IC-2 Suppresses Proliferation and Induces Apoptosis of Bladder Cancer Cells via the Wnt/β-Catenin Pathway.

BACKGROUND The Wnt/β-catenin signaling pathway participates in many important tumorigeneses processes, including bladder cancer. The inhibition of abnormal activation of Wnt pathways might provide a new approach to tumor treatment. In the present study, we investigated the role of IC-2, a novel Wnt pathways small molecular inhibitor, in bladder cancer tumorigenesis. MATERIAL AND METHODS Bladder cancer cells were treated with various concentrations of IC-2 (0-5 μM) in vitro. The proliferation ability was measured using colony formation assay and apoptosis was measured using flow cytometry analysis. The protein expression was detected using Western blot analysis. Xenograft in vivo assay was performed to assess tumor growth. RESULTS IC-2 suppressed the proliferation and aggravated the apoptosis of bladder cancer cells in dose-dependent and time-dependent manners in vitro. Moreover, high concentrations of IC-2 inhibited the Wnt pathway-related protein expression levels, including β-catenin, Cyclin D1, and TCF4. In vivo, administration of IC-2 in xenograft mice decreased the β-catenin expression and reduced the tumor volume. CONCLUSIONS Our results validate the tumor-inhibition effect of IC-2 on bladder cancer in vivo and in vitro, providing a novel therapeutic strategy for bladder cancer.

Background

Bladder cancer is one of the most common malignant tumors and is the ninth most common malignancy worldwide [1,2]. Thousands of bladder cancer patients are diagnosed in developed countries and in developing countries every year [3]. Although treatment of bladder cancer is improving with technological advances, the overall survival times of bladder cancer patients are still poor, with a 5-year survival rate of about 50% [4]. With deeper understanding of bladder cancer pathology, increasing numbers of molecular pathways are being discovered and reported, revealing the pathogenetic mechanism of bladder cancer and aiding more effective diagnosis [5].

The Wnt/β-catenin signal transduction pathway is involved in many important biological events [6]. In the normal human body environment, the Wnt signal transduction pathway is highly conserved and not activated [7,8]. Abnormal activation of Wnt pathways plays an important role in the occurrence and development of tumors; therefore, the inhibition or blockage of the abnormal activation of Wnt pathways might provide a new approach to developing antineoplastic drugs, and Wnt pathways inhibitors may also be relevant in developing new cancer drugs [9]. Abnormal activation of the Wnt/β-catenin signaling pathway is one of the main causes of many kinds of tumors, including gastrointestinal carcinoma, glioma, and other cancers [10,11].

Existing articles have illustrated its exact role in tumorigenesis, as well as bladder cancer. In the present study, we investigated and validated the function of the novel small molecular inhibitor targeting Wnt pathway in bladder cancer cells, suggesting the valuable inhibition of bladder cancer tumorigenesis.

Material and Methods

Bladder cancer cell lines and culture

Bladder cancer cell lines (SW780, T24, UMUC3) and SV-40-immortalized human uroepithelial cell line (SV-HUC-1) were purchased from the Institute of Cell Research, Chinese Academy of Sciences (Shanghai, China) for use in this study. The cells were cultured in RPMI-1640 Medium (Invitrogen, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS, Gibco, NY, USA). Plates were then placed in a 37°C incubator with a humidified atmosphere of 5% CO2.

Cell proliferation assay

Cell proliferation was evaluated using the Cell Counting Kit-8 (CCK-8, Beyotime Inst Biotech, Shanghai, China) assay and colony formation assay according to the manufacturer’s instructions. Briefly, for CCK-8 assay, bladder cancer cells (5×103 per well) were seeded in a 96-well plate and incubated for 24 h, then the absorbance was measured at 450 nm using a microplate reader.

Colony formation assays

Bladder cancer cells (1×103 per well) were treated with IC-2 (0–5 μM) and seeded into 6-well plates supplemented complete growth medium (2 ml). After 1 week, the cells were fixed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet (Beyotime, Shanghai, China) for 30 min. Colonies larger than 0.3 mm were counted. Final data were calculated from at least 3 independent experiments.

Flow cytometry of apoptosis assay

After being treated with IC-2 (0–5 μM) and fixed with 10 ml cold ethanol overnight, T24 cells were washed in PBS and treated with 500 μl PI/RNase Staining Solution (Invitrogen, Carlsbad, CA, USA) containing DNase-free RNase and reagent for 30 min at 37°C. After incubation, the cells were washed and analyzed immediately by flow cytometry analysis using a Beckman-Coulter CyAN ADP Analyzer (Beckman, CA, USA). Data were analyzed with FlowJo Version 6.1 software (TreeStar, USA). Final data were calculated from at least 3 independent experiments.

Western blot assay

Bladder cancer cells were lysed using protein extraction reagent radio-immunoprecipitation assay (RIPA) (Beyotime, Shanghai, China) with protease inhibitors cocktail and phenylmethanesulfonyl fluoride (PMSF) (Roche, Switzerland). Then, the protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. The membrane was blocked with 5% non-fat milk and incubated with primary antibodies at 4°C overnight. Primary antibodies were β/catenin (1: 1000, Abcam), Cyclin D1 (1: 1000, Abcam), and TCF4 (1: 1000, Abcam). After that, the blots were incubated with goat anti-rabbit secondary antibody (Abcam, Cambridge, MA, USA). Enhanced chemiluminescence (ECL) chromogenic substrate was used to visualize the bands. GAPDH acted as the internal control.

Immunofluorescence

For immunofluorescence studies, T24 cells (4×105 cells per well) were cultured in a 12-well culture dish and then treated with IC-2 (0 or 2 μM) for 72 h. Then, cells were fixed with paraformaldehyde (4%, 10 min), permeabilized in Triton X-100 (0.1%, 15 min), and washed using PBS. After being blocked in BSA (3%, 1 h) at room temperature, cells were incubated with anti-β-catenin antibody (1: 200) for 1 h at 37˚C and then incubated with fluorescein-labeled secondary antibody (1: 2000) for 1 h at room temperature. Then, cells were stained using nuclear staining with DAPI (Invitrogen) solution for 20 min. Lastly, cells were scanned and photographed using a Zeiss LSM510 Meta-confocal microscope.

Xenograft tumor model

BALB/c mice (4 weeks old) were purchased from the Institute of Laboratory Animal Sciences, Peking Union Medical College (Beijing, China). Animals were kept under specific pathogen-free conditions. T24 cells (3×106 per 100 μl) and IC-2 (0 or 5 mg/kg) were subcutaneously injected into the back of nude mice. All tumor volumes of mice were measured every 5 days. The tumor tissue was harvested after mice were sacrificed for further Western blot analysis and immunohistochemistry staining.

Immunohistochemistry

Neoplasm tissues excised from BALB/c mice were fixed in 10% formalin and embedded in paraffin. Briefly, after deparaffination and rehydration using Dulbecco’s phosphate-buffered saline, tissue samples were cut into 10-μm-thick sections. Then, sections were incubated overnight with anti-β-catenin antibody (dilution 1: 200; Santa Cruz) and conjugated with corresponding secondary antibody. The antigens were visualized using a SP Rabbit & Mouse HRP kit (Kangwei, Beijing, China) according to the manufacturer’s instructions. Lastly, the sections were lightly counterstained with hematoxylin. The quantification and histogram analysis of immunostained images was done under a fluorescence microscope (Axiophot; Carl Zeiss, Jena, Germany).

Statistical

All data are displayed as mean ± standard deviation (SD) and generated from 3 independent experiments. The values of results were analyzed using the t test or χ2 test using SPSS version 19.0 software (SPSS Inc. Chicago, IL, USA) and presented using GraphPad Prism 6 software (GraphPad Software Inc., La Jolla, CA, USA). P values of less than 0.05 were considered to be statistically significant.

Results

IC-2 inhibited bladder cancer cells proliferation and aggravated the apoptosis in a dose-dependent manner

To test the biologic role of IC-2 on bladder cancer cells, a series of experiments were performed. Bladder cancer cell lines SW780, T24, and MUC3 and control normal cell line SV-HUC-1 were treated with different concentrations of IC-2 (0–5 μM). After being treated with IC-2, the relative survival rate of bladder cancer cells showed that high concentration (5 μM) of IC-2 significantly decreased the survival of cells, especially in T24 cells (Figure 1A). T24 cells were chosen as the target for further research. CCK-8 assay showed that the proliferation ability was downregulated in time-dependent and dose-dependent manners (Figure 1B). Colony formation assay showed that the higher concentration of IC-2 significantly decreased the number of colonies compared with the low concentration (Figure 1C, 1D). Flow cytometry showed that the higher concentration of IC-2 promoted the apoptosis of T24 cells compared with the low concentration (Figure 1E, 1F). Overall, results showed that IC-2 inhibited bladder cancer cells proliferation and aggravated the apoptosis in a dose-dependent manner.

Figure 1

IC-2 inhibited bladder cancer cells proliferation and aggravated the apoptosis dose-dependently. (A) The relative survival rate of bladder cancer cell lines (SW780, T24, and UMUC3) and control normal cell line (SV-HUC-1) treated with different concentrations of IC-2 (0–5 μM). (B) CCK-8 assay showed the proliferation ability of bladder cancer cells in time-dependent and dose-dependent manners. (C, D) Colony formation assay showed the colonies numbers. (E, F) Flow cytometry showed the apoptosis of T24 cells. Data are presented as mean ±SD. * P<0.05, ** P<0.01 indicates a significant difference.

IC-2 inhibited activation of the Wnt/β-catenin pathway in bladder cancer cells

Activation of the Wnt/β-catenin pathway and the accumulation of β-catenin are critical processes in tumorigenesis. Thus, we proposed that IC-2, acting as a small molecular inhibitor targeting the Wnt/β-catenin pathway, would depress activation of the Wnt pathway in tumor cells. We measured the Wnt/β-catenin pathway-related protein expression in T24 cells using Western blot analysis. Results showed that protein expression levels of β-catenin, Cyclin D1, and TCF4 were decreased by treatment with a high concentration of IC-2 (2 μM) (Figure 2A–2C). Moreover, immunofluorescence assay showed that the location of β-catenin moved to cytoplasm from nuclei (Figure 2D). Our results revealed that the high concentration of IC-2 inhibited activation of the Wnt/β-catenin pathway in bladder cancer cells.

Figure 2

IC-2 inhibited the activation of Wnt/β-catenin pathway in bladder cancer cells. (A) The expression of β-catenin protein in T24 cells treated with IC-2 (0, 2 μM). (B) The expression of Cyclin D1 protein. (C) The expression of TCF4 protein. (D) Immunofluorescence assay of β-catenin showed the location in cytoplasm and nuclei. Data are presented as mean ±SD. ** P<0.01 indicates a significant difference.

IC-2 inhibited bladder cancer cells xenograft tumor growth and decreased the β-catenin expression in vivo

To test the biologic role of IC-2 on bladder cancer tumor growth, assay of xenograft mice was performed by subcutaneously transplanting T24 cells into BALB/c nude mice. Results showed that the bioluminescent intensity in tumors treated with IC-2 (5 mg/kg) was significantly lower than that of the control group (Figure 3A, 3B). Expression of β-catenin protein was decreased in mice treated with IC-2 (5 mg/kg) (Figure 3C). The tumor volumes in the group administered IC-2 (5 mg/kg) were lower than in the control group (Figure 3D). Immunohistochemistry assay showed that the stained β-catenin was decreased in the IC-2 (5 mg/kg) group compared to the control group (Figure 3E). In summary, results showed that IC-2 inhibited bladder cancer cells xenograft tumor growth in vivo and decreased the β-catenin expression.

Figure 3

IC-2 inhibited bladder cancer cells xenograft tumor growth and decreased the β-catenin expression in vivo. (A, B) Bioluminescent imaging and bioluminescent intensity in BALB/c nude mice treated with IC-2 (5 mg/kg) and blank control. (C) Expression of β-catenin protein in mice treated with blank control and IC-2 (5 mg/kg). (D) Tumor volumes in the IC-2 (5 mg/kg) group and control group measured every 5 days. (E) Immunohistochemistry assay showed the stained β-catenin. Data are presented as mean ±SD. * P<0.05, ** P<0.01 indicates a significant difference.

Discussion

The Wnt/β-catenin signaling pathway, consisting of canonical and non-canonical branches, plays important roles in multiple pathological process and tumorigenesis [12-14]. Emerging evidence indicates that inhibition of the Wnt/β-catenin signaling pathway contributes to the treatment and prevention of cancers, including bladder cancer [15,16]. In the present study, we investigated the biological role of a novel small molecular inhibitor targeting Wnt signaling pathways, IC-2, on bladder cancer cells tumorous characteristics and explored the potential therapeutic strategy for bladder cancer.

Small molecular inhibitor targeting Wnt signaling pathways are a category of novel inhibitors that can specifically integrate with β-catenin and decrease the concentration [17,18]. The abundance and nuclear location of β-catenin are critical in Wnt signaling pathways activation [19]. The persistent existence of TCF-β-catenin complex in nuclei is a common feature of cancer [20]. IC-2, a derivative of ICG-001, is a novel identified small molecular inhibitor targeting Wnt signaling pathway [21]. Using TOP/FOP flash report gene screening, we showed that, from the small molecular inhibitor library, IC-2 had the most significant inhibitory effect on the Wnt signaling pathway.

The vital role of the Wnt/β-catenin pathway in bladder cancer progression has been adequately explored and reported [22]. Using in vitro functional experiments, we performed a series of validation assays to explore the effect of IC-2 on bladder cancer cells. Results showed that IC-2 decreased the proliferation ability of T24 cells in time-dependent and dose-dependent manners. Similarly, colony formation assay indicated that the higher concentration of IC-2 significantly decreased numbers of the colonies and the higher concentration of IC-2 significantly promoted the apoptosis of T24 cells. Therefore, our experiments and results strongly support the conclusion that IC-2 significantly inhibits bladder cancer cell proliferation and aggravates the apoptosis in a dose-dependent manner. Confirming the tumor-inhibition effect of IC-2 on bladder cancer may provide an important new therapeutic strategy for bladder cancer.

The molecular mechanisms of cancer are very complex, involving multiple genes, multiple factors and multiple stages [23]. Although the coordinated regulation of the genetic and external environments plays an important role in tumorigenesis, signal transduction pathways also act as a vital regulators [24]. In the normal cellular environment, Wnt signaling pathways are not activated, but they are activated in tumorigenesis. There are 2 types of Wnt/β-catenin signaling pathways: canonical and non-canonical branches. The canonical Wnt signaling is mediated by β-catenin and activates the TCF/LEF family of transcription factors, while the non-canonical Wnt/Ca2+ and Wnt/PCP pathways are independent of β-catenin [25].

The Wnt/β-catenin signaling pathway-related proteins β-catenin, Cyclin D1, and TCF4 were assessed by Western blot analysis. Results showed that the high concentration of IC-2 (2 μM) decreased the expression levels, suggesting that IC-2 inhibits the Wnt/β-catenin signaling pathway. Moreover, immunofluorescence assay showed that β-catenin moved and located in cytoplasm when treated with IC-2. In vivo, Western blot and immunohistochemistry assay showed that expression of β-catenin protein was decreased mice treated with IC-2 (5 mg/kg) and the tumor volume was also reduced, suggesting that IC-2 inhibits bladder cancer in vivo. In CRC cells, IC-2 reduced Wnt/β-catenin transcriptional activity and decreased the expression levels of cancer stem cell marker proteins [26].

Conclusions

We investigated and validated the function of novel small molecular inhibitor targeting Wnt pathway in bladder cancer cells, suggesting the valuable inhibition of bladder cancer tumorigenesis and providing a novel therapeutic strategy for bladder cancer.