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Journal of Ethnopharmacology 135 (2011) 162–172

Contents lists available at ScienceDirect

Journal of Ethnopharmacology

journa l homepage: www.e lsev ier .com/ locate / je thpharm

aeonia lactiflora Pall inhibits bladder cancer growth involving phosphorylationf Chk2 in vitro and in vivo

ing-Tsz Oua, Cheng-Hsun Wub,1, Jeng-Dong Hsuc, Charng-Cherng Chyaud,uei-Jane Leea,∗, Chau-Jong Wanga,e,∗

Institute of Biochemistry and Biotechnology, College of Medicine, Chung Shan Medical University, Taichung, TaiwanDepartment of Anatomy, China Medical University, Taichung, TaiwanDepartment of Pathology, Chung Shan Medical University Hospital, Taichung, TaiwanInstitute of Biotechnology, College of Medicine and Nursing, Hung Kuang University, Taichung, TaiwanDepartment of Medical Research, Chung Shan Medical University Hospital, Taichung, Taiwan

r t i c l e i n f o

rticle history:eceived 1 December 2010eceived in revised form 24 February 2011ccepted 3 March 2011vailable online 9 March 2011

eywords:aeonia lactiflora Pall (RPA)poptosisell cycle2/M phaseheckpoint kinase 2

a b s t r a c t

Ethnopharmacological relevance: Extracts of Paeonia lactiflora Pall (RPA), a traditional Chinese medicineshas been shown to treat cancers.Aim of the study: The purpose of this study is to evaluate the anticancer effect of RPA in urinary bladdercarcinoma in vitro and in vivo.Materials and methods: The cell viability was analyzed with DAPI. Flow cytometry and Western blot wereused to study the apoptosis and cell cycle related mechanism. A rat model of bladder cancer was inducedby N-butyl-N-(4-hydroxybutyl) nitrosamine (OH-BBN). Tumors were analyzed with immunohistochem-ical analysis.Results: Our data suggested that RPA inhibits growth of bladder cancer via induction of apoptosis and cellcycle arrest. Treatment of TSGH-8301 cells with RPA resulted in G2-M phase arrest that was associatedwith a marked decline in protein levels of cdc2, cyclin B1, cell division cycle 25B (Cdc25B) and Cdc25 C. We

ladder carcinogenesis also reported that RPA-mediated growth inhibition of TSGH-8301 cells was correlated with activation ofcheckpoint kinase 2 (Chk2). Herein, we further evaluated urinary bladder cancer using a model of bladdercancer induced by OH-BBN. Analysis of tumors from RPA-treated rats showed significant decrease in theexpression of Bcl2, cyclin D1, and PCNA, and increase in the expression of p-Chk2 (Thr-68), Bax, andCip1/p21.

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Conclusion: Our data provbladder cancer.

. Introduction

Bladder cancer is a common malignancy annually, afflictingore than 2 million people worldwide. It ranks the second most

Abbreviations: RPA, Paeonia lactiflora Pall; OH-BBN, N-butyl-N-(4-hydroxybutyl)itrosamine; cdc2, cyclin-dependent kinase 1; Cdc25B, cell division cycle5B; Cdc25C, cell division cycle 25C; TCC, transitional urothelial cell carcino-as; HPLC, high-performance liquid chromatography; PDA, photodiode-array;

C–MS, liquid chromatography–tandem mass spectrometry; ESI, electrosprayonization; CID, collision-induced dissociation; FBS, fetal bovine serum; Z-AD-FMK, N-benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluromethylketone; DPAI, 4,-diamidino-2-phenyl-indole; PBS, phosphate buffer solution; PI, propidium iodide;CNA, proliferating cell nuclear antigen.∗ Corresponding authors at: Institute of Biochemistry and Biotechnology, Col-

ege of Medicine, Chung Shan Medical University, No. 110, Sec. 1, Jianguo N. Road,aichung 402, Taiwan. Tel.: +886 4 24730022x11675; fax: +886 4 23248195.

E-mail addresses: lhj@csmu.edu.tw (H.-J. Lee), wcj@csmu.edu.tw (C.-J. Wang).1 Contributed equally to this work.

378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2011.03.011

e experimental evidence that RPA could modulate apoptosis in models of

© 2011 Elsevier Ireland Ltd. All rights reserved.

common cause of death among genitourinary tumors, with morethan 70,980 new cases from 2009 (Jemal et al., 2009) and 95%of these tumors are transitional urothelial cell carcinomas (TCC)(Jemal et al., 2008). Approximately 75% of bladder cancer occurs inmen and 25% in women (Shen et al., 2008). The standard treatmentof bladder cancer has been radical cystectomy with urinary diver-sion. However, half of these patients subsequently develop diseaserecurrence (Stein et al., 2001).

Because of unsatisfactory outcomes associated with treatingadvanced cases of bladder cancer, the novel preventive approachesare needed to control this disease. One such preventive approachis through chemoprevention by using naturally occurring dietarysubstances (Khan et al., 2008). In recent years, there has been

considerable activity in establishing the usefulness of natu-rally occurring dietary agents for chemoprevention as well aschemotherapy of bladder cancer.

Paeonia lactiflora Pall (Radix Paeoniae Alba; RPA) is a traditionalChinese herbal medicine. The root bark is cut into thin slices and

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T.-T. Ou et al. / Journal of Ethno

ried in the sun. It has been frequently used as an important ingre-ient in many traditional prescriptions and is commonly used forourishing blood, alleviating pain, reducing irritability, as well asreating liver disease and cancer. A recent study reported that thextract of RPA inhibits the growth of hepatocellular carcinoma andL-60 leukemic cells and induces their apoptosis (Lee et al., 2002;won et al., 2006). Several constituents isolated from RPA have alsoeen found to have immunologically activities (Tomoda et al., 1993,994). However, the underlying mechanism of RPA as an anticancergent has not yet been defined.

Studies conducted by our group have also shown that polyphe-ol possesses strong anticancer efficacy against human bladderancer in vitro, where it inhibits the growth of human bladderransitional cell carcinoma cells (TSGH-8301) by causing cell cyclerrest (Ou et al., 2010). In the present study, we show that RPAnduces apoptosis and cell growth inhibition of human bladderancer TSGH-8301 cells in in vitro system and significantly inhibitsumor growth in in vivo OH-BBN-induced mouse model, and thathese effect are mediated through the Chk2 signaling.

. Materials and methods

.1. Preparation of extract of P. lactiflora Pall (RPA)

P. lactiflora Pall (RPA) was purchased in Changhwa in Taiwan.o prepare the aqueous extract of RPA, 100 g of dried material wasxtracted with 3 L of boiling water for 2 h. The filtrate was collectedfter filtration and the lyophilized. The powder obtained was storedt −20 ◦C until use. The average yield of dried extract was about2.15%.

.2. HPLC system and conditions

The HPLC apparatus consisted of a Finnigan Surveyor moduleeparation system and a photodiode-array (PDA) detector (Thermolectron Co., MA, USA). The chromatographic separation of the com-ounds was achieved at an elution flow rate of 0.2 ml/min using annalytical column (Luna 3 � C18 (2), 150 mm × 2.0 mm) equippedith a guard column [SecurityGuard C18 (ODS), 4 mm × 3.0 mm ID,

henomenex, Inc., Torrance, CA]. The elution was performed by gra-ient elution using two solvents: solvent A (water containing 0.1%ormic acid) and solvent B (acetonitrile containing 0.1% formic acid).he entire course of programmed gradient elution was carried outs follows: 0–3 min, with 10% B isocratic; 3–15 min, with 10–30%; 15–20 min, with 30% B isocratic, 20–50 min, with 30–90% B,0–60 min, with 90% B isocratic; and 60–70 min, followed by chang-

ng 90% to 10% of B. Then 20 �L of the sample extract was directlynjected into the column using a model 7725i Rheodyne injectionalve. The absorption spectra of eluted compounds were scannedithin 210–400 nm using the in-line PDA detector monitored at

30, 270 and 320 nm, respectively.

.2.1. Liquid chromatography–tandem mass spectrometryLC–MS) instrumentation and conditions

The LC elute was introduced directly into a Finnigan LCQdvantage MAX ion trap mass spectrometer operated in electro-pray ionization (ESI) with negative ionization mode. The ion trapnstrument was set as follows: capillary voltage, −4.0 V; tube lensffset, −30 V; source voltage, 3.5 kV; ion transfer capillary tem-erature, 320 ◦C; nitrogen sheath gas, 40; and auxiliary gas, 5 (inrbitrary units). Mass spectra were acquired in an m/z range of

50–1000. Furthermore, MS/MS analysis was performed on theelected molecular ions of the major peaks, i.e., m/z 169, 525 and43 to confirm the structures, where the collision-induced disso-iation (CID) fragments were produced using normalized collisionnergies with an increment of 38% and with wideband activation

acology 135 (2011) 162–172 163

“off”. Helium collision gas was introduced in accordance with themanufacturer’s recommendations.

2.3. Cell lines and reagents

Human urinary bladder cancer cells (TSGH-8301) were derivedfrom a well-differentiated human TCC of the urinary bladder (Yehet al., 1988) and purchased from the Bioresource Collection andResearch Center. Cells were maintained in RPMI 1640 medium,supplemented with 10% fetal bovine serum (FBS), 1% penicillinand 1% streptomycin at 37 ◦C in a humidified atmosphere contain-ing 5% CO2. The media was changed every other day. Bcl2, Bax,cyclin-dependent kinase 1 (cdc2), cyclin B1, caspase-3, caspase-9,and Chip1/p21 antibodies were obtained from Santa Cruz Biotech-nology (Santa Cruz, CA). Cdc25C and phosphor-Chk2 antibodieswere purchased from Cell Signaling Technology (Billerica, MA).The general caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluromethylketone (Z-VAD-FMK) was obtained from Sigma(St. Louis, MO). OH-BBN was purchased from TCI America.

2.4. DAPI staining

Cell morphological characteristic of apoptosis was examinedby fluorescence microscopy of 4, 6-diamidino-2-phenyl-indole(DPAI)-stained cells. The monolayer of cells was washed in phos-phate buffer solution (PBS) and fixed with 4% paraformaldehydefor 30 min at room temperature. The fixed cells were permeabi-lized 3 times with 0.2% Triton X 100 in PBS, and incubated with1 �g/ml of DAPI for 30 min, and then washed with PBS 3 times. Theapoptotic nuclei were examined under 400× magnification using afluorescent microscope with a 340/380 nm excitation filter.

2.5. Quantification of apoptosis by flow cytometry

For quantification of apoptosis, TSGH-8301 cells were grown at adensity of 50–60% confluence in 100-mm culture dishes and treatedwith RPA for 24 h. The cells were trypsinized, washed with PBS,and processed for labeling with FITC-conjugated Annexin V andpropidium iodide (PI) according to the manufacturer’s instructions(BD Biosciences). The labeled cells were analyzed by flow cytometry(FACS Calibur; BD Biosciences).

2.6. DNA cell cycle analysis

TSGH-8301 cells (50–60% confluent) were synchronized byovernight serum starvation, were treated with 5 �mol/L Z-VAD-FMK (a general caspase inhibitor) for 3 h, and then treated withRPA (0.5–2 mg/ml) for 24 h in complete medium. The cells weretrypsinized, washed twice with chilled PBS, and centrifuged. Thecell pellet was resuspended in 50 �l cold PBS to which ethanol (70%)was added, and the cells were incubated for 24 h at −20 ◦C. The cellswere centrifuged at 1000 rpm for 5 min; the pellet was washedtwice with chilled PBS, incubated with 500 �l propidium iodide(50 �g/ml final concentration) for 30 min and analyzed by flowcytometry. A minimum of 10,000 cells per sample was counted andDNA histograms were further analyzed by using CellQuest software(BD Biosciences) for cell cycle analysis.

2.7. Immunoblot analysis

After treatment with the desired concentration of the RPA for

24 h, the medium was removed and rinsed with PBS at roomtemperature. Then 0.5 ml of cold RIPA buffer (1% NP-40, 50 mMTris-base, 0.1% SDS, 0.5% deoxycholic acid, 150 mM NaCl, pH 7.5)with fresh protease inhibitor was added. Cells were scraped and thelysate was centrifuged at 10,000 × g for 10 min. Cell lysate (50 �g)

164 T.-T. Ou et al. / Journal of Ethnopharmacology 135 (2011) 162–172

Table 1Chromatographic and selective fragment ions of compounds detected in extracts of Radix Paeoniae Alba by LC/ESI–MS.

Rt (min) Assigned identity UV/visible �max (nm) [M−H] + m/za [M+HCOO] − m/za MS/MS m/zb

3.15 Gallic acid 272, 231 168.9 125.114.88 Epaeoniflorin sulfonat 238, 222sh, 276 543.3 421.3, 37515.62 Albiflorin 239, 222sh, 270 479.2 525.216.13 Paeoniflorin 239, 221sh, 276 479.4 525.2 449.529.57 Benzoylpaeoniflorin 238, 221sh, 276 583.3 629.330.30 Isobenzoylpaeoniflorin 238, 223sh, 276 583.3 629.332.58 Paeoniflorin derivatives 238, 221sh, 276 64735.26 Paeonol 236, 253 165.3

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t, retention time.a Selective ion monitor of the [M−H]+ and [M+HCOO]− in extracts of Radix Paeonb CID mass spectra of selected components from molecular ion.

as mixed with an equal volume of electrophoresis sample buffernd then boiled for 10 min, followed by analysis using SDS–PAGE.ransfer of protein was from the gel to nitrocellulose membraneMillipore, Bedford, MA) by using electroblotting apparatus. Thenhe proteins were supplemented with ECL Western blotting detec-ion reagents (Amersham Biosciences, USA) and analyzed using theui LAS-3000 imaging system (Japan).

.8. Urinary bladder cancer model

The animal experimental protocol used in this study waspproved by the Institutional Animal Care and Use Committee ofhung Shan Medical University (IACUC, CSMC), Taichung, Taiwan.ale Sprague–Dawley rats were purchased from the National Lab-

ratory Animal Center of Taiwan at 28 days of age and housed inolycarbonate cages (6/cage). The animals were randomly divided

nto four groups (6 mice in each group) and kept in a room lighted2 h each day and maintained at 22 ◦C. Bladder cancer was induced

n the animals of groups 2–4 by administration of OH-BBN (0.05%,/v) in the drinking water for 8 weeks. The drinking water con-

aining carcinogen was changed twice a week. After 8 weeks, micen groups 3 and 4 were also administered RPA (0.5, 1.0 g/kg) inhe drinking water, respectively, 5d/wk for 26 weeks. All groupsere maintained on a control diet for 26 weeks. Body weight andiet consumption were monitored weekly during the entire experi-ent. Animal care was in accordance with an institutional protocol

hat was approved by the animal care and use committee.

.9. Immunohistochemical analysis

Tumor samples were fixed in 10% buffered formalin. Paraffin-mbedded, 5-�m- thin sections were deparaffinized and stainedith primary antibodies anti-proliferating cell nuclear antigen

PCNA; Dako) and a phosphospecific antibody against Thr68-hosphorylated Chk2 (Cell Signaling Technology, Inc.) for 1 h at7 ◦C. The sections were then incubated with an appropriateiotinylated secondary antibody for 30 min at room temperature.hereafter, sections were incubated with 3, 3′-diaminobenzidineDako) working solution, and counterstained with hematoxylin.he immunostained cells were quantified by counting the brownells and the total number of cells at five randomly selected fields at00× magnifications. The proliferation index and phospho-Chk2-ositive cells were determined as number of positively stainedells × 100/total number of cells counted.

.10. Statistical analysis

The experiment was conducted using a completely randomesign (CRD). Data were analyzed using analysis of varianceANOVA). A significant difference was considered at the 0.05 prob-bility level and differences between treatments were tested using

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the least significant difference (LSD) test. All statistical analyses ofdata were performed using SAS.

3. Results

3.1. High-performance liquid chromatography/electrosprayionization/tandem mass spectrometry (HPLC/ESI/MS) analysis ofRPA

The components of RPA were assayed via determination ofHPLC/ESI/MS. Components belonging to monoterpene glycosides,galloylglucoses and phenolic compounds, respectively were iden-tified in the samples of RPA. Some data, such as retention timesand UV spectra of available reference compounds, were used ascomplementary data to identify components. The details of identi-fied components were summarized in Table 1, and mass spectraidentification was compared with a previous report (Li et al.,2009).

3.2. RPA inhibits the growth and causes cell death of humanbladder cancer TSHG-8301 cells

To evaluate the effect of RPA on cell viability of human blad-der cancer TSGH-8301 cells, cells were treated with increasingconcentrations of RPA (0.5, 1, and 2 mg/ml) for 24 h, and cell sur-vival was assessed by MTT assay. RPA (0.5–2 mg/ml) treatmentresulted in a concentration-dependent inhibition of the prolifer-ation of TSGH-8301 cells with an IC50 of ∼0.8 mg/ml (data notshown). Cell growth inhibition by RPA was confirmed by DAPIstain methods, and the results were shown in Fig. 1A. Proliferationof THGH-8301 cells was significantly suppressed in the pres-ence of RPA in a concentration-dependent manner. Morphologicalalterations characteristic of apoptosis included nuclear condensa-tion.

3.3. RPA induces apoptosis in TSGH-8301 cells

To test whether the RPA-mediated decrease in cell growth wasdue to induction of apoptosis, TSGH-8301 cells were stained withAnnexin V/propidium iodide and analyzed by flow cytometry. Datashowed a significant induction of apoptosis by RPA at doses of 1(12.76%) to 2 (19.7%) mg/ml in cells, which was evident from thesignificant enhancement of Annexin V/PI staining (Fig. 1B).

3.4. RPA modulates the protein levels of Bax and Bcl2

Bax and Bcl-2 belong to a multi-gene family of proteins thatplay an important role in the regulation of apoptosis. Bcl2 pro-motes cell survival, whereas Bax antagonizes this effect (Adamsand Cory, 1998). Therefore, the ratio of Bax/Bcl2 is often consid-ered as a decisive factor in determining whether cells will undergo

T.-T. Ou et al. / Journal of Ethnopharmacology 135 (2011) 162–172 165

Fig. 1. Effect of RPA on cell viability and apoptosis of TSGH-8301 cells. (A) Cells were treated with RPA (0.5–2 mg/ml) for 24 h, and the apoptotic cells were assayed by DAPIstain; the arrow indicated apoptosis cells. (B) Under the same treatment, cells were analyzed by FACS to determine the relative % of apoptotic Annexin V/PI cells. Quantitativeassessment of the percentage of cells was indicated by Anexin V/PI staining. Data are mean ± SD of the two independent experiments in triplicate. Significant difference fromcontrol (*p < 0.05, **p < 0.01) is indicated by *.

166 T.-T. Ou et al. / Journal of Ethnopharmacology 135 (2011) 162–172

Fig. 2. Effect of RPA on apoptotic proteins in TSGH-8301 cells. Cells were treated with various concentrations of RPA (0.5–2 mg/ml) for 24 h as detailed in Section 2. (A) Proteinlevels of Bcl2, Bax, caspase-3, and caspase-9 in TSGH-8301 cells were determined by immunoblot analysis. Equal loading of protein was confirmed by �-actin antibody. Resultsare representative of three independent experiments with similar results. (B) Cells were treated with 10 �mol/L concentration of the general caspase inhibitor Z-VAD-FMK for3 h, followed by the treatment with the indicated doses of RPA for 24 h. Cells were processed for PI staining, followed by flow cytomerty analysis. The position of the Sub-G1p Z-VAw in wase

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eak is integrated by apoptosis cells. (C) Cells were treated with or without RPA andhole cell lysates were determined by immunoblot analysis. Equal loading of prote

xperiments with similar results.

eath or survive. We observed that RPA treatment of cells resultedn a decrease in Bcl2 expression with a concomitant increase inhe protein level of Bax (Fig. 2A). The ratio of Bax to Bcl2 increasedfter RPA treatment in a dose-dependent manner, indicative of thepoptosis process.

.5. Activation of caspase-3 by RPA treatment in TSGH-8301 cells

Caspases are aspartate-specific cyctein proteases that playkey role in mediating apoptosis response. They are sequen-

ially activated due to cleavage of their inactive pro-caspase formThornberry and Lazebnik, 1998; Earnshaw et al., 1999). To testhether caspases were involved in apoptosis induction by RPA, werst evaluated the protein levels of procaspases and active caspases

n RPA-treated cells. Data presented in Fig. 2A showed a signif-cant increase in the levels of active caspase-3 and caspase-9 inPA-treated cells. To determine whether RPA induces apoptosisia activation of caspases, we used a general caspase inhibitor,-VAD-FMK. As a result, RPA (2 mg/ml) – treated cells exhibited

9.6% propidium iodide labeling-positive cells, which were signifi-antly reduced to 15.37% by the treatment of cells with Z-VAD-FMKFig. 2B). Immunoblot analysis showed that RPA-induced cleav-ge of pro-caspase-3 was reduced in the presence of Z-VAD-FMK,onfirming these results (Fig. 2C). Thus induction of caspases may

D-FMK for 24 h, protein levels of active caspase-3 and caspase-9 in TSGH-8301 cellsconfirmed by the blot with �-actin. Results are representative of two independent

be a mechanism by which RPA induces apoptosis in TSGH-8301cells.

3.6. RPA-treated TSGH-8301 cells are arrested in the G2/M phaseof the cell cycle

Several studies have shown that the induction of apoptosismight be due to cell cycle arrest (Hartwell and Kastan, 1994;Vermeulen et al., 2003a). The effect of RPA on cell cycle distri-bution was determined to gain insights into the mechanism of itsanti-proliferative activity, and for this we performed DNA cell cycleanalysis by flow cytometry. As shown in Fig. 3A, compared withthe control treatment, RPA treatment resulted in a dose-dependentaccumulation of cells in the G2/M phase of the cell cycle by 18.65%and 22.15% at 1 and 2 mg/ml concentration of RPA, respectively.These data suggest that the RPA induced cell cycle is arrested in theG2/M phase.

3.7. RPA modulates cell cycle regulatory proteins in TSGH-8301

cells

Molecular analysis of human cancers has revealed that cell cycleregulators are frequently mutated in most malignancies (Molinari,2000). Therefore, we next examined the effect of RPA on cell cycle

T.-T. Ou et al. / Journal of Ethnopharmacology 135 (2011) 162–172 167

Fig. 3. Effect of RPA on cell cycle modulatory proteins in TSGH-8301 cells. Cell cycle analysis was done by flow cytometry as detailed in Section 2. (A) Cell cycle analysisin TSGH-8301 cells treated with RPA, and percentage of G2/M phase cells were counted by PI staining. (B) Protein levels of Cip1/p21, cdc2 cyclinB1, Cdc25B and Cdc25C inTSGH-8301 cells as determined by immunoblot analysis. (C) Protein levels of p-Chk2 (Thr-68) and Chk2 in TSGH-8301 cells were determined by immunoblot analysis. Equalloading of protein was determined by �-actin antibody. Results are representative of two independent experiments with similar results.

168 T.-T. Ou et al. / Journal of Ethnopharmacology 135 (2011) 162–172

Fig. 4. Histopathology of the urothelium of rats in OH-BBN-induced bladder carcinogenesis. (A) 5-week-old male SD rats were randomly divided into four groups. Bladdercancer was induced in animals of groups 2–4 (6 rats in each group) by administration of OH-BBN (0.05%, w/v) in drinking water for 8 weeks. Rats in groups 3 and 4 werealso administrated RPA (0.5 and 1 g/kg) after 8 weeks during the entire experiment. (B) At the end of the study, urinary bladders were processed for H&E staining, and arepresentative picture is shown for each group. (C) Immunohistochemical staining for PCNA (magnification, 400×) in urothelium was done as detailed in Section 2. Arrowsindicate PCNA-positive cells. The proliferating cells were quantified by counting PCNA-positive cells over total cells in five randomly selected fields at 400× magnificationfor 5 different samples in each group. (D) Immunohistochemical staining for p-Chk2 (magnification, 400×) in urothelium was done. Arrows indicate p-Chk2-positive cells.The cells were quantified by counting p-Chk2-positive cells. S indicates the mean as stroma. Significant different compare with control (p < 0.01) is indicated by *.

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nhibitory protein Cip/p21, which is involved in cell cycle progres-

ion. Immunoblot analysis showed that RPA treatment at does of–2 mg/ml resulted in a significant induction of Cip1/p21 about.3 fold (Fig. 3B). We next considered the mechanism underly-

ng G2/M arrest in RPA-treated TSHG-8301 cells, and its effect onevels of proteins that regulated G2/M transition was determined

by immunoblotting. As shown in Fig. 3B, RPA treatment of cells

at doses of 1–2 mg/ml resulted in a significant reduction in theprotein levels of cyclin B1 (0.7–0.3 fold) and Cdk1 (0.8–0.6 fold).Cdc25 phosphatases play a critical role in regulation of cell cycle bydephosphorylation and activation of CDKs (Sebastian et al., 1993;Singh et al., 2004), and Cdc25B and/or Cdc25C have been shown to

T.-T. Ou et al. / Journal of Ethnopharmacology 135 (2011) 162–172 169

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e necessary for the G2/M transition (Turowski et al., 2003). Thereatment of cells with RPA at dose of 2 mg/ml resulted in a signif-cant decrease in the levels of Cdc25B (0.3 fold) and Cdc25C (0.6old) (Fig. 3B).

.8. Chk2 activation plays a critical role in RPA-induced G2/Mrrest in TSGH-8301 cells

Chk2 is a potential upstream kinase for the phosphorylation ofdc25C at the Ser216 site, which is ultimately linked to a block-de of cell cycle progression at the G2/M phase (Singh et al., 2004).e examined whether RPA affects the activated form of Chk2 that

s usually phosphorylated at the Thr68 site. Western blot analysishowed that there was a moderate to strong increase in the phos-horylation of Chk2 at Thr68 by RPA (1 and 2 mg/ml) treatment for4 h (Fig. 3C). We did not observe any considerable change in totalhk2 protein level with RPA treatments (Fig. 3C).

.9. RPA feeding decreases the incidence of OH-BBN-inducedrinary lesions in mice

Because RPA was observed to be effective for inhibiting therowth of TSGH-8301 cells in vitro, we next considered if RPA wouldnhibit bladder carcinogenesis. The extract was evaluated in theH-BBN-induced rat bladder cancer model, with the dosing sched-les shown in Fig. 4A. Briefly, rats were randomly assigned to fourroups, consisting of a control group (group 1), an OH-BBN groupgroup 2), two groups of combination treatment with OH-BBN andPA (group 3 and group 4). Rats in the control group did not receive

ny specific treatment during the entire experimental period.

A histopathologic analysis of the neoplastic progression in theH-BBN-induced urinary bladder cancer was done. H&E-stained

ections were microscopically examined and classified as (a) nor-al urothelial mucosa, characterized by epithelium of � 3 layers

nued. )

without any anaplasia; (b) mucosal dysplasia, characterized byepithelium of >3 layers with severe anaplasia with diffused pro-liferation; (c) papillary/nodular (PN) dysplasia, characterized bymoderate anaplastic epithelial lesion of localized cellular prolif-eration resulting in nodular or papillary forms. An eight-weekadministration of OH-BBN (0.05%, w/v) to SD rats resulted in theinduction of mucosal dysplasia (33%) and papillary/nodular dys-plasia (67%) of the urinary bladder at the end of the 26-week study(Table 2 and Fig. 4B). Groups 1, not induced by OH-BBN, showednormal histological characteristics. When mice were fed with RPAat doses of 0.5 g/kg and 1 g/kg body weight after OH-BBN adminis-tration and continued throughout the duration of experiment, 67%and 83% of the mice showed mucosal dysplasia with a concomitantdecrease in papillary/nodular (PN) dysplasia, respectively (Table 2and Fig. 4B).

3.10. RPA decreases urothelial cell proliferation and increasesChk2 phosphorylation in OH-BBN-treated mice

To assess the in vivo effect of RPA feeding on the prolif-eration in the urothelium of OH-BBN-treated mice, the tissuesamples were analyzed first for PCNA immunostaining. Microscopicobservation of tumors, showed less PCNA immunoreactivity inRPA-fed groups, which accounted for 36–16% decrease (p < 0.01)in PCNA-positive cells compared with the OH-BBN (53%) group(Fig. 4C). Phospho-Chk2 (Thr-68) immunostaining was next doneto assess the cell cycle arrest of RPA in tumors, showing more

phospho-Chk2-positive cells in RPA-fed groups than in the OH-BBN group. Quantification of phospho-Chk2 staining showed 26%and 44% phospho-Chk2 positive cells at doses of 0.5 g/kg and 1 g/kgRPA groups, respectively, as compared with 5.5% phospho-Chk2-positive cells in the OH-BBN group (Fig. 4D).

170 T.-T. Ou et al. / Journal of Ethnopharmacology 135 (2011) 162–172

Table 2Effect of RPA on urinary bladder cancers induced in male Sprague–Dawley rats.

Group OH-BBNa RPA treatment b Dysplasia PN dysplasia c

1 − − − −2 + − 33% 67%3 + 0.5 g/kg 67% 16%4 + 1 g/kg 83% −a OH-BBN was administered to male Sprague–Dawley rats for 8 weeks (n = 6 rats per grb Diets were supplemented with RPA after carcinogen was administrated for 8 weeks.c PN: papillary/nodular.

Fig. 5. Effect of RPA on apoptosis and antiproliferation in OH-BBN-induced urothe-lium. (A and B) Bladder tissue samples were randomly taken from each group andanalyzed for Bax, Bcl2, cyclin D1, and p-Chk2 (Thr-68) protein levels by immunoblot-ting. In each case, the densitometry data presented below the bands are fold changescompared with control. The data are representative of two independent experimentswith similar results.

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induction of Cip1/p21 (cdk inhibitors).Cellular mechanisms of genome integrity maintenance are com-

monly deregulated in cancer and numerous components of the

.11. RPA modulates the proteins levels of Bax, Bcl2, Cip1/p21,hospho-Chk2, and cyclinD1 in the urothelium of OH-BBN-treatedice

Because RPA treatment was observed to modulate the expres-ion levels of Bax and Bcl2 under in vitro conditions, we determinedhe effect of RPA administration on the expression levels of Bax andcl2 in the urothelium of OH-BBN-treated mice. The immunoblotnalysis of bladder tissue lysates exhibited significantly reducedrotein levels of Bcl2 in RPA (0.5 and 1 g/kg) groups comparedith OH-BBN alone (Fig. 5A). Inversely, a significant increase in the

xpression level of Bax was observed in the bladder urothelium ofPA (1 g/kg)-treated mice (Fig. 5A). Further more, we analyzed the

evels of Cip1/p21 and phospho-Chk2 (Thr-68) in the urotheliumf RPA-treated mice, finding a strong increase in these proteinsFig. 5B). In addition, the immunoblot analysis for cyclin D1 in

rothelium showed a significant decrease in the RPA (0.5–1 g/kg)roups as compared with OH-BBN group (Fig. 5B).

oup).

4. Discussion

Polyphenols have been proposed as potential chemopreventiveagents against cancers, primarily because of their high intake bypopulations with reduced cancer incidence and their reported abil-ity to inhibit proliferation and increase apoptosis in many cancercell types (Klein and Fischer, 2002; Lubet et al., 2007; Priego et al.,2008). RPA is a traditional medicine used for its antiinflamation andanticancer effects. The antiproliferation and anticancer activities ofRPA have been suggested to be due to the presence of polyphenoliccompounds (Madlener et al., 2007; Raina et al., 2008; Locatelli et al.,2009). Our findings suggested that in RPA, gallic acid could be oneof the bioactive compounds (Table 1). We observed the antiprolif-erative and apoptosis effects of RPA on TSGH-8301 cells in vitro andin the OH-BBN-induced rat bladder model.

Cancer progression has been suggested to involve the loss ofcell cycle checkpoint controls that regulate the passage throughcell cycles. Entry into mitosis is blocked by the G2/M checkpointmechanism when DNA is damaged (Hartwell and Weinert, 1989;Molinari, 2000). Regulation of G2/M transition is dependent onactivation of Cdk1/cyclin B1, which is maintained in an inactivestate by reversible phosphorylation on tyrosine 15 and threonine14 of Cdk1 (Deschner et al., 1991). At the onset of mitosis both ofthese residues are dephosphorylated by the Cdc25 family of phos-phatases (Molinari, 2000), and so it may be possible to block theinitiation or progression of cancer. This study indicates that RPAstrongly inhibits growth by arresting cells in the G2/M phase bothin vitro and in vivo. The observed inhibitory effects of RPA particu-larly on cyclin B1, cdc2, cdc25B, and p-Chk2 (Thr-68) in TSGH-8301cells suggest its interference in cell cycle.

The carcinogenesis process in the OH-BBN-induced murinebladder model has been well studied (McCormick et al., 1981), andOH-BBN is a well known and widely used experimental bladdercarcinogen. The dosing regimen used in the present study (0.05%OH-BBN in drinking water for 8 weeks) was highly effective ininducing bladder cancer in the animals (Table 2). Whereas no ratsin the control group developed bladder cancer, rats fed with RPAafter OH-BBN administration and continued for 26 weeks exhibitedarrested tumor progression for preinvasive lesions and a stronglydecreased incidence of papillary/nodular dysplasia with no adversehealth effects. Immunohistochemical and immunoblot analysisrevealed the inhibition of cell proliferation and induction of apo-ptotic cell death by RPA in the OH-BBN-treated rats. Overexpressionof cyclin D1 has been observed in a variety of cancers (Vinh et al.,2002; Vermeulen et al., 2003b). In our study, immunohistochemicaland immunoblot analyses for PCNA and cyclin D1 revealed over-expression of both these molecules in OH-BBN-induced bladderurothelium, which was strongly decreased with RPA treatments.Importantly, we further observed that RPA significantly reducedurinary bladder carcinogenesis in rats initiated by OH-BBN via

cell cycle checkpoint, and DNA repair pathways qualify as eithertumor suppressor or proto-oncogenes (Bartek and Lukas, 2001;

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oeijmakers, 2001; Khanna and Jackson, 2001). One of the emerg-ng tumor suppressors implicated in responses DNA damage ishk2. Prominent among the N-terminal regulatory modificationsf Chk2 is the phosphorylation of threonine 68 (p-Thr68), an ATM-ediated event and early marker of Chk2 activation, primarily in

esponse to genotoxic insults that cause DNA double-strand breaksDSBs), such as ionizing radiation and various drugs (Ahn et al.,000; Matsuoka et al., 2000; Xu et al., 2002). The fully activatedhk2 phosphorylates downstream substrates of cell cycle controlFalck et al., 2001; Ahn et al., 2002). Genetic alterations of Chk2ave been identified in a wide spectrum of human sporadic tumors

ncluding carcinomas of the breast (Sullivan et al., 2002), lungHaruki et al., 2000), prostate (Dong et al., 2003), and ovary (Liangt al., 2006). On the other hand, the role of Chk2 in tumorigene-is is far from understood in other cancers, including carcinoma ofhe urinary bladder. Here we report on an initial assessment of thehk2 tumor suppressor protein in bladder cancer. By immunohisto-hemical and immunoblot analysis, our study showed that feedingats with RPA at dosages of 0.5 g/kg and 1 g/kg resulted in signifi-ant induction of Chk2 phosphorylation (Fig. 5B). Thus, induction of-Chk2 by the extract in the bladder seems to correlate with inhibi-ion of bladder carcinogenesis, suggesting that induction of p-Chk2

ay play a role and/or act as a reliable biomarker in the inhibitionf bladder carcinogenesis by RPA.

Although the efficacy of RPA in prevention of urinary bladderancer was very clear, we observed minimal side effects of thisgent when used in the prevention of bladder cancer. High dosesf RPA (3 g/kg) were used and no effect was observed on bodyeight and tumor incidents (data not shown). In summary, ourata showed that these growth inhibitory effects of RPA could beorrelated well with induction of apoptosis and inhibition of cellroliferation in vitro and in vivo. A significant increase in caspasesleavage, Chk2-phosphorylation, an increase in Bax/Bcl2 proteination, and the inhibition of cyclin D1 and PCNA protein levelsn RPA-treated tumors. Based on the present finding, it may beuggested that RPA could receive further research as a potentialnticancer agent against human bladder cancer.

cknowledgement

This work was supported by a National Science Council GrantNSC99-2321-B-040-001), Taiwan.

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