Cytotoxic activity and DNA-binding properties of xanthone derivatives

Article abstract of DOI:10.1007/s10895-010-0680-7

In this study, the interactions of different groups substituted xanthone derivatives with calf thymus DNA (ct DNA) have been investigated by spectrophotometric methods and viscosity measurements. Results indicate that xanthone derivatives can intercalate

Full text of DOI:10.1007/s10895-010-0680-7

J Fluoresc (2010) 20:1287–1297
DOI 10.1007/s10895-010-0680-7
ORIGINAL PAPER
Cytotoxic Activity and DNA-binding Properties
of Xanthone Derivatives
Rui Shen & Peng Wang & Ning Tang
Received: 28 February 2010 /Accepted: 26 May 2010 /Published online: 8 June 2010
#
Springer Science+Business Media, LLC 2010
Abstract In this study, the interactions of different groups
substituted xanthone derivatives with calf thymus DNA (ct
DNA) have been investigated by spectrophotometric
methods and viscosity measurements. Results indicate that
xanthone derivatives can intercalate into the DNA base
pairs by the plane of xanthone ring and the various
substituents may influence the binding affinity with DNA
according to the calculated quenching constant values and
the melting temperature of DNA. Furthermore, three tumor
cell lines including esophagus squamous cancer cell line
(ECA109), stomach cancer cell line (SGC7901) and lung
cancer cell line (GLC-82) have been used to evaluate the
cytotoxic activities of xanthone derivatives by MTT
(microculture tetrazolium) method. Analysis show that the
oxiranylmethoxy or piperidinylethoxy substituted xan-
thones exhibit more effective cytotoxic activity against
three cancer cells than the other substituted xanthones. The
effects on the inhibition of tumor cells in vitro agree with
the studies of DNA-binding.
Introduction
Xanthones are secondary metabolites commonly occurring
in a few higher plant families and microorganisms. They
have a large variety of biological and pharmacological
activities including antihypertensive, antioxidative, antith-
rombotic, and anticancer activity, etc. [1–5], based on their
diverse structures (Fig. 1). Accordingly, many scientists
apply themselves to isolate or synthesize these compounds
for the development of prospective new drug candidates.
Especially, the effective inhibitory activity against human
cancer cell lines has attracted considerable attention [6–10].
The preliminary work has highlighted the high potentials of
xanthones as a promising building motif for the develop-
ment of a new class of potent anticancer drugs.
Numerous biological experiments have demonstrated
that deoxyribonucleic acid (DNA) is the primary intracel-
lular target of anticancer drugs due to the interaction
between small molecules and DNA, which cause DNA
damage in cancer cells, inhibiting the division of cancer
cells and resulting in cell death [11, 12]. Up to present, the
systematic studies of the interaction of the xanthone
compounds with DNA have been reported rarely [6, 7, 9],
and their structure-activity relationships remain unestab-
lished in the xanthones system. Lin and coworkers report
that 2,3-epoxypropoxy substituted xanthones have efficient-
ly prohibited growth of cancer cells and xanthone possess-
ing two 2,3-epoxypropoxy groups at 3 and 5 position
showed most active anticancer activity in the series
prepared [13, 14]. The structural features inherent in
xanthone derivatives make us aware that the different
substitute groups in xanthones may influence significantly
to the biological activity.
.
.
Keywords Xanthone derivatives DNA-binding Cytotoxic
activity
:
R. Shen (*) P. Wang
College of Pharmacy, Nankai University,
Tianjin 300071, People’s Republic of China
e-mail: shenr05@lzu.cn
N. Tang
College of Chemistry and Chemical Engineering,
Lanzhou University,
Lanzhou 730000, People’s Republic of China
With this rational in mind, we have investigated the
binding mode and affinity of xanthone derivatives 1–6

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adjusted to pH 7.4 with HCl; the other measurements
involving the interaction of xanthone derivatives with ct
DNA were carried out in doubly distilled water buffer
containing 5 mM Tris, 50 mM NaCl and adjusted to
pH 7.4 with HCl.
UV-Vis spectrometer was employed to check a solution
of ct DNA purity (A260: A280>1.80) and concentration (ε=
6,600 M−1cm−1 at 260 nm) in the buffer [17, 18]. The
xanthone compounds were first dissolved in a minimum
amount of ethanol (0.05% of the final volume), and then
diluted with Tris-HCl buffer at concentration 5 μM.
Fig. 1 Nucleus of xanthone
(Fig. 2) to DNA and their effect on the growth of three
human cancer cell lines, ECA109 (esophagus squamous
cancer), SGC7901 (stomach cancer) and GLC-82 (lung
cancer) in vitro. The primary aim of these experiments are
expected to gain some insight into the effect of structure
factor on the binding of various substituted xanthones to
DNA and offer an impetus for designing newer DNA
directed therapeutic agents.
Preparation of xanthone derivatives 1–6 [15, 16]
The synthetic route of dihydroxylxanthone 1 and their
alkoxy derivatives 2–6 is shown in Fig. 2. Compound 1
was synthesized in 38% yield from the condensation of
salicylic acid with phloroglucinol in the presence of
anhydrous zinc chloride and phosphorus oxychloride as a
condensing agent. Alkylation of 1 with alkyl halides in
acetone or DMF afforded 2–6 in moderate to good yields
(50–85%). All the crude products were purified by
chromatography on a silicagel column and recrystallized
to afford 1–6 as yellow solids.
Experimental
Materials
Xanthone derivatives 1–6 were prepared according to the
literatures with some improvement [15, 16]. Other chem-
icals were reagent grade and were used without further
purification. Calf thymus DNA (ct DNA) and ethidium
bromide (EB) were obtained from Sigma Chemical Co.
1, 3-Dihydroxyxanthone (1). Yield 38.25%, MS:
1
228.1[M], H NMR (400 MHz, Acetone-d6): δ 8.23–
8.20 (dd, 1 H), 7.89–7.84 (dt, 1 H), 7.57–7.55 (d, 1 H),
7.51–7.46 (t, 1 H), 6.47–6.46 (d, 1 H), 6.30–6.29 (d, 1
H) ppm.
Measurements
1-Hydroxy-3-ethoxyxanthone (2). Yield 85.16%, MS:
256.0 [M], H NMR (400 MHz, CDCl3): δ 12.87 (s, 1
Mass spectra (MS) were performed on a VG ZAB-HS
instrument and 1H NMR spectra were recorded using a
Varian Mercury Plus 400 spectrometer. The UV-Vis
absorption spectra were recorded using a Varian Cary 100
spectrophotometer and the fluorescence emission spectra
were recorded using a Hitachi F-4500 spectrofluoropho-
tometer, respectively.
The thermal denaturization of ct DNA was carried out
in doubly distilled water buffer containing 1 mM Tris
(Tris = trihydroxymethyl aminomethane), 0.1 mM
Na2EDTA (EDTA = ethylenediamine tetraacetic acid) and
1
H), 8.28–8.23 (dd, 1 H), 7.77–7.68 (m, 1 H), 7.46–7.38
(m, 2 H), 6.44–6.35 (dd, 2 H), 4.16–4.12 (dd, 2 H),
1.51–1.44 (t, 3 H) ppm.
1-Hydroxy-3-butoxyxanthone (3). Yield 80.28%,
MS: 284.0 [M], 1H NMR (400 MHz, CDCl3): δ
12.86 (s, 1 H), 8.29–8.24 (dd, 1 H), 7.77–7.69 (m, 1
H), 7.46–7.38 (m, 2 H), 6.44–6.35 (dd, 2 H), 4.10–4.04
(t, 2 H), 1.83–1.79 (m, 2 H), 1.55–1.50 (m, 2 H), 1.04–
0.97 (t, 3 H) ppm.
Fig. 2 Synthetic route for xanthone derivatives 1–6

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1-Hydroxy-3-benzyloxyxanthone (4). Yield 74.21%,
MS: 318.0 [M], H NMR (400 MHz, CDCl3): δ 12.89
(s, 1 H), 8.29–8.25 (dd, 1 H), 7.77–7.69 (m, 1 H),
7.46–7.36 (m, 7 H), 6.54–6.44 (dd, 2 H), 5.18 (s, 2 H)
ppm.
serum, penicillin G (100 U/mL) and streptomycin (100 μg/
mL) at 37 °C in a humidified atmosphere containing 5%
CO2. After a confluent cell layer was formed, the cells were
harvested from the adherent cultures using 0.25% trypsin +
0.02% EDTA in phosphate buffered saline or D-Hank’s
buffer for 5 min. Suspensions were adjusted to cell
densities of 5×104 cells/mL in order to ensure exponential
growth throughout drug exposure. Aliquots of these
suspensions were seeded into 96-well microcultures
(90 μL/well). After incubation for 24 h, cells were exposed
to the tested compounds of serial concentrations. Xanthone
derivatives were dissolved in ethanol and diluted with
RPMI-1640 to the required concentrations prior to use.
After addition, the different concentration of xanthones
(10 μL/well) and incubation for 68 h, 10 μL of aqueous
MTT solution (5 mg/mL) was added to each well, and
the cells were incubated continually for another 4 h. The
medium and MTT mixtures were removed and the
formazan crystals were dissolved in 100 μL DMSO/cell.
The absorbance of each cell at 570 nm was determined by
analysis with a microplate spectrophotometer (ELX800, U.
S.A.), and the percentage cell viability was determined by
dividing the average absorbance of each column of
xanthones-treated wells by the average absorbance of the
control wells. The growth inhibitory rate of treated cells
was calculated by ðODcontrol ꢀ ODtestÞ=ODcontrol ꢁ 100%.
The IC50 values were determined by plotting the percentage
viability versus concentration on a logarithmic graph and
reading off the concentration at which 50% of cells remain
viable relative to the control. Each experiment was repeated
at least three times to get the mean values.
1
1-Hydroxy-3-(2-(1-piperidinyl)ethoxy)xanthone (5).
1
Yield 69.61%, MS: 339.1 [M], H NMR (400 MHz,
CDCl3): δ 12.86 (s, 1 H), 8.28–8.24 (dd, 1 H), 7.77–
7.68 (m, 1 H), 7.46–7.34 (m, 2 H), 6.46–6.36 (dd, 2
H), 4.26–4.20 (t, 2 H), 2.88–2.82 (t, 2 H), 2.56 (br, 4
H), 1.65–1.63 (br, 4 H), 1.49–1.47 (br, 2 H) ppm.
1-Hydroxy-3-(2-oxiranylmethoxy)xanthone (6).
1
Yield 51.41%, MS: 283.9 [M], H NMR (400 MHz,
CDCl3): δ 12.89 (s, 1 H), 8.27–8.25 (dd, 1 H), 7.75–
7.71 (m, 1 H), 7.46–7.26 (m, 2 H), 6.48–6.37 (dd, 2
H), 4.37–4.34 (m, 1 H), 4.04–3.99 (m, 1 H), 3.42–3.39
(m, 1 H), 2.97–2.95 (m, 1 H), 2.81–2.79 (m, 1 H) ppm.
DNA interactions
The competitive binding experiment was carried out by
maintaining the EB and ct DNA concentration at 2 μM and
30 μM, respectively, while increasing the concentration of
xanthone derivatives. Fitting was completed using an Origin
6.0 spreadsheet, where values of the quenching constant Kq
were calculated. Fluorescence spectra experiment was
performed by fixing the xanthones concentration as constant
at 5 μM while varying the concentration of ct DNA.
Viscosity experiments were carried on an Ubbelodhe
viscometer, immersed in a thermostated water-bath main-
tained to 35.0 0.5 °C. Titrations were performed for EB or
xanthone derivatives (1–6 μM), and the compounds were
introduced into DNA solution (50 μM) present in the
viscometer. Flow time was measured with a digital
stopwatch. The average time was calculated from the
triplicate measurement results.
Results and discussion
Competitive binding experiment
Thermal denaturization experiments were performed on
a UV-Vis spectrophotometer equipped with a Peltier
temperature controller in 1 mM Tris-HCl buffer. The
temperature of the cell containing the cuvette was ramped
from 50 °C to 100 °C at 1 °C /min rate, and the absorbance
at 260 nm was measured every 2 °C. The Tm values were
determined from the maximum of the first derivative or
tangentially from the graphs at midpoint of the transition
curves. ΔTm values were calculated by subtracting Tm of
the compound with ct DNA from Tm of the free ct DNA.
By measuring the ability of a compound to affect the
ethidium bromide (EB) fluorescence intensity in the DNA-
EB adduct, the binding mode and affinity of the compound
for DNA can be determined. If a compound can replace EB
from DNA-EB, the fluorescence of the solution will
decrease due to the fact that free EB molecules are readily
to be quenched by the surrounding water molecules [19].
Figure 3 shows the emission spectra of DNA-EB system
upon the increasing amount of xanthone derivatives. The
emission intensity of DNA-EB system at 587 nm decreases
as the concentration of xanthones increased and an
isobathic point appears around 550–560 nm for the alkoxy
substituted xanthones. This indicates that xanthone deriva-
tives could displace EB from the DNA-EB system and
induce the translocation of EB from a hydrophobic
environment to an aqueous environment [20]. Such a
Cell culture and cytotoxicity assay
Cells were supplied by the School of Pharmacy, Lanzhou
University (Lanzhou, China). Cells were routinely kept in
RPMI-1640 medium supplemented with 10% fetal bovine

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Fig. 3 Fluorescence emission spectra of DNA-EB in the absence and
2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5, 25 μM. The inset is Stern-
presence of increasing amounts of xanthone derivatives 1–6 (λex
=
Volmer quenching plots
500 nm, λem=520–720 nm) CEB=2 μM, CDNA=20 μM, Ccompound=0,

J Fluoresc (2010) 20:1287–1297
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Table 1 The Kq and IC50 values of xanthone derivatives 1–6 in EB
shows the fluorescent emission spectra of xanthone
derivatives in the presence and absence of DNA. The
results suggest that the xanthones can be protected
efficiently from solvent water molecules by the hydropho-
bic environment inside the DNA helix. This indicates that
the xanthones can insert between DNA base pairs deeply,
which is consistent with the above competitive binding
experiment results. Since the hydrophobic environment
inside the DNA helix reduces the accessibility of solvent
water molecules to the compound and the compound
mobility is restricted at the binding site, a decrease of the
vibrational modes of relaxation results. The binding of the
xanthones to DNA leads to a marked increase in emission
intensity, which also agrees with those observed for other
intercalators [24].
displacement experimentsa
1
2
3
4
5
6
Kq×104
IC50×10−4 (M)
1.51
0.66
1.09
0.92
0.89
1.12
1.59
0.63
1.91
0.52
2.29
0.43
a IC50 represents the concentration of quencher that is required for 50%
quenching
characteristic change is often observed in intercalative
DNA interactions [21].
According to the classical Stern-Volmer Eq. (1) [22]:
F0=F ¼ 1 þ Kq½Qꢂ
ð1Þ
where F0 and F represent the emission intensity in the
absence and presence of quencher, respectively, Kq is a
linear Stem-Volmer quenching constant and [Q] is the
quencher concentration. The quenching plots illustrate that
the quenching of EB bound to DNA by xanthone
derivatives are in good agreement with the linear Stern-
Volmer equation (Fig. 3, inset). In the plots of F0/F versus
[Q], Kq is given by the ratio of the slope to the intercept.
The Kq and IC50 values for the xanthones are summarized
in Table 1, so the order of the binding affinity is 3 < 2 < 1 <
4 < 5 < 6 by comparing quenching constant values.
Moreover, as these changes indicate only one quenching
process, it may be concluded that the compounds bind to
DNA solely by intercalation mode.
Because EB is useful as a DNA structure probe, further
support for xanthone derivatives binding to DNA by
intercalation mode is shown in Fig. 4. The maximal
absorption of EB at 480 nm decreased and shifted to
525 nm in the presence of DNA, which is characteristic of
intercalation. The dotted curve in Fig. 4 shows the
absorption spectrum of a solution containing EB, xanthones
and DNA. It is found that the absorption at about 525 nm
increased in comparison with that shown in dashed curve of
Fig. 4. It could result from two reasons: (1) EB bound to the
compounds strongly, resulting in the decrease of the
amount of EB intercalated into DNA; (2) there exist
competitive intercalation between the compounds and EB
with DNA, so releasing some free EB from DNA-EB
complex [23]. However, the former reason could be
precluded because no new absorption peak was seen.
Viscosity measurements
Optical photophysical probes generally provide necessary,
but not sufficient, clues to support a binding model.
Hydrodynamic measurements that are sensitive to length
change (i.e. viscosity and sedimentation) are regarded as
the least ambiguous and the most critical tests of binding
model of small molecule to DNA in solution in the absence
of crystallographic structural data. A classical intercalation
model results in lengthening the DNA helix as base pairs
are separated to accommodate the binding small molecules,
leading to the increase of DNA viscosity. In contrast, a
partial and non-classical intercalation of small molecules
could bend or kink the DNA helix, reduce its effective
length and, concomitantly, its viscosity [25, 26].
To confirm the above inferences, viscosity measure-
ments are carried out and the results are presented as (η/
η0)1/3 versus binding ratio, where η is the viscosity of DNA
in the presence of xanthone derivatives, and η0 is the
viscosity of DNA alone. Viscosity values are calculated
from the observed flow time of DNA-containing solution
corrected from the flow time of butter alone (t0), h ¼ t ꢀ t0
[27, 28]. As can be seen from Fig. 6, EB, compound 5 and
6 can increase the relative viscosity of DNA greatly, and the
extent of the viscosity increase caused by EB is more
obvious, which is consistent with the literature reports [29,
30]. Upon increasing the amounts of compound 1, 2, 3 and
4, the relative viscosity of DNA increases steadily similarly
to the behavior of EB, compound 5 and 6. The increased
degree of viscosity, which may depend on the binding
affinity to DNA, follows the order of 3 < 2 < 1 < 4 < 5 <
6 < EB. These agree with the results of the competitive
binding experiment. The results demonstrate that the
xanthones can intercalate between adjacent DNA base
pairs, causing an extension in the helix, and the binding
affinity of compound 5 and 6 is higher than that of the other
compounds.
Fluorescence spectra experiment
In Tris-HCl buffer, xanthone derivatives have fluorescence
around 410 nm when excited at 305 nm. If DNA solution is
added to the xanthones solution, enhanced fluorescence is
observed when excited under the given conditions. Figure 5

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Fig. 4 The visible absorption spectra of 10 μM EB (solid curve); 10 μM EB + 10 μM DNA (dashed curve); 10 μM EB +10 μM DNA + 5 μM
xanthone derivatives 1–6 (dotted curve)

J Fluoresc (2010) 20:1287–1297
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Fig. 5 Fluorescence emission spectra of xanthones in the presence of increasing amounts of ct DNA. (λex=305 nm, λem=360–480 nm) CCu
5 μM, CDNA=0, 5, 10, 15, 20, 25 μM
=

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Fig. 6 Effects of increasing amounts of EB (■), 1 (△), 2 (▼), 3 (□), 4
(◆) 5 (○) and 6 (▲) on the relative viscosities of ct DNA ([DNA] =
50 μM), shown as a function of the concentration ratios of
[Compound]/[DNA]. η is the viscosity of DNA in the presence of
compounds and η0 is the viscosity of DNA alone
Fig. 7 Thermal denaturization curves of ct DNA ([DNA] = 50 μM) at
the concentration ratios of [Compound]/[DNA] = 1/10. (DNA (▽),
DNA+1 (△), DNA+2 (□), DNA+3 (▲), DNA+4 (▼), DNA+5 (○)
and DNA+6 (■))
Thermal denaturization of DNA
ECA109 (esophagus squamous cancer), SGC7901 (stom-
ach cancer) and GLC-82 (lung cancer) are incubated for
72 h with varying concentrations of them and the cell
viability is determined by the MTT assay in vitro.
Intercalation of small molecules into the double helix is
known to increase the helix melting temperature, the
temperature at which the double helix denatures into
single-strand DNA. The extinction coefficient of DNA
bases at 260 nm in the double-helical form is much less
than that in the single strand form, hence, melting of the
helix leads to an increase in the absorption at this
wavelength. Thus, the helix to coil transition temperature
can be determined by monitoring the absorbance of the
DNA bases at 260 nm as function of temperature [18, 31–
33]. The thermal denaturization profiles of ct DNA in the
absence and presence of xanthone derivatives 1–6 are
shown in Fig. 7. A0, Af and A are the initial absorbance, the
final absorbance and the apparent absorbance, respectively.
The Af maxima is about 0.51. In the absence of the
xanthones, ct DNA shows a main transition at Tm=69.3 °C.
A slight increase in the melting temperature is observed in
the presence of 1, 2 and 3, while the Tm of ct DNA is
distinctly increased upon addition of 4, 5 and 6 (Table 2).
Compared with some DNA intercalators [34], the large
increased ΔTm suggest an intercalative binding of xan-
thones to DNA. This result is also consistent with the
notion that the compounds binds to DNA by intercalation
mode and the intercalative binding of the compound 5 and
6 is stronger than that of the others.
The cell viability is decreased in response to xanthone
derivatives 1–6 in a dose-dependent manner as illustrated
in Fig. 8. These compounds exhibit certain cytotoxic
activity against three human tumor cell lines. The results
demonstrate that the tree tumor cell lines are susceptible to
the xanthones. It may because the compounds intercalate
into the base group pairs of DNA, which induce damage to
DNA in the cancer cells, inhibiting the division of cancer
cells and resulting in cell death [11]. Combining with the
DNA-binding experiment, it’s possible that the different
substitute groups could influence the electron cloud
density change of xanthone ring by different level. And
the increased aromaticity and the concentrated negative
charge centers would cause the molecular shows a stronger
polarity. The molecular polarity changes relate to DNA
binding affinity. The flexible chain with active group
linked at rigid xanthone plane may enhance the DNA
binding affinity and facilitate the DNA selective binding,
which imply that the various substituent flexible side
chains in xanthone derivatives might contribute to the
Table 2 The ΔTm values of ct-DNA in the presence of xanthone
derivatives 1–6
Cell culture and cytotoxicity assay
Xanthones derivatives
1
2
3
4
5
6
To evaluate the potential cytotoxic activity of xanthone
derivatives 1–6, three human cancer cell lines including
ΔTm (°C)
5.8
3.8
3.5
9.8
10.9
14.6

J Fluoresc (2010) 20:1287–1297
1295
120
ECA109 cells
100
80
60
40
20
0
2.5 µM
5.0 µM
7.5 µM
10.0 µM
12.5 µM
15.0 µM
1
2
3
3
3
4
4
4
5
5
5
6
6
6
120
100
80
60
40
20
0
SGC7901 cells
2.5 µM
5.0 µM
7.5 µM
10.0 µM
12.5 µM
15.0 µM
1
2
120
100
80
60
40
20
0
GLC-82 cells
2.5 µM
5.0 µM
7.5 µM
10.0 µM
12.5 µM
15.0 µM
1
2
Fig. 8 Cytotoxicities of xanthone derivatives 1–6 against various human cancer cells
increase of their anticancer activity or to the decrease of
their toxicity.
Comparing to the IC50 values of xanthone derivatives
1–6 against various human cancer cells, the oxiranylme-
thoxy or piperidinylethoxy substituted xanthones have
efficiently prohibited growth of three cancer cells; the
other group substituted xanthones have inhibited growth of
different cancer cells (Table 3). This suggests that the
cytotoxic activity of the same compound against one tumor
cell line differ from another. It is likely that the exact
action mechanisms of xanthones against tumor cell lines
are different from each other because of the multiple
Table 3 The IC50 values of
IC50 (μ M)
Cell
1
2
3
4
5
6
xanthone derivatives 1–6
against various human cancer
cells
ECA109 (esophagus)
SGC7901 (stomach)
GLC-82 (lung)
13.87
16.78
98.49
>100
>100
9.58
>100
12.06
6.42
8.17
8.25
7.84
8.25
>100
10.38
67.35
12.06
10.07

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Conclusion
The interaction mode between DNA and a series different
substituted xanthones have been investigated by spectro-
photometric methods and viscosity measurements. The
results suggest that xanthones can intercalate into the
base group pairs of DNA because of the good planarity
of their ring. They all have strong binding affinity with
DNA. Comparing the binding extents of them, it is
concluded that binding affinity of oxiranylmethoxy or
piperidinylethoxy substituted xanthones is stronger than
the other group substituted xanthones. The result of
inhibitory effect in vitro is consistent with the result of
DNA binding study. And each compound would show
the cytotoxic activity varying according to the various
tumor cells. We conclude that the xanthones intercalate
between DNA base pairs and cause DNA damage in
cancer cells, thus inhibiting the division of cancer cells.
Information obtained from the present work provides
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