Novel Quinazolinone MJ-29 Triggers Endoplasmic
Reticulum Stress and Intrinsic Apoptosis in Murine
Leukemia WEHI-3 Cells and Inhibits Leukemic Mice
Chi-Cheng Lu1, Jai-Sing Yang2, Jo-Hua Chiang1, Mann-Jen Hour3, Kuei-Li Lin4, Jen-Jyh Lin5,6, Wen-
Wen Huang7, Minoru Tsuzuki8,9, Tsung-Han Lee1,7*., Jing-Gung Chung7,10*.
1Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan, 2Department of Pharmacology, China Medical University, Taichung, Taiwan, 3School
of Pharmacy, China Medical University, Taichung, Taiwan, 4Department of Radiation Oncology, Chi Mei Medical Center, Tainan, Taiwan, 5Graduate Institute of Chinese
Medicine, China Medical University, Taichung, Taiwan, 6Division of Cardiology, China Medical University Hospital, Taichung, Taiwan, 7Department of Biological Science
and Technology, China Medical University, Taichung, Taiwan, 8Department of Biochemistry, Nihon Pharmaceutical University, Saitama, Japan, 9Tsuzuki Institute for
Traditional Medicine, China Medical University, Taichung, Taiwan, 10Department of Biotechnology, Asia University, Taichung, Taiwan
The present study was to explore the biological responses of the newly compound, MJ-29 in murine myelomonocytic
leukemia WEHI-3 cells in vitro and in vivo fates. We focused on the in vitro effects of MJ-29 on ER stress and mitochondria-
dependent apoptotic death in WEHI-3 cells, and to hypothesize that MJ-29 might fully impair the orthotopic leukemic mice.
Our results indicated that a concentration-dependent decrease of cell viability was shown in MJ-29-treated cells. DNA
content was examined utilizing flow cytometry, whereas apoptotic populations were determined using annexin V/PI, DAPI
staining and TUNEL assay. Increasing vital factors of mitochondrial dysfunction by MJ-29 were further investigated. Thus,
MJ-29-provaked apoptosis of WEHI-3 cells is mediated through the intrinsic pathway. Importantly, intracellular Ca2+release
and ER stress-associated signaling also contributed to MJ-29-triggered cell apoptosis. We found that MJ-29 stimulated the
protein levels of calpain 1, CHOP and p-eIF2a pathways in WEHI-3 cells. In in vivo experiments, intraperitoneal
administration of MJ-29 significantly improved the total survival rate, enhanced body weight and attenuated enlarged
spleen and liver tissues in leukemic mice. The infiltration of immature myeloblastic cells into splenic red pulp was reduced in
MJ-29-treated leukemic mice. Moreover, MJ-29 increased the differentiations of T and B cells but decreased that of
macrophages and monocytes. Additionally, MJ-29-stimulated immune responses might be involved in anti-leukemic activity
in vivo. Based on these observations, MJ-29 suppresses WEHI-3 cells in vitro and in vivo, and it is proposed that this potent
and selective agent could be a new chemotherapeutic candidate for anti-leukemia in the future.
Citation: Lu C-C, Yang J-S, Chiang J-H, Hour M-J, Lin K-L, et al. (2012) Novel Quinazolinone MJ-29 Triggers Endoplasmic Reticulum Stress and Intrinsic Apoptosis in
Murine Leukemia WEHI-3 Cells and Inhibits Leukemic Mice. PLoS ONE 7(5): e36831. doi:10.1371/journal.pone.0036831
Editor: Zhengqi Wang, Emory University, United States of America
Received December 12, 2011; Accepted April 7, 2012; Published May 25, 2012
Copyright: ? 2012 Lu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors thank the grant-in-aid (NSC 95-2320-B-039-049-MY2) from the National Science Council, Republic of China (Taiwan) and the grant support
by the Taiwan Department of Health, China Medical University Hospital Cancer Research Center of Excellence (DOH101-TD-C-111-005). The funders had no role in
study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com (JGC); firstname.lastname@example.org (THL)
. These authors contributed equally to this work.
Leukemia, a group of hematologic malignancies disorder in
leukocytes, is characterized by the uncontrolled proliferation and
blocked in differentiation of hematopoietic cells [1,2] and
subdivided into acute and chronic forms . Among most human
leukemias, they exhibit the blockage of differentiation, enhance-
ment of viability and dysregulation of cell cycle control that is
necessary for occurrences of malignant transformation . In the
United States, leukemia is the largest number of cases of childhood
cancer (approximately 2,000 cases per year) . In Taiwan, a
2010 report from the Department of Health, R.O.C. (Taiwan)
indicated that approximately 4.2 per 100,000 individuals die
annually from leukemia . The current clinical trials for
leukemia include the pharmaceutical medications, debilitating
radiation, and a bone marrow transplant therapy but these
strategies have not proven to be satisfied. Hence, new targets for
treating leukemia are necessary and the best functions for agents
are carried out through promoting differentiation or trigging
apoptotic death in leukemia cells [7,8]. Apoptosis, a process of
programmed cell death type I, is a major method of anticancer
properties to eliminate cancer cells . The mitochondrial
depolarization and activations of caspase family proteases are
the central steps when the development of apoptosis , and
their associated signaling pathways include intrinsic (mitochon-
dria-dependent) and ER stress (unfolded protein response) signals
Numerous phytochemicals are known to present in many herbal
based dietary supplements or herbal medicines, which might be
effective in clinical applications and used as cancer suppressors;
these molecules are invaluable contributions of nature [13,14]. It
has been reported that the microtubule-targeting agents (MTAs)
are one of the most effective drugs in leukemia  but they exert
PLoS ONE | www.plosone.org1May 2012 | Volume 7 | Issue 5 | e36831
side effects and high toxicity on normal tissues after treating to
patients [16,17]. However, the current effective chemotherapeutic
agents, such as taxanes and vinca have limitations and are not
satisfying leukemic therapies because of toxic side effects and drug
resistance . Seeking novel agents for chemotherapy-induced
apoptotic death is not only becoming more important and essential
but has received increasing attention in the leukemia patients .
The previous reports have shown that alkaloids with 4-
quinazolinone nuclei possess various biological functions (anti-
inflammatory, anti-bacterial and anti-malarial) and antitumor
effects [20,21]. In our cooperative laboratory, a series of 2-phenyl
6-pyrrolidinyl-4-quinazolinone derivatives have been designed and
synthesized, and which are found to have anti-mitotic functions
and anticancer activities in many types of tumor cell lines,
including colorectal, lung, ovarian, oral, prostate and breast
cancer as well as glioblastoma, osteosarcoma, melanoma and
leukemia [20,22,23]. This novel agent, 6-pyrrolidinyl-2-(2-hydro-
xyphenyl)-4-quinazolinone (MJ-29) exhibits the most potent
cytotoxicity against leukemia cell lines . Our earlier study
also indicated that MJ-29 inhibited tubulin polymerization,
induced mitotic arrest and provoked apoptosis in a human
leukemic monocyte lymphoma cell line (U937), and that
attenuated U937 xenograts tumor growth in vivo . Until
now, the anticancer actions of the newly quinazolinone com-
pound, MJ-29 on murine leukemia cells in vitro and in vivo are not
yet completely understood. The objectives of this study are to
verify the hypothesis that MJ-29 might influence the murine
myelomonocytic leukemia cell line (WEHI-3), as was the
underlying mechanisms by MJ-29 might induce ER stress and
mitochondria-mediated apoptosis, and further evaluate anti-
leukemic activity in orthotopic model of leukemic mice.
MJ-29 induces cytotoxicity and morphological changes
in murine leukemia WEHI-3 cells
Cells were exposed to MJ-29 at the concentrations of 0, 0.5, 1, 5
or 10 mM for a 24-h treatment. The potential cytotoxic effects of
MJ-29 on WEHI-3 cells were investigated for cell viability by a
propidium iodide (PI) exclusion method and using flow cytometric
analysis. Results in Figure 1A showed that MJ-29 decreased the
percentage of viable cells in WEHI-3 cells in a concentration-
dependent response. We also confirmed that MJ-29 concentration-
dependently reduced the cell viability by MTT assay (Figure S1A
and Method S1). Figure 1B indicates that WEHI-3 cells were
morphologically-altered by MJ-29 treatment (such as cell rounding
and shrinkage) and these effects were concentration-dependent.
The half-maximal effective concentration (EC50) value of MJ-29
for 24-h exposure was 1.0360.29 mM after the non-linear dose-
response regression curve was fitted by SigmaPlot 10 (Systat
Software, Inc. San Jose, CA, USA) [24,25]. Therefore, MJ-29 at
the concentration of 1 mM was selected for further experiments in
this study. Importantly, our earlier study has reported that MJ-29
exhibited less toxicity in normal cells, including peripheral blood
mononuclear cells (PBMC) and human umbilical vein endothelial
cells (HUVECs) in comparison to that in the higher sensitive
WEHI-3 cells .
MJ-29 triggers G2/M phase arrest and provokes
apoptosis in WEHI-3 cells
To verify MJ-29-induced cell death through G2/M phase arrest
and apoptotic death, cells were treated with MJ-29 before analyses
with sub-G1 population (apoptosis), Annexin V FITC/PI kit, 49,6-
diamidino-2-phenylindole (DAPI) staining and terminal DNA
transferase-mediated dUTP nick end labeling (TUNEL) assays.
The results revealed that MJ-29 induced G2/M phase arrest from
23.31% to 77.89%, and it increased the sub-G1 group from 2.63%
to 49.7% in WEHI-3 cells (Figure 1C and Figure S1B). Figure 1D
and Figure S1C show that the apoptotic cells (annexin V positive
cells) increased from 2.0% to 39.5% within 24 h between the
control sample and MJ-29-treated cells. Also, these effects are to
undergo a time-dependent association in MJ-29-treated WEHI-3
cells. Moreover, MJ-29 caused chromatin condensation (a
characteristic of apoptosis) in WEHI-3 cells as shown by an
increase in mean fluorescence intensity (MFI) (Figure 1E). As
demonstrated in Figure 1F, MJ-29 exposure for 0, 6, 12 and 24 h
time-dependently stimulated the appearance of TUNEL positive
cells, causing that the DNA fragmentation occurred in WEHI-3
MJ-29 stimulates mitochondrial dysfunction in WEHI-3
To evaluate whether MJ-29 influences crucial factors in
mitochondria and investigate the roles of mitochondria-regulated
death pathways, our results showed that MJ-29 depolarized the
level of mitochondrial membrane potential (DYm) (Figures 2C
and D), promoted the opening of the mitochondrial permeability
transition (MPT) pores (Figures 2E and F) and triggered level of
cardiolipin oxidation (Figure 2G) in WEHI-3 cells. The responses
occurred in a time-course effect. These data indicated that
treatment of WEHI-3 cells by MJ-29 which induced the cell
apoptosis, disrupted the DYm and provoked mitochondrial
depolarization. It is reported that mitochondrial dysfunction
might result from oxidative stress, leading to cardiolipin oxidation
[26,27]. We further investigated that if oxidative stress influences
the upstream of mitochondrial dysfunction, and our findings
demonstrated that MJ-29 increased ROS levels up to 24-h
treatment in WEHI-3 cells as shown in Figures 2A and B.
MJ-29 triggers cell death in WEHI-3 cells through the
intrinsic apoptotic pathway
Our data in Figure 3 indicated that MJ-29-induced apoptosis
was mediated by stimulating caspase-9 (Figures 3A and B) and
caspase-3 (Figures 3C and D) activities in a time-dependent effect.
Figure 3E indicates that MJ-29 up-regulated the protein levels of
Bax, cytochrome c, Endo G, AIF, cleaved caspase-3 (p17) and
cleaved caspase-9 (p35) but it down-regulated that of Bcl-2 and
Bcl-xL (Figure 3C) in WEHI-3 cells, reflecting the apoptotic states
of WEHI-3 cells. Additionally, the trafficking of cytochrome c from
mitochondria to cytosol was stimulated in MJ-29-treated WEHI-3
cells as illustrated in Figure 4A. To confirm if MJ-29-induced
apoptosis is involved in caspase-9 and caspase-3-mediated
mitochondria-dependent signaling, cells were individually pre-
treated with specific caspase-9 (Z-LEHD-FMK) and caspase-3 (Z-
DEVD-FMK) inhibitors before 1 mM of MJ-29 for 24 h. Results
in Figure 3F showed that both specific inhibitors substantially
reduced the effects of viability (cell death) on WEHI-3 cells,
resuljting in more viable cells when compared to the MJ-29-
treated alone sample. The current evidence suggests that the
activations of caspase-9 and caspase-3 might fully contribute to
MJ-29-triggered apoptotic death in WEHI-3 cells. Therefore, MJ-
29-enhanced apoptosis in murine leukemia WEHI-3 cells was
carried out mainly by the activations of caspase-9 and caspase-3-
mediated mitochondrial signaling pathways.
MJ-29 Inhibits Leukemia Cells In Vitro and In Vivo
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Intracellular Ca2+release and unfolded protein response
are associated with the induction of apoptosis in MJ-29-
treated WEHI-3 cells
To elucidate the upstream possible signaling pathways of MJ-
29-induced cell death of WEHI-3 cells, we tested whether
induction contributes to MJ-29-activated
apoptotic signaling. Figures 5A and B display that cells were
incubated with 1 mM of MJ-29 for 3 h to 24 h and it is found that
MJ-29 significantly increased cytosolic Ca2+level in WEHI-3 cells.
Many reports stated that activation of calpain, a member of
calcium-dependent proteases, is implicated with ER stress and
perturbations intercellular Ca2+release in mammalian cells
[28,29]. As a result shown in Figure 5B, the expressions of these
ER stress-related protein levels modulated by intercellular Ca2+
and caspase signals, including calpain 1, calpain 2 and caspase-12
in WEHI-3 cells were time-dependently induced after MJ-29
treatment. However, the protein level of casepase-4 was not
dramatically increased in MJ-29-treated WEHI-3 cells (Figure 5C).
To determine whether MJ-29 could induce ER stress, we
investigated several vital hallmarks of UPR, including C/EBP
homologous protein (CHOP), immunoglobulin heavy chain
binding protein (BiP), glucose-regulated protein 94 (GRP94),
alpha subunit of eukaryotic initiation factor 2 (eIF2a) and PRK
(RNA-dependent protein kinase)-like ER kinase (PERK) proteins
levels. Results in Figure 5D demonstrated that the increased
expressions of CHOP, BiP and GRP94 at 6 to 24-h exposure in
WEHI-3 cells after MJ-29 exposure. Also, treatment with MJ-29
promoted the translocation of CHOP/GADD153 level to nucleus
in WEHI-3 cells (Figure 4B). Additionally, we examined whether
Figure 1. MJ-29 decreases the viability and induces apoptotic death in WEHI-3 cells. Cells were treated with or without 0.5, 1, 5 or 10 mM
of MJ-29 for 24 h and exposed to 1 mM of MJ-29 for indicated durations. (A) Cell viability was determined by a PI exclusion method and analyzed by
flow cytometry (B) before the investigations for the cells’ morphological changes were observed (q reveals the shrinkage and rounding of apoptotic
cells) and photographed under phase-contract microscopy (scale bar, 15 mm). (C) Cells with G2/M phase and hypodiploid DNA contents (%) represent
the fractions undergoing apoptotic DNA degradation. (D) Quantification of annexin V positive cells was measured using Annexin V FITC/PI kit and
examined by flow cytometry. (E) DAPI staining and a fluorescent microscope were used to analyze chromatin condensation (a catachrestic of
apoptosis) in MJ-29-treated cells. The arrow bar (q) shows chromatin condensations in apoptotic cells due to their higher fluorescent intensity
compared to the vehicle control group (scale bar, 15 mm). MFI of DAPI was measured and quantified. (F) TUNEL positive cells were determined and
quantified by flow cytometry. Each assay described in the ‘‘Materials and Methods’’. Each point is a mean 6 S.D. of three independent experiments.
*p,0.05 is significantly different compared with the 0.1% (v/v) DMSO-treated vehicle control by Tukey’s HSD test.
MJ-29 Inhibits Leukemia Cells In Vitro and In Vivo
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MJ-29 affects the phosphorylation of eIF2a and PERK, which are
the ER stress-associated important protein levels. Results revealed
that MJ-29 induced an increase in the protein levels of p-eIF2a
and p-PERK during the time period of 6–24 h in WEHI-3 cells
(Figure 5D). Moreover, we determined if intracellular Ca2+and
UPR-related signals are involved in modulating the MJ-29-
induced ER stress through activating calpain 1 and CHOP/eIF2a
signaling pathways. We compared the effects of MJ-29-treated
WEHI-3 cells on the levels of calpain 1, p-eIF2a and CHOP in the
absence and presence of BAPTA or salubrinal, respectively.
Results in Figure 5E showed that BAPTA (a Ca2+chelator) that is
effective at suppressing MJ-29-induced calpain 1 protein level
compared with only treated WEHI-3 cells. Alternatively, pretreat-
ment with salubrinal (an eIF2a dephosphorylation inhibitor)
resulted in the decreased MJ-29-activated the levels of p-eIF2a
and CHOP in WEHI-3 cells (Figure 5F). As seen in Figure 5G,
reduction of cell viability in WEHI-3 cells by MJ-29 was
significantly decreased not only by BAPTA but also by salubrinal
in comparison to MJ-29-treated only cells. Overall, data in Figure 5
clearly demonstrated that stimulation of unfolded protein response
might be responsible for increased intracellular Ca2+release as
well as calpain 1 and CHOP/eIF2a signaling pathways in MJ-29-
treated WEHI-3 cells.
Figure 2. MJ-29 enhances ROS productions and promotes mitochondrial dysfunctions in WEHI-3 cell apoptosis. Cells were incubated
with 1 mM of MJ-29 for indicated periods of time (3, 6, 12 and 24 h) and then were harvested for examining the (A) ROS productions, (C) the level of
DYm and (E) MPT pores opening as described in the ‘‘Materials and Methods’’. (B, D, F) Quantification of these responses was displayed and
determined by BD CellQuest Pro software. The results are shown as means 6 S.D. (n=3) and significant different (*p,0.05) was considered when
compared to the 0.1% (v/v) DMSO vehicle control (0 h) by Tukey’s HSD test. (G) NAO fluorescence by flow cytometry was performed for cardiolipin
oxidation to evaluate a left shift. Representative images are taken from three independent experiments.
MJ-29 Inhibits Leukemia Cells In Vitro and In Vivo
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MJ-29 prolongs the survival rate and enhances the body
weight of WEHI-3 leukemic BALB/c mice
We have previously established the leukemic animal model to
test the effects of MJ-29 on the survival rate in WEHI-3 leukemic
mice [30,31]. Our experimental design and protocol of the
different treatment groups are shown in Figure 6A. We
intraperitoneally administrated with MJ-29 (10 and 20 mg/kg,
respectively) in mice after being inoculated with WEHI-3 cells. As
Figure 3. MJ-29 triggers apoptotic death in WEHI-3 cells through the intrinsic signaling pathway. Cells were pretreated in the presence
or absence of the specific inhibitors of caspase-9 (Z-LEHD-FMK) and caspase-3 (Z-DEVD-FMK) for 2 h and then were exposed to 1 mM of MJ-29 for 6,
12 or 24 h. Flow cytometry analysis was used to determine caspase-9 (A) and caspase-3 (C) activity and respective profiles were shown using
CellQuest Pro software (B and D). MFI indicates mean fluorescence intensity. (E) Cell extracts were prepared to determine by Western blotting analysis
for protein levels of Bax, Bcl-2, Bcl-xL, cytochrome c, Endo G, AIF, cleaved caspase-3 and cleaved caspase-9. b-Actin was used as a loading control. (F)
Pretreatments with Z-LEHD-FMK and Z-DEVD-FMK followed exposure to MJ-29 were determined by a PI exclusion method as described in the
‘‘Materials and Methods’’. Columns, mean (n=3); bars, SD. *, p,0.05, is significantly different compared with 0.1% (v/v) DMSO vehicle control and
#, p,0.05 significantly greater than values obtained for cells treated with MJ-29 alone by Tukey’s HSD test.
MJ-29 Inhibits Leukemia Cells In Vitro and In Vivo
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displayed in Figure 6B, MJ-29 significantly prolonged the survival
time of mice with median survival time (MST) by 23.8% and
33.3% (median, 21 days for control mice versus 26 and 28 days for
mice treated with MJ-29 at 10 and 20 mg/kg, respectively) using
Kaplan-Meier estimator and there was 5- and 7-days prolonga-
tions in average life span of leukemic mice until 28 days.
Furthermore, MJ-29 (10 and 20 mg/kg) promoted body weight
in leukemic mice (WEHI-3/BALB/c: 20.3261.82 g; WEHI-3/
BALB/c/MJ-29 10 mg/kg: 25.6360.76 g; WEHI-3/BALB/c/
MJ-29 20 mg/kg: 26.7161.24 g) for 16-days exposure with
intraperitoneal treatment (Figure 6C).
MJ-29 alters the CD markers of leukocytes from PBMC in
The normal and leukemic mice were treated with or without
MJ-29 (10 and 20 mg/kg) for 16 days. Blood samples were
collected from individual animals of each group, and then
leukocytes were stained with anti-CD3 for T cells, anti-CD19 for
B cells, anti-Mac-3 for macrophages and anti-CD11b for
monocytes, respectively. Results shown in Figure 6D and Figure
S3 indicated that MJ-29 induced the levels of CD3 (10 mg/kg: an
increase of 12.3%; 20 mg/kg: an increase of 28.3%) and CD19
(10 mg/kg: an increase of 3.6%; 20 mg/kg: an increase of 14.3%).
Based on these observations, both of the effects were dose-
dependent in leukemic mice. However, MJ-29 reduced the levels
of Mac-3 (10 mg/kg: a decrease of 2.5%; 20 mg/kg: a decrease of
9.0%) and CD11b (10 mg/kg: a decrease of 18.1%; 20 mg/kg: a
decrease of 14.4%) in comparison to the only WEHI-3 cells-
injected mice group. Therefore, intraperitoneal administration
with MJ-29 to leukemic mice altered the specific surface markers
from PBMC in vivo.
MJ-29 affects the weights of spleen and liver tissues as
well as splenic histopathology in leukemic mice
Each spleen or liver tissue isolated from the normal and
leukemic mice groups was weighed. As can be seen in Figures 7A
and B, both doses (10 and 20 mg/kg) of MJ-29 treatment
significantly decreased the weights of spleen (the decreases of
16.7% and 52.8%, respectively) between WEHI-3 leukemic mice
with or without MJ-29 intraperitoneal administration. Also, MJ-29
at 20 mg/kg significantly reduced the weight of liver tissues (a
decrease of 30.3%) in comparison to only WEHI-3 cells-injected
mice in vivo (Figures 7C and D). Moreover, the representative
results of histopathological examination are presented in Figure 7E,
which indicates that the infiltration of immature myeloblastic cells
into splenic red pulp (R) in spleen section was eliminated in MJ-29
(20 mg/kg)-treated leukemic mice (Figure 7E, right panel) when
compared to untreated-mice after intravenous injection with
WEHI-3 cells. Little differences were shown that marked
expansion in the R area rather than that in the white pulp. The
neoplastic cells contained large irregular nuclei accompanied with
clumped chromatin and prominent nucleoli versus abundantly
clear and light eosinophilic cytoplasm. Also, the number of
megakaryocytes increased in MJ-29-treated leukemic animals
MJ-29 enhances the phagocytosis by macrophages as
well as the T- and B-cell proliferations and NK cell
cytotoxicity in leukemic mice
To explore if MJ-29 affects phagocytosis, the leukocytes from
MJ-29-treated or untreated mice were isolated and phagocytic
activity by macrophages was determined which can be seen in
Figures 8A and B. Our results revealed that MJ-29 (10 and
20 mg/kg, respectively) enhanced phagocytosis from PBMC
(10 mg/kg: 18.4%; 20 mg/kg/day: 20.6%) (Figure 8A) but it did
not significantly influence that from peritoneal cavity (Figure 8B)
by comparison to untreated leukemic mice. Thereafter, we further
investigated that whether MJ-29 promotes T- and B-cell
proliferations from splenocytes in leukemic mice. Our results in
Figure 8C indicated that both doses of MJ-29 increased T-cell
proliferation after concanavalin A (Con A) stimulation but it only
promoted B-cell proliferation by co-treatment with lipopolysac-
charide (LPS) when leukemic mice after 20 mg/kg MJ-29
exposure. Furthermore, splenocytes from MJ-29-treated or
Figure 4. MJ-29 stimulates the translocations of cytochrome c and CHOP/GADD153 levels to cytosol or nucleus in WEHI-3 cells. Cells
(56104cells/well) plated on 4-well chamber slides were incubated in the presence or absence with 1 mM of MJ-29 for 24 h before being stained by
the antibodies against (A) cytychrome c and (B) CHOP/GADD153, and then FITC-labeled secondary antibodies were applied (green fluorescence).
Cytosol and nucleus were counterstained with CellTracker Red CMTPX and PI, respectively (red color). 0.1% (v/v) DMSO alone served as a vehicle
control. The images were visualized under a confocal microscope as described in the ‘‘Materials and Methods’’. Scale bar, 15 mm. Data are
representative of three independent experiments that yielded similar results.
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untreated mice were isolated and determined NK cell cytotoxicity
in vivo. MJ-29 at 20 mg/kg was effective at both target ratios of 50/
1 and 25/1 but only MJ-29 at 10 mg/kg-tested dose showed a
significant effect at the ratio of 25/1 when compared to the
untreated control leukemic mice (Figure 8D).
Numerous reports have described that novel agents for therapies
based on targeting on interference of microtubule assembling
provided a promising insight toward a cure for leukemia
[14,32,33]. Recently, we have synthesized the 2-phenyl-4-
quinazolinone analogs which are found to have anti-mitotic
functions and bioactivities in many types of cancer cell lines,
including human leukemia cells [20,22,23]. The previous study
has shown that MJ-29 possessed the suppression of the prolifer-
ation and induction of apoptosis in human leukemia U937 cells
. Also, the in vivo study showed that MJ-29 inhibited tumor
growth of xenografts in nude mice. In the current study, our results
presented here clearly demonstrated that MJ-29 could modulate
antiproliferative effects and trigger apoptosis caused by ER stress
and intrinsic pathways in WEHI-3 cells. Furthermore, MJ-29
could prolong the survival rate in leukemic mice that might be
involved in levels of specific cell surface markers and alterations of
immunemodulation in vivo. Therefore, this study strongly suggests
that the newly quinazolinone compound, MJ-29 triggers cell
Figure 5. MJ-29 provokes intracellular Ca2+release and unfolded protein response-related hallmark protein expressions in WEHI-3
cells. (A) Cells were incubated with or without 1 mM of MJ-29 at the indicated intervals of time for measuring the fluorescence intensity of Fluo-3 by
flow cytometry, and (B) data were analyzed using BD CellQuest Pro software. Cells were harvested and the indicated proteins levels [(C): calpain 1,
calpain 2, caspase-12 and caspase-4; (D): CHOP, BiP, GRP97, p-eIF2a, eIF2a, p-PERK and PERK] were subjected to Western blotting. To confirm the
effects of BAPTA or salubrinal on MJ-29-induced cell death, cells were individually pretreated with or without 5 mM BAPTA or 10 mM salubrinal for 2 h
and further treated with 1 mM of MJ-29 following a 24-h exposure. Whole-cell protein lysates were prepared and performed for detecting the protein
levels of (E) calpain 1 and (F) p-eIF2a by immunoblotting. b-Actin served for loading control. (G) Cell viability was determined by a PI exclusion
method and flow cytometry. Data are expressed as overall means 6 S.D. from three independent experiments. Statistical significance was determined
by Tukey’s HSD test. *, p,0.05, shows significant difference compared with 0.1% (v/v) DMSO vehicle control; #, p,0.05, is significantly different
compared to MJ-29 treatment alone.
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apoptosis in murine leukemia WEHI-3 cells in vitro and suppresses
orthotopic leukemic mice in vivo.
We therefore examined the cytotoxicity of MJ-29 in WEHI-3
cells and investigated its molecular mechanisms in vitro. Our data
revealed that MJ-29 at the concentrations of 0.5–10 mM had the
concentration-dependent cytotoxic effect (Figure 1A and Figure
S1A) and induced morphological changes, such as cell shrinkage
and rounding (Figure 1B) on WEHI-3 leukemia cells. Additionally,
our earlier study indicated that MJ-29 exerted a less cytotoxicity in
normal cells (PBMC and HUVECs) and EC50value of both is
more than 10 mM of MJ-29 for a 24-h treatment . It is notable
that MJ-29 could represent a promising agent for anti-leukemia,
and there are more safety properties, fewer side effects and
selectivity of actions on normal cells as compared to that of
paclitaxel and vincristine based on the critical factors, such as
doubling time and EC50value of PBMC and HUVECs .
It has been generally accepted that induction of apoptosis is a
best strategy or option by many chemotherapeutic agents for
antitumor and anti-leukemia treatments [18,34], and the apoptotic
characteristics include oligonucleosomal DNA fragmentation,
membrane blebbing, etc . In the present study, we examined
antitumor effects of MJ-29 on WEHI-3 cells by investigating
mitochondrial dysfunction-associated apoptotic signaling pathway.
The apoptotic WEHI-3 cells were determined with four different
assays, containing DNA content analysis, annexin V population
assessment, DAPI staining and TUNEL assay. Results showed that
MJ-29 treatment promoted G2/M phase arrest and increased the
number of cells in the sub-G1 peak (apoptotic cells) (Figure 1C).
Furthermore, MJ-29-triggered apoptotic populations were con-
firmed by annexin V/PI assay, DAPI staining and TUNEL assay.
Our data demonstrated that MJ-29-increased the early (annexin V
positive and PI negative) and late (annexin V positive and PI
positive) apoptotic death can be distinguished in WEHI-3 cells
(Figure 1D and Figure S1C), suggesting that the phospholipid
phosphatidylserine (PS) is trafficking the outer leaflet since cell
apoptosis . Also, we found that MJ-29 caused chromatin
condensation (a characteristic of cell apoptosis) (Figure 1E) and
DNA fragmentations (Figure 1F) in WEHI-3 cells. These findings
provided vital insights showing that MJ-29-induced cytotoxicity is
mediated by inducing apoptotic death in WEHI-3 cells. This is
Figure 6. Anti-leukemic activity of MJ-29-impaired WEHI-3 leukemic mice in vivo. (A) The experimental design and protocol of orthotopic
leukemic mice model. Efficacy of intraperitoneal (i.p.) treatment with MJ-29 was investigated the survival rate and anti-leukemic responses on
leukemic mice [BALB/c mice after intraperitoneal (i.v.) with WEHI-3 cells]. (B) Whole survival rate of leukemic mice was counted after MJ-29 exposure
for 28-days administration. The animals were given intraperitoneally and anti-leukemic activity was determined as the survival rate by Kaplan-Meier
estimator every day in all groups. There is a significant overall survival difference in comparison to the leukemic mice groups in the presence and
absence of MJ-29 exposure versus control mice. (C) Mice were intravenously injected with WEHI-3 cells (16106cells/100 ml per mouse) in PBS, and
then treated with or without MJ-29 (10 and 20 mg/kg) by intraperitoneal injection for 16 days. During the treatment, each animal was measured the
body weight once every four days for 16-days intervals as described in the ‘‘Materials and Methods’’. (D) Whole blood was collected from individual
mice and leukocytes were analyzed the with specific cell surface markers by flow cytometry. The CD markers include CD3 for T lymphocytes, CD19 for
B cells, Mac-3 for macrophages and CD11b for monocytes. The results are expressed as means 6 S.D. and samples were obtained from at least five
mice per group, and * p,0.05 is found significantly different by Tukey’s HSD test when compared with the leukemic (only injection with WEHI-3 cells)
and experimental (WEHI-3 cells injected before intraperitoneal treatment with MJ-29 at 10 and 20 mg/kg) mice groups.
MJ-29 Inhibits Leukemia Cells In Vitro and In Vivo
PLoS ONE | www.plosone.org8 May 2012 | Volume 7 | Issue 5 | e36831
also in agreement with our previous study addressing MJ-29-
induced apoptosis in human leukemia cells, which was involved in
the dysfunction of mitotic spindles, induction of mitotic arrest and
cell apoptosis . Importantly, MJ-29-stimulated CDK1 activa-
tion appears to be acting the phosphorylation of Bcl-2 (Ser70),
resulting in promotion of the intrinsic apoptotic signaling in
human leukemia U937 cells .
Mitochondria are one of primary key targets and ROS caused
an increase of mitochondrial depolarization, leading to caspase
cascades activations when tumor cell apoptosis [27,36]. In this
study, we displayed that MJ-29 increased DYm dissipation
(Figure 2D), MPT pores opening (Figure 2F), and activations of
caspase-9 and caspase-3 (Figure 3), following inductions of
mitochondria-mediated apoptosis in WEHI-3 cells. Also, the
effects of cell death were significantly blocked after individual
pretreatment with specific caspase-9 and caspase-3 inhibitors
(Figure 3F) in WEHI-3 cells. Additionally, oxidative stress could
promote a decrease the level of cardiolipin oxidation which is
known to lead to an event of mitochondrial dysfunction and, in
consequence, cytochrome c release [26,27]. Therefore, we
[DiOC6(3)] and 10-nonyl bromide acridine orange (NAO)
Figure 7. Effects of the weights in spleen and liver as well as histopathological examination of spleen tissues on MJ-29-treated
leukemic mice. Animals were intravenously injected with WEHI-3 cells (16106cells/100 ml per mouse) in PBS, and then treated intraperitoneally
with MJ-29 (10 and 20 mg/kg alternate day for 8 times). Weights and representative images of spleen (A and B) and liver (C and D) tissues from
leukemic mice were determined and measured individually. Each point is mean 6 S.D. (at least five samples). * p,0.05 indicates significant difference
by Tukey’s HSD test between the WEHI-3 leukemic mice and experimental (normal or intraperitoneal treatment with MJ-29 at 10 and 20 mg/kg,
respectively) groups. (E) Dissected leukemic mice and hematoxylin-eosin stain for the paraffin sections of spleens from MJ-29-treated and un-treated
leukemic mice as described in the ‘‘Materials and Methods’’. Arrows (q) shows infiltration of immature myeloblastic cells (leukemia cells) into red
pulp of the spleen. R, red pulp. The data are performed with representative experiment in triplicate and three independent experiments with similar
MJ-29 Inhibits Leukemia Cells In Vitro and In Vivo
PLoS ONE | www.plosone.org9 May 2012 | Volume 7 | Issue 5 | e36831
fluorescence in treated cells, and it now seems likely that MJ-29
induced the level of ROS increase (Figure 2B), caused cardiolipin
peroxidation (Figure 2G) and promoted the trafficking of
cytochrome c release from mitochondria to cytosol (Figure 4A)
in WEHI-3 cells. These results suggest that MJ-29-triggered
apoptosis in WEHI-3 cells attributed to ROS production and the
mitochondria-dependent signaling pathways.
To further demonstrate our notion that cellular ER stress
responses the accumulation of unfolded or misfolded proteins
triggered by chemotherapeutic agents [11,37]. CHOP is a typical
and important member of pro-apoptotic transcription factor
related to ER stress . The hallmarks of ER stress-regulated
protein levels are up-regulated, which induced intracellular Ca2+
release, increased calpain level and activated caspase-12 and/or
caspase-4 signals . The results from our current study also
clearly showed that UPR occurred in WEHI-3 cells, leading to
cytosolic Ca2+generation (Figure 5A) and triggering cell apoptosis.
To clarify this evidence that by pre-treating with a Ca2+chelator
(BAPTA) in MJ-29-treated WEHI-3 cells, our results indicated the
decrease of calpain 1 protein level (Figure 5E) and increased MJ-
29-reduced viability (Figure 5G). Also, down-regulation of p-eIF2a
expression using an ER stress inhibitor (salubrinal, which targets
the eIF2a dephosphorylation) (Figure 5F) significantly blocked MJ-
29-induced apoptosis in WEHI-3 cells (Figure 5G), suggesting the
critical roles of eIF2a and CHOP in MJ-29-induced ER stress-
mediated apoptotic death. Induction of CHOP expression is
mediated through an eIF2a phosphorylation manner in MJ-29-
triggered UPR (Figure 5F). Our novel findings regarding ER
stress-modulated eIF2a phosphorylation and intracellular Ca2+in
WEHI-3 cells are critical events, leading to apoptotic cell death
caused by MJ-29 treatment. Strikingly, cells were pre-incubated
with N-acetylcysteine (NAC), an intracellular ROS scavenger,
prior to MJ-29 treatment and we found that NAC significantly
attenuated MJ-29-stululated the activations of CHOP and BiP
protein expressions (Figure S2A) and attenuated cell death (Figure
S2B) induced by MJ-29 in comparison to the treated only cells. We
suggest that ROS generation plays an essential influence on MJ-
29-triggered ER stress in WEHI-3 cells, and this finding is also
agreement with other reports [39,40]. Based on our functional in
vitro study, it is demonstrated that MJ-29 not only disrupted the
mitochondrial dysfunction and triggered intrinsic apoptosis but
also activated the UPR in the murine myelomonocytic leukemia
cell line (WEHI-3) in vitro.
In in vivo study, despite of MJ-29-chemosensitized tumor nude
mice xenografts bearing subcutaneous human leukemia U937 cells
, the present study emphasized that the establishment of an
orthotopic leukemia model and elevations for the biological
functions for MJ-29 regarding anti-leukemic activities and
immunemodulations in vivo. BALB/c mice were intravenously
transplanted with WEHI-3 cells, a murine monomyelocytic
leukemia cell line originally derived from the BALB/c mouse
[41,42], which is an ideal system to test anti-leukemic chemother-
apeutic agents [30,31].
Figure 8. MJ-29 alters phagocytosis by macrophages from PBMC as well as T- and B-cell proliferations and NK cell cytotoxicity from
splenocytes in leukemic mice. Mice were intravenously injected with WEHI-3 cells (16106cells/100 ml per mouse) and intraperitoneally treated
with MJ-29 (10 and 20 mg/kg, respectively) for 16 days. Macrophages were isolated from (A) PBMC and (B) peritoneal cavity of each group. The
percentage of phagocytic leukocytes that was ingesting FITC-E. coli, and the samples were determined using flow cytometry. Splenocytes were
isolated from each mouse of groups for (C) T- and B-cell proliferation examinations and (D) NK cell cytotoxicity as described in the ‘‘Materials and
Methods’’. Each point is mean 6 S.D. for at least five samples per group. * p,0.05 is considered significant by Tukey’s HSD test when compared with
the untreated WEHI-3 leukemic mice group.
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Consequently, we further verified striking anti-leukemic func-
tions and found that MJ-29 inhibited WEHI-3 cells in vivo. As
shown in Figure 6B, MJ-29 had longer the survival rate of BALB/
c leukemic mice for at least 5 days by a 28-days treatment. Also,
MJ-29 is able to prevent the loss of body weight when compared
with the WEHI-3/leukemic mice group (Figure 6C) after 16-days
exposure. The spleen or liver tissues from MJ-29-treated or
untreated animals were weighed and histopathologically examined
as presented in Figure 7. Our results revealed that MJ-29
significantly suppressed the enlargement of spleen (Figure 7A)
and liver (Figure 7C), and the reductions of the infiltration in
immature myeloblastic cells into splenic red pulp (Figure 7E) were
also observed in MJ-29-treated leukemic mice. It is reported that
immature myeloblastic cells entering the leukocytes can be
discovered in case of the leukemic cells metastasized to the liver
tissues . Neoplastic cells are not easy to discover in the red
pulp of the spleens from chemotherapeutic leukemic mice . It
is noteworthy that regarding the side effects and toxicity of MJ-29
on normal and leukemic mice, and no significant difference in
blood chemistry values within treated animals was found
compared to both doses of MJ-29 and control groups (Table S1
and Method S2). Moreover, there are significant differences in the
increases in the amount of T and B cells (CD3 and CD19,
respectively) rather than the decreases of monocytes and
macrophages (CD11b and Mac-3, respectively) between the
leukemic mice treated or un-treated groups (Figure 6D and Figure
S3). These results might be involved in the reason why WEHI-3
cells, originally designated as a myelomonocytic cell line ,
stimulated that of T and B cells but decreased in the levels of
macrophages and monocytes from MJ-29-treated leukemic mice.
Also, we proposed that MJ-29 modulated immune responses
through not only increasing T- and B-cells proliferations and
phagocytotic activity by macrophages but also promoting NK cell
cytotoxicity in leukemic mice in vivo (Figure 8). It is well known that
that macrophages are major roles to innate immunity [44,45], and
stimulation of NK cell cytotoxicity could contribute to the increase
immune response . Thus, we suggest that MJ-29 might possess
anti-leukemic activity partially through modulating immune
responses in BALB/c mice. However, MJ-29 if directivity affects
immunemodulations to reach anti-leukemia is indeed for further
In conclusions, our study is the first report to provide an
approach regarding that the newly quinazolinone MJ-29 tends to
inhibit murine myelomonocytic leukemia WEHI-3 cells in vitro and
in vivo. The induction of cell death in MJ-29-treated WEHI-3 cells
has therefore been proven by an assessment of apoptosis in vitro
culture through not only intrinsic apoptotic pathway but also ER
stress signaling. Strikingly, the orthotopic leukemic mice were
exposed to MJ-29 in in vivo model, and it is shown that anti-
leukemic responses occurred when the increased immune response
in vivo. For that reason, we presented our novel findings and the
efficacy of MJ-29 might be sufficient to investigate the potential of
leukemia treatment in the future.
Materials and Methods
The murine myelomonocytic leukemia cell line (WEHI-3) was
purchased from the Bioresource Collection and Research Center
(BCRC), Food Industry Research and Development Institute
(FIRDI) (Hsinchu, Taiwan). Cells plated in 75-cm2cell culture
flasks were grown in RPMI 1640 medium with supplements plus
10% (v/v) fetal bovine serum, 2 mM L-glutamine, 100 Units/ml
penicillin and 100 mg/ml streptomycin at 37uC under a humid-
ified 5% (v/v) CO2one atmosphere in an incubator. Cells were
split and centrifuged every two days to maintain cell growth before
Chemicals and reagents
DMSO, PI, RNase A and Triton X-100 were obtained from
Sigma-Aldrich Corp. (St. Louis, MO, USA). Cell culture materials
were purchased from Gibco/Life Technologies (Carlsbad, CA,
(H2DCFDA), DiOC6(3), MitoProbe Transition Pore Assay kit,
NAO, Fluo-3/AM and BAPTA were purchased from Molecular
Probes/Life Technologies (Eugene, OR, USA). Caspase-9 and
caspase-3 substrate reagent kits (CaspaLux-9 M1D2and PhiPhi-
Lux G1D2) were bought from OncoImmunin, Inc. (Gaithersburg,
MD, USA). The specific caspase inhibitors (Z-LEHD-FMK for
caspase-9 and Z-DEVD-FMK for caspase-3) were purchased from
BioVision, Inc. (Mountain View, CA, USA). Anti-Bax, anti-Bcl-2,
anti-cleaved caspase-3, anti-cleaved caspase-9, anti-eIF2a and
anti-phospho-eIF2a (Ser51) were obtained from Cell Signaling
Technology (Beverly, MA, USA). The other primary antibodies
used in this study and salubrinal were bought from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA, USA). MJ-29 as shown in
the top of Figure 1A was synthesized and provided by Mann-Jen
Hour, Ph.D. (School of Pharmacy, College of Pharmacy, China
Determination of cell viability and morphological
Cells at a density of 26105cells/well were placed in 24-well
plates and treated with DMSO alone [0.1% (v/v) in media served
as a vehicle control] and different concentrations (0.5, 1, 5 or
10 mM) of MJ-29 for 24 h. For measuring the viability, cells from
each sample were collected and labeled with PI (4 mg/ml). Live
and dead cells were determined by a PI exclusion method as
previously described [21,26]. Cells were immediately analyzed by
a Becton Dickinson FACSCalibur flow cytometer (BD Biosciences,
Franklin Lakes, NJ, USA) and calculated utilizing BD CellQuest
Pro software. After incubation, treated cells were photographed
under a phase-contrast microscope before being harvested .
Analysis for DNA content and sub-G1 populations by
Approximately 26105cells per well were seeded in 24-well
plates and then exposed to 1 mM of MJ-29 for 12, 18 and 24 h.
The trypsinized cells were washed with PBS and fixed with ice-
cold 70% (v/v) ethanol at 220uC overnight. After being washed,
cells were stained with PI at a concentration of 40 mg/ml in the
presence of 0.1% (v/v) Triton X-100 and RNase A (20 mg/ml) for
30 min in a dark room. The apoptotic cells were quantified by
measuring the sub-G1 DNA content using the PI method. Each
sample was analyzed, and fluorescence intensity of DNA content
in the FL-2 channel was determined and monitored by flow
cytometry as described elsewhere .
Assay for apoptosis by annexin V/PI double staining
Cells (26105cells/well) after exposure to 1 mM of MJ-29 for 0,
12 and 24 h were trypsinized and harvested before incubation
with annexin V and PI. Apoptotic cells were determined utilizing
an Annexin V-FITC Apoptosis Detection kit (BD Biosciences
Pharmingen, San Diego, CA, USA) according to the manufac-
turer’s protocol. Ten thousand cells were measured per sample by
flow cytometry, and the analysis of apoptotic cells was performed
using BD CellQuest Pro software as previously described [35,49].
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Nuclear staining with DAPI for apoptosis
Cells (16105cells per well) cultured in 24-well plates were
treated with or without 1 mM of MJ-29 following a 24-h treatment.
Cells were washed with PBS, fixed in 4% (v/v) formaldehyde
(Sigma-Aldrich Corp.) for 15 min and permeabilized by sequen-
tially treating with 0.1% (v/v) Triton X-100 for another 15-min
exposure. A 200-ml DAPI solution (1 mg/ml) was added into each
well for 30 min at 37uC in the dark and thereafter visualized using
a fluorescence microscope (Nikon Inc., Tokyo, Japan) [35,50].
Quantification of apoptotic cells was performed utilizing Meta-
morph Imaging System (Universal Imaging Corp., Downingtown,
PA, USA) in three random fields from each well.
TUNEL assay by flow cytometry
TUNEL assay was used to determine apoptotic DNA breaks
utilizing the in situ Cell Death Detection Kit, Fluorescein (Roche
Diagnostics, Boehringer Mannheim, Mannheim, Germany).
Briefly, cells at a density of 26105cells/well in 24-well plates
were treated with 1 mM of MJ-29 for 0, 6, 12 and 24 h. After the
end of treatment, cells were harvested and followed the protocol
provided by the manufacturer. TUNEL positive cells were
measured by flow cytometry as previously described [30,51].
Determinations of ROS productions, DYm, MPT pores
and level of cardiolipin oxidation by flow cytometry
Cells at an initial density of 26105cell/ml in 24-well plates were
incubated with 1 mM of MJ-29 for 3, 6, 12 and 24 h or vehicle
control to detect if this compound would affect ROS, DYm, the
opening of MPT pores and cardiolipin oxidation which were
measured as previously described [35,50,52]. Cells were harvested,
washed with PBS twice and re-suspended in 500 ml of H2DCFDA
(10 mM) for ROS, DiOC6(3) (500 nM) for DYm and NAO
(500 nM) for cardiolipin oxidation at 37uC for an additional
30 min. The opening of MPT pores in MJ-29-treated WEHI-3
cells was monitored using the MitoProbe Transition Pore Assay kit
(Molecular Probes/Life Technologies) and performed according
procedures provided by the manufacturer [35,53].
Flow cytometric analysis for caspase-9 and caspase-3
activities and intracellular Ca2+level
Cells (26105cells/well) in 24-well plates were treated with MJ-
29 (1 mM) for 6, 12 and 24-h treatments or vehicle only. Cells were
then harvested from each treatment, and activities of caspase-9
(CaspaLux-9 M1D2kit) and caspase-3 (PhiPhiLux G1D2kit) were
determined according to the manufacturer’s protocol (OncoIm-
munin, Inc., Gaithersburg, MD, USA) as previously described
. For determining intracellular Ca2+level, cells were stained
with Ca2+indicator Fluo-3/AM (2.5 mg/ml) at 37uC for 40 min
after 1 mM of MJ-29 exposure for 3, 6, 12 and 24 h. Flow
cytometric analysis was used to detect the influences on the level of
intracellular Ca2+as described elsewhere .
Western blotting analysis for protein levels
Cells in 6-well plates at a density of 16106cells/well were
treated in the presence or absence of MJ-29 (1 mM) for 6, 12 and
24 h. At the end of each incubation, cells were harvested, washed
twice with cold PBS and centrifuged at 1,0006g for 5 min. Cell
protein was extracted into the PRO-PREP protein extraction
solution (iNtRON Biotechnology, Seongnam-si, Gyeonggi-do,
Korea), and protein concentration was determined by the Bio-
Rad protein assay (Bio-Rad Laboratories, Hercules, CA, USA).
Protein samples (30 mg) were loaded on a 10% (w/v) polyacryl-
amide gel and subjected to sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) [35,48]. Samples were then
transferred to Immobilon-P transfer membrane (Millipore, Bed-
ford, MA, USA) and the membranes blocked in PBST [16PBS
and 0.1% (v/v) Tween 20 containing 5% (w/v) nonfat dry milk for
2 h at room temperature, and hybridized with appropriate
primary antibodies (1:1000 dilution) overnight at 4uC followed
by horseradish peroxidase (HRP)-conjugated secondary antibod-
ies. Blots were detected using the enhanced chemiluminescence
(ECL) western blotting detection kit (Immobilon Western HRP
substrate, Millipore) and autoradiography utilizing X-ray film (GE
Healthcare, Piscataway, NJ, USA) [26,35]. The band densities
were quantified using the NIH ImageJ 1.45 program (Bethesda,
MI, USA). The blots were reprobed with an anti-b-Actin antibody
(Sigma-Aldrich Corp.) for equal protein loading.
Effects of inhibitors of apoptosis and ER stress on
cytotoxicity and abundance of protein levels induced by
Cells were pretreated with or without 10 mM of specific caspase
inhibitors (Z-LEHD-FMK for caspase-9 and Z-DEVD-FMK for
caspase-3, respectively) for 2 h before the finish of MJ-29
treatment (1 mM) for 24 h, and cell viability was determined using
a PI exclusion method as described above. For inhibition of ER
stress-mediated apoptosis, both of Ca2+chelator (BAPTA) and ER
stress inhibitor (salubrinal, an eIF2a dephosphorylation inhibitor)
were applied. Cells were pre-incubated with the BAPTA (5 mM) or
salubrinal (10 mM) for 2 h and thereafter exposed to 1 mM of MJ-
29. After a 24-h treatment, cells were harvested for measuring
viability using flow cytometric analysis and determining ER stress-
related protein levels by immunoblotting as previously described
Immunofluorescence and co-localization of proteins by
Cells plated on 4-well chamber slides at a density 56104cells/
well were treated with 1 mM of MJ-29 for 24 h. Cells were then
fixed in 3% (v/v) formaldehyde (Sigma-Aldrich Corp.) in PBS for
15 min, permeabilized in 0.1% (v/v) Triton X-100 in PBS for
30 min and stained with the primary antibodies (anti-cytochrome c
and anti-CHOP/GADD153 diluted at 1:200) at 4uC overnight,
followed utilizing FITC-conjugated secondary antibodies (green
fluorescence, Invitrogen/Life Technologies) at 1:100 dilutions for
1 h at room temperature. Cells were washed twice with PBS and
individually stained with CellTracker Red CMTPX (Molecular
Probes/Life Technologies) for cytosol and with PI for nucleus (red
fluorescence). Coverslips were mounted, and photomicrographs
were obtained using a Leica TCS SP2 confocal spectral
microscope (Leica Microsystems, Heidelberg, Mannheim, Ger-
many) as previously described [26,35].
Eighty five-week-old male BALB/c mice (approximately 20–
25 g body weight) were obtained from the National Laboratory
Animal Center (Taipei, Taiwan). Mice were housed in a regular
12-h light/12-h dark cycle, bred with clean water and fed
commercial diet ad libitum in standard conditions of constant
temperature and humidity. The start of our experiment worked
after keeping at least one week. All animal studies were conducted
according to institutional guidelines (Affidavit of Approval of
Animal Use Protocol, No. 97-33-N) approved by the Institutional
Animal Care and Use Committee (IACUC) of China Medical
University (Taichung, Taiwan).
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Establishment of the orthotopic leukemic mice and MJ-
All mice were randomly divided into 8 groups, and each group
contains 10 animals. Group I and V are a normal control group;
and group II and VI were intravenously injected with WEHI-3
cells (16106cells in 100 ml PBS per mouse) only as a model of
leukemic mice. Groups III and IV as well as VII and VIII were
intravenously injected with WEHI-3 cells (16106cells/mouse) for
a 7-day incubation periods, and the animals were then treated
with MJ-29 (10 and 20 mg/kg body weight, respectively) by
intraperitoneal administration once every two days .
The in vivo studies are divided into two parts
Part I. survival analysis for MJ-29-treated orthotopic
The animals in groups V, VI, VII and VIII
were treated with or without MJ-29 at 10 and 20 mg/kg. These
groups were administrated for 21–28 days for measuring the
survival rate. The Kaplan-Meier estimator for the survival
function was expressed as previously described [47,54,55]. Anti-
leukemic activity was performed as the ratio of median survival
time (MST) of treated or untreated leukemic mice (T) groups
versus that of the control (C) group. Data for survival rate (%) was
undertaken as the following formula: life span T/C (%)=(MST of
T group/MST of C group)6100.
Part II. Evaluations for anti-leukemic activity.
was administered for every other day up to 16 days by
intraperitoneal injection at 10 and 20 mg/kg in leukemic mice.
At the end of the experiment, all animals were sacrificed by
euthanasia with carbon dioxide (CO2) followed by cervical
Assessments for the weights of body, spleen and liver
Body weight of each mouse during treatment was recorded once
every four days for 16 days. After finishing treatment, each animal
was sacrificed on day 16 before blood was collected, and spleen
and liver samples were obtained and weighed individually as
previously described [31,47].
Histopathology of spleen tissue by hematoxylin-eosin
The spleen tissues from leukemic mice were isolated and
subjected to histopathological analysis. Spleen was fixed in 4% (v/
v) formaldehyde, embedded in paraffin, sectioned at 5 mm, and
stained with hematoxylin and eosin according to the previous
Blood samples collection and immunofluorescence
About 500 ml blood was collected from each mouse in different
groups and then immediately exposed to 16 Pharm Lyse lysing
buffer (BD Biosciences, San Jose, CA, USA) for lysing of the red
blood cells followed by centrifugation for 5 min at 1500 rpm at
4uC. The isolated leukocytes were examined for cell markers based
on being stained with FITC-conjugated anti-mouse CD3, PE-
conjugated anti-mouse CD19, PE-conjugated anti-mouse Mac-3
and FITC-conjugated anti-mouse CD11b antibodies (BD Phar-
Mingen Inc, San Diego, CA, USA). Subsequently, cells were
determined for the levels of specific cell surface markers by flow
cytometry as described elsewhere [31,47].
Detection for phagocytic activity by macrophages
The heparinized blood samples or peritoneal macrophages from
each mouse in MJ-29-treated or un-treated groups were isolated as
previously described . Approximately 16105leukocytes in
100 ml of samples were incubated for 1 h at 37uC with FITC-
labeled opsonized Escherichia coli (E. coli) (at a density of 26107
bacteria per 20 ml 16 solution from the PHAGOTEST kit,
Glycotope Biotechnology GmbH, Czernyring, Heidelberg, Ger-
many). The reaction was stopped by the addition of quenching
solution (100 ml) according to the manufacturer’s instruction, and
the whole blood is then lysed with 2 ml of 16lysing solution for
20 min at room temperature. After the completion of phagocy-
tosis, DNA was stained and cells from each sample were analyzed
by flow cytometery as previously described [47,57]. Fluorescent
particles were collected on 10,000 cells and data were analyzed
using the BD CellQuest Pro software.
Assay for NK cell cytotoxicity
The fresh spleens from all experimental mice were processed to
isolate splenocytes, and about 16105splenocytes were cultured in
each well of 24-well culture plates. YAC-1 cells obtained from the
BCRC (Hsinchu, Taiwan) were stained according to the
manufacturer’s protocols (PKH67 Fluorescent Cell Linker Kits,
Sigma-Aldrich Corp.) [47,58]. The labeled YAC-1 cells (about
16106cells per 100 ml) were placed on 96-well plates before the
addition of the splenocytes from each treatment to the wells after a
12-h incubation and determination of NK cell cytotoxicity by a PI
exclusion assay and flow cytometry as previously described
Assessments of T- and B-cell proliferation
Splenocytes (16105cells/well) from each mouse seeded in
100 ml of RPMI 1640 medium with 10% (v/v) fetal bovine serum
in 96-well plates were stimulated with Con A (1 mg/ml; Sigma-
Aldrich Corp.) for T-cell and LPS (1 mg/ml; Sigma-Aldrich Corp.)
for B-cell for 3 and 5 days incubations, respectively. T- and B-cell
proliferation was determined by the CellTiter 96 AQueous One
Solution Cell Proliferation Assay kit (Promega, Madison, WI,
USA) as previously described [47,58].
Data are represented as means 6 standard deviation (S.D.)
from at least three independent experiments. The values are
analyzed by one-way analysis of variance (ANOVA) followed by
Tukey’s HSD test. Cases in which p-value of less than 0.05 was
accepted to have a pronounced statistically difference between
experimental and control samples. In in vivo experiment, survival of
the mice was measured from the date of pair matching to sacrifice
(event) or end of study (censored). The Kaplan-Meier method was
used to estimate survival when comparing survival between
leukemic mice and the respective treatment groups.
cols for Figure S1A and Table S1.
Expanded description of evaluation proto-
apoptotic death in WEHI-3 cells. Cells were treated with
different concentrations (0.5, 1, 5 or 10 mM) of MJ-29 for 24 h or
1 mM of MJ-29 for 12 and 24 h. (A) MJ-29 concentration-
dependently inhibited the cell viability, which was determined by
MTT assay and the percent viabilities were plotted as the means
MJ-29 reduces cell viability and triggers
MJ-29 Inhibits Leukemia Cells In Vitro and In Vivo
PLoS ONE | www.plosone.org13May 2012 | Volume 7 | Issue 5 | e36831
6 S.D. of at least three experiments. *p,0.05 compared with
0.1% (v/v) DMSO-treated vehicle-treated control cells by Tukey’s
HSD test. (B) The representative profiles from BD CellQuest Pro
software indicated that DNA content for distribution of cell cycle
by PI-stained assay and (C) apoptotic cells (annexin V-FITC
positive) by annexin V/PI staining in the presence of 1 mM of MJ-
29 for 12 and 24 h were determined utilizing flow cytometry.
Quantifications of annexin V positive cells were measured as
described in the ‘‘Materials and Methods’’. The data conducted
three times with similar results.
curs in MJ-29-treated WEHI cells. Cells were pretreated with
or without 10 mM of NAC (Sigma-Aldrich Corp.), a ROS
scavenger for 1 h and then exposed to 1 mM of MJ-29 for 24 h. At
the end of treatment, cells were collected and determined the
hallmark protein levels of ER stress and viability in MJ-29-treated
cells as described in the ‘‘Materials and Methods’’. (A) The protein
expressions of CHOP and BiP were performed by Western
blotting. b-Actin was an internal control. Results shown are
representative of three independent experiments. (B) Abrogation
of MJ-29-reduced cell viability by NAC was detected by flow
cytometric analysis and analyzed utilizing BD CellQuest Pro
software. Results are shown as means 6 S.D. in triplicate and
determined by Tukey’s HSD test. *, p,0.05, shows significant
difference compared with 0.1% (v/v) DMSO vehicle control;
#, p,0.05, is significantly different compared to only MJ-29-
ROS and ER-stress-mediated apoptosis oc-
in leukemic mice. Animals were intravenously injected with
WEHI-3 cells (16106cells/100 ml) and intraperitoneally treated
with MJ-29 (10 and 20 mg/kg) every other day for 16 days. Whole
blood was collected from individual mice, and leukocytes were
analyzed the with specific cell surface markers by flow cytometry.
(A) The profiles of anti-CD3-PE for T lymphocytes and anti-
CD19-FITC for B cells from BD CellQuest Pro software were
shown, and (B) that of anti-Mac-3-PE for macrophages and anti-
CD11b-FITC for monocytes were revealed as described in the
‘‘Materials and Methods’’.
MJ-29 alters the levels of CD surface markers
mice with or without WEHI-3 cells by intravein trans-
plantation following treatment of MJ-29 by intraperito-
Blood biochemical profiles in the BALB/c
The authors appreciate Dr. Hsiu-Maan Kuo and Dr. Ya-Ling Lin
(Department of Parasitology, China Medical University) for providing
partial constructs used in this study. We are also grateful to the members of
Dr. Chung’s laboratory for technical assistance and support in animal
Conceived and designed the experiments: CCL JSY THL JGC. Performed
the experiments: CCL JSY JHC. Analyzed the data: CCL JSY JHC.
Contributed reagents/materials/analysis tools: MJH KLL JJL WWH MT.
Wrote the paper: CCL JGC.
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MJ-29 Inhibits Leukemia Cells In Vitro and In Vivo
PLoS ONE | www.plosone.org15 May 2012 | Volume 7 | Issue 5 | e36831