Paeonia lactiflora Pall inhibits bladder cancer growth involving phosphorylation of Chk2 in vitro and in vivo

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Journal of Ethnopharmacology

j o u r n a l h o m e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / j e t h p h a r m

Paeonia lactiﬂora Pall inhibits bladder cancer growth involving phosphorylation

of Chk2 in vitro and in vivo

Ting-Tsz Ou

a

_{, Cheng-Hsun Wu}

b,1

_{, Jeng-Dong Hsu}

c

_{, Charng-Cherng Chyau}

d

_,

Huei-Jane Lee

a,∗

_{, Chau-Jong Wang}

a,e,∗

a_{Institute of Biochemistry and Biotechnology, College of Medicine, Chung Shan Medical University, Taichung, Taiwan} b_{Department of Anatomy, China Medical University, Taichung, Taiwan}

c_{Department of Pathology, Chung Shan Medical University Hospital, Taichung, Taiwan}

d_{Institute of Biotechnology, College of Medicine and Nursing, Hung Kuang University, Taichung, Taiwan} e_{Department of Medical Research, Chung Shan Medical University Hospital, Taichung, Taiwan}

a r t i c l e i n f o

Article history:

Received 1 December 2010

Received in revised form 24 February 2011 Accepted 3 March 2011

Available online 9 March 2011 Keywords:

Paeonia lactiﬂora Pall (RPA) Apoptosis Cell cycle G2/M phase Checkpoint kinase 2 Bladder carcinogenesis

a b s t r a c t

Ethnopharmacological relevance: Extracts of Paeonia lactiﬂora Pall (RPA), a traditional Chinese medicines has been shown to treat cancers.

Aim of the study: The purpose of this study is to evaluate the anticancer effect of RPA in urinary bladder carcinoma in vitro and in vivo.

Materials and methods: The cell viability was analyzed with DAPI. Flow cytometry and Western blot were used to study the apoptosis and cell cycle related mechanism. A rat model of bladder cancer was induced by 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 cell cycle arrest. Treatment of TSGH-8301 cells with RPA resulted in G2-M phase arrest that was associated with a marked decline in protein levels of cdc2, cyclin B1, cell division cycle 25B (Cdc25B) and Cdc25 C. We also reported that RPA-mediated growth inhibition of TSGH-8301 cells was correlated with activation of checkpoint kinase 2 (Chk2). Herein, we further evaluated urinary bladder cancer using a model of bladder cancer induced by OH-BBN. Analysis of tumors from RPA-treated rats showed signiﬁcant decrease in the expression of Bcl2, cyclin D1, and PCNA, and increase in the expression of p-Chk2 (Thr-68), Bax, and Cip1/p21.

Conclusion: Our data provide the experimental evidence that RPA could modulate apoptosis in models of bladder cancer.

1. Introduction

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

Abbreviations: RPA, Paeonia lactiﬂora Pall; OH-BBN, N-butyl-N-(4-hydroxybutyl) nitrosamine; cdc2, cyclin-dependent kinase 1; Cdc25B, cell division cycle 25B; Cdc25C, cell division cycle 25C; TCC, transitional urothelial cell carcino-mas; HPLC, high-performance liquid chromatography; PDA, photodiode-array; LC–MS, liquid chromatography–tandem mass spectrometry; ESI, electrospray ionization; CID, collision-induced dissociation; FBS, fetal bovine serum; Z-VAD-FMK, N-benzyloxycarbonyl-Val-Ala-Asp (OMe)-ﬂuromethylketone; DPAI, 4, 6-diamidino-2-phenyl-indole; PBS, phosphate buffer solution; PI, propidium iodide; PCNA, proliferating cell nuclear antigen.

∗ Corresponding authors at: Institute of Biochemistry and Biotechnology, Col-lege of Medicine, Chung Shan Medical University, No. 110, Sec. 1, Jianguo N. Road, Taichung 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.}

common cause of death among genitourinary tumors, with more than 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 in men and 25% in women (Shen et al., 2008). The standard treatment of bladder cancer has been radical cystectomy with urinary diver-sion. However, half of these patients subsequently develop disease recurrence (Stein et al., 2001).

Because of unsatisfactory outcomes associated with treating advanced cases of bladder cancer, the novel preventive approaches are needed to control this disease. One such preventive approach is through chemoprevention by using naturally occurring dietary substances (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 as chemotherapy of bladder cancer.

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

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dried in the sun. It has been frequently used as an important ingre-dient in many traditional prescriptions and is commonly used for nourishing blood, alleviating pain, reducing irritability, as well as treating liver disease and cancer. A recent study reported that the extract of RPA inhibits the growth of hepatocellular carcinoma and HL-60 leukemic cells and induces their apoptosis (Lee et al., 2002; Kwon et al., 2006). Several constituents isolated from RPA have also been found to have immunologically activities (Tomoda et al., 1993, 1994). However, the underlying mechanism of RPA as an anticancer agent has not yet been deﬁned.

Studies conducted by our group have also shown that polyphe-nol possesses strong anticancer efﬁcacy against human bladder cancer in vitro, where it inhibits the growth of human bladder transitional cell carcinoma cells (TSGH-8301) by causing cell cycle arrest (Ou et al., 2010). In the present study, we show that RPA induces apoptosis and cell growth inhibition of human bladder cancer TSGH-8301 cells in in vitro system and signiﬁcantly inhibits tumor growth in in vivo OH-BBN-induced mouse model, and that these effect are mediated through the Chk2 signaling.

2. Materials and methods

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

P. lactiflora Pall (RPA) was purchased in Changhwa in Taiwan. To prepare the aqueous extract of RPA, 100 g of dried material was extracted with 3 L of boiling water for 2 h. The filtrate was collected after filtration and the lyophilized. The powder obtained was stored at−20◦_{C until use. The average yield of dried extract was about}

22.15%.

2.2. HPLC system and conditions

The HPLC apparatus consisted of a Finnigan Surveyor module separation system and a photodiode-array (PDA) detector (Thermo Electron Co., MA, USA). The chromatographic separation of the com-pounds was achieved at an elution ﬂow rate of 0.2 ml/min using an analytical column (Luna 3␮ C18 (2), 150 mm × 2.0 mm) equipped with a guard column [SecurityGuard C18 (ODS), 4 mm× 3.0 mm ID, Phenomenex, Inc., Torrance, CA]. The elution was performed by gra-dient elution using two solvents: solvent A (water containing 0.1% formic acid) and solvent B (acetonitrile containing 0.1% formic acid). The entire course of programmed gradient elution was carried out as follows: 0–3 min, with 10% B isocratic; 3–15 min, with 10–30% B; 15–20 min, with 30% B isocratic, 20–50 min, with 30–90% B, 50–60 min, with 90% B isocratic; and 60–70 min, followed by chang-ing 90% to 10% of B. Then 20␮L of the sample extract was directly injected into the column using a model 7725i Rheodyne injection valve. The absorption spectra of eluted compounds were scanned within 210–400 nm using the in-line PDA detector monitored at 230, 270 and 320 nm, respectively.

2.2.1. Liquid chromatography–tandem mass spectrometry (LC–MS) instrumentation and conditions

The LC elute was introduced directly into a Finnigan LCQ Advantage MAX ion trap mass spectrometer operated in electro-spray ionization (ESI) with negative ionization mode. The ion trap instrument was set as follows: capillary voltage,−4.0 V; tube lens offset,−30 V; source voltage, 3.5 kV; ion transfer capillary tem-perature, 320◦C; nitrogen sheath gas, 40; and auxiliary gas, 5 (in arbitrary units). Mass spectra were acquired in an m/z range of 150–1000. Furthermore, MS/MS analysis was performed on the selected molecular ions of the major peaks, i.e., m/z 169, 525 and 543 to conﬁrm the structures, where the collision-induced disso-ciation (CID) fragments were produced using normalized collision energies with an increment of 38% and with wideband activation

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

2.3. Cell lines and reagents

Human urinary bladder cancer cells (TSGH-8301) were derived from a well-differentiated human TCC of the urinary bladder (Yeh et al., 1988) and purchased from the Bioresource Collection and Research Center. Cells were maintained in RPMI 1640 medium, supplemented with 10% fetal bovine serum (FBS), 1% penicillin and 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 antibodies were 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 examined by 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% paraformaldehyde for 30 min at room temperature. The fixed cells were permeabi-lized 3 times with 0.2% Triton X 100 in PBS, and incubated with 1␮g/ml of DAPI for 30 min, and then washed with PBS 3 times. The apoptotic nuclei were examined under 400× magnification using a fluorescent 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 a density of 50–60% confluence in 100-mm culture dishes and treated with RPA for 24 h. The cells were trypsinized, washed with PBS, and processed for labeling with FITC-conjugated Annexin V and propidium 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 by overnight serum starvation, were treated with 5␮mol/L Z-VAD-FMK (a general caspase inhibitor) for 3 h, and then treated with RPA (0.5–2 mg/ml) for 24 h in complete medium. The cells were trypsinized, washed twice with chilled PBS, and centrifuged. The cell 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 cells}

were centrifuged at 1000 rpm for 5 min; the pellet was washed twice with chilled PBS, incubated with 500␮l propidium iodide (50␮g/ml ﬁnal concentration) for 30 min and analyzed by ﬂow cytometry. A minimum of 10,000 cells per sample was counted and DNA 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 room temperature. Then 0.5 ml of cold RIPA buffer (1% NP-40, 50 mM Tris-base, 0.1% SDS, 0.5% deoxycholic acid, 150 mM NaCl, pH 7.5) with fresh protease inhibitor was added. Cells were scraped and the lysate was centrifuged at 10,000× g for 10 min. Cell lysate (50 ␮g)

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Table 1

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

Rt (min) Assigned identity UV/visiblemax(nm) [M−H] + m/za _{[M+HCOO]− m/z}a _{MS/MS m/z}b

3.15 Gallic acid 272, 231 168.9 125.1 14.88 Epaeoniflorin sulfonat 238, 222sh, 276 543.3 421.3, 375 15.62 Albiflorin 239, 222sh, 270 479.2 525.2 16.13 Paeoniflorin 239, 221sh, 276 479.4 525.2 449.5 29.57 Benzoylpaeoniflorin 238, 221sh, 276 583.3 629.3 30.30 Isobenzoylpaeoniflorin 238, 223sh, 276 583.3 629.3 32.58 Paeoniflorin derivatives 238, 221sh, 276 647 35.26 Paeonol 236, 253 165.3 Rt, retention time.

a_{Selective ion monitor of the [M−H]+ and [M+HCOO]− in extracts of Radix Paeoniae Alba.} b _{CID mass spectra of selected components from molecular ion.}

was mixed with an equal volume of electrophoresis sample buffer and then boiled for 10 min, followed by analysis using SDS–PAGE. Transfer of protein was from the gel to nitrocellulose membrane (Millipore, Bedford, MA) by using electroblotting apparatus. Then the proteins were supplemented with ECL Western blotting detec-tion reagents (Amersham Biosciences, USA) and analyzed using the Fui LAS-3000 imaging system (Japan).

2.8. Urinary bladder cancer model

The animal experimental protocol used in this study was approved by the Institutional Animal Care and Use Committee of Chung Shan Medical University (IACUC, CSMC), Taichung, Taiwan. Male Sprague–Dawley rats were purchased from the National Lab-oratory Animal Center of Taiwan at 28 days of age and housed in polycarbonate cages (6/cage). The animals were randomly divided into four groups (6 mice in each group) and kept in a room lighted 12 h each day and maintained at 22◦C. Bladder cancer was induced in the animals of groups 2–4 by administration of OH-BBN (0.05%, w/v) in the drinking water for 8 weeks. The drinking water con-taining carcinogen was changed twice a week. After 8 weeks, mice in groups 3 and 4 were also administered RPA (0.5, 1.0 g/kg) in the drinking water, respectively, 5d/wk for 26 weeks. All groups were maintained on a control diet for 26 weeks. Body weight and diet consumption were monitored weekly during the entire experi-ment. Animal care was in accordance with an institutional protocol that was approved by the animal care and use committee.

2.9. Immunohistochemical analysis

Tumor samples were fixed in 10% buffered formalin. Paraffin-embedded, 5-␮m- thin sections were deparaffinized and stained with primary antibodies anti-proliferating cell nuclear antigen (PCNA; Dako) and a phosphospecific antibody against Thr68-phosphorylated Chk2 (Cell Signaling Technology, Inc.) for 1 h at 37◦C. The sections were then incubated with an appropriate biotinylated secondary antibody for 30 min at room temperature. Thereafter, sections were incubated with 3, 3-diaminobenzidine (Dako) working solution, and counterstained with hematoxylin. The immunostained cells were quantified by counting the brown cells and the total number of cells at five randomly selected fields at 400× magnifications. The proliferation index and phospho-Chk2-positive cells were determined as number of phospho-Chk2-positively stained cells× 100/total number of cells counted.

2.10. Statistical analysis

The experiment was conducted using a completely random design (CRD). Data were analyzed using analysis of variance (ANOVA). A signiﬁcant difference was considered at the 0.05 prob-ability level and differences between treatments were tested using

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

3. Results

3.1. High-performance liquid chromatography/electrospray ionization/tandem mass spectrometry (HPLC/ESI/MS) analysis of RPA

The components of RPA were assayed via determination of HPLC/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 times and UV spectra of available reference compounds, were used as complementary data to identify components. The details of identi-fied components were summarized inTable 1, and mass spectra identification was compared with a previous report (Li et al., 2009).

3.2. RPA inhibits the growth and causes cell death of human bladder 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 increasing concentrations 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) treatment resulted in a concentration-dependent inhibition of the prolifer-ation of TSGH-8301 cells with an IC50 of ∼0.8 mg/ml (data not shown). Cell growth inhibition by RPA was conﬁrmed by DAPI stain methods, and the results were shown inFig. 1A. Proliferation of THGH-8301 cells was signiﬁcantly suppressed in the pres-ence of RPA in a concentration-dependent manner. Morphological alterations 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 was due to induction of apoptosis, TSGH-8301 cells were stained with Annexin V/propidium iodide and analyzed by flow cytometry. Data showed 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 the significant 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 that play an important role in the regulation of apoptosis. Bcl2 pro-motes cell survival, whereas Bax antagonizes this effect (Adams and Cory, 1998). Therefore, the ratio of Bax/Bcl2 is often consid-ered as a decisive factor in determining whether cells will undergo

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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 DAPI stain; 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. Quantitative assessment 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 from control (*p < 0.05, **p < 0.01) is indicated by *.

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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 Section2. (A) Protein levels 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. Results are 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 for 3 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-G1 peak is integrated by apoptosis cells. (C) Cells were treated with or without RPA and Z-VAD-FMK for 24 h, protein levels of active caspase-3 and caspase-9 in TSGH-8301 cells whole cell lysates were determined by immunoblot analysis. Equal loading of protein was confirmed by the blot with␤-actin. Results are representative of two independent experiments with similar results.

death or survive. We observed that RPA treatment of cells resulted in a decrease in Bcl2 expression with a concomitant increase in the protein level of Bax (Fig. 2A). The ratio of Bax to Bcl2 increased after RPA treatment in a dose-dependent manner, indicative of the apoptosis process.

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

Caspases are aspartate-specific cyctein proteases that play a key role in mediating apoptosis response. They are sequen-tially activated due to cleavage of their inactive pro-caspase form (Thornberry and Lazebnik, 1998; Earnshaw et al., 1999). To test whether caspases were involved in apoptosis induction by RPA, we first evaluated the protein levels of procaspases and active caspases in RPA-treated cells. Data presented inFig. 2A showed a signif-icant increase in the levels of active caspase-3 and caspase-9 in RPA-treated cells. To determine whether RPA induces apoptosis via activation of caspases, we used a general caspase inhibitor, Z-VAD-FMK. As a result, RPA (2 mg/ml) – treated cells exhibited 29.6% propidium iodide labeling-positive cells, which were signifi-cantly reduced to 15.37% by the treatment of cells with Z-VAD-FMK (Fig. 2B). Immunoblot analysis showed that RPA-induced cleav-age of pro-caspase-3 was reduced in the presence of Z-VAD-FMK, confirming these results (Fig. 2C). Thus induction of caspases may

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

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

Several studies have shown that the induction of apoptosis might 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 its anti-proliferative activity, and for this we performed DNA cell cycle analysis by ﬂow cytometry. As shown inFig. 3A, compared with the control treatment, RPA treatment resulted in a dose-dependent accumulation 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 the G2/M phase.

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

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

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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 Section2. (A) Cell cycle analysis in 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 in TSGH-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. Equal loading of protein was determined by␤-actin antibody. Results are representative of two independent experiments with similar results.

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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. Bladder cancer 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 were also 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 a representative picture is shown for each group. (C) Immunohistochemical staining for PCNA (magnification, 400×) in urothelium was done as detailed in Section2. Arrows indicate PCNA-positive cells. The proliferating cells were quantified by counting PCNA-positive cells over total cells in five randomly selected fields at 400× magnification for 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 *.

inhibitory protein Cip/p21, which is involved in cell cycle progres-sion. Immunoblot analysis showed that RPA treatment at does of 1–2 mg/ml resulted in a signiﬁcant induction of Cip1/p21 about 1.3 fold (Fig. 3B). We next considered the mechanism underly-ing G2/M arrest in RPA-treated TSHG-8301 cells, and its effect on levels of proteins that regulated G2/M transition was determined

by immunoblotting. As shown inFig. 3B, RPA treatment of cells at doses of 1–2 mg/ml resulted in a signiﬁcant reduction in the protein 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 by dephosphorylation and activation of CDKs (Sebastian et al., 1993; Singh et al., 2004), and Cdc25B and/or Cdc25C have been shown to

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Fig. 4. (Continued. )

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

3.8. Chk2 activation plays a critical role in RPA-induced G2/M arrest in TSGH-8301 cells

Chk2 is a potential upstream kinase for the phosphorylation of Cdc25C at the Ser216 site, which is ultimately linked to a block-ade of cell cycle progression at the G2/M phase (Singh et al., 2004). We examined whether RPA affects the activated form of Chk2 that is usually phosphorylated at the Thr68 site. Western blot analysis showed that there was a moderate to strong increase in the phos-phorylation of Chk2 at Thr68 by RPA (1 and 2 mg/ml) treatment for 24 h (Fig. 3C). We did not observe any considerable change in total Chk2 protein level with RPA treatments (Fig. 3C).

3.9. RPA feeding decreases the incidence of OH-BBN-induced urinary lesions in mice

Because RPA was observed to be effective for inhibiting the growth of TSGH-8301 cells in vitro, we next considered if RPA would inhibit bladder carcinogenesis. The extract was evaluated in the OH-BBN-induced rat bladder cancer model, with the dosing sched-ules shown inFig. 4A. Brieﬂy, rats were randomly assigned to four groups, consisting of a control group (group 1), an OH-BBN group (group 2), two groups of combination treatment with OH-BBN and RPA (group 3 and group 4). Rats in the control group did not receive any speciﬁc treatment during the entire experimental period.

A histopathologic analysis of the neoplastic progression in the OH-BBN-induced urinary bladder cancer was done. H&E-stained sections were microscopically examined and classiﬁed as (a) nor-mal urothelial mucosa, characterized by epithelium of 3 layers

without any anaplasia; (b) mucosal dysplasia, characterized by epithelium of >3 layers with severe anaplasia with diffused pro-liferation; (c) papillary/nodular (PN) dysplasia, characterized by moderate anaplastic epithelial lesion of localized cellular prolif-eration resulting in nodular or papillary forms. An eight-week administration of OH-BBN (0.05%, w/v) to SD rats resulted in the induction of mucosal dysplasia (33%) and papillary/nodular dys-plasia (67%) of the urinary bladder at the end of the 26-week study (Table 2andFig. 4B). Groups 1, not induced by OH-BBN, showed normal histological characteristics. When mice were fed with RPA at 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 concomitant decrease in papillary/nodular (PN) dysplasia, respectively (Table 2

andFig. 4B).

3.10. RPA decreases urothelial cell proliferation and increases Chk2 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 tissue samples were analyzed ﬁrst for PCNA immunostaining. Microscopic observation of tumors, showed less PCNA immunoreactivity in RPA-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 done to 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/kg RPA groups, respectively, as compared with 5.5% phospho-Chk2-positive cells in the OH-BBN group (Fig. 4D).

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Table 2

Effect 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 group).} b _{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 and analyzed 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 changes compared with control. The data are representative of two independent experiments with similar results.

3.11. RPA modulates the proteins levels of Bax, Bcl2, Cip1/p21, phospho-Chk2, and cyclinD1 in the urothelium of OH-BBN-treated mice

Because RPA treatment was observed to modulate the expres-sion levels of Bax and Bcl2 under in vitro conditions, we determined the effect of RPA administration on the expression levels of Bax and Bcl2 in the urothelium of OH-BBN-treated mice. The immunoblot analysis of bladder tissue lysates exhibited significantly reduced protein levels of Bcl2 in RPA (0.5 and 1 g/kg) groups compared with OH-BBN alone (Fig. 5A). Inversely, a significant increase in the expression level of Bax was observed in the bladder urothelium of RPA (1 g/kg)-treated mice (Fig. 5A). Further more, we analyzed the levels of Cip1/p21 and phospho-Chk2 (Thr-68) in the urothelium of RPA-treated mice, finding a strong increase in these proteins (Fig. 5B). In addition, the immunoblot analysis for cyclin D1 in urothelium showed a significant decrease in the RPA (0.5–1 g/kg) groups as compared with OH-BBN group (Fig. 5B).

4. Discussion

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

Cancer progression has been suggested to involve the loss of cell cycle checkpoint controls that regulate the passage through cell cycles. Entry into mitosis is blocked by the G2/M checkpoint mechanism when DNA is damaged (Hartwell and Weinert, 1989; Molinari, 2000). Regulation of G2/M transition is dependent on activation of Cdk1/cyclin B1, which is maintained in an inactive state by reversible phosphorylation on tyrosine 15 and threonine 14 of Cdk1 (Deschner et al., 1991). At the onset of mitosis both of these residues are dephosphorylated by the Cdc25 family of phos-phatases (Molinari, 2000), and so it may be possible to block the initiation or progression of cancer. This study indicates that RPA strongly inhibits growth by arresting cells in the G2/M phase both in vitro and in vivo. The observed inhibitory effects of RPA particu-larly on cyclin B1, cdc2, cdc25B, and p-Chk2 (Thr-68) in TSGH-8301 cells suggest its interference in cell cycle.

The carcinogenesis process in the OH-BBN-induced murine bladder model has been well studied (McCormick et al., 1981), and OH-BBN is a well known and widely used experimental bladder carcinogen. The dosing regimen used in the present study (0.05% OH-BBN in drinking water for 8 weeks) was highly effective in inducing bladder cancer in the animals (Table 2). Whereas no rats in the control group developed bladder cancer, rats fed with RPA after OH-BBN administration and continued for 26 weeks exhibited arrested tumor progression for preinvasive lesions and a strongly decreased incidence of papillary/nodular dysplasia with no adverse health effects. Immunohistochemical and immunoblot analysis revealed the inhibition of cell proliferation and induction of apo-ptotic cell death by RPA in the OH-BBN-treated rats. Overexpression of cyclin D1 has been observed in a variety of cancers (Vinh et al., 2002; Vermeulen et al., 2003b). In our study, immunohistochemical and immunoblot analyses for PCNA and cyclin D1 revealed over-expression of both these molecules in OH-BBN-induced bladder urothelium, which was strongly decreased with RPA treatments. Importantly, we further observed that RPA signiﬁcantly reduced urinary bladder carcinogenesis in rats initiated by OH-BBN via induction of Cip1/p21 (cdk inhibitors).

Cellular mechanisms of genome integrity maintenance are com-monly deregulated in cancer and numerous components of the cell cycle checkpoint, and DNA repair pathways qualify as either tumor suppressor or proto-oncogenes (Bartek and Lukas, 2001;

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Hoeijmakers, 2001; Khanna and Jackson, 2001). One of the emerg-ing tumor suppressors implicated in responses DNA damage is Chk2. Prominent among the N-terminal regulatory modifications of Chk2 is the phosphorylation of threonine 68 (p-Thr68), an ATM-mediated event and early marker of Chk2 activation, primarily in response to genotoxic insults that cause DNA double-strand breaks (DSBs), such as ionizing radiation and various drugs (Ahn et al., 2000; Matsuoka et al., 2000; Xu et al., 2002). The fully activated Chk2 phosphorylates downstream substrates of cell cycle control (Falck et al., 2001; Ahn et al., 2002). Genetic alterations of Chk2 have been identified in a wide spectrum of human sporadic tumors including carcinomas of the breast (Sullivan et al., 2002), lung (Haruki et al., 2000), prostate (Dong et al., 2003), and ovary (Liang et al., 2006). On the other hand, the role of Chk2 in tumorigene-sis is far from understood in other cancers, including carcinoma of the urinary bladder. Here we report on an initial assessment of the Chk2 tumor suppressor protein in bladder cancer. By immunohisto-chemical and immunoblot analysis, our study showed that feeding rats with RPA at dosages of 0.5 g/kg and 1 g/kg resulted in signifi-cant induction of Chk2 phosphorylation (Fig. 5B). Thus, induction of p-Chk2 by the extract in the bladder seems to correlate with inhibi-tion of bladder carcinogenesis, suggesting that inducinhibi-tion of p-Chk2 may play a role and/or act as a reliable biomarker in the inhibition of bladder carcinogenesis by RPA.

Although the efficacy of RPA in prevention of urinary bladder cancer was very clear, we observed minimal side effects of this agent when used in the prevention of bladder cancer. High doses of RPA (3 g/kg) were used and no effect was observed on body weight and tumor incidents (data not shown). In summary, our data showed that these growth inhibitory effects of RPA could be correlated well with induction of apoptosis and inhibition of cell proliferation in vitro and in vivo. A significant increase in caspases cleavage, Chk2-phosphorylation, an increase in Bax/Bcl2 protein ration, and the inhibition of cyclin D1 and PCNA protein levels in RPA-treated tumors. Based on the present finding, it may be suggested that RPA could receive further research as a potential anticancer agent against human bladder cancer.

Acknowledgement

This work was supported by a National Science Council Grant (NSC99-2321-B-040-001), Taiwan.

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