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
PLoS ONE | www.plosone.org2May 2012 | Volume 7 | Issue 5 | e36831
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
PLoS ONE | www.plosone.org3 May 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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PLoS ONE | www.plosone.org15 May 2012 | Volume 7 | Issue 5 | e36831