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Anti-proliferative and apoptosis-
inducing activities of juglone in LS-
174T cells 

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Introduction 

Quinones represent a broad category of widely distri-
buted quinoid compounds in nature. Many quinones 
have been associated with a range of biological activi-
ties, including anti-cancer activity (Babula et al., 2007). 
Juglone (5-hydroxy-1,4-naphtha-quinone) (Figure 1) is a 
quinone found in the roots, leaves and bark of walnut 
trees (Inbaraj et al., 2004; Varga et al., 1996).  

The bark, branches and exocarp of the immature green 
fruit of walnut trees have been used to treat gastric 
cancer, liver cancer, lung cancer and other types of 
cancer for a long time in China (Liu et al., 2004). Juglone 
has cytotoxic properties when administered to cell 
cultures (Inbaraj et al., 2004; Kamei et al., 1998; 
Rippmann et al., 2000; Kiran et al., 2009; Ji et al., 2009.) 
and it possesses antiviral, antibacterial and antifungal 
properties (Clark et al., 2006; Inbaraj et al., 2004). In 
addition, juglone promotes generation of hydrogen 
peroxide (Inbaraj et al., 2004), block of K+ channel 
(Varga et al., 1996) and inhibition of transcription (Chao 
et al., 2001). Furthermore, benzobijuglone, a novel 
cytotoxic compound from Juglans mandshurica, could 

induce apoptosis in HeLa cells (Li et al., 2007). 

In this paper, we investigated the anti-proliferative and 
apoptosis-inducing effects of juglone in LS-174T cells. 
The work reported here is focused on which compo-
nents of apoptosis pathway were involved in cell death, 
and whether reactive oxygen species (ROS) have a role 
in LS-174T cells death induced by juglone. Although 
some groups have studied the in vitro cytotoxic activity 
of juglone against cancer cell lines (Kamei et al., 1998; 
Segura-Aguilar et al., 1992), the exact mechanism 
remains doubtful. Therefore, we investigated the cyto-
toxic potential of juglone and its underlying mecha-
nisms. 
 

Materials and methods 

Materials 

Juglone was kindly gifted by Dr. Ma Zhiqiang. The 
purity of jugone was measured by HPLC and deter-
mined to be 97%. Juglone was dissolved in dimethyl 
sulfoxide (DMSO) to make a stock solution. The concen-
tration of DMSO was kept below 0.3% in all the cell 

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Anti-proliferative and apoptosis-inducing activities of juglone in 
LS-174T cells 
Qiang Li1, Xue Ling Zhao1, Juan Sun1, Shou Gang Jiang2 and Xian Feng Gong1 
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)RUHVW�3ODQW�(FRORJ\��0LQLVWU\�RI�(GXFDWLRQ��1RUWKHDVW�)RUHVWU\�8QLYHUVLW\��+DUELQ���������35�&KLQD� 

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Anti-proliferative and apoptosis-inducing effects of juglone in LS-174T cells 
were investigated. In this study, we showed that juglone inhibited the 
proliferation of LS-174T cells in a time and dose-dependent manner, treatment 
of juglone resulted in the activation of caspase-9 and 3, decrease of Bcl-2. N-
acetylcysteine significantly attenuate LS-174T cell death induced by juglone 
(p<0.001). In addition, NAC could reverse caspase-3 and 9 activation, increase 
expression of Bcl-2 protein. Taken together, these findings indicated that 
juglone-mediated oxidative injury may act as upstream change, trigger ROS 
release, Bcl-2 modulation, caspase activation, and consequently leading cell 
apoptosis in LS-174T cells. In conclusion, these findings suggest that juglone 
may be an effective way for treating human cancers. 

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Received:  2 January 2013 
Accepted:  14 January 2013 
Available Online:   27 January 2013  
 

DOI: 10.3329/bjp.v8i1.13174 
 
 

Cite this article: 
Li Q, Zhao XL, Sun J, Jiang SG, Gong 
XF. Anti-proliferative and apoptosis-
inducing activities of juglone in LS-
174T cells. Bangladesh J Pharmacol. 
2013; 8: 65-72. 

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cultures and did not exert any detectable effect on cell 
growth. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide (MTT), Hoechst 33258, RNase A, 
Proteinase K and N-acetyl-L-cysteine (NAC) were pur-
chased from Sigma Chemical (USA). Cell Death Detec-
tion ELISAPLUS was purchased from Roche (Roche 
Molecular Biochemicals, Germany). Western blotting 
antibodies against caspase-3, 9 and alkaline phospha-
tase conjugated secondary antibodies were purchased 
from Santa Cruz Biotechnology (USA). Antibody 
against Bcl-2 was purchased from Abacam (Cambridge, 
MA). Antibodys against PARP and caspase-3 colori-
metric activity assay kit were purchased from Beyotime 
Institute of Biotechnology (Haimen, Jiangsu). 

Cell culture 

LS-174T, human colon adenocarcinoma cell line (ATCC 
CL-188), were cultured in RPMI-1640 medium 
(Hyclone, Logan, UT) supplemented with 10% heat 
inactivated (56°&, 30 min) fetal calf serum (Hangzhou 
Sijiqing Biological Engineering Materials Co., Ltd., 
Zhejiang), 2 mM glutamine (Gibco, Grand Island, NY), 
and maintained at 37°&�with 5% CO2 in a humidified 
atmosphere.  

Cell growth inhibition test 

The inhibition of cultured cells was determined by MTT 
test (Rasu Azhar et al., 2012). In brief, after incubation 
with juglone (0-200 µM) for indicated time period, LS-
174T cells (5 ´ 104/well) in 96-well plate were washed 
once with PBS and MTT (20 µL of 5 mg/mL in PBS) was 
added to each well. The cells were further incubated at 
37°& for 4 hours, and DMSO (150 µL) was added to 
dissolve the formazan crystals. Absorbance was 
measured at 490 nm with micoplate reader (BioRad 680, 
Hercules, CA). 

Nuclear damage observed by Hoechst 33258 staining 

After treated by 50 µM juglone for 36 hours, LS-174T 
cells were harvested by centrifugation at 1,000 ´ g for 5 
min, washed two times with PBS and fixed with 3.7% 
paraformaldelyde at room temperature for 2 hours. The 
fixed cells were washed with PBS and stained with 
Hoechst 33258 solution for 10 min at room temperature, 
then observed with fluorescence microscopy. 

Determination of DNA fragmentation by agarose gel 
electrophoresis 

LS-174T cells, both adherent and floating, were 
collected by centrifugation at 1,000 ´ g for 5 min. The 
cell pellets were suspended in cell lysis buffer (Tris-HCl 
10 mM pH 7.4, edetic acid 10 mM pH 8.0, Triton-100 
0.5%) and kept at 4°& for 30 min. The lysate was 
centrifuged at 25,000 ´ g for 20 min. The supernatant 
was incubated with 20 mg/mL RNase A (2 µL) at 37°& 
for 1 hours, then incubated with 20 mg/mL proteinase 
K (2 µL) at 37°& for 1 hours. The supernatant was mixed 
with 5 M NaCl (20 µL) and isopropanol (120 µL) at –20°
& overnight, then centrifuged at 25,000 ´ g for 15 min. 
After drying, DNA was dissolved in TE buffer (Tris-
HCl 10 mM pH 7.4, edetic acid 1 mM pH 8.0) and 
separated by 2%agarose gel electrophoresis at 100 V for 
50 min. 

Enzyme-linked immunosorbent assay for apoptosis 

The Cell Death Detection ELISA kit (Roche Molecular 
Biochemicals) was employed to quantify DNA 
fragmentation on the basis of antibody detection of free 
histone according to the protocol of manufactrure. 

Western blot analysis of protein expression 

Western blot analysis was performed as follows. 
Briefly, LS-174T cells treated with different 
concentrations of juglone for 36 hours were harvested, 
rinsed twice with cold PBS, and resuspended in lysis 
buffer, including Hepes 50 mM  pH 7.4, 1%Triton-X 100, 
sodium orthovanada 2 mM, sodium fluoride 100 mM, 
edetic acid 1 mM, egtazic acid 1 mM, PMSF 1 mM, 
aprotinin 0.1 g/L, leupeptin 0.01 g/L, then lysed in 4°& 
for 1 hours. After 13,000 ´ g centrifugation for 10 min, 
the protein content of supernatant was determined 
using Bio-Rad protein assay reagent (Bio-Rad, Hercules, 
CA). Equal amounts of protein (30-40 µg) was subjected 
to 12% SDS polyacrylamide gels and transferred to 
nitrocellulose membranes. Membranes were blocked 
with 5% defatted milk, and then incubated overnight 
with the appropriate primary antibody at dilutions 
specified by the manufacturer, followed by incubation 
at room temperature for 1 hour with the corresponding 
alkaline phosphatase conjugated secondary antibody at 
1:500-1000 dilution in TBST. Bound secondary antibody 
was detected using an BCIP/NBT Alkaline Phosphatase 
Color Development Kit (Beyotime Institute of 
Biotechnology, Haimen, Jiangsu, China) 

Determination of caspase-3 activity 

The activity of caspase-3 was determined using the 
caspase-3 activity kit. Briefly, LS-174T cells were seeded 
in culture flasks (1 × 106 cells/flask) and allowed to 
attach overnight. After treating the cells with juglone 
(12, 25, 50 and 100 μmol/L) or medium for 36 hours, 
cell lysates were prepared by incubating 2 × 106 cells in 
100 mL lysis buffer for 15 min on ice. Cell lysates were 
centrifuged at 20,000 × g for 15 min at 4°&. 
Supernatants were collected and added to an ice-cold 

66 Bangladesh J Pharmacol 2013; 8: 65-72 

2 

2 2+ 

Figure 1: The chemical structure of juglone� 



 

 

centrifuge tube. A blank solution containing 90 mL 
reaction buffer and 10 mL Ac-DEVD-pNA and the 
sample solution for each group including 80 mL 
reaction buffer, 10 mL sample and 10 mL Ac-DEVD-
pNA were incubated in a 96-well microplate for 2 hours 
at 37°&. Caspase-3 activity was measured at 405 nm 
using a microplate reader (Bio-Rad 680, Hercules, CA). 
The active unit of caspase-3 was calculated. One unit is 
the amount of enzyme that will cleave 1.0 nmol/L of 
the colorimetric substrate Ac-DEVD-pNA per hour at 
37°& under saturated substrate concentrations. 

Statistical analysis 

All experiments were repeated at least three times. The 
results from treated and untreated control cells were 

compared using the Student’s t-test to assess statistical 
significance. 
  

Results 

To determine the inhibition of LS-174T cells exposed to 
juglone, cells were treated with juglone of different 
concentrations, ranging from 0 to 200 µM for 24, 36 and 
48 hours. Inhibition of LS-174T cell proliferation was in 
a time- and dose-dependent manner (Figure 2). The IC50 
dose of juglone for 24, 36 and 48 hours was appro-
ximately 56, 30 and 22 µM, respectively. 

Morphological changes were observed by fluorescence 
microscopy. Nuclear morphological changes were 
observed by Hoechst 33258 staining. In control group, 
cells were round in shape and stained homogeneously. 
After 24 hours treatment with 50 µM juglone, blebbing 
nuclei and granular apoptotic bodies appeared (Figure 
3A).  

To further verify apoptosis induced by juglone, the Cell 
Death Detection ELISA kit was employed to quantify 
cytoplasmic histone-associated-DNA-fragments (by 
measuring optical density). The results showed that 
juglone induced significant apoptotic cell death at 25 
and 50 μM (Figure 3B). In addition, DNA fragmenta-
tion became obvious after 25, 50 and 100 µM juglone 
treatment for 36 hours on agarose gel electrophoresis 
(Figure 3C), which further confirmed juglone induced 
cell apoptosis. 

To investigate whether caspase-3 and 9 participated in 
juglone-induced cell apoptosis, expressions of 
procaspse-3 and -9 by western blot analysis were 

 Bangladesh J Pharmacol 2013; 8: 65-72                                                                                    67 

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Figure 2: Juglone induces growth inhibition 
The cells were exposed to juglone at concentrations ranging from 0-200 
μM and incubated for 24 hours (◆), 36 hours (■) and 48 hours (▲). The 
inhibitory ratio was measured by MTT assay. Data represent the mean ± 
SD of three independent experiments� 

Figure 3A:  Juglone-induced apoptotic morphological changes 
LS-174T were incubated in the medium alone for 36 hours (A) or in the medium containing 50 µM juglone for 36 hours (B). Arrows indicates frag-
mented nuclei 

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performed. After incubation with juglone of indicated 
concentrations, expression of procaspse-3 and -9 protein 
was decreased, indicating caspase-3 and -9 activation 
(Figure 4A).  

To further determine whether caspase 3 participated 
juglone induced apoptosis in LS-174T cells, Western 
blot analysis was performed to examine the expressions 
of caspase 3 substrate, PARP, and caspase-3 activity 

was also determined. Figure 4B showed that the 
expression of minor PARP (85 kDa) protein after 
cleavage was up-regulated by juglone, indicating that 
caspase-3 is activated. Caspase-3 activity was increased 
in a dose-dependent manner (Figure 4C). These results 
correspond to the activation of caspase-3 as shown in 
Figure 4A.  

Mitochondrial Bcl-2 family is a series of proteins that 
regulate apoptosis. We measured the expressions of Bcl
-2 by Western blot analysis. After incubation with jug-
lone, expression of Bcl-2 protein was decreased (Figure 
5).  

The generation of ROS may be relate to apoptosis 
induced by juglone, therefore, we investigated the 
induction of apoptosis by juglone to determine if it was 
associated with ROS generation. To accomplish this, LS-
174T cells were pre-treated for 1 hour with 0.4, 2 and 10 
mM NAC, a commonly used reactive oxygen 
intermediate scavenger. The cells were then treated 
with 50 μM juglone for 36 hours. As shown in Figure 
6A, pre-treatment with NAC (0.4, 2 and 10 mM) 
significantly blocked the inhibition induced by juglone 
(p<0.001). Furthermore, pre-treatment with 5 mM NAC 
inhibited procaspase-3 and -9 activation that was 
observed in LS-174T cells in response to juglone 
treatment. Additionally, we also found that pre-
treatment with 5 mM NAC reversed juglone-induced 
Bcl-2 down-regulation (Figure 6B) 

 

Discussion 

Walnut has been used in traditional medicines for 
various ailments and some extracts of walnut are also 

68 Bangladesh J Pharmacol 2013; 8: 65-72 

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Figure 3B: Pro-apoptotic effect of juglone determined by ELISA quantification of DNA fragmentation expressed as optical density 
(OD) values 
LS-174T cells were incubated in the medium alone or in the medium containing 12.5,25,50 and 100 µM juglone for 36 hours (** and  
*** stands for p<0.01 and p<0.001 compared with 0 µM, respectively) 

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Figure 3C: DNA fragmentation induced by juglone in LS-174T 
cells 
a: marker. b-f: LS-174T cells were treated with juglone 0, 12, 25, 50, 100 
µM for 36 h. DNA was isolated by agarose gel electrophoresis and 
analyzed by ethidium bromide staining 



 

 

reported to possess anticancer properties (Duke et al., 
1998). However little has been reported about the 
mechanisms underlying the cytotoxic potential of 
juglone in LS-174T cells. In the present study, we found 
juglone, a main active ingredient in walnut, strongly 
inhibited the growth of LS-174T cells in a concentration- 
and time-dependent manner. Earlier, Segura-Aguilar 
and co-workers (1992) compared the effect of juglone 
and other quinones on human leukemic (HL-60) cells 
and doxorubicin-resistant human leukemic (HL-60R) 
cells and concluded that multidrug resistance that 
develops in the doxorubicin-resistant HL-60R cells had 
no effect on the cytotoxicity of juglone indicating its 
therapeutic possibilities (Segura-Aguilar et al., 1992). In 
addition, we did observe juglone-induced typical 
chromatin condensation and DNA fragmentation 
shown by Hoechst 33258 staining and ethidium 

bromide staining. 

Caspases play an important role in apoptosis (Kwon et 
al., 2003). Caspase-3 is activated either by extrinsic 
pathway (death receptor mediated) or by intrinsic 
pathway (mitochondria dependent pathway) 
(Verhagen et al., 2000). Caspases are proteases that are 
activated during apoptosis, and cleave substrates such 
as PARP (Lazebnik et al., 1994). PARP (116 kDa), a 
DNA repair enzyme, is probably best characterized 
caspase substrate, which is cleaved during apoptosis to 
a 24 kDa and a 85 kDa fragment representing the N-
terminal DNA-binding domain and the C-terminal 
catalytic subunit, respectively. During apoptosis, PARP 
is selectively cleaved by several caspases, especially by 
caspase-3 (Lazebnik et al., 1994; Kaufmann et al., 1993). 
Detection of a 85 kDa or 24 kDa caspase cleavage 

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Figure 4A: The expression of procaspase-3 and procaspase-9 in 0, 12, 25, 50, 100 μM juglone-treated LS-174T cells. The cells were 
treated with juglone 36 hours. Procaspase-3 and procaspase-9 were analysed by western blot. GAPDH was used as an equal load-
ing control  

Figure 4B: The expression of PARP in 0, 12, 25, 50, 100 μM juglone-treated LS-174T cells. The cells were treated with juglone 36 
hours. PARP were analysed by Western blot. GAPDH was used as an equal loading control  

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Figure 4C: Caspase-3 activity of juglone-treated LS-174T cells 
The cells were treated with 0, 12, 25, 50, 100 μM juglone for 36 hours (**stands for p<0.01 compared with 0 µM) 



 

 

fragment of PARP was shown to be a hallmark of 
apoptosis. In this paper, PARP minor 85 kDa fragment 
expression was increased in a time dependent manner, 
and caspase-3 activity was also up-regulated in a 
concentration-dependent manner after treatment with 
juglone, which further confirmed that caspase-3 is 
activated in jugone induced cell apoptosis.  

It is widely accepted that alteration in mitochondrial 
structure and function play an important role in caspase
-9 dependent apoptosis through releasing cytochrome c, 
which interacts with Apaf-1 and procaspase-9 to form 
the apoptosome. Then caspase-9 was activated, which 
in turn cleaves and activated caspase-3, the executioner 
caspase, which cleaves PARP and activates 
endonucleases leading to DNA fragmentation. To verify 
whether mitochondrial pathway participates in juglone-
induced cell apoptosis, procaspase-9 protein was 
determined. The activation of caspase-9 in juglone-
induced cell apoptosis strongly suggests an 
involvement of a mitochondrial pathway.  

The Bcl-2 family of proteins serves as critical regulators 
of pathways involved in apoptosis (Adams et al., 1998). 

The main protagonists are suggested to be anti-
apoptotic Bcl-2 and pro-apototic Bax. If the 
concentration of Bcl-2 is enough to complex with at 
least half of Bax, then apoptosis is prevented (Burlacu et 
al., 2003). In this paper, the Bcl-2 protein in juglone-
treated LS-174T cells was down-regulated. This result 
suggested that the mitochondrial pathway of cell death 
might be involved in juglone -induced LS-174T cells 
death.  

Induction of apoptosis by compounds such as arsenic 
trioxide (As2O3) (Jing et al., 1999; Chen et al., 1998), 
tumor necrosis factor-α (TNF-α)( Larrick et al., 1990), 
ceramide (Quillet-Mary et al., 1997) and erbstatin 
(Shimizu et al., 1996) involves the production of ROS. In 
order to determine whether ROS participates juglone-
induced cells apoptosis, a water-soluble antioxidant, N-
acetylcysteine (NAC) was used. NAC, a sulfhydryl 
group donor, serves as a precursor of GSH synthesis 
(Lauteburg et al., 1983) and inhibits the formation of 
extracellular reactive oxygen intermediates (Nakano et 
al., 1995). In this paper, juglone-induced cytotoxicity 
was markedly decreased by ROS scavenger NAC. In 
addition, activation of caspase-3 and caspase-9 was 

70 Bangladesh J Pharmacol 2013; 8: 65-72 

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Figure 5: The expression of Bcl-2 in 0, 12, 25, 50, 100 μM Juglone-treated LS-174T. The cells were treated with ju-
glone 36 hours. Bcl-2 were analysed by western blot. GAPDH was used as an equal loading control� 

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Juglone 50 μM + - - - + + + 

NAC 10 mM - + - - + - - 

NAC 2 mM - - + - - + - 

NAC 0.4 mM - - - + - - + 

Figure 6A: Effect of NAC on juglone -induced cell death 
One hour prior to the addition of 50 μM juglone, LS-174T cells were treated with NAC (0.4, 2 and 10 mM), then incubated for 36 
hours. The inhibition was measured by MTT method. `x ± s, n = 3 (***stands for p<0.001)  



 

 

reversed by NAC and down-regulation of bcl-2 protein 
was also reversed, suggesting plausible role of ROS in 
juglone-induced apoptosis. 

 

Conclusion 

Our in vitro studies suggested that juglone could inhibit 
growth of LS-174T cells in a dose- and time-dependent 
manner. In addition, juglone was shown to induce 
apoptosis. This apoptotic response was associated with 
the down-regulation of Bcl-2 and activation of caspase-3 
and -9. Taken together, juglone may be a promising 
chemopreventive and chemotherapeutic agent against 
colon adenocarcinoma. 

 

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 Bangladesh J Pharmacol 2013; 8: 65-72                                                                                    71 

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Figure 6B: Effect of NAC on juglone-induced procaspase-3 and procaspase-9 activation and Bcl-2 degradation. The cells were 
treated with 50 μM juglone for 36 h in the presence or absence of 5 mM NAC, followed by western blot analysis for detection of 
procaspase-3, procaspase-9 and Bcl-2 expressions. GAPDH was used as an equal loading control  

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Xian Feng Gong (Principal contact) 
e-mail: gongxianfeng@yahoo.com.cn 

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