Inhibition of Wnt/b-catenin signaling mediates ursolic ...€¦ · Inhibition of Wnt/b-catenin...

Inhibition of Wnt/b-catenin signaling mediates

ursolic acid-induced apoptosis in PC-3 prostate

cancer cells

Ji-Hyuk Park1+, Hee-Young Kwon1+, Eun Jung Sohn1+, Kyung A Kim2,

Bonglee Kim1, Soo-Jin Jeong1, Jun ho Song1, Jin Suk Koo3, Sung-Hoon Kim1

1College of Oriental Medicine, Kyung Hee University, Seoul 130-701, South Korea

2Department of Orthodontics, Kyung Hee University School of Dentistry, South Korea

3College of Natural Sciences, Andong National University, Andong, 760-749, South Korea

Correspondence: Sung-Hoon Kim, e-mail: [email protected]

Abstract:

Background: Ursolic acid, a pentacyclic triterpenoid, is known to exert antitumor activity in breast, lung, liver and colon cancers.

Nonetheless, the underlying mechanism of ursolic acid in prostate cancer cells still remains unclear. To investigate the antitumor

mechanism, the apoptotic mechanism of ursolic acid via Wnt/b-catenin signaling was examined in PC-3 prostate cancer cells.

Methods: Cytotoxicity assay, flow cytometry, immunofluorescence assay and western blotting were performed.

Results: Ursolic acid showed cytotoxicity against PC-3, LNCaP and DU145 prostate cancer cells with IC50 of 35 µM, 47 µM and

80 µM, respectively. Also, ursolic acid significantly increased the number of ethidium homodimer stained cells and apoptotic bod-

ies, and dose-dependently enhanced the sub-G1 apoptotic accumulation in PC-3 cells. Consistently, western blotting revealed that

ursolic acid effectively cleaved poly (ADP-ribose) polymerase (PARP), activated caspase-9 and -3, suppressed the expression of sur-

vival proteins such as Bcl-XL, Bcl-2 and Mcl-1, and upregulated the expression of Bax in PC-3 cells. Interestingly, ursolic acid sup-

pressed the expression of Wnt5a/b and b-catenin, and enhanced the phosphorylation of glycogen synthase kinase 3 b (GSK3b).

Furthermore, the GSK3b inhibitor SB216763 or Wnt3a-conditioned medium (Wnt3a-CM) reversed the cleavages of caspase-3 and

PARP induced by ursolic acid in PC-3 cells.

Conclusions: Our findings suggest that ursolic acid induces apoptosis via inhibition of the Wnt5/b-catenin pathway and activation

of caspase in PC-3 prostate cancer cells. These results support scientific evidence that medicinal plants containing ursolic acid can be

applied to cancer prevention and treatment as a complement and alternative medicine (CAM) agent.

Key words:

ursolic acid, apoptosis, caspase, Wnt/GSK3b/b-catenin signaling

1366 Pharmacological Reports, 2013, 65, 1366�1374

Pharmacological Reports2013, 65, 1366�1374ISSN 1734-1140

Copyright © 2013by Institute of PharmacologyPolish Academy of Sciences

+Contributed equally to this work

Introduction

Prostate cancer is internationally the second most

common cancer diagnosed in men and the sixth most

common cause of cancer death among men. Accord-

ing to the NCI report, the incidence rate of prostate

cancer is approximately 16.22% in men born today [2,

6]. Recent clinical practice shows that medicinal

herbs with antitumor activities have chemopreventive

potential with few side effects [4, 35].

Ursolic acid as a pentacyclic triterpene acid is

mainly contained in several fruits such as hawthorn,

prunes, peppermint, apples, rosemary, lavender, and

also medicinal plants including Rosmarinus officina-

lis, Oldenlandia diffusa, Eriobotrya japonica, and

Glechoma hederaceae [31]. As a whole, ursolic acid

has been suggested as a non-toxic chemopreven-

tive/chemoprotective agent in clinical practice [20],

even though there are some evidences that ursolic

acid causes the DNA damages in human endothelial

cells [18] and reduces the sperm motility [3].

Previous evidence shows that ursolic acid induces

apoptosis in several cancers such as colon cancer [1],

breast cancer [11], leukemia [15], melanoma [12] and

prostate cancer [24]. Several papers studied the effect

of ursolic acid in vivo models. Ursolic acid signifi-

cantly inhibited tumor growth in transgenic mouse

prostate model [24] and prostate cancer xenograft in

nude mice [23]. Although ursolic acid exerts antitu-

mor activity via inhibition of CXCR4/CXCL12 [23],

matrix metalloproteinase 2 (MMP2) [12], P2Y2/Src/

p38/COX-2 [13] and the AKT pathway [37] as well as

autophagy [25], the antitumor mechanism of ursolic

acid still remains unclear in prostate cancers. Thus, in

the present study, the role of Wnt/b-catenin signaling

was elucidated in ursolic acid-induced apoptosis in

PC-3 prostate cancer cells using XTT assay, cell cycle

analysis, and western blotting.

Materials and Methods

Cell culture

PC-3, DU145, LNCaP prostate cancer cells, Raw

264.7 (leukemic monocyte macrophage cells) and

HEK293 (human embryonic kidney 293) cells were

obtained from American Type Culture Collection

(ATCC). HEK293, PC-3 and DU145 cells were main-

tained in Roswell Park Memorial Institute (RPMI)

1640 supplemented with 10% fetal bovine serum

(FBS) (Gibco, Carlsbad, CA, USA) and 1% antibiot-

ics at 37°C in a humidified atmosphere containing 5%

CO2. LNCaP cells were cultured in RPMI 1640 sup-

plemented with 10% FBS and 2 µmol/l L-glutamine,

10 µmol/l HEPES, 1 µmol/l sodium pyruvate, and

4.5% D-glucose without antibiotics at 37°C in a hu-

midified atmosphere containing 5% CO2. Wnt3a CM

was prepared as previously described [21].

Cytotoxicity assay

To investigate the cytotoxicity, 3-[4,5-dimethylthia-

zol-2-yl]-2,5-diphenyltetrazolium (MTT) bromide col-

orimetric assay was used. Prostate cancer cells were

plated at 104 cells in 96-well plates and incubated over-

night. Cells were treated with various concentrations of

ursolic acid. After 24-h incubation, 100 ml of MTT

(Sigma Chemical Co., St. Louis, MO, USA) was added

to each well and incubated for 4 h at 37°C. Formazan

crystals were dissolved by addition of 100 ml DMSO

solution. The microplate reader at 570 nM was used to

determine the absorbance of each well.

Ethidium homodimer assay

To measure cell death, we used the DAPI and ethid-

ium homodimer dye following the manufactures’ in-

structions (Molecular Probes). In brief, PC-3 cells

were treated with 30 µM ursolic acid for 24 h. After

incubation, cells were fixed in 4% methanol-free for-

maldehyde solution and stained with the 5 µM ethid-

ium homodimer and then incubated at 37°C for

30 min in the dark. Then, the cells were mounted with

mounting medium containing DAPI and visualized

under an Axio vision 4.0 fluorescence microscope

(Carl Zeiss Inc., Weimar, Germany).

Cell cycle analysis

PC3 cells treated with ursolic acid or GSK3b inhibitor

SB216763 (10 µM) for 24 h were fixed in 75% etha-

nol at –20°C, resuspended in PBS containing RNase

A (1 mg/ml), and incubated for 1 h at 37°C. The fixed

cells were stained with propidium iodide (50 µg/ml)

for 30 min at room temperature. The DNA contents

were analyzed using CellQuest Software with the

FACSCalibur flow cytometry (Becton Dickinson,

Franklin Lakes, NJ, USA).

Pharmacological Reports, 2013, 65, 1366�1374 1367

Wnt signaling mediates ursolic acid-induced apoptosisJi-Hyuk Park et al.

Western blotting

Whole cell lysates from the prostate cancer cells

exposed to ursolic acid or SB216763 (10 µM) or

Wnt3a-CM for 24 h were prepared using lysis buffer

(50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton

X-100, 0.1% SDS, 1 mM EDTA, 1 mM Na3VO4,1 mM NaF and protease inhibitor cocktail). The pro-

tein contents in the supernatants were measured by

using a Bio-Rad DC protein assay kit II (Bio-Rad,

Hercules, CA, USA), separated on 4–12% NuPAGE

Bis-Tris gels (Invitrogen, Carlsbad, CA, USA) and

electro-transferred onto a Hybond ECL transfer mem-

brane (GE Health Are Bio-Science, Piscataway, NJ,

USA). The membranes were blocked with 5% nonfat

dry milk and immunoblotted with anti-cleaved

caspase-3, caspase-3, cleaved caspase-9, caspase-9,

PARP, Bax, Bcl-2, Bcl-xL, Mcl-1L, Wnt5a/b, p-

GSK3b, GSK3b or b-catenin (Cell Signaling, Dan-

vers, MA, USA) antibodies.

Immunofluorescence assay

PC 3 cells in the absence or presence of ursolic acid

were fixed with 4% paraformaldehyde and permeabi-

lized in cold methanol for 15 min at –20°C. Fixed

cells were then washed twice with 1 × PBS, followed

by blocking with 10% normal goat serum blocking

solution (Zymed Laboratories, Carlsbad, CA, USA)

for 30 min. Cells were incubated with the primary an-

tibodies against b-catenin (Cell Signaling, Danvers,

MA, USA) for overnight at 4°C. The cells were

washed three times in 1 × PBS and incubated with Al-

exa Fluor 594 goat anti-rabbit IgG (Invitrogen, Carls-

bad CA, USA) for 45 min at room temperature in

a humidified chamber. Cells were washed in 1 × PBS,

mounted with Vectashield/DAPI (Vector Laborato-

ries, Burlingame, CA, USA) and visualized by a Carl

Zeiss LSM5 confocal microscope.

Statistical analysis

Statistical analysis of the data was conducted using

Sigmaplot version 12 software (Systat Software Inc.,

San Jose, CA, USA). All data were expressed as the

means ± standard deviation (SD). The statistically sig-

nificant differences between control and ursolic acid-

treated cells were calculated by the Student’s t-test.

Results

Cytotoxic effects of ursolic acid on PC-3, DU145and LNCaP prostate cancer cells

The cytotoxicity of ursolic acid (Fig. 1A) was evalu-

ated against PC-3, DU145 and LNCaP prostate cancer

cells using MTT assay. Cells were treated with vari-

ous concentrations of ursolic acid (0, 5, 10, 20, 40 or

80 µM) for 24 h. As shown in Figure 1B, ursolic acid

exerted cytotoxicity in PC-3, DU145 more than in

LNCaP cells. In contrast, ursolic acid did not show

significant cytotoxicity against Raw 264.7 leukemic

monocyte macrophage cells, while only high concen-

tration of ursolic acid (80 µM) affected the viability of

the cells.


Fig. 1. Cytotoxic effect of ursolic acid in prostate cancer cells.(A) Chemical structure of ursolic acid (MW = 456). (B) Human pros-tate cancer cell lines PC-3, DU145, LNCaP or leukemic monocytemacrophage cell line Raw 264.7 were treated with various concentra-tions of ursolic acid (0, 5, 10, 20, 40 or 80 µM) for 24 h. Cell viabilitywas determined by XTT assay. The data represent the means ± SD.(C) Morphological feature of PC-3 cells were observed in inverted mi-croscope (100´). Cells were treated with ursolic acid (0, 7.5, 15 or30 µM) for 24 h. (D) PC-3 cells were treated with 30 µM of ursolic acidand then stained with ethidium bromide homodimer dye. Signalswere visualized under an Axio vision 4.0 fluorescence microscope

Apoptotic bodies were also observed in ursolic

acid-treated PC-3 cells under inverted microscope, in-

dicating apoptotic feature of ursolic acid, while intact

morphology was shown in untreated control (Fig. 1C).

Similarly, cell death was confirmed in ursolic acid

treated PC-3 cells by ethidium homodimer assay. As

shown in Figure 1D, ethidium homodimer assay re-

vealed apoptotic features in ursolic acid treated PC-3

cells compared to untreated control.

Ursolic acid increased the sub-G1 apoptotic

portion in PC-3 cells

To confirm whether the cytotoxicity of ursolic acid

against PC-3 cells was due to apoptosis induction, cell

cycle analysis was carried out. As shown in Figure 2,

ursolic acid increased sub-G1 apoptotic portion by 1.30

± 0.02, 5.95 ± 1.43, 24.33 ± 0.16, and 49.93 ± 4.09% at

the concentrations of 7.5, 15, 30 and 60 µM, respec-

tively, compared to untreated control (1.45 ± 0.23%).



Fig. 2. Effect of ursolic acid on thesub-G1 apoptotic portion in PC-3 cells.Cells were untreated or treated withvarious concentraions of ursolic acid(7.5, 15 or 30 µM) for 24 h. After fixingin 75% ethanol, cells were stained withpropidium iodide (PI) and cell cycle wasanalyzed by flow cytometry. (A) Histo-grams of flow cytometry analysis re-veal cell cycle distribution of eachsample. (B) Graphs represent per-centages of apoptotic portion. Datarepresent the means ± SD. * p < 0.05vs. untreated control

Ursolic acid activated caspase cascades andregulated the Bcl-2 family proteins in PC-3 cells

Generally, apoptosis is induced through two distinctive

pathways such as cell death extrinsic pathway and mi-

tochondrial dependent intrinsic pathway [8]. Western

blotting showed that ursolic acid activated caspase-9

and -3 and cleaved PARP in PC-3 cells as shown in

Figure 3A, implying that ursolic acid induces apoptosis

via mitochondrial dependent pathway in PC-3 cells.

Anti-apoptotic Bcl-2 family proteins such as Bcl-2

and Bcl-xL are frequently overexpressed in cancers [22].

In the present study, western blotting revealed that urso-

lic acid suppressed the expression of Bcl-XL, Bcl 2, and

Mcl-1L as anti-apoptotic genes and also up-regulated the

expression of Bax in PC-3 cells as shown in Figure 3B.

To further compare the apoptotic activity of ursolic

acid in other prostate cancer cells, the effect of ursolic

acid on apoptosis related proteins such as PARP, cas-

pase 3, Bcl-xL, Bcl-2 in Du145 and LNCaP cells was

examined by western blotting. Here, ursolic acid acti-

vated PARP and Bax as well as suppressed the expres-

sion of Bcl-xL and Bcl-2 in DU145 cells, while ursolic

acid cleaved PARP and suppressed the expression of

Bcl-xL in LNCaP cells (Figs. 3C and D).

Ursolic acid induced apoptosis through theWnt5/GSK3b/b-catenin signaling in PC-3 cells

Glycogen synthase kinase 3b (GSK3b), a regulator of

glycogen metabolism is involved in protein synthesis,

cell proliferation, cell differentiation, microtubule dy-

namics, cell motility and apoptosis [9]. GSK3b plays

a critical role in Wnt/b-catenin signaling pathway

[34]. Here, ursolic acid suppressed the expression of

Wnt5a/b and b-catenin, and enhanced the phosphory-

lation of GSK3b at Ser 9 in PC-3 cells (Fig. 4A).

GSK3b selective inhibitor SB216763 blocked the de-

creased expression of Wnt5a/b and b-catenin, and the

activated phosphorylation of GSK3b at Ser 9 induced

by ursolic acid (Fig. 4B). Likewise, GSK3 inhibitor

SB216763 prevented the cleavages of caspase-3 and

PARP induced by ursolic acid in PC-3 cells (Fig. 4C),

implying that ursolic acid-induced apoptosis is con-

trolled through the Wnt5/GSK3b/b-catenin signaling.

Wnt (Wnt1, Wnt3a, and Wnt8) signalings promote

the dissociation of b-catenin from Axin and inhibit

b-catenin phopsphorylation and subsequently b-ca-

tenin degradation [14, 16]. As shown in Figure 4D,

ursolic treatment induced the degradation of b-catenin

(Fig. 4D lower panel), while b-catenin exhibited sub-


Fig. 3. Effect of ursolic acid on activa-tion and expression of apoptosis-related protein in PC-3 cells. Cellswere treated with various concentra-tions of ursolic acid (0, 3.75, 7.5, 15 or30 µM) for 24 h. Western blotting wasperformed to see apoptosis-relatedproteins on PC-3 (A, B), DU145 (C)and LNCaP (D) cells

cellular localization in PC-3 cells (Fig. 4D upper

panel). HEK 293 cells treated with 20 µM of ursolic

acid in the presence of Wnt3a-conditioned medium

blocked the apoptotic signaling such as PARP and

caspase 3 (Fig. 4E). Also, ursolic acid attenuated the

expression of Wnt5a and b-catenin in DU145 and

LNCaP cells (Figs. 4 F and G).

Several studies reported that Wnt3a activates b-ca-

tenin dependent canonical Wnt signaling and inhibits

the proliferation [19, 26]. Thus, we investigated whether

or not Wnt3a-CM affected the apoptosis induced by ur-

solic acid. HEK 293 cells were treated with ursolic acid

in the absence and presence of Wnt3a-CM. As shown

in Figure 4E, Wnt3-CM treatment reversed the de-

creased b-catenin and increased cleavages of PARP

and caspase 3 induced by ursolic acid in HEK 293

cells, implying that Wnt signaling mediates the apopto-

sis induced by ursolic acid in HEK 293 cells.



Fig. 4. Effect of ursolic acid on theWnt5/GSK3b/b-catenin signaling inPC-3 cells. (A) Cells were treated withvarious concentrations of ursolic acid(0, 3.75, 7.5, 15 or 30 µM) for 24 h.Western blotting was performed forWnt5a/b, GSK3b and b-catenin. (B, C)Cells were treated with ursolic acid (30µM) and/or GSK3b inhibitor SB216763(10 µM) for 8 h. Western blotting wascarried out with indicated antibodiessuch as Wnt5a/b, GSK3b and b-ca-tenin (B), cleaved PARP and cleavedcaspase-3 (C). (D) Cellular localizationof b-catenin was evaluated by indirectimmunofluorescence using b-cateninantibody. Immunofluorescence label-ing of b-catenin (red) in ursolic aciduntreated or treated PC-3 cells for 24 hare shown. Nuclei were shown withDAPI (blue). (E) HEK 293 cells weretreated with 20 µM of ursolic acid in theabsence or presence of Wnt3a-condi-tioned medium (Wnt3a-CM). Westernblotting was carried out with b-catenin,PARP and cleaved caspase 3. (F, G)Various concentrations of ursolic acid(0, 3.75, 7.5, 15 or 30 µM) were treatedin DU145 and LNCaP cells for 24 h.Western blotting was carried out to de-tect Wnt5a/b, GSK3b and b-catenin inDU145 (F) and LNCaP cells (G)

GSK3b inhibitor blocked the sub G-1 population inursolic acid treated PC-3 cells

To determine whether ursolic acid induces apoptosis

via GSK3b signaling, we used a GSK3b inhibitor. The

cells treated by ursolic acid were cultured in the ab-

sence or presence of GSK3b inhibitor and then flow

cytometry analysis was performed. As shown in Figure

5, GSK3b inhibitor significantly blocked the sub G-1

population to 11.18 ± 0.61% compared to ursolic acid

treated cells (23.31 ± 0.13%), indicating that UA in-

duced the apoptosis through GSK3-b activation.

Discussion

Apoptosis, also known as programmed cell death, is

one of cell death type distinct from necrosis. More re-

cently, apoptosis induction has been regarded as the

major therapeutic target for cancer chemotherapy [5,

32]. In the current study, ursolic acid showed more

significant cytotoxic effects in PC-3 cells than DU145

or LNCaP prostate cancer cells. Thus, we performed

subsequent experiments with PC-3 cells. Here we

found that the cytotoxic effect of ursolic acid was in-

duced by apoptosis, not necrosis, by observing apop-

totic bodies in ursolic acid-treated PC-3 cells. Consis-

tently, ursolic acid significantly increased cell death

biomarker ethidium homodimer stained cells and the

sub-G1 apoptotic portion in PC-3 cells, implying the

apoptotic activity of ursolic acid. Western blotting

showed that ursolic acid effectively induced PARP

cleavages and also activated caspase-9 and -3 in PC-3

cells. Furthermore, ursolic acid suppressed the ex-

pression of survival genes such as Bcl-XL, Bcl-2 and

Mcl-1L in PC-3 cells, indicating that the regulation of

anti-survival genes mediates ursolic acid-induced

apoptosis in PC-3 cells.


Fig. 5. Effect of ursolic acid in the pres-ence of GSK3b inhibitor on the sub-G1apoptotic portion in PC 3 cells. (A)Cells were untreated or treated with ur-solic acid (30 µM) or GSK3b inhibitor(10 µM) for 24 h and stained withpropidium iodide (PI). Cell cycle distri-bution was analyzed by flow cytome-try. (B) Bar graphs represent the per-centages of sub-G1 DNA contents un-dergoing apoptosis. Data representthe means ± SD. * p < 0.05 vs. un-treated control. ### p < 0.001 vs. urso-lic acid

The Wnt/b-catenin pathway is involved in regulat-

ing the cell proliferation and cell differentiation of

neural stem/progenitor cells [30]. Also, there are ac-

cumulating evidences that GSK3b is closely associ-

ated with b-catenin signaling [27, 36]. The axin/

GSK3/APC complex normally promotes the prote-

olytic degradation of the b-catenin intracellular sig-

naling molecule [10]. GSK3b is involved in a wide

range of cellular processes including differentiation,

growth, motility and apoptosis. Thus, the aberrant

regulation of GSK3b has been implicated in various

diseases including Alzheimer’s disease, non-insulin-

dependent diabetes mellitus and cancer [7, 17]. Since

the balance of inhibitory phospho-serine9 (pSer9; in-

active form) and stimulatory phospho-tyrosine216

(pY216; active form) of GSK3b is considered a hall-

mark of its transient kinase regulation in normal

CD34+ cells and several human cell types, previous

studies demonstrated that GSK3b phosphorylation at

serine enhances apoptosis in breast cancer [29], me-

dulloblastoma [33] and SK-OV-3 cells [28]. Here, ur-

solic acid suppressed the expression of Wnt5a/b and

b-catenin, and induced the phosphorylation of GSK3b

at Ser 9, which is an indicator of GSK3b deactivation

in PC-3 cells. These data imply that ursolic acid-

induced apoptosis can be mediated by Wnt5a/b sup-

pression, b-catenin degradation and GSK3b inactiva-

tion in PC-3 cells. Consistently, GSK3b inhibitor

SB216763 or Wnt3a-CM blocked the cleavages of

caspase- 3 and PARP in ursolic acid-treated PC-3

cells, further confirming the involvement of GSK3b

phosphorylation in ursolic acid-induced apoptosis in

PC-3 cells. These results support scientific evidence

that medicinal plants such as Oldenlandia diffusa,

Eriobotrya japonica, Rosmarinus offýcinalis and Gle-

choma hederaceae containing ursolic acid as a lead-

ing compound can be applied to cancer prevention

and treatment as a complementary and alternative

medicine (CAM) agent.

Conflict of interest:

The authors declare no conflict of interest.

Acknowledgment:

This work was supported by the National Research Foundation of

Korea (NRF) grant funded by the Korea government [MEST] (No.

2012-0005755).

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Received: October 8, 2012; in the revised form: May 10, 2013;

accepted: June 7, 2013.


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