MOLECULAR CARCINOGENESIS

Kaempferol Suppresses Bladder Cancer Tumor Growth by Inhibiting Cell Proliferation and Inducing Apoptosis Qiang Dang,1 Wenbin Song,1 Defeng Xu,2 Yanmin Ma,1 Feng Li,1 Jin Zeng,1 Guodong Zhu,1 Xinyang Wang,1 Luke S Chang,1 Dalin He,1* and Lei Li1** 1 2

Department of Urology, The First Affiliated Hospital of the Medical College of Xi’an Jiaotong University, Xi’an, PR, China School of Pharmaceutical and Life Sciences, Changzhou University, Changzhou, Jiangsu, PR, China

The effects of the flavonoid compound, kaempferol, which is an inhibitor of cancer cell proliferation and an inducer of cell apoptosis have been shown in various cancers, including lung, pancreatic, and ovarian, but its effect has never been studied in bladder cancer. Here, we investigated the effects of kaempferol on bladder cancer using multiple in vitro cell lines and in vivo mice studies. The MTT assay results on various bladder cancer cell lines showed that kaempferol enhanced bladder cancer cell cytotoxicity. In contrast, when analyzed by the flow cytometric analysis, DNA ladder experiment, and TUNEL assay, kaempferol significantly was shown to induce apoptosis and cell cycle arrest. These in vitro results were confirmed in in vivo mice studies using subcutaneous xenografted mouse models. Consistent with the in vitro results, we found that treating mice with kaempferol significant suppression in tumor growth compared to the control group mice. Tumor tissue staining results showed decreased expressions of the growth related markers, yet increased expressions in apoptosis markers in the kaempferol treated group mice tissues compared to the control group mice. In addition, our in vitro and in vivo data showed kaempferol can also inhibit bladder cancer invasion and metastasis. Further mechanism dissection studies showed that significant down-regulation of the c-Met/p38 signaling pathway is responsible for the kaempferol mediated cell proliferation inhibition. All these findings suggest kaempferol might be an effective and novel chemotherapeutic drug to apply for the future therapeutic agent to combat bladder cancer. © 2014 Wiley Periodicals, Inc. Key words: kaempferol; bladder cancer; cell growth; apoptosis; c-met/p38

INTRODUCTION Bladder cancer is the second most common malignancy of the genitourinary after prostate cancer, even though the early diagnosis and treatment have been well developed, the bladder cancer related mortality is still high. An estimated 386 300 new cases and 150 200 deaths from bladder cancer occurred in 2011 worldwide [1]. Most commonly, bladder cancer is of the transitional cell type, and 60–70% of newly diagnosed bladder cancers are non-muscle invasive bladder cancers, which can be treated well with Transurethral Resection of Bladder Tumor (TURBT). However, there many cases recur and progress to higher stages and metastases [2], so the post-operative chemotherapy is still important and meaningful. Kaempferol is a kind of flavonoid and can be commonly found in plant-derived foods and Chinese herbal plants. Like other flavonoids, kaempferol has a diphenylpropane structure (C6-C3-C6) [3]. Some epidemiological studies found that consumption of foods containing kaempferol can prevent or reduce the risk of developing several types of tumors, including lung cancer [4], gastric cancer, pancreatic cancer [5], and epithelial ovarian cancer [6]. Kaempferol was shown to inhibit tumor growth and angiogenesis, and induce cell death through apoptosis, either via caspase-dependent pathways [7] or caspase-independent pathways [8]. In addition, kaempferol was also shown to activate some proß 2014 WILEY PERIODICALS, INC.

apoptotic genes [9] or inhibit some oncogenes [10]. However, whether this compound will inhibit cell growth and/or induce apoptosis of bladder cancer cells remains unclear. Hepatocyte growth factor (HGF) and its receptor cMet, became the research hot spot when it was cloned by Nakamura et al. [11], and their expressions were found up-regulated in most human solid tumors [12]. c-Met is essential for cell proliferation, migration, and tissue regeneration in normal tissues, but its abnormally up-regulated level in cancer was shown to be

Abbreviations: TURBT, transurethral Resection of Bladder Tumor; KAP, Kaempferol; HGF, hepatocyte growth factor; MTT, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling; MAPK, mitogen-activated protein kinase; IHC, immunohistochemical. Qiang Dang and Wenbing Song have equal contribution to this paper. Grant sponsor: National Natural Science Foundation of China; Grant number: 81101936 *Correspondence to: Dalin He, Department of Urology, The First Affiliated Hospital of the Medical College of Xi'an Jiaotong University, Xi'an, PR China. **Correspondence to: Lei Li, Department of Urology, The First Affiliated Hospital of the Medical College of Xi'an Jiaotong University, Xi'an, PR China. Received 9 September 2013; Revised 26 February 2014; Accepted 10 March 2014 DOI 10.1002/mc.22154 Published online in Wiley Online Library (wileyonlinelibrary.com).

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correlated with poor prognosis since c-Met can trigger tumor cell growth, formation new blood vessels, and increase the capability of invasion and metastasis in vitro and in vivo [13–15]. HGF binding to c-Met can activate multiple signaling cascades, and promote cell proliferation mainly through the MAP kinase pathway [16]. Interestingly, it was found that inhibition of c-Met suppressed bladder cancer cell proliferation [17]. In this study we investigated the effect of kaempferol on bladder cancer. Its effects on bladder cancer cells cytotoxicity, cell cycle arrest, and apoptosis were tested and in vivo mice studies were also elucidated in parallel. MATERIALS AND METHODS Cell Culture and Animals Human bladder cancer cell lines (5637, T24) were purchased from ATCC, TCC-SUP, and 253J and T24-L were obtained from Dr jer-tsong hsien of university of southwestern medical center. The 5637 cell line was maintained in RPMI1640, and T24, TCC-SUP, 253J, and T24-L. They utilized the T24 cells to develop an in vivo experimental model of bladder cancer metastasis [18]. The development of spontaneous lung metastases after intravesical instillation was monitored using bioluminescent imaging (BLI). In the previous studies, they had demonstrated that T24-L cells exhibit more invasive, metastatic, and chemoresistant abilities in vitro and in vivo [19]) cell lines were maintained in DMEM, both containing 10% fetal bovine serum (10% FBS), 2 mM Lglutamine, 100 IU/ml penicillin, 50 mg/ml streptomycin, and cultured at 378C, in a humidified atmosphere containing 5% CO2. Six to 8 wk old athymic BALB/c nu/ nu male mice were obtained from the Experimental Animal Center of Xi’an Jiaotong University. Animal care and protocols were approved by the Institutional Animal Care and Use Committee of Xi’an Jiaotong University and all the animal experiments were performed in adherence with the NIH Guidelines on the Use of Laboratory Animals. Reagents Kaempferol was a gift from Professor Xu defeng at Shanghai Jiaotong University. Kaempferol was dissolved in N,N-dimethylacetamide (DMA) and diluted with Kolliphor EL (Sigma–Aldrich, St.Louis, MO) and 0.9% normal saline but use in animal studies. Antibodies against Chk1,Chk2, p35, c-Met, c-Fos, GAPDH, and cyclinB1 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) Antibodies against caspase7, cleaved caspase7, p-ERK1/2, cleaved PARP, p-c-Met Tyr1234/1235, p-p38, and p21 waf1/ cip1 were purchased from Cell signaling Technology, Inc. (Boston, MA) The inhibitors, sb203580 and PHA665752 were purchased from Cell signaling Technology and Tocris Bioscience, respectively, caspase3/7 inhibitor1 was purchased from APEXBIO Molecular Carcinogenesis

(Houston, TX). Tunnel assay kit was purchased from Roche (Indianapolis, IN). DNA ladder and Annexin VFITC Apoptosis Detection kits were from Jiancheng Bioengineering Institute (Nanjing, China), pCDNA3Flag MKK6(glu) was purchased from Addgene (Cambridge, MA). Cell Viability Test Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. 1
103 cells in 50 ml of media per well were plated in 96-well plates. Cells treated with different doses of kaempferol were incubated for the indicated times, and then incubated with 0.5 mg/ml of MTT at 378C for 1 h. At the end of incubation, DMSO was added to dissolve the precipitates. Colorimetric analysis using a 96-well microplate reader was performed at wavelength 490 nm. The experiments were performed in triplicate. Detection of Apoptosis by Flow Cytometric Analysis Cells (5637 and T24) were exposed to different doses of kaempferol (50, 100, and 150 mM) for 48 h. Cells were then collected and subjected to Annexin V and propidium iodide (PI) staining using an Annexin V-FITC Apoptosis Detection Kit, following the protocols provided by the manufacturer. Apoptotic cells were then analyzed by flow cytometry (BD FACSCalibur, San Jose, CA). TUNEL Assay Cells, treated with either kaempferol or vehicle and the paraffin embedded sections of tumor tissues samples were used in terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. Staining was carried out according to the protocol provided by the company. DNA Ladder Assay Cells were treated with different doses of kaempferol, DNA were extracted by lysis buffer, and then subjected to agarose gel electrophoresis run. All of procedures were carried out according to the protocols supplied by company. Cell Cycle Detection Assay After cells reach to 60–80% confluence, cells were treated with different doses of kaempferol (50, 100, and 150 mM). After 48 h of incubation, cells were washed twice with PBS, fixed with 70% ethanol for 1 h at 48C, and resuspended with PI solution (0.05 mg/ml) containing RNase and incubated at room temperature in the dark for 30 min. DNA content was then analyzed using the flow cytometry (BD FACSCalibur). Western Blot Analysis Cells were lysed in RIPA buffer (50 mM Tris–HCl/pH 7.4, 1% NP-40, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM Na3VO4, 1 mM NaF, 1 mM okadaic acid,

KAEPMFEROL CAN INHIBIT BLADDER CANCER GROWTH AND METASTASIS

and 1 mg/ml aprotinin, leupeptin, and pepstatin) with cocktail (Roche). Cell extracts (25 mg) were applied to electrophoresis run on 12–15% SDS/ PAGE gel and then transferred onto PVDF membranes (Millipore, Billerica, MA). After blocking the membranes with 5% BSA in PBS for 1 h at room temperature, the membranes were incubated with appropriate dilutions of specific primary antibodies overnight at 48C. After washing, the blots were incubated with anti-rabbit, anti-mouse, anti-goat IgG HRPs for 1 h. The blots were developed in ECL mixture (Thermo Fisher Scientific, Waltham, MA). Animal Studies The 5637 cells were trypsinized, washed, and resuspended in the medium. Cells (5
105 cells, 100 mL), as a mixture with matrigel (BD) (1:1, v/v) were then injected subcutaneously into the flanks of mice to develop tumors. After tumors develop, tumorbearing mice were randomized to control and various treatment groups (n ¼ 8) and intraperitoneally injected with kaempferol (described in Reagent Section). All treatments were administered for 4 wk. Body weights were recorded weekly throughout the study. Immunohistochemical (IHC) Staining Tumor tissues obtained from euthanized xenografted mice were fixed in 10% (v/v) formaldehyde in PBS, embedded in paraffin, and cut into 5-mm sections. When stained, tumor tissue sections were deparaffinized in xylene solution and rehydrated using gradient ethanol concentrations. The staining was according to the DAKO system protocol. The tissue sections were stained with specific antibodies against c-Met (1:100), cyclinB1 (1:100), or c-Fos (1:100). Hematoxylin was used for counterstaining. Slides were viewed and photographed under a light microscope. The sections incubated with secondary antibodies in the absence of primary antibodies were used as negative control. Statistical Analysis All statistical analyses were performed using SPSS 16.0 software. Quantitative data are presented as mean  SE and the differences among the control and test groups were compared by one-way ANOVA, followed by Dunnett’s t test for separate comparisons. In the comparison involving only two groups, Student’s t test was used. P < 0.05 was considered statistically significant. RESULTS Kaempferol Increases Cytotoxicity of Bladder Cancer Cells We first investigated the effects of kaempferol (structure is shown in Figure 1A) on bladder cancer cell survival. We used four bladder cancer cell lines, 5637, T24, 253J, and TCCSUP representing high risk non-muscle invasive TCC, high grade TCC, invasive Molecular Carcinogenesis

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metastatic TCC, and high-grade invasive TCC, respectively, were used as the model system. We treated cells with various doses of kaempferol for 48 h and at the end of treatment, the cell viability was analyzed by MTT tests. As shown in Figure 1B, significantly increased cytotoxicities were detected in every cell line tested in a dose dependent manner. We then fixed the kaempferol dose (100 mM), and varied the incubation time and cell viability for similar analysis. As Figure 1C shows, the kaempferol induced-enhanced cytotoxicities were observed in every cell line in a time-dependent manner. These results suggest that kaempferol is effective in inducing cytotoxicity of bladder cancer cells at all stages. Kaempferol Induces Apoptosis of Bladder Cancer Cells We then used 5637 and T24 bladder cancer cells and investigated the effects of kaempferol on inducing apoptosis by applying three different apoptosis analysis methods. Cells were treated with 50, 100, and 150 mM of kaempferol for 48 h. Figure 2A and B showed the flow cytometric analysis data with the Annexin V positive cells increased after kaempferol treatments. As a golden standard method for apoptosis analysis, we also performed the DNA ladder analysis. As shown in Figure 2C, we were able to detect apoptotic DNA fragments in 5637 cells at all doses of kaempferol, whereas we could observe DNA fragments only at high dose of kaempferol in T24 cells. The results of 5637 cells showing DNA fragments at lower doses of kaempferol is consistent with the previous data of the viability test and flow cytometric analysis demonstrating more sensitivity to this drug. As a 3rd method to test apoptosis, we performed TUNEL assay. As clearly demonstrated in Figure S1, the apoptotic 5637 cells (green fluorescent cells) were increased upon kaempferol treatment. The induction of apoptosis upon kaempferol treatment was further confirmed in Western blot analysis using antibodies against the well-known apoptosis markers, cleaved caspase-7 [20], and cleaved-PARP [21]. We found that expression of these two molecules were markedly increased in 5637 and T24 cells upon kaempferol treatment (Figure 2D). Furthermore, caspase 7 inhibitor did not completely reverse kaempferol-induced apoptosis, suggesting the potential involvement of other caspases. The specificity and efficacy of caspase 7 inhibitor activity was confirmed by caspase 7 activity assay that clearly demonstrated that pretreatment with caspase 7 inhibitor inhibit caspase 7 activity (Figure 2E and F). Taken together, these results indicate that kaempferol induced bladder cancer apoptosis through caspase-dependent pathway and independent pathway. Kaempferol Induces Cell Cycle Arrest of Bladder Cancer Cells We next investigated the effect of kaempferol on cell cycle profiles. After treating 5637 and T24 bladder

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Figure 1. Effect of kaempferol on bladder cancer cell viability. (A) Chemical structure of kaempferol. (B) Bladder cancer cells (5637, T24, 253J, TCCSUP) were treated with indicated doses of kaempferol (kap), 50, 100, and 150 mM, for 48 h and cell viability was analyzed by MTT assay. (C) The same bladder cancer cells as in B were treated with 100 mM kaempferol for different time periods (0, 24, 48, and 72 h) and cell viability was analyzed as in B.  P < 0.05.

cancer cells with kaempferol for 24 h, cell cycle profiles were analyzed by flow cytometric analysis. As shown in Figure 3A and B, we observed cell cycle arrest at G2-M stage by 15.22%, 36.05%, and 39.37%, at 50, 100, and 150 mM kaempferol treatment, respectively, in 5637 cells while 10.52%, 19.53%, and 25.49% arrest was observed at 50, 100, and 150 mM kaempferol treatment, respectively, in T24 cells. We also investigated expressions of the cell cycle associated genes in bladder cancer cells upon kaempferol treatment. We found expressions of several cell cycle related genes, including CHK1, CHK2, and p21waf1/Cip1, that up-regulated while expression of the p35 and cyclinB1 genes were decreased in 5637 cells upon kaempferol treatment (Figure 3C). We observed similar results in studies with T24 cells (Figure 3D), except for the p21 expression, which is lacking in T24 cells. These results indicate that Molecular Carcinogenesis

kaempferol can induce bladder cancer cell cycle arrest. Kaempferol Inhibits Bladder Cancer Cell Proliferation Via Modulation of the c-Met/MAPK Pathway Since we showed that kaempferol can inhibit bladder cancer cell proliferation, we next determined to dissect the underlying mechanism of kaempferol action. We investigated the expressions of several cell proliferation associated proteins using cell extracts obtained from the 5637 cells (Figure 4A) and T24 cell (Figure 4B), with or without kaempferol treatment, and found significant down-regulation of the phosphorylated mitogen-activated protein kinases (pMAPK, also known as p38) upon kaempferol treatment although expression of p38 molecule itself remained unchanged (data not shown). We also investigated upstream signaling that can modulate the MAPK pathway. Several signaling

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Figure 2. Kaempferol effect on inducing apoptosis of bladder cancer cells. (A) After treating with indicated doses of kaempferol (50,100, and 150 mM) for 48 h, 5637 and T24 cells were collected, stained with Annexin V and PI, and the positively stained cells were analyzed by flow cytometry. (B) Quantification is shown in lower panel. (C) DNA ladder assay. After the 5637 and T24 cells were treated with kaempferol as in A, DNAs were extracted and subjected to agarose gel electrophoresis. (D) After the 5637 and T24 cells were treated with kaempferol as in the previous experiments, total cell lysates were obtained and expressions

of caspase 7 (1:1000) and cleaved PARP (1:1000) were analyzed by western blot analyses using appropriate antibodies. Cells were treated with 100 mM of kaempferol (kap 100 mM) and/or 100 mMM of caspase 7 inhibitor 1 for 48 h; in combination treatment, caspase 3/7 inhibitor 1 was added 1 h prior to kaempferol treatment. (E) At the end of treatment, the cells were analyzed for apoptosis by flow cytometry and (F) cell extracts were prepared for caspase-7 activity assay. Error bars represent SEs.  P < 0.05.

pathways, such as epidermal growth factor and its receptor (EGF/EGFR) and hepatocyte growth factor and its receptor (HGF/HGFR, also known as c-Met) can regulate MAPK signaling [22]. When we investigated possible down-regulation of these two signaling pathways upon kaempferol treatment in the 5637 and T24 cell, we found down-regulation of c-Met and p-cMet molecules (Figure 4A and B), but not the p-EGFR level (data not shown). We also found expression of one of the p38 downstream molecules, c-Fos, was significantly down-regulated after kaempferol treatment (Figure 4A and B). c-Met can modulate the MAPK pathway to affect cell proliferation [23]. To delineate whether the proliferation inhibition we observed in bladder cancer cells upon kaempferol treatment was mainly through c-Met/MAPK signaling pathway, we added c-Met

inhibitor PHA665752 [24] and p38 inhibitor SB203580 [25] into the culture and examined their effects on cell growth. As shown in Figure 4C and D, kaempferol blocked HGF induced up-regulation of cMet and p38 activations and the effects were comparable to their inhibitor effects. We next used inhibitors of the c-Met (PHA665752) and p-38 (SB203580) signaling and compared these inhibitor effects with the kaempferol effect on suppressing cell growth. We added these inhibitors or kaempferol into the HGF induced cell culture system and cell growth was analyzed by MTT assay. As shown in Figure 4E and F, the kaempferol inhibitory effect on cell growth was similar to the effects obtained with the inhibitors. But we also found kaempferol can further decrease p38 activity after added p38 inhibitor, and cell growth also can be

Molecular Carcinogenesis

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Figure 3. Kaempferol effect on inducing bladder cancer cell cycle arrest. (A) Cell cycle analysis. After treating with indicated doses of kaempferol, bladder cancer cells were collected, fixed, stained with PI, and then cell cycle profiles were investigated using flow cytometry (FACS). (B) Percentage distribution of each cells stage was shown. (C) After treating with indicated doses of kaempferol, the 5637 bladder cancer cells lysates were obtained and expressions of the cell cycle markers, Chk 1, Chk 2, p35, cyclin B1, and p21 were analyzed by Western blot analysis. GAPDH was used as loading control. (D) Expressions of the cell cycle markers in T24 cells were investigated as in B.  P < 0.05.

further suppressed (supplementary Figure S2A,B). MKK6 is a dual-specificity protein kinase of the STE7 family, which can continuely activate p38 MAP kinase by phosphorylating a Thr and a Tyr residue in the activation loop. And we transfect MKK6 into cell to rescue the p38 and cell growth, but like Figure S2C,D shown, MKK6 just can partially reverse the cell growth. Molecular Carcinogenesis

From the kaempferol can inhibit bladder cancer cell growth via inhibiting the c-Met/p38 signaling pathway. Kaempferol Suppresses Bladder Tumor Growth and Metastasis Previously we demonstrated that kaempferol induces cell cytotoxicity, promotes apoptosis, and mediates cell cycle arrest in vitro, meanwhile, we

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Figure 4. Kaempferol inhibited p38 phosphorylation through c-Met/p38 signaling pathway. (A) After treating with indicated doses of kaempferol, the 5637 bladder cancer cells lysates were obtained and expressions of p-c-Met, p-p38, p-ERK, and c-Fos were examined by Western blot analysis. GAPDH was used as loading control. (B) Expressions of p-c-Met, p-p38, p-ERK, and c-Fos in T24 cells were examined as in A. (C) The 5637 cells were treated with kaempferol at indicated doses, in the absence and presence of the c-Met inhibitor, PHA665752 (c-Met inhibitor, 10 mM) and incubated for 48 h. Two h before collecting cells, HGF was added into the culture to activate cMet signaling. At the end of experiment, cell extracts were obtained and expression of p-c-Met was investigated by Western blot analysis. (D) 5637 cells were treated are similar to C, but cells were treated with the p-38 inhibitor, SB203580 (20 mM) instead of c-Met inhibitor, and HGF was added 30 min before collecting cells and expression of pp38 was analyzed. (E) Seed cell in 24 well plates, pretreat with PHA and kaempferol for 2 h, and added HGF to culture for 48 hr, use MTT assay to detect cell proliferation. (F) Similar with E, but use p38 inhibitor SB203580 instead of PHA.  P < 0.05.

also found kaempferol significantly inhibit motility and invasion of bladder cancer cells in vitro as determined by wound healing assay (Figure S3) and Transwell invasion assay (Figure S4). We then determined to confirm these in vitro results in in vivo mouse studies. Firstly we developed bladder cancer xenograft mouse model by injecting 5637 bladder cells subcutaneously into the nude mice. When tumors develop, we divided mice into four groups, for treatment as follows: Group 1, injected with vehicle (DMA); Group 2, injected with 50 mg/kg kaempferol; Group 3, injected with 100 mg/kg kaempferol; Group 4, injected with 150 mg/kg kaempferol. (i.p. injection, daily, for 4 wks). Molecular Carcinogenesis

At the end of treatment, mice were sacrificed and tumor weights in each group mice were compared. Surprisingly, we observed significant differences in tumor weights between control and test group (kaempferol treated) mice. All three test groups mice showed reduction in tumor weights and the reduction was more significant with the high dose drug treatment (Figure 5A), confirming the positive effect of this drug in effectively reducing tumor growth. During the treatment, body weights of each group mice were monitored and as shown in Figure 5B, there were no noticeable difference observed. Gross and microscopic examination of the liver, lung, spleen, and kidney did not reveal

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any evidence of toxicity (data not shown). These results suggested effective in vivo antitumor efficacy of kaempferol in treated doses against human bladder tumor without host toxicity, and also our in vitro data showed low toxicity to norma human uroepithelial cell-SVHUC (Figure S5). We next performed IHC tissue staining using the tumor tissues obtained from each groups mice. The TUNEL assay (Figure 5C upper panel) result showed apoptotic cells increase from 7% in control group mice to 70% in the 150 mg/kg kaempferol treated mice. The IHC staining of c-Met, cyclin B1, and c-Fos showed the consistent results with the in vitro data obtained previously by demonstrating decreased expressions in these molecules in the kaempferol treated mice tissues compared to the control mice (Figure 5C, lower 3 sets of panels). Secondly, we developed bladder cancer matastasis mouse model by tail vein injecting T24-luciferae cells. We started injection of 100 mg/kg kaempferol and vehicle control before 3 d injection of T24 cells, until the end of 3 wk, and monitored the tumor matastasis status by IVIS system. As the Figure S6 shown, kaempferol can significantly inhibit the tumor matastasis in vivo. Taken together, the in vivo mice studies results confirmed the in vitro kaempferol effects suppressing bladder cancer cell growth and matastsis. DISCUSSION Kaempferol is a polyphenol, which is a type of flavonoid (flavonol), and is a natural compound and present in a wide variety of fruits and vegetables [26]. Therefore, research has been focused on use of this compound for protection of many diseases, although Garcia et al. reported high intake of specific carotenoids and flavonoids did not reduce the risk of bladder cancer [27], our present preclinical study did not focus on the effects of kaempferol on bladder cancer recurrence, the data we obtained strongly suggested the potential anti-cancer effects of kaempferol against bladder cancer, providing a basis for future clinical trials of kaempferol used in patients with primary or recurrent bladder cancer. Indeed, this compound has shown a protective effect against various cancers [28]. In addition to this protective effect, this compound was also reported to be effective in inhibiting cell growth and proliferation of various types of cancer cells and also known to induce cancer cells cytotoxicity [29,30]. In addition, kaempferol effects on sensitizing cancer cells in response to other drugs, such as cisplatin, has been shown [31]. In this study, for the first time, we have demonstrated kaempferol effects on inducing cytotoxicity, apoptosis, and modulating cell cycle of bladder cancer cells. Interestingly, this compound showed effects on all bladder cell lines we have tested, which represent different stages of bladder cancer although each cell line exhibited a small variation in sensitivity to the Molecular Carcinogenesis

compound. We speculate that kaempferol can be used in therapeutic approach to bladder cancer of all stages although testing this drug effect in different mouse models representing different stages of bladder cancers will be necessary to confirm this hypothesis. The result of kaempferol effect on bladder cancer cells we showed here is exciting since it could trigger both apoptosis and cell cycle arrest at the same time. Considering not many drugs can target apoptosis and growth together, the result we have shown here may have a clinical value. The effect of this compound on inducing apoptosis was shown clearly in three different apoptosis assays and its effect on cell cycle arrest was proved by the flow cytometric analysis and by expression differences in cell cycle marker proteins. Kaempferol effect on affecting cell cycle has been demonstrated previously in other cancers. However, our result was different from the previous studies on other cancers in that the previous studies showed this compound effect on inducing G2-M phase arrest [32–35] while our result showed its effect on inducing G1-S phase arrest. We further showed that this arrest is associated with p21 waf1/cip1 upregulation. We also elucidated the mechanism by which kaempferol mediates cell cycle arrest. We have shown that kaempferol arrested cell cycle, thereby suppressed cell growth via down-regulation of c-Met/ p38 signaling pathway. Interestingly, we found p-ERK expression was increased in bladder cancer cells upon kaempferol treatment (date not shown), which is opposite to the previous report suggesting downregulation of this molecule in mediating apoptosis increase [36]. When we used an inhibitor of this pathway, we could not influence apoptosis, either (data not shown), so we believe that this pathway may not be involved in apoptosis induction in bladder cancer cells. This result indicates that the mechanism of the kaempferol action shown in bladder cancer may be different from other cancers. As we all known, most of the recrudescent cases progress to higher stages and metastases [2], so in our study, we also test the effect of kaempferol on bladder cancer cell invasion and metastasis, and our data clearly show kaempferol can inhibit bladder cancer invasion and metastasis, which consistent with feng xie’ results [37], but we are first to show the effect of kaempferol on bladder cancer by metastasis mouse model. We have not studied the mechanism by which kaempferol induces bladder cancer cell apoptosis. Other laboratories showed that kaempferol can induce apoptosis through generation of reactive oxygen species. For example, Sharma et al. [38] reported that kaempferol can induce apoptosis in glioblastoma cells through induction of oxidative stress. Further investigation will be needed to determine whether the kaempferol effect on bladder cancer is also through this pathway.

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Figure 5. Kaempferol suppressed bladder tumor growth and induced apoptosis of bladder cancer cells. (A) The 5637 cells were subcutaneously injected into flanks of mice and when tumors develop, mice were divided into control and text groups and were i.p. injected daily with either vehicle (control group mice) or kaempferol (50, 100, and 150 mg/kg) for 4 wk. At sacrifice of each group of mice, tumors were dissected and weighed. (B) Mice body weight comparison

between control and test group mice. (C) TUNEL staining (upper panel) and IHC staining of tumor tissues for examining levels c-Met (upper middle panel), cyclin B1(lower middle panel), and c-Fos (lower panel) proteins expressions. Magnifications, 400
. Quantification for TUNEL and IHC staining were shown on right. Data are shown as mean of 8 mice results in each group.  P < 0.05,  P < 0.01.

Our in vivo xenograft mouse model studies confirmed our in vitro results. Interestingly, the tumor growth suppression effect was more significant when we increased the kaempferol dose. We believe that more animal studies should be performed to determine the kaempferol dose that can yield the best efficacy without resulting in serious toxicity and side effects.

by the National Natural Science Foundation of China (NSFC NO. 81101936 to J.Z.).

ACKNOWLEDGMENTS We thank Dr. Soo Ok Lee for preparing discussion and editing the manuscript. This work was supported Molecular Carcinogenesis

REFERENCES 1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin 2011;61:69–90. 2. Schenk-Braat EA, Bangma CH. Immunotherapy for superficial bladder cancer. Cancer Immunol Immunother 2005;54:414– 423. 3. Calderon-Montano JM, Burgos-Moron E, Perez-Guerrero C, Lopez-Lazaro M. A review on the dietary flavonoid kaempferol. Mini Rev Med Chem 2011;11:298–344.

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4. Cui Y, Morgenstern H, Greenland S, et al. Dietary flavonoid intake and lung cancer—A population-based case-control study. Cancer 2008;112:2241–2248. 5. Nothlings U, Murphy SP, Wilkens LR, Henderson BE, Kolonel LN. Flavonols and pancreatic cancer risk: The multiethnic cohort study. Am J Epidemiol 2007;166:924–931. 6. Gates MA, Vitonis AF, Tworoger SS, et al. Flavonoid intake and ovarian cancer risk in a population-based case-control study. Int J Cancer 2009;124:1918–1925. 7. Marfe G, Tafani M, Indelicato M, et al. Kaempferol induces apoptosis in two different cell lines via Akt inactivation, Bax and SIRT3 activation, and mitochondrial dysfunction. J Cell Biochem 2009;106:643–650. 8. Leung HW, Lin CJ, Hour MJ, Yang WH, Wang MY, Lee HZ. Kaempferol induces apoptosis in human lung non-small carcinoma cells accompanied by an induction of antioxidant enzymes. Food Chem Toxicol 2007;45:2005–2013. 9. Siegelin MD, Reuss DE, Habel A, Herold-Mende C, von Deimling A. The flavonoid kaempferol sensitizes human glioma cells to TRAIL-mediated apoptosis by proteasomal degradation of survivin. Mol Cancer Ther 2008;7:3566–3574. 10. Luo H, Rankin GO, Liu L, Daddysman MK, Jiang BH, Chen YC. Kaempferol inhibits angiogenesis and VEGF expression through both HIF dependent and independent pathways in human ovarian cancer cells. Nutr Cancer 2009;61:554–563. 11. Nakamura T, Nishizawa T, Hagiya M, et al. Molecular cloning and expression of human hepatocyte growth factor. Nature 1989;342:440–443. 12. Martin TA, Jiang WG. Hepatocyte growth factor and its receptor signalling complex as targets in cancer therapy. Anticancer Agents Med Chem 2010;10:2–6. 13. Danilkovitch-Miagkova A, Zbar B. Dysregulation of Met receptor tyrosine kinase activity in invasive tumors. J Clin Invest 2002;109:863–867. 14. Gao CF, Vande Woude GF. HGF/SF-Met signaling in tumor progression. Cell Res 2005;15:49–51. 15. Puri N, Khramtsov A, Ahmed S, et al. A selective small molecule inhibitor of c-Met, PHA665752, inhibits tumorigenicity and angiogenesis in mouse lung cancer xenografts. Cancer Res 2007;67:3529–3534. 16. Delehedde M, Sergeant N, Lyon M, Rudland PS, Fernig DG. Hepatocyte growth factor/scatter factor stimulates migration of rat mammary fibroblasts through both mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt pathways. Eur J Biochem 2001;268:4423–4429. 17. Todeschini AR, Dos Santos JN, Handa K, Hakomori SI. Ganglioside GM2/GM3 complex affixed on silica nanospheres strongly inhibits cell motility through CD82/cMet-mediated pathway. Proc Natl Acad Sci USA 2008;105:1925–1930. 18. Karam JA, Huang S, Fan J, et al. Upregulation of TRAG3 gene in urothelial carcinoma of the bladder. Int J Cancer 2011;128:2823–2832. 19. Zhang T, Fan J, Wu K, et al. Roles of HIF-1a in a novel optical orthotopic spontaneous metastatic bladder cancer animal model[C]. Urol Oncol 2012;30:928–935. 20. Walsh JG, Cullen SP, Sheridan C, Luthi AU, Gerner C, Martin SJ. Executioner caspase-3 and caspase-7 are functionally distinct proteases. Proc Natl Acad Sci USA 2008;105:12815– 12819. 21. Yu SW, Andrabi SA, Wang H, et al. Apoptosis-inducing factor mediates poly(ADP-ribose) (PAR) polymer-induced cell death. Proc Natl Acad Sci USA 2006;103:18314–18319. 22. Day RM, Cioce V, Breckenridge D, Castagnino P, Bottaro DP. Differential signaling by alternative HGF isoforms through c-

Molecular Carcinogenesis

23. 24. 25. 26. 27. 28. 29.

30. 31.

32.

33.

34.

35.

36.

37. 38.

Met: Activation of both MAP kinase and PI 3-kinase pathways is insufficient for mitogenesis. Oncogene 1999;18:3399– 3406. Marshall CJ. Specificity of receptor tyrosine kinase signaling: Transient versus sustained extracellular signal-regulated kinase activation. Cell 1995;80:179–185. Yang Y, Wislez M, Fujimoto N, et al. A selective small molecule inhibitor of c-Met, PHA-665752, reverses lung premalignancy induced by mutant K-ras. Mol Cancer Ther 2008;7:952–960. Clerk A, Sugden PH. The p38-MAPK inhibitor, SB203580, inhibits cardiac stress-activated protein kinases/c-Jun N-terminal kinases (SAPKs/JNKs). FEBS Lett 1998;426:93–96. Bosetti C, Rossi M, McLaughlin JK, et al. Flavonoids and the risk of renal cell carcinoma. Cancer Epidemiol Biomarkers Prev 2007;16:98–101. Garcia R, Gonzalez CA, Agudo A, Riboli E. High intake of specific carotenoids and flavonoids does not reduce the risk of bladder cancer. Nutr Cancer 1999;35:212–214. Gonzalez CA, Riboli E. Diet and cancer prevention: Where we are, where we are going. Nutr Cancer 2006;56:225–231. Adhami VM, Malik A, Zaman N, et al. Combined inhibitory effects of green tea polyphenols and selective cyclooxygenase2 inhibitors on the growth of human prostate cancer cells both in vitro and in vivo. Clin Cancer Res 2007;13:1611–1619. Plochmann K, Korte G, Koutsilieri E, et al. Structure-activity relationships of flavonoid-induced cytotoxicity on human leukemia cells. Arch Biochem Biophys 2007;460:1–9. Luo H, Daddysman MK, Rankin GO, Jiang BH, Chen YC. Kaempferol enhances cisplatin's effect on ovarian cancer cells through promoting apoptosis caused by down regulation of cMyc. Cancer Cell Int 2010;10:16. Huang WW, Tsai SC, Peng SF, et al. Kaempferol induces autophagy through AMPK and AKT signaling molecules and causes G2/M arrest via downregulation of CDK1/cyclin B in SKHEP-1 human hepatic cancer cells. Int J Oncol 2013;42:2069– 2077. Naowaratwattana W, De-Eknamkul W, De Mejia EG. Phenoliccontaining organic extracts of mulberry (Morus alba L.) leaves inhibit HepG2 hepatoma cells through G2/M phase arrest, induction of apoptosis, and inhibition of topoisomerase IIalpha activity. J Med Food 2010;13:1045–1056. Zhang Q, Zhao XH, Wang ZJ. Cytotoxicity of flavones and flavonols to a human esophageal squamous cell carcinoma cell line (KYSE-510) by induction of G2/M arrest and apoptosis. Toxicol In Vitro 2009;23:797–807. Mu JJ, Zeng YY, Huang XY, Zhao XH, Song B. Effects of Kaempferol on activation, proliferation and cell cycle of mouse T lymphocytes in vitro. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi 2009;25:1106–1108. Nguyen TTT, Tran E, Ong CK, et al. Kaempferol-induced growth inhibition and apoptosis in A549 lung cancer cells is mediated by activation of MEK-MAPK. J Cell Physiol 2003;197:110–121. Xie F, Su M, Qiu W, et al. Kaempferol promotes apoptosis in human bladder cancer cells by inducing the tumor suppressor, PTEN. Int J Mol Sci 2013;14:21215–21226. Sharma V, Joseph C, Ghosh S, Agarwal A, Mishra MK, Sen E. Kaempferol induces apoptosis in glioblastoma cells through oxidative stress. Mol Cancer Ther 2007;6:2544–2553.

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