A novel carbazole derivative, MHY407, sensitizes cancer cells to doxorubicin-, etoposide-, and radiation treatment via DNA damage

Article abstract of DOI:10.1016/j.ejphar.2012.10.001

In this study, we synthesized a novel carbazole derivative, MHY407, as a sensitizer of cancer cells to increase DNA damage. We then evaluated the anticancer effects of MHY407 and identified the molecular mechanism for the sensitization of breast cancer ce

Full text of DOI:10.1016/j.ejphar.2012.10.001

European Journal of Pharmacology 697 (2012) 24–31
Contents lists available at SciVerse ScienceDirect
European Journal of Pharmacology
journal homepage: www.elsevier.com/locate/ejphar
Molecular and cellular pharmacology
A novel carbazole derivative, MHY407, sensitizes cancer cells to
doxorubicin-, etoposide-, and radiation treatment via DNA damage
Sungpil Yoon a,n, Ju-Hwa Kim a, Young Ju Lee b, Mee Young Ahn b, Gayoung Choi a, Won Ki Kim a,
nn
Zunhua Yang b, Hye Jin Lee b, Hyung Ryong Moon b, Hyung Sik Kim b,
a Research Institute, National Cancer Center, Ilsan-gu, Goyang-si, Gyeonggi-do, Republic of Korea
b MRC Center, College of Pharmacy, Pusan National University, Busan, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, we synthesized a novel carbazole derivative, MHY407, as a sensitizer of cancer cells to
increase DNA damage. We then evaluated the anticancer effects of MHY407 and identified the
molecular mechanism for the sensitization of breast cancer cell lines. MHY407 significantly increased
DNA damage as determined by DNA breakage, levels of damage-responsive proteins, and DNA foci. In
addition, MHY407 increased p21 and decreased cyclin D1 protein levels. MHY407 also involved
increased cell cycle arrest at the S phase. Furthermore, in doxorubicin and etoposide-treated breast
cancer cells, co-treatment with MHY407 reduced cell viability and increased apoptosis. Co-treatment of
MHY407 with doxorubicin or etoposide increased DNA damage-related proteins and foci formation,
suggesting that increased DNA damage by MHY407 plays an important role in the sensitization.
In addition, MHY407 also sensitized the cancer cells to DNA damaging radiation treatment. These
results may contribute to the development of MHY407-based treatments for cancer patients receiving
DNA-damage therapy.
Received 15 May 2012
Received in revised form
25 September 2012
Accepted 8 October 2012
Available online 16 October 2012
Keywords:
MHY407
Doxorubicin
Etoposide
Radiation
p21
DNA damage
& 2012 Elsevier B.V. All rights reserved.
1. Introduction
Carbazole derivatives have antitumor activity in various can-
cer cells by inhibiting telomerase and topoisomerase activity
Increased DNA damage to cancer cells is an important improve-
ment in cancer treatment. Currently, doxorubicin and etoposide are
widely used for the treatment of numerous types of cancers and act
by damaging the DNA of cancer cells (Biganzoli et al., 2004; Dillman
et al., 2005; Llovet, 2005). The DNA damage is caused through the
inhibition of topoisomerases as well as by triggering of apoptosis
(Bidwell and Raucher, 2006; Hsiao et al., 2008; Nitiss, 2002). Radia-
tion is another option for increasing DNA damage in cancer cells
(Inskip et al., 2009; Shin et al., 2010). As patients develop resistance to
these treatments, research has been aimed at providing better DNA-
damaging treatment of cancer that increases apoptosis (Crivellari
et al., 2007; Gluck, 2005; Humber et al., 2007; Kaklamani and
Gradishar, 2005; Leary et al., 2007; Thomadaki and Scorilas, 2008;
Van Valen et al., 2003). Identifying novel drugs that damage DNA
would be an important step in the development of new pharmaco-
logical treatments for cancers. Furthermore, combination therapy
with these novel DNA damaging drugs may be an important option
for overcoming resistance and sensitizing cells to doxorubicin, etopo-
side, or radiation treatments.
(Asche and Demeunynck, 2007; Nagarapu et al., 2010; Patra
et al., 2011). Here, we synthesized novel carbazole derivatives
by adding epoxypropoxy or thioepoxypropoxy groups to the
carbazole scaffold. Based on previous studies, we hypothesized
that the addition of epoxypropoxy or thioepoxypropoxy groups
would increase the ability of carbazole to cause DNA damage. We
selected MHY407 from among the carbazole derivatives because
it demonstrated greater DNA damaging ability than the others.
We then investigated its mechanism in breast cancer cell lines.
We found that MHY407 damages DNA through a different
mechanism than doxorubicin or etoposide. In addition, MHY407
sensitized doxorubicin, etoposide, and radiation-treated cancer
cells. These results may contribute to the development of
carbazole-based therapy for cancer patients who are treated with
doxorubicin, etoposide, or radiation.
2. Materials and methods
2.1. Reagents
Doxorubicin (Boryung Pharmacy, Seoul, Korea) was obtained from
the National Cancer Center in Korea. Etoposide, verapamil, and
5(6)-carboxyfluorescein diacetate (CFDA) were purchased from
n Corresponding author. Tel.: þ82 31 920 2361; fax: þ82 31 920 2468.
nn Corresponding author. Tel.: þ82 51 510 2816; fax: þ82 51 513 6754.
E-mail addresses: yoons@ncc.re.kr (S. Yoon), hkims@pusan.ac.kr (H.S. Kim).
0014-2999/$ - see front matter & 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ejphar.2012.10.001

S. Yoon et al. / European Journal of Pharmacology 697 (2012) 24–31
25
Sigma-Aldrich (St. Louis, MO). MHY403 (2-(thiiran-2-ylmethoxy)-
2.6. Cell proliferation assay
9 H-carbazole), and MHY404 (2-(oxiran-2-ylmethoxy)-9H-carbazole)
were prepared by previously described methods (Asche and
Demeunynck, 2007; Nagarapu et al., 2010; Patra et al., 2011).
Briefly, the reaction of 2-hydroxycarbazole with epichlorohydrin
(1.0 eq.) in acetone and in the presence of K2CO3 (2.5 eq.) at room
temperature (RT) resulted in a 49% yield of MHY403. Reaction of
2-hydroxycarbazole with epithiochlorohydrin (3.0 eq.) in dimethyl-
formamide (DMF) and in the presence of K2CO3 (2.5 eq.) at 70 1C
produced a 19% yield of MHY404. MHY407 (9-methyl-2-(oxiran-2-
ylmethoxy)-9H-carbazole) was synthesized from MHY403 after
NaOH (3.0 eq.) was added to a stirred solution of MHY403 in DMF
at 0 1C and stirred for 10 min; methyl iodide (10 eq.) was then added
and the reaction mixture was stirred for 24 h, producing a 79% yield
of MHY407 with the following nuclear magnetic resonance (NMR)
Cell proliferation was determined in a colorimetric assay
using the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,
5-diphenyltetrazolium bromide (MTS) kit (Promega, Madison,
WI, USA) according to a previously described method (Kim et al.,
2009, 2010).
2.7. Fluorescence-activated cell sorting (FACS) analysis
Cells grown in 60-mm-diameter dishes were treated with the
indicated drugs for the prescribed times. The cells were then
dislodged by trypsin, and the entire volume was centrifuged to
collect the cells. The pelleted cells were washed thoroughly with
PBS, suspended in 75% ethanol for at least 1 h at 4 1C, washed
again with PBS, and re-suspended in a cold propidium iodide (PI)
values: 1H NMR (400 MHz, CDCl3)
d 8.01 (d, 1H, J¼7.6 Hz), 7.96
(d, 1H, J¼8.8 Hz), 7.42 (t, 1H, J¼7.6 Hz), 7.33 (d, 1H, J¼8.0 Hz), 7.23
(t, 1H, J¼7.6 Hz), 6.88–6.85 (m, 2H), 4.32 (dd, 1H, J¼3.2, 10.8 Hz),
4.06 (dd, 1H, J¼5.2, 10.8 Hz), 3.73 (s, 3H), 3.42 (m, 1H), 2.94 (t, 1H,
staining solution (100 mg/mL RNase A and 10 mg/mL PI in PBS) for
40 min at 4 1C. The stained cells were analyzed for relative DNA
content using a FACSCalibur flow cytometry system (BD, Franklin
Lakes, NJ, USA).
J¼4.8 Hz), 2.81 (dd, 1H, J¼2.8, 4.8 Hz); 13C NMR (100 MHz, CDCl3)
d
158.2, 142.5, 141.4, 124.8, 123.1, 121.3, 119.8, 119.3, 117.3, 108.5,
107.8, 94.2, 69.6, 50.6, 45.1, 29.3.
2.8. Western blot analysis
Total cellular proteins were extracted using a previously
described trichloroacetic acid (TCA) method (Kim et al., 2010;
Lee et al., 2008). Briefly, cells grown in 60-mm-diameter dishes
2.2. Antibodies
Antibodies against p53, p21, pBRCA1, pChk1, pChk2, cyclin D1,
and cleaved poly-ADP ribose polymerase (C-PARP) were pur-
chased from Cell Signaling Technology (Danvers, MA, USA).
Antibodies against cyclin B1, cyclin A, pRb, Rb, cyclin E, Cdk4,
p27, p16, p18, and glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) were purchased from Santa Cruz Biotechnology (Santa
Cruz, CA, USA). Antibodies against p53 and pH2AX were obtained
from Abcam (Cambridge, UK). In addition, HRP-conjugated goat
rabbit- and goat mouse-secondary antibodies were purchased
from Cell Signaling Technology.
were washed three times with 5 mL PBS. Next, 500 mL of 20% TCA
was added to each plate. The cells were then dislodged by
scraping and transferred to Eppendorf tubes. Proteins were
precipitated by centrifugation for 5 min at 800g and resuspended
in 1 M Tris–HCl (pH 8.0) buffer. The protein concentrations were
quantified using the Bio-Rad protein Assay Reagent (Bio-Rad
Laboratories, Hercules, CA, USA) according to the manufacturer’s
protocol. The proteins were resolved by sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to
Western blot analysis.
2.3. Cell culture and drug treatment
2.9. Comet assay
Previously described human cancer cell lines (Kim et al.,
2011a, 2011b) were used. MCF7 was obtained from the American
Type Culture Collection (Manassas, VA, USA). Hs578T breast
cancer cell line was obtained from the Korean Cell Line Bank
(Seoul, Korea). All cell lines were cultured in DMEM or RPMI1640
containing 10% fetal bovine serum (FBS), 100 U/mL penicillin, and
Residual DNA damage following doxorubicin or MHY407
treatment was detected using the Comet assay single gel electro-
phoresis assay kit (Trevigen, Gaithersburg, MD, USA) according to
the manufacturer’s instructions. Briefly, cells grown in 6-well
plates to approximately 30% confluence were treated for 4 h with
doxorubicin or Sal. The treated cells were trypsinized and pelleted
by centrifugation. The cells were washed and resuspended in
100 mg/mL streptomycin (WelGENE, Daegu, Korea).
200 mL of ice-cold 1X PBS. The cell suspension was mixed 1:10
2.4. Radiation treatment
(v/v) with low melting point agar at 42 1C and immediately
pipetted onto the CometSlideTM area. Slides were incubated at
4 1C in the dark for 30 min, immersed in chilled lysis solution,
and incubated again at 4 1C for 30 min. Slides were placed in a
horizontal electrophoresis chamber and electrophoresed with
buffer (0.3 N NaOH, 1 mM EDTA) at 1 V/cm for 20 min. Samples
were dried and stained with ethidium bromide (EtBr). Comet tails
were imaged using a model DM 2500 fluorescent microscope
(Leica, Wetzlar, Germany) and quantified by Komet 5.5 software
(Andor Technology, Belfast, UK). A minimum of 50 cells were
scored per treatment. All experiments were performed indepen-
dently at least 3 times.
Cells grown in 60-mm-diameter dishes or chamber slides were
irradiated with graded doses of g-rays using a Gammacell 1000
Elan system (MDS Nordion, Ottawa, Ontario, Canada) with a dose
of 4 Gy as determined by the thermoluminescence dosimetry for
the system. After irradiation, cells were treated with the MHY407
for the prescribed times.
2.5. Trypan blue dye exclusion test
Trypan blue staining was performed to determine the number
of viable cells and cell proliferation status. Briefly, cells grown in
60-mm-diameter dishes were washed with 5 mL of phosphate
buffered saline (PBS), trypsinized, and pelleted by centrifugation
2.10. Immunocytochemistry
for 1 min at 450g. The pellet was suspended in 500
m
L of cell
Cells were plated in chamber slides, incubated for 24 h at
37 1C, and treated with etoposide or MHY407. After incubation,
the cells were fixed with 4% paraformaldehyde for 10 min, and
were then permeabilized by placing them in PBS containing
culture medium and Trypan blue was added to the cell suspen-
sion. The number of dye-excluding (viable) cells and stained
(dead) cells were counted using a hematocytometer.

26
S. Yoon et al. / European Journal of Pharmacology 697 (2012) 24–31
Triton X-100 and bovine serum albumin (BSA) for 20 min at RT.
The permeabilized cells were incubated with pH2AX and p53BP1
primary antibodies overnight at 4 1C. They were then incubated
with Green-fluorescent Alexa Fluor 488 secondary antibody
(Invitrogen, Carlsbad, CA, USA), Orange-Red-fluorescent Alexa
Fluor 568 secondary antibody (Invitrogen), and stained with
40-6-diamidino-2-phenylindole (DAPI) for 1 h at 37 1C. The cells
were examined using a LSM 510 META inverted confocal micro-
scope (Carl Zeiss, Oberkochen, Germany) with a 40 ꢀ water
immersion objective lens (Carl Zeiss C-Apochromat NA 1.2)
coupled with three optical zoom lenses.
variance (ANOVA) followed by a multiple-comparison test.
Results were considered to be statistically significant compared
to the control (*) when Po0.05. All samples were tested in
triplicate, and all experiments were performed at least twice.
3. Results
3.1. MHY407 increases DNA damage-response proteins and reduces
cyclin D1
The structures of the three novel carbazole derivatives,
MHY403, MHY404, and MHY407, are shown in Fig. 1A and
Supplementary Fig. 1A. Two 2- and 9-positions of the basic
carbazole structure were modified. The addition of epoxypropoxy
or thioepoxypropoxy groups at the 2-position was designed for
efficient cytotoxicity through DNA damage to cancer cells (Asche
and Demeunynck, 2007; Nagarapu et al., 2010; Patra et al., 2011).
We studied the effects of the carbazole derivatives on the MCF7 and
Hs578T human breast cancer cell lines. When compared to MHY403
and MHY404 at the same concentration, MHY407 showed better
sensitizing effects as determined by FACS, C-PARP production, and
DNA damaging-related proteins (Supplementary Fig. 1B–D). MCF7
and Hs578T cells treated with MHY407 had higher levels of the
tumor suppressor, p21 (Abukhdeir and Park, 2008), than those treated
with the other derivatives (Supplementary Fig. 1D). We therefore
selected the MHY407 for further study as a cancer sensitizer.
2.11. CFDA uptake tests
Tests to measure the inhibition of P-glycoprotein (P-gp) were
performed using a previously described method (Kim et al., 2011a).
Briefly, cells grown in wells of 6-well plates were treated with
verapamil or MHY407 for 24 h at 37 1C. The cells were then incubated
with 10 mM CFDA for 90 min at 37 1C. The medium was removed and
the cells were washed twice with PBS. The stained cells were
subsequently examined using an inverted fluorescence microscope
and analyzed using a FACSCalibur flow cytometry system.
2.12. Statistical analysis
The data are presented as the mean 7S.D. Statistical analysis
was conducted using Student’s t-test and a one-way analysis of
Fig. 1. MHY407 increases DNA damage-response proteins and reduces cyclin D1. (A) The structure of MHY407 [9-methyl-2-(oxiran-2-ylmethoxy)-9H-carbazole] derived
from the carbazole backbone. (B) MCF7 cells were plated in 60 mm dishes. Cells were then stimulated for 48 h with 5 M MHY407, 10 M MHY407, 20 M MHY407, or
were DMSO-treated (black bar). The trypan blue exclusion test was performed to count cell numbers as described in Materials and methods. The data represent the
mean7S.D. of at least three independent experiments;
P o0.05 compared to the control. Statistical analysis was conducted using Student’s t-test. (C–E) MCF7 cell
extracts were collected 6 or 20 h after treatment with 0 M, 10 M, or 20 M of MHY407. The cell extracts were used for Western blot analyses using antibodies against
pH2AX, pBRCA1, C-PARP, GAPDH, Cyclin A, Cyclin D1, Cyclin B1, Cyclin E, Cdk4, pRb, Rb, p16, p18, p21, p27, and p53.
m
m
m
n
m
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m

S. Yoon et al. / European Journal of Pharmacology 697 (2012) 24–31
27
We further analyzed the mechanisms of the sensitizing effects of
MHY407 in the breast cancer cells. MHY407 significantly inhibited
cellular proliferation in a concentration-dependent manner and
to anti-cancer drugs (Maddika et al., 2007; Sherr and McCormick,
2002). This experiment was performed because alterations in the
level of these proteins may reduce cell viability and increase DNA
damage by MHY407. Cyclin D1 and pRb levels were reduced by
MHY407, whereas tumor suppressor p21 levels were increased
(Fig. 1E). The changes in these proteins were confirmed in Hs578T
cells (Supplementary Fig. 2C and D). This indicated that MHY407
targets cyclin D1, pRb, and p21 proteins. It also suggested that
increased DNA damage by MHY407 contributes to changes in the
levels of cell cycle arrest proteins. Cyclin D1 seems to be most
important when compared between early (6 h) and late (20 h) time,
as it was the only protein that was reduced by MHY407 until the
late time (Fig. 1E).
proliferation was reduced by approximately half at 5 mM MHY407
(Fig. 1B). As shown in Fig. 1C, MHY407 treatment increased C-PARP
levels, suggesting that apoptosis was also increased. We also tested
the DNA damaging ability. DNA damage-response proteins such as
pH2AX and pBRCA1 levels (Kawabe, 2004; Solier et al., 2009) were
also increased in a concentration-dependent manner (Fig. 1C). This
suggests that DNA damage and apoptosis were proportionate to
MHY407 concentration. MHY407 caused DNA damage relatively
soon after exposure (at 6 h), suggesting that MHY407 increases DNA
damage at a relatively early time. As shown in Supplementary
Fig. 2A and B, we also confirmed the DNA damaging ability of
MHY407 in another breast cancer cell line, Hs578T. When C-PARP
production of between MCF7 and Hs578T cells was compared
(Fig. 1C vs. Supplementary Fig. 2A), Hs578T cells showed a higher
rate of apoptosis than MCF7, suggesting that MHY407 has specificity
and selectivity for apoptosis among cancer cell types.
3.2. MHY407 increases G1/S arrests
Since doxorubicin and etoposide are widely used DNA dama-
ging anti-cancer drugs, we compared the sensitizing effects of
MHY407 and doxorubicin or etoposide. In FACS analysis, doxor-
ubicin and etoposide increased S and G2/M phases, whereas
MHY407 only increased S phase (Fig. 2A and B). The increased
We also assessed whether MHY407 influences the levels of cell
cycle proteins or tumor suppressors, which are important responses
Fig. 2. MHY407 has a different pathway for DNA damage than that of doxorubicin and etoposide. (A and B) MCF7 cells (4 ꢀ 105) were plated on 60-mm-diameter dishes.
The cells were then incubated for 24 h with 3.5 mM doxorubicin, 50 mM etoposide, 10 mM MHY407, or were DMSO-treated (Control). FACS analysis was performed to
determine the cell distributions at each phase of the cell cycle. (C) MCF7 cell extracts were collected 6 h after treatment with doxorubicin (3.5 mM), MHY407 (10 mM), or
after DMSO-treatment (Control, Con). The cell extracts were used for Western blot analyses using antibodies against pH2AX, pBRCA1, pRb, p27, cyclin D1, p21, and GAPDH.
Control cells were treated with DMSO alone.

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S. Yoon et al. / European Journal of Pharmacology 697 (2012) 24–31
G1 and S phases after MHY407 treatment were also detected in
Hs578T cells (Supplementary Fig. 3A). The results suggest that
MHY407 causes DNA damage through a different signaling path-
way than the well-known topoisomerase inhibitors, doxorubicin
and etoposide. To confirm this difference at the molecular level,
we measured the levels of both DNA damage-responsive proteins
and cell cycle arrest-related proteins. Expression of pH2AX levels
was much higher in doxorubicin-treated cells than in MHY407-
treated cells, whereas expression of pRb was lower and p21 level
was higher in MCF7 and Hs578T cells against MHY407 treatment
(Fig. 2C and Supplementary Fig. 2D). The results indicated that
MHY407 has a different pathway for DNA damage than doxor-
ubicin and etoposide.
treated with doxorubicin or MHY407 for 24 h showed a reduced
cell growth (Fig. 3A). The degree of cellular proliferation was
lower in cells co-treated with doxorubicin and MHY407 relative
to cells treated with either doxorubicin or MHY407 alone. Cell
proliferation assays indicated that doxorubicin and MHY407 co-
treated cells had reduced cell viability compared to corresponding
cells treated solely with either doxorubicin or MHY407 (Fig. 3B).
This reduction in cell proliferation and viability after co-treatment
with doxorubicin and MHY407 was confirmed in Hs578T cells
(Supplementary Fig. 4A and B). To further analyze the reduced
viability of MCF7 cells after co-treatment with MHY407 and
doxorubicin, apoptosis was assessed by the production of cleaved
PARP. Co-treatment with doxorubicin and MHY407 greatly
increased C-PARP production (Fig. 3C), suggesting that apoptosis
was increased by co-treatment.
3.3. MHY407 sensitizes doxorubicin-treated cancer cells
We also assessed whether co-treatment with doxorubicin and
MHY407 influenced the levels of cell cycle-related p21, cyclin D1,
and pRb proteins, all of which showed changes after treatment
with MHY407 alone (Fig. 1E). Cyclin D1 and p21 were reduced in
cells co-treated with MHY407 and doxorubicin in both MCF7 and
Hs578T cells (Fig. 3D and Supplementary Fig. 4C). This suggested
that cyclin D1 and p21 changes contribute to sensitization by
MHY407 in doxorubicin-treated cells.
As MHY407 and doxorubicin seem to affect cell cycles differ-
ently and have different DNA damaging pathways, their combina-
tion may have a greater cancer cell sensitization effect than either
drug alone. Therefore, we investigated whether co-treatment
with MHY407 sensitizes cancer cells to doxorubicin-treatment.
Microscopic examination of MCF7 cells revealed that untreated
cells grew and divided in a side-by-side fashion, whereas cells
Fig. 3. MHY407 sensitizes doxorubicin-treated cancer cells. (A) MCF7 cells were grown in wells of a 6-well plate and treated with 3.5
3.5 M doxorubicin with 10
M MHY407 (DOXþMHY407), or DMSO (Control, Con). After 24 h, cells were observed using an inverted microscope with a 10X objective
lens. (B) MCF7 cells were plated in wells of 96-well plates and grown to 30–40% confluence. The cells were then stimulated for 48 h with 3.5 M doxorubicin, 5
MHY407, 3.5 M doxorubicin with 5
M MHY407 (DOXþMHY407), or DMSO (Control, Con). A cell proliferation assay was performed as described in Materials and
methods. The data represent the mean7S.D. of at least three independent experiments; *P o0.05 compared to the corresponding control. Statistical analysis was
conducted using Student’s t-test and a one-way analysis of variance (ANOVA) followed by a multiple-comparison test. (C and D) MCF7 cells were treated with 3.5
doxorubicin, 10 M MHY407, 3.5 M doxorubicin with 10
M MHY407 (DOXþMHY407), or DMSO (Control, Con) for 6 and 20 h, respectively. The cell extracts were used
for Western blot analyses using antibodies against C-PARP, p21, cyclin D1, pRb, and GAPDH.
mM doxorubicin, 10 mM MHY407,
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S. Yoon et al. / European Journal of Pharmacology 697 (2012) 24–31
29
3.4. MHY407 increases DNA damage in doxorubicin-treated cells
after exposure to doxorubicin and MHY407 (Fig. 4D). In addition,
C-PARP, pChk2, and pH2AX levels were all elevated at 20 h
(Fig. 3C vs. Fig. 4C and D), indicating that apoptosis and DNA
damage were positively correlated. The results were consistent
with the idea that MHY407 increases the rate of apoptosis and
DNA damage, and that the DNA damage continues for a relatively
late time.
To determine whether MHY407 prevents the efflux of doxor-
ubicin, thereby increasing cellular damage, we tested whether
MHY407 could inhibit P-gp. MHY407 was compared to verapamil,
a
well-known P-gp inhibitor (Kim et al., 2007). Verapamil
increased the inhibition of CFDA efflux (Supplementary Fig. 3B),
which is a well-known substrate of P-gp (Kim et al., 2007; Kweon
et al., 2010). However, MHY407 did not result in increased CFDA
staining (Supplementary Fig. 3B). This implied that MHY407
utilizes other mechanism to sensitize doxorubicin-treated cells,
independent of P-gp inhibition.
As both MHY407 and doxorubicin have DNA damaging ability
(Fig. 2A–C), we thought that their combination might have
additive effects in increasing DNA damage. Therefore, we tested
the ability of MHY407 to increase both DNA breakage and damage
in doxorubicin-treated cells. Using an alkaline Comet assay
(Devlin et al., 2008), the contribution of MHY407 to increased
DNA breakage was measured (Fig. 4A and B). Increased DNA
breakage was evident in cells co-treated with MHY407 than in
cells treated with doxorubicin alone (Fig. 4A and B).
3.5. MHY407 also reduces viability of etoposide- and radiation-
treated cells via increasing DNA damage
Etoposide is an anti-cancer drug that, similar to doxorubicin,
induces DNA breakage (Biganzoli et al., 2004; Dillman et al., 2005;
Llovet, 2005). We tested whether MHY407 reduced the viability
of etoposide-treated cells. Viability decreased in co-treated MCF7
cancer cells compared to corresponding cells treated with
either etoposide or MHY407 alone (Fig. 5A). Furthermore, cells
co-treated with etoposide and MHY407 had higher levels of DNA
damage response proteins pChk1, pChk2, pH2AX than cells
treated with etoposide alone (Fig. 5B). Because pChk1 was the
highest elevated in cells co-treated with doxorubicin and
MHY407, sensitization by MHY407 seems to have different path-
ways for doxorubicin and etoposide.
We assessed the influence of co-treatment with doxorubicin
and MHY407 on the activation status of DNA damage-related
proteins such as pH2AX, pChk2, and pBRCA1 (Devlin et al., 2008).
We confirmed that MHY407 increased pH2AX, pChk2, and
pBRCA1 proteins levels after 6 h co-treatment with doxorubicin
(Fig. 4C). In addition, pH2AX and pChk2 levels were still elevated
Immunocytochemistry demonstrated that MHY407 increased
the number of pH2AX foci (Fig. 5C), a marker for DNA damage
(Kawabe, 2004; Solier et al., 2009). Moreover, we found that
MHY407 increased p53BP1 foci staining in the etoposide-treated
Fig. 4. MHY407 increases DNA damage in doxorubicin-treated cells. (A) Relative tail moment in a Comet assay in MCF7 cells for 4 h with 3.5
10 M MHY407 (white bar with dots), 3.5 M doxorubicin with 10
mM doxorubicin (grey bar),
mM MHY407 (DOXþMHY407: white bar), or with DMSO (black bar: Control, Con). The Comet assay was
m
m
performed as described in the section ‘‘Materials and methods’’. The data represent the mean7S.D. of at least three independent experiments; *P o0.05 compared to the
corresponding control. Statistical analysis was conducted using Student’s t-test and a one-way analysis of variance (ANOVA) followed by a multiple-comparison test. (B) A
representative Comet assay result. (C and D) MCF7 cells were grown on 60-mm-diameter dishes and then incubated with 3.5
doxorubicin with 5
M MHY407 (DOXþMHY407), or DMSO (Control, Con). After 6 or 20 h, Western blot analysis was performed using antibodies against pH2AX, pChk2,
pBRCA1, and GAPDH.
mM doxorubicin, 5 mM MHY407, 3.5 mM
m

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S. Yoon et al. / European Journal of Pharmacology 697 (2012) 24–31
Fig. 5. MHY407 reduces viability of etoposide- and radiation-treated cells via increased DNA damage. (A) MCF7 cells were plated in wells of 96-well plates and grown to
30–40% confluence. The cells were then stimulated for 48 h with 25 M etoposide, 5 M MHY407, 25 M etoposide with 5
m
m
m
mM MHY407 (ETOþMHY407), or DMSO
(Control, Con). A cell proliferation assay was performed as described in the section ‘‘Materials and methods’’. The data represent the mean7S.D. of at least three
independent experiments; *Po0.05 compared to the corresponding control. Statistical analysis was conducted using Student’s t-test and a one-way analysis of variance
(ANOVA) followed by a multiple-comparison test. (B) MCF7 cells were grown on 60-mm-diameter dishes and then incubated with 3.5
m
M doxorubicin, 50
M MHY407 (ETOþMHY407), or DMSO (Control, Con). After 6 or 20 h,
Western blot analysis was performed using antibodies against pChk1, pChk2, pH2AX, and GAPDH. (C) MCF7 cells were grown to 50% confluence and stimulated for 3 h
with 25 M etoposide, 10 M MHY407, 25 M etoposide with 10
M MHY407 (ETOþMHY407). DMSO-treated cells served as control (Con). Immunocytochemistry was
performed as described in the section ‘‘Materials and methods’’. Immunocytochemical staining for pH2AX is indicated by red fluorescence. Green staining is p53BP1. DAPI
nuclear staining (blue) is also shown. (D) MCF7 cells were grown on 60-mm-diameter dishes and then treated with 4 Gy radiation, 10 M MHY407, 4 Gy radiation with
10
M MHY407 (4 GyþMHY407), or DMSO (Control, Con). After 1 h, Western blot analysis was performed using antibodies against pH2AX, p21, cyclin D1, and GAPDH.
(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
mM etoposide,
10 mM MHY407, 3.5 mM doxorubicin with 5 mM MHY407 (DOXþMHY407), 50 mM etoposide with 5 m
m
m
m
m
m
m
MCF7 cells, confirming that the sensitization by MHY407 in
etoposide-treated cells resulted from the cytotoxic effects of
increased DNA damage. We only found that pH2AX foci formation
increased after co-treatment with etoposide and MHY407 in
Hs578T cells, but not in MCF7 (Fig. 5C vs. Supplementary
Fig. 5), suggesting that MHY407 can increase pH2AX DNA foci
with cell-line specificity. As seen in Fig. 5D, MHY407 can also
increase pH2AX and reduce cyclin D1 levels for radiation-treated
cells. The results suggest that MHY407 can be used in combina-
tion therapy with any kind of DNA damaging agent. We demon-
strated that MHY407 can sensitize cancer cells to doxorubicin,
etoposide, and radiation-treatment cells, resulting in increased
DNA damage.
present study were to identify and improve novel carbazole deriva-
tives that increase sensitivity of cancer cells, as the DNA damaging
properties of substances have been used for chemotherapy drugs.
In addition, we hypothesized that the identified novel carbazole
derivatives might sensitize cancer cells by enhancing DNA damage
in combination with DNA damaging anti-cancer agents such as
doxorubicin, etoposide, or radiation.
We modified the carbazole module and identified a derivative,
MHY407, with a relatively high DNA damaging ability. MHY407
caused more cell cycle arrest, C-PARP production, and DNA
damage than other derivatives. We explored the mechanism of
action in more detail. MHY407 can inhibit topoisomerase II
catalytic activity (data not shown), suggesting that its mechanism
involves DNA damage. Doxorubicin, etoposide, and MHY407 have
the ability to inhibit a common target, topoisomerase II. But, the
cell cycle in MHY407-treated cells was mainly arrested in the S
phase. It suggests that MHY407 might have different unknown
targets or molecules for sensitization than the well-known
topoisomerase inhibitors, doxorubicin and etoposide. Further
study is needed to describe how MHY407 makes DNA breaks.
Based on a more detailed analysis of molecular levels, we
determined that MHY407 targets proteins (cyclin D1, pRb, and
p21), confirming that MHY407 increases S phase arrest by
4. Discussion
Carbazole has been shown to be a potential anti-cancer drug that
causes DNA damage (Asche and Demeunynck, 2007; Nagarapu et al.,
2010; Patra et al., 2011). In general, the carbazole derivatives are
found in many natural compounds of biological interest such as
radical scavenging, cytotoxicity, and anti-microbial activities (Bandgar
et al., 2012; Hsu et al., 2005; Nagappan et al., 2011). The aims of the

S. Yoon et al. / European Journal of Pharmacology 697 (2012) 24–31
31
regulating cyclin D1, pRb, and p21. Our results demonstrate that
MHY407 represents a novel type of DNA damaging agent that has
multiple effects on cancer cells and has the potential for future
anticancer drug developments.
Gluck, S., 2005. Adjuvant chemotherapy for early breast cancer: optimal use of
epirubicin. Oncologist 10, 780–791.
Hsiao, C.J., Li, T.K., Chan, Y.L., Hsin, L.W., Liao, C.H., Lee, C.H., Lyu, P.C., Guh, J.H.,
2008. WRC-213, an l-methionine-conjugated mitoxantrone derivative, dis-
plays anticancer activity with reduced cardiotoxicity and drug resistance:
identification of topoisomerase II inhibition and apoptotic machinery in
prostate cancers. Biochem. Pharmacol. 75, 847–856.
Since the common DNA damaging drugs, doxorubicin and etopo-
side caused S and G2/M phases arrest (Kim et al., 2011a, 2011b, 2009,
2010; Patra et al., 2011), we hypothesized that the different actions of
MHY407 have benefits, if used as combination with doxorubicin,
etoposide, or radiation. We then demonstrated that MHY407 sensi-
tizes cancer cells to doxorubicin-, etoposide-, and radiation-
treatment. It may be possible that MHY407 can be used in
combination-therapy to sensitize resistant cancer cells for S phase
arrest drugs, including anti-mitotic drugs. We also conclude that
MHY407 can sensitize cells to any form of DNA damaging agents
when used in combination therapy with doxorubicin-, etoposide-,
and radiation. We also found consistent results for MHY407 in both
MCF7 and Hs578T breast cancer cells. Therefore, we assume that the
findings of MHY407 can be generally applied to breast cancer cells.
We are currently preparing a series of experiments to demonstrate
MHY407 effects in an in vivo tumor xenograft mouse model. The
planned study should also examine how adverse effects of MHY407
to normal organs and cells.
In conclusion, we synthesized carbazole derivative, and then
their anti-cancer activities against cancer cells were evaluated.
Among them, MHY407 showed an obvious growth inhibitory
activity against breast cancer cells by modulating DNA damage
and cell cycle arrest. We also suggest that MHY407 may be used
to improve various combination-chemotherapeutic treatments
such as doxorubicin, etoposide, or radiation, and is therefore
considered to be a promising candidate drug of tumors.
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Acknowledgements
Leary, A.F., Sirohi, B., Johnston, S.R., 2007. Clinical trials update: endocrine and
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This work was supported by grants the National Research
Foundation of Korea (NRF) funded by the Korea Government
(no. 2011-0030652 and KRF-2011-0026174).
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Appendix A. Supplementary materials
Maddika, S., Ande, S.R., Panigrahi, S., Paranjothy, T., Weglarczyk, K., Zuse, A.,
Eshraghi, M., Manda, K.D., Wiechec, E., Los, M., 2007. Cell survival, cell death
and cell cycle pathways are interconnected: implications for cancer therapy.
Drug Resist. Update 10, 13–29.
Supplementary data associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.ejphar.2012.10.001.
Nagappan, T., Ramasamy, P., Wahid, M.E., Segaran, T.C., Vairappan, C.S., 2011.
Biological activity of carbazole alkaloids and essential oil of Murraya koenigii
against antibiotic resistant microbes and cancer cell lines. Molecules 16,
9651–9664.
Nagarapu, L., Gaikwad, H.K., Sarikonda, K., Mateti, J., Bantu, R., Raghu, P.S., Manda,
K.M., Kalvendi, S.V., 2010. Synthesis and cytotoxicity evaluation of 1-[3-(9H-
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