectively. (1) 9f +
MBTH; (2) 9f + tyrosinase; (3) MBTH + tyrosinase; (4) 9f + MBTH +
tyrosinase. (B) Absorption spectra of 9f (25 µM) and MBTH (50 µM) before
and after reaction with tyrosinase (0.2 units/mL) for 1 h at 37 °C. The spectra
were measured using a phosphate buffer solution (pH 6.4) as a blank.
Since the oxidation of the dihydroxyphenyl groups occurs in
the cytoplasm, the oxidation product o-quinone might react with
other nucleophiles in cells (e.g., reduced glutathione,14 a thiol-
containing tripeptide) rather than nucleus DNA. Glutathione is
well-known for its redox capabilities and is widely distributed
in cells.15 In vitro, we found that the fluorescence of BODIPY-
modified catechol 13 increased after addition of the tyrosinase/
reduced glutathione mixture (Figure S4 in the Supporting
Information). Thus, the faintness of the fluorescence in the
nucleus was not due to quenching of the o-quinone by
glutathione but instead resulted from DNA alkylation or cross-
linking in the nucleus.
in the Supporting Information) as a fluorescent dye to monitor
the spatiotemporal distribution of the dihydroxyphenyl groups
and cellular processes of DNA cross-linking.
The fluorescence of 13 was weak. After oxidation by
tyrosinase in a phosphate buffer solution, the fluorescence of
13 increased ∼2-fold (Figure S2 in the Supporting Information).
B16F1 cells loaded with 13 elicited bright-green fluorescence
in cytoplasm, whereas Hela and CHO cells gave weak fluores-
cence responses (Figure 4). This implies that the dihydroxyphe-
nyl group can be activated by the tyrosinase in B16F1 cells.
The catechol dye 13 had little effect on Hela and CHO cells
because of the nonoxidizing environment.
Figure 3. Proposed mechanism of tyrosinase oxidation between DNA and catechol.
9
15324 J. AM. CHEM. SOC. VOL. 132, NO. 43, 2010

Suppression of Melanoma Cells by DNA Cross-Linking Agents
A R T I C L E S
Figure 4. Confocal microphotographs of three different cells after treatment with compound 13 (500 nM) followed by staining with Hoechst-33324 (blue).
bis(catechol) 9f resulted in a shorter comet tail. This result
indicates that (1) 9f reached its target DNA inside the cells; (2)
at low doses, 9f alkylated chromosomal DNA; and (3) at higher
concentrations, 9f displayed efficient cross-linking ability, which
was observed as a tail with a wide range of lengths. However,
less damage and less DNA migration were observed in the Hela
and CHO cells treated with high concentrations of compound
9f (Figure S5 in the Supporting Information).
Immunofluorescence Microscope Detection of γ-H2AX Foci.
γ-H2AX immunofluorescence microscopy is a reliable and
sensitive method for measuring DNA damage. The histone
H2AX is rapidly phosphorylated following exposure of cells
to DSB-inducing agents.18 Within minutes of DSB generation,
γ-H2AX molecules are phosphorylated in large chromatin
domains that flank the DNA DSBs; these domains can be
observed by immunofluorescence microscopy and are termed
γ-H2AX foci.19
It has been reported that the formation of DNA interstrand
cross-links can be converted to DSBs when a DNA replication
fork is encountered or during its repair.16 As shown in Figure
6A, treatment with compound 9f triggered DNA damage in the
nucleus. The DNA damage induced by 9f was evidenced by
the presence of γ-H2AX foci, which were absent in the control
cells. In contrast, treatment of Hela and CHO cells with 20 µM
9f for 48 h did not induce noticeable DNA damage (Figure S6
in the Supporting Information). Furthermore, treatment with
compound 9f caused nucleus budding in B16F1 cells (Figure
S7 in the Supporting Information). Nuclear condensation and
fragmentation, the typical characteristics of apoptosis, were
observed only in B16F1 cells. These results indicate that the
tyrosinase-activated cross-linking induces cytotoxic DNA dam-
age and give further evidence that these catechol derivatives
may specifically interact with the B16F1 cells as a result of
DNA alkylation only in the B16F1 cells.
Figure 5. Alkaline comet assay for B16F1 cells treated with compound
9f. Cells were incubated with increasing concentrations of compound 9f
(0, 5, 10, and 20 µM) for 48 h.
Bis(catechol) Quaternary Ammonium Derivative 9f Produces
DNA Alkylation in Cell Systems. DNA interstrand cross-links are
absolute blocks to replication and transcription and may also
give rise to DSBs during its repair. DNA DSBs are considered
to be the most dangerous form of DNA damage.16 We
performed an alkaline comet assay17 to confirm the DNA
damage induced by bis(catechol) quaternary ammonium deriva-
tive 9f in the cellular environment (Figure 5).
In the control untreated cells, DNA damage was not detected,
and the high-molecular-weight supercoiled DNA remained
intact. As the drug concentration was increased, a fixed number
of random DNA strand breaks produced short DNA fragments
that were observed as comet images. At a dose of 20 µM,
Conclusion
In summary, we have successfully synthesized a series of
bis(catechol) quaternary ammonium derivatives that efficiently
9
J. AM. CHEM. SOC. VOL. 132, NO. 43, 2010 15325

A R T I C L E S
Bai et al.
Figure 6. γ-H2AX immunofluorescence in B16F1 cells after incubation (A) with and (B) without treatment with 5 µM 9f for 48 h. The arrows indicate
γ-H2AX foci (red) and the Hoechst-33258 dye (blue) localizes the nuclear DNA.
chem), and propidium iodide (Sigma-Aldrich). UV spectra were
measured on a Shimadzu 2550 UV-vis double-beam spectropho-
tometer. Fluorescence spectra were collected on PerkinElmer LS
55 fluorimeter. The excitation and emission slit widths were both
set to 10 nm. All of the cell imaging was performed with a Nikon
confocal laser scanning microscope (TE2000, Japan). Images and
merges were obtained with EZ-C1 software.
All other chemicals were purchased from Sigma-Aldrich and used
as received. All new compounds were fully characterized by HRMS
and 1H NMR and 13C NMR spectroscopy. 1H and 13C NMR spectra
were recorded on Varian Mercury 300 and 600 spectrometers,
respectively. Chemical shifts were reported as δ values relative to
the internal standard tetramethylsilane. HRMS spectra were recorded
on a Bruker APEX IV (7.0 T) ESI-QTPF mass spectrometer (Bruker
Daltonik GmbH, Bremen, Germany) and an APEX II FT-ICR
instrument.
General Procedure for the Synthesis of 2,3-Dihydroxy Deriva-
tives 6a-f and 3,4-Dihydroxy Derivatives 9a-f. The symmetrical
bis(catechol) quaternary ammonium derivatives 5a-f and 8a-f
were prepared through condensation of the corresponding dibromo
compounds with (2,3-dimethoxybenzyl)dimethylamine (4) and (3,4-
dimethoxybenzyl)dimethylamine (7), respectively. Demethylation
reactions of compounds 5a-f and 8a-f were performed in 1:1
HOAc/40% HBr cosolvents, affording the compounds 6a-f and
9a-f in good yields. Compounds 4 and 7 were synthesized
according to our previously published procedure.6 4,4′-Bis(bro-
momethyl)biphenyl was synthesized according to a previously
published procedure.20
Alkaline Agarose Gel Electrophoresis Assay. The duplex DNA
was linearized by restriction endonuclease digestion with EcoR I.
DNA cross-linking experiments were carried out in 10 mM
phosphate buffer (pH 6.4). Samples were incubated with 40 units
of mushroom tyrosinase in the presence of atmospheric O2 at 37
°C for 30 min. The crude reaction mixtures were loaded onto a
denaturing 0.9% alkaline agarose gel. The gel was stained in
ethidium bromide (0.5 µg/mL) for 30 min and subsequently washed
in water for 10 min. Gels were visualized by UV and photographed
using a Vilber Lourmat video system.
cross-link DNA through the formation of bis(o-quinone) by
tyrosinase-dependent oxidation. We have shown that DNA
cross-linking may lead to specific and potent targeting of
tyrosinase-expressing cells. This novel therapeutic strategy
induces cytotoxicity to melanoma cells. This may well provide
a possible chemotherapy for melanoma that employs tyrosinase-
activated agents that can induce cytotoxic DNA double-stand
breaks.
Experimental Section
Materials and Apparatus. The following compounds and
reagents were commercially available: tyrosinase mushroom (Sigma),
MBTH (Sigma), reduced glutathione (Sigma), MEM (HyClone,
Thermo Scientific), RPMI 1640 medium (GIBCO), fetal bovine
serum (FBS, HyClone), penicillin and streptomycin (Invitrogen),
MTT (Sigma), γ-H2AX (phosphor S139) rabbit polyclonal (ab11174,
Abcam), Hoechst-33258 (Calbiochem), Hoechst-33324 (Calbio-
(13) (a) Domaille, D. W.; Zeng, L.; Chang, C. J. J. Am. Chem. Soc. 2010,
132, 1194–1195. (b) Devaraj, N. K.; Hilderbrand, S.; Upadhyay, R.;
Mazitschek, R.; Weissleder, R. Angew. Chem., Int. Ed. 2010, 49, 2869–
2872.
(14) (a) Pallardo´, F. V.; Markovic, J.; Garc´ıa, J. L.; Vin˜a, J. Mol. Aspects
Med. 2009, 30, 77–85. (b) Gad, S. C. Glutathione 1998, 2, 53–54.
(15) (a) Potters, G.; Jansen, M. A. K.; Horemans, N.; Guisez, Y.; Pasternak,
T. In Vitro Cell. DeV. Biol.: Plant 2010, 46, 289–297. (b) Kudugunti,
S. K.; Vad, N. M.; Whiteside, A. J.; Naik, B. U.; Yusuf, M. A.;
Srivenugopal, K. S.; Moridani, M. Y. Chem. Biol. Interact. 2010, 188,
1–14. (c) Manini, P.; Napolitano, A.; Westerhof, W.; Riley, P. A.;
d’Ischia, M. Chem. Res. Toxicol. 2009, 22, 1398–1405.
(16) (a) Muniandy, P. A.; Liu, J.; Majumdar, A.; Liu, S.; Seidman, M. M.
Crit. ReV. Biochem. Mol. Biol. 2010, 45, 23–49. (b) Sczepanski, J. T.;
Jacobs, A. C.; Houten, B. V.; Greenberg, M. M. Biochemistry 2009,
48, 7565–7567. (c) Wood, R. D. EnViron. Mol. Mutagen. 2010, 51,
510–526. (d) Hinz, J. M. EnViron. Mol. Mutagen. 2010, 51, 582–
603.
(17) (a) Doria, F.; Richter, S. N.; Nadai, M.; Colloredo-Mels, S.; Mella,
M.; Palumbo, M.; Freccero, M. J. Med. Chem. 2007, 50, 6570–6579.
(b) Cederberg, H.; Henriksson, J.; Binderup, M. L. Mutagenesis 2010,
¨
25, 133–138. (c) Eren, K.; Ozmeric¸, N.; Sardas¸, S. Clin. Oral InVest.
2002, 6, 150–154.
BMTH Assay. Compound 9f (500 µM) was incubated with 40
units of mushroom tyrosinase in the presence of atmospheric O2
and 1 mM MBTH for 1 h. MBTH solutions were freshly prepared
before use. The spectrum was measured on UV-vis spectropho-
tometer.
(18) (a) Kinner, A.; Wu, W. Q.; Staudt, C.; Iliakis, G. Nucleic Acids Res.
2008, 36, 5678–5694. (b) Cowell, I. G.; Sunter, N. J.; Singh, P. B.;
Austin, C. A.; Durkacz, B. W.; Tilby, M. J. PLoS One 2007, 10, e1057.
(19) (a) Gomez, D.; Wenner, T.; Brassart, B.; Douarre, C.; O’Donohue,
M. F.; Khoury, V. E.; Shin-ya, K.; Morjani, H.; Trentesaux, C.; Riou,
J. F. J. Biol. Chem. 2006, 281, 38721–38729. (b) Yao, K.; Wu, W.;
Yu, Y. B.; Zeng, Q. L.; He, J. L.; Lu, D. Q.; Wang, K. J. InVest.
Ophthalmol. Visual Sci. 2009, 49, 2009–2015.
(20) Helms, A.; Heiler, D.; McLendon, G. J. Am. Chem. Soc. 1992, 114,
6227–6238.
9
15326 J. AM. CHEM. SOC. VOL. 132, NO. 43, 2010

Suppression of Melanoma Cells by DNA Cross-Linking Agents
A R T I C L E S
Cell Culture. B16F1, Hela, and CHO cell lines were purchased
from China Center for Type Culture Collection (CCTCC). Hela
and CHO cell lines were grown in MEM medium, and B16F1 cells
were grown in RPMI 1640 medium supplemented with 10% FBS
and 1% penicillin and streptomycin.
X-100/PBS. The cells were then treated with permeabilization buffer
[20 mmol/L Tris-HCl (pH 8), 0.5% Triton X-100, 50 mmol/L NaCl,
3 mmol/L MgCl2, and 300 mmol/L sucrose] for 15 min at 37 °C,
washed twice with PBS, and then antibody-stained with 1 µg/mL
γ-H2AX (phosphor S139) rabbit polyclonal at 4 °C overnight. The
nuclear DNA was stained with 5 µg/L Hoechst-33258. Secondary
antibodies raised against rabbit were labeled with tetramethyl-
rhodamine isothiocyanate, after which images were analyzed using
a Nikon confocal laser scanning microscope.
Intracellular Dihydroxyphenyl Imaging. Cells were plated on
a 35 mm × mm glass-bottom cell culture dish and then treated
with BODIPY-modified catechol 13 for 12 h. After the incubation
period, the 13-treated cells were rinsed with phosphate buffer, and
5 µg/L Hoechst-33324 was added to stain the cell nuclei for 30
min at room temperature. Images were then analyzed using a Nikon
confocal laser scanning microscope.
Cell Survival Assay. Cells were harvested using trypsin and
washed with PBS. The cells were added to the wells of a 96-well
plate and incubated overnight for attachment at 37 °C in a 5% CO2
incubator. The following day, cells were treated with the compounds
(10 nM-50 mM) for 48 h. Control cells (without any compound)
were treated under the exact same conditions. After the incubation
period, cell survival was evaluated using an MTT assay: 10 µL of
a freshly prepared solution of MTT (5 mg/mL in PBS) was added
to each well, and after 4 h of incubation, the medium was removed;
after addition of 100 µL of DMSO to each well, the optical density
values were detected at 570 nm. Cytotoxicity data were expressed
as IC50 values. Data were expressed as mean values of three
individual experiments conducted in triplicate.
Acknowledgment. This work was supported by the National
Science of Foundation of China (90813031, 30973605, 21072155,
20802055); the National Key Foundation for Infectious Diseases
(Protection and treatment of AIDs, virus hepatitis, 2008ZX10003-
005); the Fundamental Research Funds for the Central Universities;
the State Key Laboratory of Applied Organic Chemistry, Lanzhou
University; and the State Key Laboratory of Bioorganic and Natural
Products Chemistry, Shanghai Institute of Organic Chemistry, the
Chinese Academy of Sciences. We thank the editor and reviewers
for their critical comments. We also thank Mr. Yangyang Zhou
for his assistance in drawing the TOC graphic.
Alkaline Comet Assay. Cells were plated (4 × 104 cells/well)
onto six-well plates to a final volume of 2 mL and allowed an
overnight period for attachment. Next, 1 µL of each dilution of the
tested compounds (0-20 µM) was added per well, and the mixtures
were incubated at 37 °C for 48 h. After 48 h of incubation, the
medium was removed and replaced with HBSS solution, after which
the cells were scraped off. A total of 10 µL of cells was suspended
in 65 µL of 0.5% low-melting-point agarose and precoated with
0.5% normal-melting-point agarose. Cells were lysed overnight at
4 °C in cold lysis buffer (1% fresh Triton X-100, 2.5 M NaCl, 100
mM EDTA, and 10 mM Tris-HCl, pH 10.0). The agarose gels were
then washed in water, incubated in alkaline electrophoresis buffer
(300 mM NaOH and 1 mM EDTA at pH >13) for 30 min, and
then subjected to electrophoresis in the same buffer at 25 V and
300 mA for 40 min. The gels were then rinsed with neutralization
buffer (0.4 M Tris, pH 7.5) and left to air-dry, after which they
were stained with 10 µg/mL propidium iodide and incubated for
20 min. Finally, images were analyzed using a Nikon confocal laser
scanning microscope.
Supporting Information Available: Full synthesis procedures,
characterization data for individual compounds, UV-vis and
fluorescence spectra data, alkaline comet assay for Hela and
CHO cells, γ-H2AX immunofluorescence in CHO and Hela
cells, microphotographs of three different cells after treatment
with the compounds, and proposed mechanism of the oxidative
addition reaction between DNA and catechol. This material is
available free of charge via the Internet at http://pubs.acs.org.
γ-H2AX Immunofluorescence Assay. In the γ-H2AX immu-
nofluorescence assay, cells plated on a 35 mm × 10 mm glass-
bottom cell culture dish were fixed and then washed in 0.5% Triton
JA106637E
9
J. AM. CHEM. SOC. VOL. 132, NO. 43, 2010 15327