Food and Chemical Toxicology 65 (2014) 242–251

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Licochalcone B inhibits growth of bladder cancer cells by arresting cell cycle progression and inducing apoptosis Xuan Yuan a, Tao Li a, Erlong Xiao a, Hong Zhao b, Yongqian Li a, Shengjun Fu a, Lu Gan d, Zhenhua Wang c, Qiusheng Zheng b,c,⇑, Zhiping Wang a,⇑ a

Institute of Urology, Second Hospital, Lanzhou University, 730030 Lanzhou, China Key Laboratory of Xinjiang Endemic Phytomedicine Resources, Ministry of Education, School of Pharmacy, Shihezi University, 832002 Shihezi, China Life Science School, Yantai University, 264000 Yantai, China d Institute of Modern Physics, Chinese Academy of Sciences, 730000 Lanzhou, China b c

a r t i c l e

i n f o

Article history: Received 16 September 2013 Accepted 21 December 2013 Available online 31 December 2013 Keywords: Licochalcone B Bladder cancer S phase arrest Apoptosis

a b s t r a c t To examine the mechanisms by which licochalcone B (LCB) inhibits the proliferation of human malignant bladder cancer cell lines (T24 and EJ) in vitro and antitumor activity in vivo in MB49 (murine bladder cancer cell line) tumor model. Exposure of T24 or EJ cells to LCB significantly inhibited cell lines proliferation in a concentration-dependent and time-dependent manner, and resulted in S phase arrest in T24 or EJ cells, respectively. LCB treatment decreased the expression of cyclin A, cyclin-dependent kinase (CDK1 and CDK2) mRNA, cell division cycle 25 (Cdc25A and Cdc25B) protein. In addition, LCB treatment down-regulated Bcl-2 and survivin expression, enhanced Bax expression, activated caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP) protein. Consistently, the tumorigenicity of LCB-treated MB49 cells was limited significantly by using the colony formation assay in vitro and the MB49 tumor model performed in C57BL/6 mice in vivo. These findings provide support for the use of LCB in chemoprevention and bladder cancer therapy. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Bladder cancer is one of the most common urologic malignancies in economically advanced countries, and nearly all malignant bladder cancers are transitional cell carcinoma (TCC), which arise from the transitional epithelium (Jemal et al., 2007). Two types of TCC have been histopathologically classified: non-muscle invasive bladder cancer (NMIBC) and muscle invasive bladder cancer (MIBC). Approximately 70–80% of patients are NMIBC that is restricted to the mucosa (Lee et al., 2012). The remainder of the cases presents MIBC with invasion of the muscular layers of the bladder. The patients with NMIBC can be treated with endoscopic resection, while the most deaths occur in patients with deeply invasive tumors and regional or distant metastases, and the high rates of recurrence for tumors treated with resection alone (Tian et al., 2008). Therefore, much effort has been focused on the use of

⇑ Corresponding authors. Addresses: Key Laboratory of Xinjiang Endemic Phytomedicine Resources, Ministry of Education, School of Pharmacy, Shihezi University, 832002 Shihezi, China. Tel.: +86 0993 2057003; fax: +86 0993 2057005 (Q. Zheng). Institute of Urology, Second Hospital, Lanzhou University, 730030 Lanzhou, China. Tel.: +86 0931 8942821; fax: +86 0931 8942821 (Z. Wang). E-mail addresses: [email protected] (Q. Zheng), [email protected] (Z. Wang). 0278-6915/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2013.12.030

adjuvant therapy with chemotherapeutic agents. However, the conventional chemotherapeutic regimens are often intolerable because of the strong systemic toxicity, local irritation and the low efficacy (Herr et al., 1987; Huncharek et al., 2001; Pichu et al., 2012). These factors highlight the urgent need to find some novel concepts and identify novel drugs for the treatment of bladder cancer to reduce the rate of recurrence and mortality. Herbal therapy treatment have been regarded as a precious alternative to modern medicine and investigations on active components with anticancer potential and less side effects have opened up newer avenues (Tan et al., 2011). Chalcones are ubiquitous natural compounds with a wide variety of reported biological activities, such as antiviral, antimicrobial effects, and antitumoral effects (Espinoza-Hicks et al., 2013; Sawle et al., 2008). Chalcones have also been reported to inhibit cell proliferation, induce cell cycle arrest and/or induce apoptosis in cancer cells (Hseu et al., 2012; Tang et al., 2008; Zi and Simoneau, 2005). Licochalcone B (LCB, Fig. 1), a chalcone existing the root of Glycyrrhiza species (Haraguchi et al., 1998), but biological activities of this chalcone has not been well examined. In this study, we investigated the inhibitory effects of LCB on bladder cancer cells, and demonstrated the cellular mechanisms underlying the action of LCB.

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X. Yuan et al. / Food and Chemical Toxicology 65 (2014) 242–251 2.6. Apoptosis analysis by flow cytometry

LCB induced apoptosis in T24 or EJ cells was determined by flow cytometry using the Annexin V-FITC Apoptosis Detection Kit following the manufacturer’s instructions. Briefly, 1.5  105 cells/mL were treated with LCB (0, 40, 80 lM) for 72. Afterwards, the cells were washed twice with ice-cold PBS, and then 5 lL of Annexin V-FITC and 1 lL of PI (1 mg/mL) were applied to stain cells. The stained cells were analyzed using a flow cytometer (BD, NJ, USA) (Hockenbery et al., 1990). Fig. 1. Chemical structure of licochalcone B. 2.7. Cell cycle analysis 2. Materials and methods 2.1. Chemicals and reagents LCB (purity P 98%) was purchased from Shanghai Lichen Biotechnology Co., Ltd. (Shanghai, China). RPMI 1640 and fetal bovine serum (FBS) were purchased from Hyclone (Hyclone, UT, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and dimethyl-sulfoxide (DMSO) were purchased from Sigma Chemical Company (St. Louis, Missouri, USA). The Annexin V-FITC Apoptosis Detection Kit was purchased from Invitrogen Corporation (Carlsbad, California, USA). Penicillin and streptomycin were obtained from Shandong Sunrise Pharmaceutical Co., Ltd. (Shandong, China). LCB was dissolved in DMSO and diluted with fresh medium to achieve the desired concentration. The final concentration of DMSO did not exceed 0.2% in the fresh medium, and DMSO at this concentration had no significant effect on the cell viability. Unless indicated otherwise, the other reagents were purchased from Sigma. 2.2. Cell culture Human bladder carcinoma cells (T24 and EJ cells) were obtained from China Center for Type Culture Collection (Wuhan, China) and the murine transitional cell carcinoma cell line MB49 were obtained from the Southern Medical University (Guangdong, China). All the cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 lg/mL streptomycin in a humidified atmosphere with 5% CO2 at 37 °C. 2.3. Cell growth curve and determination of doubling time Cells in logarithmic growth phase were seeded into 6-well plates at a density of 6  104 cells/well. One day after seeding, 3 wells were taken to calculate the number of cells as the starting cell number, then LCB was added in three replicates of 0 mM, 5 mM, 10 mM, 20 mM for each cell population. Trypan blue staining was performed once daily since the 1st day of culture, and the viable cells in 3 wells of each group were counted for five consecutive days. The doubling time (Td) of cell was calculated according to the Patterson formula as follow: Td = T  lg2 / (lg N2 lg N1), where N1 is cell number on the 1st day, and N2 is cell number at T h after culture; T (h) is the time from N1 to N2. 2.4. Cell viability Cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, 96-well plates (Nunc, Roskilde, Denmark) were plated in triplicate with 6  103 cells per well. After a 24 h incubation, the cells were treated with various concentrations of LCB (0, 10, 20, 40, 60, 80 lM) for 24, 48 or 72 h. MTT solution (20 lL of 5 mg/mL) was added, and the cells were incubated for 2 h at 37 °C. The medium was then removed, and 150 lL of DMSO was added to each well. Absorbance at 490 nm was determined using a fluorescence plate reader (Bio-Rad Laboratories, CA, USA). The data were expressed as percent cell viability compared with control (DMSO). The experiment was repeated three times under the same conditions (Hou et al., 2008). 2.5. Morphological assay The cells were placed on a six-well chamber slides at a density of 20,000 cells/ slide, and treated with increasing concentrations of LCB for 72 h to examine whether LCB induced apoptosis in T24 or EJ cells. The cells were fixed in formaldehyde with 40 g/L in phosphate buffered saline (PBS) for 20 min followed by Hoechst 33258 (10 mg/L) staining for 30 min in the dark at 37 °C. The cells in the slides were then inspected using fluorescence microscopy (Jung et al., 2006).

Cells (1.5  105 cells/mL) were treated with LCB (0, 40, 80 lM) for 72. Afterwards, the cells were harvested and fixed in 70% ethanol for 30 min on ice. After washing with PBS, the cells were labeled with propidium iodide (PI, 0.05 mg/mL) in the presence of RNaseA (0.5 mg/mL) and incubated at room-temperature in the dark for 30 min. DNA contents were analyzed using the flow cytometer (BD, NJ, USA) equipped with CellQuest Pro Software (Samuelsson et al., 1999). ModFit LT cell cycle analysis software was used to determine the percentage of cells in the different phases of the cell cycle.

2.8. Quantitative real-time polymerase chain reaction Cells at a density of 1.5  105 cells/mL were incubated with LCB (0, 40, 80 lM) for 72. Total RNA was extracted using Trizol reagent (Sangon Biotechnology Co. Ltd., Shanghai, China). After extraction 3 lL of total RNA were reverse transcribed to cDNA using reverse transcription reagents (Fermentas China Co. Ltd., Shenzhen, China) in a 20 lL volume. The resulting cDNA samples were subjected to real-time PCR using the Rotor-Gene Q (QIAGEN, Germany) with SYBR Green as a fluorescent reporter using SYBR Premix EX TaqTM (TaKaRa, Dalian, China). The amplification reaction mixture (25 lL) contained cDNAs, forward primers, reverse primers, and SYBR Premix EX TaqTM. The thermocycler parameters were as follows: 95 °C for 30 s; 35 cycles of 95 °C for 5 s and 61 °C for 30 s; and 72 °C for 20 s. Results were collected and analyzed to determine the PCR cycle number that generated the first fluorescence signal above a threshold (threshold cycle number, CT), after which a comparative CT method was used to measure relative gene expression. The following formula was used to calculate the relative amount of the transcript of interest in the treated sample (X) and the control sample (Y), both of which were normalized to an endogenous reference value (GAPDH): 2 DDCT, where DCT is the difference in CT between the gene of interest and GAPDH, with the DDCT for sample X = DCT (X) DCT (Y) (Chowdhury et al., 2010). The primers sequences were as follows (Table 1).

2.9. Western blot analysis Cells at a density of 1.5  105 cells/mL were incubated with LCB (0, 40, 80 lM) for 72. Soluble lysates (15 lL per lane) were subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), transferred onto nitrocellulose membranes (Amersham Biosciences, New Jersey, USA), and blocked with 5% nonfat milk in Tris-buffered saline with Tween (TBST) for 2 h at room temperature. Membranes were incubated with anti-Bcl-2, anti-Survivin, anti-Bax, anti-PARP, antiCdc25A and anti-Cdc25C antibody (1:500) and anti-b-actin (1:2000) (Amersham Biosciences) in 5% milk/TBST at 4 °C overnight. After washing five times with TBST, the membranes were incubated with horseradish peroxidase-conjugated antibody for 1 h at room temperature. Western blots were developed using enhanced chemiluminescence (Thermo, NY, USA) and were exposed on Kodak radiographic film.

2.10. Clonal formation assay Approximately 500 cells of MB49 were plated in 2.5 mL complete medium onto 60 mm tissue culture dishes. Cells were allowed to attach for 24 h before the addition of 2.5 mL complete medium containing LCB (0, 40, 80, 160 lM). Dishes were placed in the incubator for 24 h, and then gently rinsed with RPMI 1640 three times to remove the LCB. Complete culture medium was added to each dish. Dishes were returned to the incubator for up to 14 days and surviving tumor cells were allowed to form colonies. After 14 days colonies were fixed with methanol, stained with Giemsa stain and the numbers of colonies per dish were counted. The assay was repeated three times using each cell line. Each experiment was repeated at least for three times to establish the association (Sun et al., 2013).

Table 1 Primers used for quantitative real-time polymerase chain reaction (qRT-PCR). Gene

Forward primer

Reverse primer

Cyclin A CDK1 CDK2 GAPDH

5-TCCAAGAGGACCAGGAGAATATCA-3 5-CGTGGGGGAGCGGATTT-3 5-ACGTACGGAGTTGTGTACAAAGCC-3 5-ACCACAGTCCATGCCATCAC-3

5-TCCTCATGGTAGTCTGGTACTTCA-3 5-CGGAGGGCGAGTATTGAGGA-3 5-GCTAGTCCAAAGTCTGCTAGCTTG-3 5-TCCACCACCCTGTTGCTGTA-3

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Fig. 2. Effects of LCB on cells proliferation of human bladder cancer T24 or EJ cells. LCB inhibited the proliferation of human bladder cancer T24 and EJ cells, after exposure to LCB at various concentrations and incubation for 24, 48, or 72 h. All data are reported as the percentage change in comparison with the LCB-untreated control group, which were arbitrarily assigned 100% viability. ⁄P < 0.05, ⁄⁄P < 0.01 compared with the LCB-untreated control group cells.

Fig. 3. Effects of LCB on cell cycle distribution of human bladder cancer T24 or EJ cells. Cells were exposed to LCB at concentrations of 40 lM or 80 lM for 72 h. Values are expressed as percentage of the cell population in the G1, S, and G2/M, sub-G1 phase of cell cycle. ⁄P < 0.05, ⁄⁄P < 0.01 compared with the LCB-untreated control group cells. 2.11. Animal preparation Male C57BL/6 mice, aged 6–8 weeks and body weight 13–15 g, were obtained from the Medical Laboratory Animal Center (SDXK (Xin) 2011-004), Xinjiang Medical University, Xinjiang, China. The mice were maintained under standard animal care conditions (22 ± 3 °C and 60% humidity) for 14 days, with a commercial standard mouse cube diet (Shihezi University Laboratory Animal Center, Xinjiang, China) and water ad libitum. All studies were carried out in accordance with the protocol of local animal care and use committee. 2.12. MB49 tumor model The MB49 cell line, grown as a s.c. implant, provides a rapid method for assessing the ability of interventions to alter the growth of an invasive, poorly

differentiated bladder tumor (Zhou et al., 1998). The murine transitional cell carcinoma cell line MB49 (2  106 /mL, 100 lL) was injected subcutaneously into the flank of C57BL/6 mice. Tumor formation in C57BL/6 mice was monitored. Subcutaneous tumors induced by MB49 cells in C57BL/6 mice were randomly divided into two treatment groups (10 of each group). One week after inoculation, the mice were given of 100 lL of LCB (160 lM) by intratumoral injection every two day. Control mice were given the same volume of normal saline. The mice weight was measured by laboratory electronic balance every week. The animals were sacrificed at 30 days after cancer cells inoculation. The blood was collected by removalling eyeball after anesthesia. The serum was collected by centrifuger and submitted for measuring the serum glutamate oxaloacetate aminotranferase (GOT) and glutamate pyruvate aminotransferase (GPT). The tumors were weighted, then fixed in 10% formaldehyde at least 24 h and embedded in paraffin after and submitted for hematoxylin and eosin (HE) staining.

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Fig. 4. Effects of LCB on mRNA and protein expression related S cycle arrest of human bladder cancers T24 or EJ cells. Cells were exposed to LCB at 40 lM or 80 lM for 72 h. (A) Quantitative analysis of CyclinA, CDK1 and CDK2 mRNA levels via qRT-PCR. (B) Cdc25A and Cdc25B expressions were analyzed via Western blot. (C) Quantitative analysis of Cdc25A and Cdc25B protein levels. Control group (LCB-untreated group) level was accepted to be ‘‘1.0’’. ⁄P < 0.05, ⁄⁄P < 0.01 compared with the LCB-untreated control group cells.

2.13. TdT-mediated dUTP nick end labeling (TUNEL)

2.14. Statistical analysis

The TUNEL staining kit was purchased from Roche Group (Basel, Switzerland). Staining precedures were as following: (1) The tissue sections were deparaffinized by immersing slides in xylene, and rehydrated by sequentially immersing the slides through graded ethanol washes. (2)The activity of endogenous peroxidase was quenched by adding 0.3% H2O2 for 10 min and then washed in 10 mmol/L PBS (pH 7.4), 3 times for 5 min each. (3) The sections were digested with 20 lg/mL proteinase K for 25 min at room temperature, and washed in 10 mmol/L PBS (pH 7.4), 3 times for 5 min each. (4) The TUNEL reaction mixture was added to each section followed by incubating in a humid chamber for 60 min at 37 °C. (5) They were then washed in 10 mmol/L PBS (pH 7.4), 3 times for 5 min each, and incubated with peroxidase conjugated anti-fluorescein antibody in a humid chamber for 30 min at 37 °C. Then they were washed in 10 mmol/L PBS (pH 7.4), 3 times for 5 min each. (6) The tissue sections were stained wit h DAB for 5 min, counterst ained with hematoxylin, dehydrated, cleared in xylene and coverslipped.

Data were presented as means ± S.D. from at least three independent experiments and evaluated by analysis of variance (ANOVA) followed by Bonferroni method. The values of P < 0.05 were considered statistically significant. The analyses were performed using SPSS 17.0 statistical software program.

3. Results 3.1. LCB inhibited cell proliferation in T24 and EJ cells The T24 and EJ cells were treated with increasing concentrations of LCB for five days, the growth curve was shown in Supplementary data 1. The data showed that the growth of LCB-treated cells was

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Fig. 5. Effects of LCB on apoptosis of human bladder cancers T24 or EJ cells. Cells were exposed to LCB at 40 lM and 80 lM for 72 h. (A) Morphology measurements in T24 and EJ cells were carried out by Hoechst dye 33258. (B) Detection of apoptotic rates by flow cytometry.⁄P < 0.05, ⁄⁄P < 0.01 compared with the vehicle control group.

relatively slower than that of untreated cells in a concentrationdependent manner, the doubling time of LCB-treated T24 and EJ cells were significantly longer than that of untreated (Supplementary data 2). LCB cytotoxicity was determined using a MTT assay. The data indicated that LCB (20, 40, 60 or 80 lM) inhibited the proliferation of T24 and EJ in a concentration- and time-dependent manner (Fig. 2). After LCB treatment, a significant inhibition of proliferation on both cell lines was observed. Treatment for 72 h, compared with untreated cells, the cell viability of LCB-treated (40 or 80 lM) T24 cells was decreased by 59.91% and 73.51% at the concentration of 40 or 80 lM, respectively. The cell viability of EJ cells was decreased by 51.36% and 81.92% at concentration of 40 or 80 lM, respectively. The concentrations of 40 lM and 80 lM were used for most of the subsequent assays, and the two concentrations were close to or higher than the IC50 value of each cell type. 3.2. LCB induced S-phase arrest in T24 and EJ cells In order to find out the possible mechanisms of the significant proliferation inhibition in human bladder cancer T24 and EJ cells induced by LCB, we investigated the effects of LCB on cell cycle progression for treatment 72 h. Significant cell cycle arrest at the S phase with LCB treatment was observed in both cell lines. Of note,

LCB led to the EJ cells undergoing apoptosis after 72 h of treatment, as detected in a prominent sub-G1 apoptotic peak (Fig. 3). 3.3. Effect of LCB on levels of mRNA and proteins that regulate S transition in T24 and EJ cells Eukaryotic cell cycle progression is regulated by sequential activation of CDKs, whose activity is dependent upon their association with regulatory cyclins, and cyclin A, CDK1 and CDK2 play critical roles in the regulation of the S transition in the cell cycle, so we examined the effects of LCB on the expression of these mRNA. As shown in Fig. 4A, treatment with LCB resulted in a substantial reduction in the expression of cyclin A, CDK1 and CDK2 in T24 and EJ cells. Cdc25 dual specificity phosphatases are prominent regulators of cell cycle transition through the activation of cyclin-dependent kinases (CDKs). Cdc25A and Cdc25B also play essential roles in the regulation of cell cycle S phase transition. A marked decrease in Cdc25A and Cdc25B protein levels were observed in LCB-treated cells (Fig. 4B and C). 3.4. LCB induced apoptosis in T24 and EJ cells To determine whether the LCB-induced proliferation inhibition in human bladder cancer T24 and EJ cells was associated with the

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Fig. 6. Effects of LCB on protein expression related apoptosis of bladder cancers T24 or EJ cells. Cells were exposed to LCB at 40 lM or 80 lM for 72 h. (A) Bcl-2, Bax, Caspase3, PARP and survivin expressions were analyzed via Western blot. (B) Quantitative analysis of Bcl-2, Bax, Caspase-3, PARP and survivin protein levels. (C) The ratio of Bcl-2/ Bax. Control group (LCB-untreated group) level was accepted to be ‘‘1.0’’. ⁄P < 0.05, ⁄⁄P < 0.01 compared with the LCB-untreated control group cells.

induction of apoptosis, Hoechst dye staining was carried out. The typical apoptosis morphology, such as nuclear condensation and fragmentations, was observed in 80 lM LCB-treated groups (Fig. 5A). Extracellular phosphatidylserine (PS) flux from the inner to the outer leaflet of the plasma membrane represents an early marker of apoptosis that can be detected by Annexin V staining. However, it is essential to measure annexin staining only on cells within the viable gate to avoid the artifactual staining of dying cells that have lost membrane integrity and expose PS irrespective of whether the cells were caused to die by apoptotic, necrotic, or other uncharacterized processes. So, the cell size profile by the forward scatter parameter of flow cytometry in Annexin-V experiment was assessed (Supplementary data 3). LCB-induced

apoptosis was further quantified by using flow cytometry, after treatment with 80 lM of LCB for 72 h, representative results for T24 and EJ cells were shown in Fig. 5B. The percentage of apoptotic cells increased from 5.27% in control cells to 42.18% in T24 cells, and increased from 8.65% to 57.32% in EJ cells, respectively. 3.5. Effect of LCB on levels of apoptosis-related proteins in T24 and EJ cells The proteins of the Bcl-2 and Bax family play critical roles in regulation of apoptosis; the levels of Bcl-2 and Bax in cells treated with LCB were estimated. The both bladder cancer cell lines exposed to LCB showed a concentration-dependent reduction in the

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Fig. 7. Effects of LCB on colony formation of murine bladder carcinoma MB49 cells. Cells were treated with indicated amounts of LCB 24 h and allowed to grow for 14 days. ⁄⁄ P < 0.01 compared with the LCB-untreatedcontrol group cells.

level of Bcl-2 protein, with a concomitant increase in the level of Bax, when compared with the control cells. With the elevated of Bax expression and the reduced of Bcl-2 expression, the downstream casepase-3 for apoptosis was activated. As was shown, the expression of survivin, unactivated caspase-3 and full length of PARP was decreased (Fig. 6). 3.6. LCB reduced colony formation of MB49 cells in vitro MB49 cell is a 7, 1 2-dimethylbenz(a)anthracene-transformed mouse bladder epithelial cell line, and the murine model MB49 has been used as one of the most widely studied models of bladder cancer. The colony formation of tumor cells is closer to its physiology and growth in vivo, so we investigated the effect of LCB on suppression of long-term colony formation. Treatment of the cells with LCB (0, 40, 80 and 160 lM) resulted in a greater inhibition of colony formation. At 160 lM visual inspection of the all dishes revealed absent cells or colonies, indicating that LCB was completely lethal to MB49 cell (Fig. 7). 3.7. Inhibited tumor growth and induced tumor cells apoptosis in MB49 tumor model in vivo Based on the encouraging findings that LCB inhibited bladder cancer cells growth in vitro, we used MB49 tumor models to investigate whether LCB could suppress tumor progression in vivo. The murine MB49 bladder carcinoma in C57BL/6 mice was used as an in vivo model to evaluate the effects of LCB on tumor growth. Tumor growth inhibition was distinct in mice treated with LCB at 160 lM, compared with mice treated with normal saline (Fig. 8A). To gain insight whether LCB could induce apoptosis in vivo, paraffin sections of MB49 tumor from C57BL/6 mice were applied to the HE staining. Altered cells showed microscopic signs of apoptosis, i.e. cellular shrinking, a condensed bright eosinophilic cytoplasm and pycnotic dark small nuclei as a result of chromatin conden sation (Fig. 8B). The increased number of TUNEL-positive cells clearly demonstrated that the LCB could induce apoptosis of MB49 cells in vivo (Fig. 8C). Furthermore, there was no significant toxicity to mice treated with LCB (160 lM) by assessing mice weight of 2 groups, measuring the serum GOT and GPT (Fig. 8D). 4. Discussion Bladder cancer is a common but serious malignancy. It is widely accepted that chemoprevention may be an effective way to decrease the rate of recurrence and mortality (Shen et al., 2008). However, the overall response rate to combination chemotherapy for bladder cancer is intolerable, with a high incidence of side effects.

Therefore, more attention has focused on the potential application of natural products in the treatment of bladder cancer (Tian et al., 2008). In the current study, we demonstrated for the first time that LCB caused a concentration- and time-dependent cancer cell growth inhibition in vitro, and this anti-proliferative effect appears to be due to its ability to induce S-phase arrest and apoptotic cell death. Furthermore, we examined the S-phase regulated mRNA, proteins, and apoptotic markers after exposure of different concentrations of LCB, the results indicated that LCB could decrease cyclin A, CDK1, CDK2, Cdc25A and Cdc25B expression, cleavage of caspase-3 and PARP, down-regulate the anti-apoptotic Bcl-2 and survivin as well as up- regulate the pro-apoptotic Bax. Accordingly, we observed the growth inhibitory and pro-apoptotic effect of LCB on bladder cancer cells with MB49 tumor model in vivo. Deregulation of the cell cycle is one of the most frequent alterations during tumor development (Park and Lee, 2003). Therefore, Inhibition of cell cycle progression is considered an effective strategy for the control of tumor growth (Deep and Agarwal, 2008). The results obtained in the present study provided convincing evidence that LCB induced significant cell cycle arrest in both T24 and EJ cells (Fig. 3). In T24 cells, LCB (40 or 80 lM) caused S phase cycle arrest. The cell population in the S phase increased by over 1.31 or 1.63-fold compared to the control. A similar pattern was observed for EJ cells, with a 1.18 or 1.56-fold increase compared to the control. The cell cycle control system is based on two protein families: the cyclin-dependent protein kinases (CDKs) and the cyclins. Cyclin A accumulates at the G1/S phase transition and persists through S phase. Cyclin A initially associates with CDK2 and then, in late S phase, associates with CDK1. Cyclin A-associated kinase activity is required for entry into S phase, completion of S phase, and entry into M phase (Johnson and Walker, 1999). The decrease of cyclin A, CDK1, and CDK2 mRNA expression provided an additional explanation for the S phase arrest induced by LCB (Fig. 4A). Cdc25 dual specificity phosphatases are another mechanism regulating cell cycle transition. Whereas Cdc25A controls both the transit from G1 to S phase as well as that through G2-M phase (Hoffmann et al., 1994; Kiyokawa and Ray, 2008), Cdc25B seems to promote the entry from S phase into G2-M (Lammer et al., 1998; Unger et al., 2012). Cdc25C enables G2-M transit that can be compensated by Cdc25A (Ferguson et al., 2005). Accordingly, the down-regulated of Cdc25A and Cdc25B protein expression also may account for S-phase arrest (Fig. 4B and C). Chalcones are a group of plant-derived polyphenolic compounds belonging to the flavonoids family and possess a wide variety of cytoprotective and modulatory functions. Some chalconoids were demonstrated the ability to block the S-G2 transition has been reported previously in MDA-MB-435 human mammary adenocarcinoma cells (Gerhauser et al., 2002). However, other investigators

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Fig. 8. LCB inhibited the growth and induced cell apoptosis in MB49 tumor models in vivo. (A) Average tumor weight was measured by laboratory electronic balance. (B) HE staining. (C) TUNEL staining. (D) Average body weight, serum GOT and GPT activities. ⁄P < 0.05, compared with LCB-untreated control group cell.

have reported an arrest in the G1 phase with A549 cells (Rao et al., 2010). A G2-M phase cell cycle arrest was demonstrated in the human osteosarcoma cells (Ji et al., 2013). Therefore, the effects of chalcones on cell cycle progression can vary in different experimental systems. The present study demonstrated LCB induced S phase cell cycle arrest in both T24 and EJ cells. However, the underlying mechanisms of this phenomenon are not clear and further investigations are needed to address.

The induction of apoptosis is now considered to be an attractive strategy for cancer therapy (Bremer et al., 2006; Lawen, 2003). Bcl-2 family proteins are known to play a pivotal role in the induction of caspases activation and in the regulation of apoptosis (Adams and Cory, 1998; Burlacu, 2003). Caspase-3, the executioner caspase, cleave various substrates, including inhibitor of caspase-activated DNase (ICAD), PARP (a DNA repair enzyme), and lamin and others, that ultimately cause the morphological and biochemical changes

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seen in apoptotic cells (Slee et al., 2001). Thus, therapeutic strategies designed to stimulate apoptosis by regulating Bcl-2 family proteins or activating caspase-3 may help in combating cancer caused by apoptosis deficiency. In the present study, we have traced from PARP cleavage and activation of caspase-3 to anti-apoptotic Bcl-2 and survivin decrease and pro-apoptotic Bax increase (Fig. 6). The activation of Bax and caspase-3 is critical for initiation and amplification of apoptosis, respectively, their activation is normally blocked by Bcl-2 and Bcl-xL or inhibitors of apoptosis proteins (e.g., survivin and XIAP) at caspases levels (Zi and Simoneau, 2005). Unlike XIAP, survivin did not reveal the similar binding sites of other inhibitors of apoptosis proteins with caspases, the antiapoptotic function of survivin is well established (Altieri, 2003; Riedl et al., 2001). In bladder cancer, its expression correlates with tumor grade, recurrence risk, and survival. So, it has been suggested as a novel diagnostic/prognostic marker of bladder cancer (Ma et al., 2006). In this current study, the lower expression of survivin protein occurred after LCB treatment for 72 h. Because the pro-apoptotic effects and the down-regulation of bladder cancer associated protein were observed in LCB-treated T24 and EJ cells, LCB has the potential to be used for treatment of bladder cancer. Further, the MB49 tumor models were established to investigate whether LCB could suppress tumor progression in vivo. Results revealed LCB could induce apoptosis of in vivo, and did not cause any side effect (Fig. 8). 5. Conclusions Our results show that LCB caused a concentration-dependent bladder cancer cells proliferation inhibition, and this antiproliferative effect appears to be due to its ability to induce S-phase arrest and apoptotic cell death. Furthermore, we have identified cyclin A, Cdc25A, Cdc25B and apoptotic index possibly as candidate biomarkers for use as surrogate intermediate end points. The present study provided evidence to support the use of this compound in bladder cancer prevention and therapy trials. Conflict of Interest There is no conflict interest to disclose for all authors. Transparency Document The Transparency documents associated with this article can be found in the online version. Acknowledgements This study was supported by the Science and Technology Major Project of Gansu Province, China (No. 1203FKDA032), the Traditional Chinese Medicine Scientific Research Projects of Gansu Province, China (No. GZK-2009-5) to Zhiping Wang; and the National Natural Science Foundation of China (No. 81260338), the Xinjiang Production and Construction Corps Funds for Distinguished Young Scientists (2011CD006), and International Cooperation Projects (2012BC001) to Qiusheng Zheng. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.fct.2013.12.030. References Adams, J.M., Cory, S., 1998. The Bcl-2 protein family: arbiters of cell survival. Science 281 (5381), 1322–1326. Altieri, D.C., 2003. Validating survivin as a cancer therapeutic target. Nat. Rev. Cancer 3 (1), 46–54.

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