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Biological Evaluation of 2,3-Dichloro-5,8-Dimethoxy-1,4-Naphthoquinone

as an Anti-breast Cancer Agent

Article  in  Anticancer research · February 2009

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ANTICANCER RESEARCH 29: 191-200 (2009)
Biological Evaluation of 2,3-Dichloro-5,8-Dimethoxy-1,4-
Naphthoquinone as an Anti-breast Cancer Agent
YASMINE M. KANAAN3, JHARNA R. DAS1, OLADAPO BAKARE2, NKECHI M. ENWEREM2,
SOLOMON BERHE2, DESTA BEYENE3, VONITA WILLIAMS3, YANFEI ZHOU4 and ROBERT L. COPELAND Jr.1
Departments of 1Pharmacology, 2Chemistry and 3Microbiology, College of Medicine and Cancer Center, and
4Oral Diagnosis, College of Dentistry, Howard University, Washington, D.C. 20059, U.S.A.
Abstract. Background: Breast cancer is the most frequent
cancer and the second leading cause of cancer deaths in
women today. A number of 1,4-naphthoquinone derivatives
have been found to possess significant pharmacological
effects associated with marked antimicrobial and antitumor
activities. In the present study, the in vitro effect of 2,3-
dichloro-5,8-dimethoxy-1,4-naphthoquinone (DCDMNQ)
was evaluated on estrogen-positive MCF-7 and estrogen-
negative MDA-MB-436 and Hs-578T human breast cancer
cell lines. Moreover, the in vitro activity of this compound on
cell cycle regulation and apoptosis were evaluated. Materials
and Methods: Established methods of cell viability, cell cycle,
Western blot and apoptosis were used. Results: The effect of
DCDMNQ on MCF-7, MDA-MB-436 and Hs-578T cells
revealed significant antitumor activities with IC50s, of
0.6±0.02, 1.4±0.25 and 3.1±0.4 μM respectively. Cell cycle
analysis showed that DCDMNQ inhibited progression
through the cell cycle in MCF-7 and MDA-MB-436 cell lines
in a time-dependent manner. DCDMNQ arrested cells in the
S-phase of the cell cycle with the greatest proportion of cells
in the S-phase by day 5. This cell-cycle arrest was
corroborated by inhibition of topoisomerase I induced by
DCDMNQ. These findings were further validated using
Western blot analysis of retinoblastoma protein time-
dependent phosphorylation. Furthermore, DCDMNQ induced
apoptosis in both estrogen-positive and -negative cell lines
in a time-dependent manner. However, the highest
Abbreviations: 2,3-Dichloro-5,8-dimethoxy-1,4-naphthoquinone
(DCDMNQ), mitogen-activated protein kinase (MAPK), epidermal
growth factor receptor (EGFR).
Correspondence to: Dr. Robert L. Copeland Jr, Department of
Pharmacology, College of Medicine, Howard University, Adams
Bldg., 520 W. Street NW, Washington, D.C. 20059, U.S.A. Tel: +1
2028066311, Fax: +1 2028064453, e-mail: rlcopeland@howard.edu
Key Words: 2,3-Dichloro-5,8-dimethoxy-1,4-naphthoquinone, estrogen
positive/negative breast cancer cell lines, cytotoxicity, cell cycle,
apoptosis.
0250-7005/2009 $2.00+.40
percentages of apoptotic cells were observed in the MDA-
MB-436 cell line. Conclusion: Although the mechanism of
action of DCDMNQ has not been completely elucidated, it
appears that this compound can inhibit topoisomerase I in a
concentration-dependent manner. These promising results to
explore novel naphthoquinone analogues as potential breast
cancer agents. This study suggests that DCDMNQ may have
an impact on treatment of estrogen-positive and -negative
breast cancer while protecting the bone marrow.
Breast cancer is a leading cause of morbidity and mortality
in women, not only in developed countries but increasingly
also in developing countries (1). It is estimated that in 2008
there would be approximately 182,460 women diagnosed
with the disease and 40,480 deaths from breast cancer (2).
In the new millennium, the expected number of annual new
cases of breast cancer worldwide could exceed 1.5 million
(3). These statistics emphasize the urgent need for
improvements in detection, diagnosis and treatment of breast
cancer. Several studies have shown that the mortality rate
with breast cancer is about three times higher in African-
American women than in other populations (4). In addition,
the available data also indicate that the tumors are very
aggressive and poorly differentiated, with a very low
frequency of hormone receptors, higher S-phase fraction and
tumor necrosis (5).
Breast cancer is highly curable if diagnosed at early stage.
It is now well established that adjuvant systemic therapy
improves survival in patients with early-stage breast cancer
(5). Current treatment methods of breast cancer, depending
on the stage of cancer upon diagnosis, include surgery,
radiation therapy, biological therapy, hormone therapy (e.g.
tamoxifen, aromatase inhibitor) and chemotherapy (e.g.
anthracyclines, taxanes). Recent progress in diagnosis and
therapy has increased the survival of women with estrogen-
dependent breast cancer. However, the treatment options
available for estrogen-independent tumors are far from
satisfactory and consequently carry a poorer prognosis.
The intricate details of breast cell growth control have not
been completely elucidated. Estrogens are well recognized
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 ANTICANCER RESEARCH 29: 191-200 (2009)
as playing the predominant role in breast cancer development
and growth and much effort has been devoted to block
estrogen formation and action (6). The most widely used
therapy for breast cancer is the use of antiestrogen such as
tamoxifen. However, the present breast cancer therapies
achieve meaningful clinical results in only 30-40% of
patients (7) because drug resistance is linked to the presence
of estrogen-independent pathways for breast cancer cell
growth (8). Therefore, more potent anti-breast cancer agents
that combine the desired, tissue-selective effects with novel
structures or new mechanism(s) of action must be developed.
Naphthoquinones are widely distributed in nature and play
important physiological roles in animals and plants. Quinone
derivatives may be toxic to cells by a number of mechanisms
including redox cycling, arylation, intercalation, induction of
DNA strands breaks, generation of free radicals and alkylation
via quinone methide formation (9). As a consequence, the
molecular framework of a great number of pharmaceuticals
and biologically important compounds contain a quinone
moiety. Representative examples of this class of compounds
are the well-known anticancer drugs of the anthracycline
series, doxorubicin and mitoxanthrone, the action of which is
believed to occur via topoisomerase II inhibition (10, 11). In
addition, a number of naphthoquinone analogs such as
plumbagin, shikonin and naphthazarin, as well as β-lapachone,
have also been found to inhibit topoisomerase (12-14). DNA
topoisomerases are a class of enzymes that alter DNA
conformation through a concerted breaking and rejoining of
DNA molecule, thereby controlling the topological state of
DNA. There are two major categories of topoisomerases,
topoisomerase I (Topo I) and topoisomerase II (Topo II). They
are reported to be involved in many important processes of
DNA metabolism including replication, transcription,
recombination and chromosome segregation (15). They have
been identified as important antitumor targets because of their
essential physiological functions.
A number of 1,4-naphthoquinone derivatives have been
found to possess powerful pharmacological effects associated
with marked antimicrobial and antitumor activities (16, 17).
Bakare et al. (18) reported the MEK-1 specific inhibitory
activity of 2-chloro-3-(N-succinimidyl)-1,4-naphthoquinones.
In our earlier paper, we reported the synthesis of 2,3-
dichloro-5,8-dimethoxy-1,4-naphthoquinone (DCDMNQ)
which showed significant cytotoxicity against prostate cancer
cell lines and at the same time protected bone marrow cells
(19). Therefore, the present study was undertaken to
determine the cytotoxic and cell cycle regulatory effects of
DCDMNQ on human breast cancer cell lines.
Materials and Methods
Synthesis of DCDMNQ. This compound was synthesized as
described elsewhere (19).
192
Reagents. The BCA Protein assay kit, PVDF membrane and Super
Signal West Dura were purchased from Pierce, Rockford IL, U.S.A.
Propidium iodide (PI), annexin V and primary (Rb) antibody were
from BD Biosciences, CA, U.S.A. MTT, trypan blue dye and all other
reagents were purchased from Sigma Chemical Company, St. Louis,
MO, U.S.A. An early passage of human breast cancer cells were
obtained from the American Type Culture Collection (ATCC),
Manassas, VA, U.S.A.
Cell culture. Stock cultures of the human breast cancer (estrogen-
positive MCF-7 and estrogen-negative MDA-MB-436 and
Hs-578T) cell lines were grown in 75 cm3 flasks and incubated in
RPMI-1640 medium (Cellgro, Mediatech Inc. VA, USA) with 10%
fetal bovine serum incubated in 5% CO2/95% air at 37˚C. Upon
reaching confluence, the cells were trypsinized, stained with 0.2%
trypan blue dye and counted in a hemaocytometer.
Cell viability. Cell viability was quantitatively determined using a
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)
colorimetric assay as described elsewhere (19). Briefly, estrogen-
positive MCF-7 and estrogen-negative MDA-MB-436 and Hs-578T
(1.5×104 cells) were grown in 96-multi-well plates in RPMI-1640
media in the presence or absence of DCDMNQ in a 3-log
concentration range (20 nM to 20 μM). The formazan dye produced
by viable cells was quantified by measuring the absorbance of the dye
solution at 440 nm using a microtiter plate reader.
Cell cycle analysis. Cell cycle perturbations induced by DCDMNQ
were analyzed by propidium iodide (PI) DNA staining as described
elsewhere (19). The cells were grown in 6-well plates (1×105 cells
per well) in the presence or absence of the DCDMNQ. After 48 h,
of exposure cells were collected and evaluated after propidium
iodide staining and cell cycle profiles were obtained using a BD
FACS Calibar Cell flow Cytometer (Becton Dickinson San Jose,
CA, USA). Data were analyzed by ModFit LT software (Verity
Software House, Inc., Topsham, ME, USA).
Western blot analysis. The retinoblastoma protein (Rb) is an
important regulator of cell cycle when phosphorylated cells progress
from the G1- to the S-phase. To quantify the total Rb protein in
breast cancer cells, Western blot analysis was performed.
Approximately 1×106 cells were plated in 60-mm Petri-dishes and
allowed to attach overnight and then cells were exposed to IC50s
(the concentration which kills 50% of cells calculated from cell
viability experiment) of DCDMNQ for 3 or 5 days of exposure; the
control was without any treatment. Cells were collected by scraping
and homogenized using lysis buffer containing protease inhibitor
(Roche, IN, USA). The cell lysates were centrifuged at 5000× g for
10 min at 4˚C. The supernatant was collected and protein
concentration was measured using BCA Protein assay with bovine
serum albumin as standard. Equal amounts of protein (20 μg) were
loaded onto a 4-12% SDS-PAGE and then transferred to a
polyvinylidenedifluoride (PVDF) membrane and cut according to
the size of total Rb protein and actin. The membranes were probed
with primary antibody (mouse anti-human retinoblastoma protein
Rb monoclonal antibody, 1:1,000) and β-actin (mouse monoclonal
antibody, 1:5,000) to correct for unequal loading and followed by
horseradish peroxidase-conjugated secondary antibody (goat anti-
mouse IgG, 1:10,000). Antibody detection and densitometric
analysis were performed as described elsewhere (19).
 Kanaan et al: Cytotoxic Effect of DCDMNQ on Human Breast Cancer Cell Lines
Figure 1. The effect of 2,3-dichloro-5,8-dimethoxy-1,4-naphthoquinone
(DCDMNQ) on cell viability of human breast cancer (estrogen-positive
MCF-7 and estrogen-negative MDA-MB-436, Hs-578T) cell lines.
Determination of apoptotic cells. To quantify drug-induced
apoptosis, annexin V/propidium iodide staining was performed using
flow cytometry. Briefly, after DCDMNQ treatment, both floating and
attached cells were combined and subjected to annexin V/propidium
iodide staining annexin V-FITC apoptosis detection kit according to
the protocol provided by the manufacturer. Untreated control cells
were maintained in parallel to the DCDMNQ treatment group. The
cells were exposed to IC80s (the concentration which kills 80% of
cells calculated from cell viability experiment) of DCDMNQ for 3 or
5 days. Double staining was used to distinguish between viable, early
apoptotic and necrotic or late apoptotic cells. The resulting
fluorescence was measured using flow cytometry using a BD FACS
Caliber Cell Flow Cytometer (Becton Dickinson). Data were
analyzed by ModFit LT software (Verity Software House, Inc.).
Topoisomerase I assay. The inhibition of topoisomerase I was
determined by Topoisomerase I assay kit (TopoGEN, Columbus, OH,
USA) according to the manufacturer’s instructions. Supercoiled pRYG
DNA (0.5 μg) was incubated with 4 units of human topoisomerase I in
cleavage buffer (30 mM Tris-HCl pH 7.8, 50 mM KCl, 10 mM MgCl2,
3 mM ATP, 15 mM mercaptoethanol), in the presence of different
concentrations of DCDMNQ; absence of drug served as the control. The
reactions were carried out at 37˚C for 1 h and then terminated by the
addition of 0.25% sodium dodecyl sulphate (SDS) and proteinase K
(50 μg/ml). The reaction mixture was subjected to electrophoresis in a
1% agarose gel containing 0.5 mg/ml ethidium bromide in TBE buffer
(90 mM Tris-borate and 2 mM EDTA). The gels were stained with
ethidium bromide and photographed under UV light.
Statistical analyses. Data were expressed as mean±standard error.
Statistical differences within and between treatments groups were
determined by one-way ANOVA followed by Dunnett’s comparison
test to determine statistical differences between control and
treatment groups. P<0.05 was considered statistically significant.
Data were analyzed using Graphpad InStat (Graphpad Software Inc,
San Diego, CA, USA).
Results
Effect of DCDMNQ on the cell viability, cell cycle
progression, apoptosis and topoisomerase assay of human
breast cancer cell lines.
Cell viability. 2,3-Dichloro-5,8-dimethoxy-1,4-naphthoquinone
inhibited the growth of the estrogen-positive MCF-7 and
estrogen-negative MDA-MB-436 and Hs-578T human breast
cancer cell lines in a dose-dependent manner following
treatment for 5 days. These in vitro studies of DCDMNQ on
human breast cancer cell lines revealed significant antitumor
activities with IC50s for MCF-7, MDA-MB-436 and Hs-578T
cells of 0.6±0.02, 1.4±0.25 and 3.1±0.4 μM respectively
(Figure 1).
Cell flow cytometric analysis. Cell flow cytometry was used
to determine the effect of the DCDMNQ compound on the
progression of the cell cycle. The cell cycle profile in Figure
2A is representative of three independent experiments
inclusive of the three treatment groups in all the cell lines.
Figure 2B shows the percentage of cells in the S-phase at
different time points under each treatment. Analysis revealed
that the highest number of cells progressing to the S-phase of
the cell cycle was found in MCF-7 cells (55% ±7.2% )
followed by MDA-MB-436 cells (44% ±5.6% ) and Hs-578T
(38% ±5.4% ) cells. The proportions of S-phase cells in
treated groups were significantly higher than the
corresponding control values.
Western blot analysis. The retinoblastoma protein (Rb), a cell
cycle regulator which when phosphorylated allows the
progression of cells from the G1- to the S-phase, was used as
a marker to determine the effects of DCDMNQ on cellular
progression at the molecular level. The Western blot shown in
Figure 3A is representative of five independent experiments
of the two treatment groups. The relative optical density
(ROD) is shown in Figure 3B. The highest density was
observed in MCF-7 cells, followed by MDA-Mb-436 and Hs-
578T, indicating higher phosphorylation. Significant
phosphorylation was observed in all the cell lines when
compared to the control results. Similar results were observed
with the cell flow cytometric analysis.
Apoptosis. The cells with lower DNA content, as shown by PI
staining less than G1, were defined as apoptotic (sub G0/G1
population). The maximum sub-G0/G1 population was observed
in cells of MDA-MB-436 (70% ±8.3% ) followed by MCF-7
(42% ±5.2% ) and the lowest number of apoptotic cells were
observed in Hs-578T (17% ±3.4% ) as shown in Figure 4. The
percentage of apoptotic cells increased in a time-dependent
manner when compared with their respective controls.
To quantify drug-induced apoptosis, annexin V/propidium
iodide staining was performed using flow cytometry. The
193
 ANTICANCER RESEARCH 29: 191-200 (2009)

Figure 2. Effect of 2,3-dichloro-5,8-dimethoxy-1,4-naphthoquinone (DCDMNQ) on the cell cycle progression of human breast cancer cell lines.
Cells were exposed for 3 and 5 days in presence and absence of drug at their respective IC50s. Control cells shown were measured at 3 days and as
expected no significant changes were observed in the control cells at 5 days. A, The cell cycle profile is representative of three independent
experiments. B, Percentage of cells in S-phase. Results represent mean ± SEM of three independent experiments. Analysis of variance indicated a
significant increase in DCDMNQ-treated cells compared with the control (**p<0.01, ***p<0.001).

194
 Kanaan et al: Cytotoxic Effect of DCDMNQ on Human Breast Cancer Cell Lines

Figure 3. Effect of 2,3-dichloro-5,8-dimethoxy-1,4-naphthoquinone (DCDMNQ) on the level of retinoblastoma protein (Rb) and β-actin in human breast
cancer cells. Cells were exposed for 3 and 5 days. A, The gel image is representative of five independent experiments. B, Relative optical density of Rb
protein. Analysis of variance indicated a significant increase in DCDMNQ-treated cells compared with the control (*p<0.05, **p<0.01, ***p<0.001).

195
 ANTICANCER RESEARCH 29: 191-200 (2009)

Figure 4. Percentage of apoptosis calculated by measuring the sub-G0/G1 population using cell flow cytometry in all the cell lines mentioned. The
cells with lower DNA content showing less PI staining than G1 were defined as apoptotic. A, The cell cycle profile with M1 gate is representative of
three independent experiments. B, Percentage of apoptotic cells in the M1 (sub-G0/G1 population). Analysis of variance indicated a significant
increase in DCDMNQ-treated cells compared with the control (*p<0.05, **p<0.01, ***p<0.001).

196
 Kanaan et al: Cytotoxic Effect of DCDMNQ on Human Breast Cancer Cell Lines

Figure 5. Apoptosis analysis of human breast cancer cell lines treated at their respective IC80s. Cells were exposed for 3 and 5 days. Control cells
shown were measured at 3 days and as expected no significant changes were observed in the control cells at 5 days. Double staining was used to
distinguish between viable, early apoptotic, necrotic and late apoptotic cells. The lower left quadrant shows the viable cells, the upper left quadrant
shows cell debris, the lower right quadrant shows the early apoptotic cells and the upper right quadrant shows the late apoptotic and necrotic cells.

Figure 6. Effect of 2,3-dichloro-5,8-dimethoxy-1,4-naphthoquinone (DCDMNQ) on the topoisomerase I activity. A, The gel image is representative
of three independent experiments. B, Relative optical density of supercoiled DNA and linear DNA. Analysis of variance indicated a significant
decrease in the supercoiled DNA ( +p<0.05) and an increase (*p<0.05) in the linear DNA of DCDMNQ-treated cells as compared with control.

combination of early and late apoptotic cells was significantly Topoisomerase I assay. To determine DCDMNQ as an inhibitor
higher in MDA-MB-436 cells following DCDMNQ treatment of topoisomerase I, we tested its effect on the catalytic activity
for 5 days as shown in Figure 5. The lowest number of of topoisomerase I. Figure 6A is representative of three
apoptotic cells was observed in Hs-578T cells. independent experiments and Figure 6B shows the relative

197
 ANTICANCER RESEARCH 29: 191-200 (2009)
optical density of supercoiled DNA and linear DNA. Results
indicated that DCDMNQ inhibits topoisomerase I in a
concentration-dependent manner. The significant increase in
linear DNA and reduction of supercoiled DNA was observed
with 50 and 100 μM of DCDMNQ.
Discussion
The cytotoxicity results revealed that DCDMNQ is a potent
inhibitor of the growth of both estrogen-positive and -
negative human breast cancer cell lines. The results of cell
flow cytometry and Western blot showed significantly higher
numbers of cells entering the S-phase of the cell cycle in
MDA-MB-436 cells followed by MCF-7 then Hs-578T,
increasing in a time-dependent manner. Therefore, DCDMNQ
arrested cells in the S-phase of the cell cycle in both estrogen-
positive and -negative human breast cancer cells.
It is known that apoptosis-signaling pathways and cellular
events controlling them have a profound effect both on cancer
progression and on response to chemotherapy (20, 21). Based
on annexin V/propidium iodide staining, it is clear that the
highest proportion of apoptotic cell death was observed in
MDA-MB-436 cells followed by MCF-7 cells. DCDMNQ
significantly inhibited the growth of breast cancer cells in
vitro, possibly by either up-regulation of apoptotic Bax and
down-regulation of anti-apoptotic Bcl-XL or by increasing the
p21waf1/cip1 protein, which is involved in cell cycle arrest in
G1 or G2 (22). It has also been reported that bis-type
aziridinylnaphthoquinone induced cell death in human breast
adenocarcinoma BC-M1 cell line by mediating the apoptotic
pathway. Yang et al. (23) reported that apoptosis induced by
aziridinylnaphthoquinone was initially triggered by the
activation of p53 protein followed by up-regulation of the p21
protein to inhibit cyclin kinase cdk2 expression to arrest the
BC-M1 cell cycle, leading finally to progression into the
apoptotic process. Hou et al. (24) reported that the cytotoxic
effect of shikonin (naphthoquinone) on MCF-7 cells occurred
by an apoptotic process. The cell cycle alterations indicate
that cell cycle arrest is one of the primary mechanisms
responsible for the antiproliferative action of DCDMNQ in
breast tumor cells in vitro.
Topoisomerase I catalyzes changes in the linkage of DNA
strands via the formation of covalent enzyme-DNA
intermediates. A phosphotyrosyl linkage of Topo I to the 3’ -
end of the cleaved DNA strand allows the free 5’ DNA end to
rotate. The covalent Topo I-DNA intermediate is the cellular
target of Topo I cytotoxins which reversibly stabilize the
covalent Topo I-DNA intermediate by inhibiting DNA
religation. During S-phase, these ternary Topo I-DNA-drug
intermediates are transformed into potentially lethal lesions,
which induce cell cycle arrest and cell death (25). The cell-
cycle arrest induced by DCDMNQ in the S-phase is
corroborated by the concentration-dependent inhibition of
198
Topo I. It thus appears that DCDMNQ inhibits Topo I activity
by stabilizing the cleavable complex.
However, Topo II inhibitors do not exhibit the same S-phase
specificity as Topo I inhibitors (25). Other antineoplastic drugs
dramatically increase levels of Topo II cleaved-DNA complexes,
resulting in permanent DNA damage followed by G2/M-phase
cell cycle arrest and apoptotic cell death (26). Earlier reports
have suggested that 1,2-naphthoquinone thiosemicarbazone and
its metal derivatives inhibit DNA Topo II by stabilizing the
intermediate forms of enzyme-DNA complexes (cleavable
complexes) in MCF-7 human breast cancer cells (27, 28). The
stabilizing effect is mainly due to the alkylation of thiol residues
on the Topo II-DNA complex (28). Protein denaturation of this
cleavable complex results in single-stranded and double-
stranded DNA breaks that lead to apoptosis. It is through this
cellular process that Topo II inhibitors exert their antitumor
activity (29). DCDMNQ appeared to have only weak Topo II
inhibitor activity (data not shown).
Conclusion
2,3-Dichloro-5,8-dimethoxy-1,4-naphthoquinone showed
potent cytotoxicity against both estrogen-positive and
-negative human breast cancer cell lines as confirmed in cell
cycle and apoptosis experiments. Moreover, HS-5 bone
marrow cells were reported to be less sensitive to DCDMNQ
(19). It is well known that late-stage breast cancer
metastasizes to bone.
Therefore, considering the cytotoxicity profile in these
different breast cancer cell lines and the ability to arrest cell
cycle progression and induce apoptosis, this compound
represents the first in a class of specific compounds which
may be useful in the treatment of estrogen-positive and
-negative breast cancer.
Acknowledgements
This work was supported in part by grant number 5-U54-CA914-31
(Howard University/Johns Hopkins Cancer Center Partnership) and
the RCMI/NIH grant G12RR003048. We are grateful to the
Department of Pharmacology, College of Medicine, Howard
University for allowing us to use their core facility to conduct the
research work.
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Received March 18, 2008
Revised August 21, 2008
Accepted October 10, 2008
199