NF-κB suppresses apoptosis and promotes bladder cancer cell proliferation by upregulating survivin expression in vitro and in vivo

Nuclear factor kappa-B (NF-κB) activation is a common phenomenon in cancers, which results in the aberrant expression of NF-κB target genes and leads to malignant transformation, metastatic dissemination, abnormal cell proliferation or resistance to cell death. Survivin is a unique member of the IAP family, a well-known cancer-specific molecule and a molecular marker of poor clinical outcome in several cancer types, including bladder cancer. YM-155, a potent survivin suppressor, has been shown to have anti-tumor activity in preclinical cell lines, xenograft models and phase I/II studies. In the present study, we investigated the function of the NF-κB/survivin pathway in bladder cancer. We found that NF-κB can promote cell cycle progression and reduce apoptosis by upregulating survivin expression, thereby increasing cellular proliferation. We further confirmed the tumorigenic function of the NF-κB/survivin pathway in vivo using a xenograft tumor model of stable NF-κB-overexpressing 5637 cells. Moreover, we found that YM-155 significantly induced apoptosis and decreased cellular proliferation as well as tumor growth in mice. Our results demonstrate the carcinogenic function of the NF-κB/survivin pathway in bladder cancer and the role of YM-155 as a promising agent for the strategic treatment of bladder cancer.

Scientific RepoRts | 7:40723 | DOI: 10.1038/srep40723 predictor of bladder cancer recurrence and cancer-specific survival 17,20,21 . Therefore, an in-depth study targeting the survivin regulation network is being pursued for novel anti-cancer therapeutics.
Many studies have focused on the role of IAPs as modulators of nuclear factor kappa-B (NF-κ B), which has been proposed to be a crucial regulatory family for inflammation, immunity and cell survival and is hallmark of tumorigenesis 10,22 . For example, it has been established that c-IAP1 and c-IAP2 form a complex with TNF receptor 1 (TNFR1) and promote TNFα -induced NF-κ B activation 23,24 . Moreover, the assembly of a survivin-XIAP complex also functions as an effective activator of NF-κ B 25 . On the other hand, the importance of the regulatory function of NF-κ B in cancer is in the transcription of growth-promoting and anti-apoptotic genes. As reported earlier, in some cancer types, NF-κ B inhibits apoptosis by targeting BCL2 and/or IAPs [26][27][28] . Nonetheless, the precise mechanisms of NF-κ B activation and its regulatory role in cell survival and anti-apoptosis in bladder cancer remain unclear.
In this study, we investigated the mechanisms of cell survival in bladder cancer cells. We confirmed that NF-κ B activation contributes to the upregulation of the survivin gene in bladder cancer, and we revealed that by upregulating survivin expression, NF-κ B enhances the proliferation and suppresses the apoptosis of bladder cancer cell lines both in vitro and in vivo. In addition, YM-155, a selective inhibitor of survivin, efficiently inhibited survivin expression and xenograft tumor growth.

Results
Expression of NF-κB and survivin is upregulated in bladder cancer cell lines and tissue specimens, and a positive correlation is detected in tumor tissue specimens. We first investigated the survivin and NF-κ B subunit p65/RelA expression profiles in one immortalized ureter urothelial cell line and seven bladder cancer cell lines by western blotting. As shown in Fig. 1A,B, bladder cancer cell lines exhibited significantly increased survivin and nuclear p65/RelA expression compared to the ureter urothelial cell line. Next, we detected the expression of the above two proteins in four pairs of bladder cancer and adjacent normal tissue specimens. Bladder cancer tissue specimens revealed significant upregulated survivin and nuclear RelA expression compared with that of adjacent normal mucosa tissue specimens (Fig. 1C,D). Then, we detected survivin and p65/RelA expression in one normal bladder mucosa tissue and 4 bladder cancer tissues which were diagnosed with bladder urothelial carcinoma staging from pT1 to pT4 by western blot. The results suggested that with the pathological progression of bladder cancer, expression of survivin and p65/RelA revealed significant elevation ( Fig. 1E-G). To further confirm the expression pattern and correlation of these two proteins in clinical tissue specimens, immunostaining analysis ( Fig. 2A) was performed in 5 normal bladder mucosa tissue specimens and 40 bladder cancer tissue specimens which were diagnosed with bladder urothelial carcinoma staging from pT1 to pT4 (10 cases/stage). The results (Fig. 2B,C) showed that with the pathological progression of bladder cancer, survivin and p65/RelA expression significantly increased. Furthermore, Pearson's correlation coefficient analysis (Fig. 2D) suggested a significant positive correlation between expression of survivin and p65/RelA in 40 bladder cancer tissue specimens (R = 0.6708, p < 0.01). We also assessed the association of the survivin and p65/RelA expression level with the clinical and pathological characteristics of these patients. As shown in Table 1, survivin and NF-κ B p65 were highly expressed in high-grade tumors, tumors with metastases and Lymphatic invasion. These results suggest that the expression of survivin and NF-κ B p65/RelA is not only remarkably upregulated in bladder cancer cell lines and tissues but also closely correlated with the clinical progression of bladder cancer, moreover, expression of the two proteins is positively correlated in bladder cancer tissue specimens. NF-κB activation enhances survivin expression in bladder cancer cell lines. We next asked whether NF-κ B activation contributes to the upregulation of survivin expression in bladder cancer. To test this hypothesis, we successively activated or deactivated NF-κ B signaling using tumor necrosis factor-α (TNF-α ) or BAY 11-7082, respectively. TNF-α activates the classical NF-κ B signaling pathway, whereas BAY 11-7082 selectively inhibits the phosphorylation of Iκ B kinase (IKK) and leads to the deactivation of NF-κ B signaling 29 . First, we treated the 5637 and T24 bladder cancer cell lines with TNF-α (50 ng/ml) for 0, 2, 3, 4 and 6 h. Next, we measured survivin and nuclear p65/RelA protein expression by western blotting analysis. As shown in Fig. 3A,B, NF-κ B was activated by TNF-α in a time-dependent manner, and survivin expression was relatively upregulated. Next, we treated the tested cells with BAY 11-7082 using increasing concentrations of 0.25, 0.5, 1 and 2 μ M for the 5637 cells and 0.1, 0.2, 0.5 and 1 μ M for the T24 cells (Fig. 3C,D). Our results suggest that survivin expression exhibits significant downregulation, which is a general reflection of the level of inhibition of NF-κ B signaling. Next, to avoid the influence that TNF-α or BAY 11-7082 may cause on survivin expression through other pathways rather than NF-κ B, we knocked down p65/RelA with siRNA oligo, then the cells were treated with TNF-α in the same method. Gene knockdwon efficiency was measured by western blot and real-time quantitative PCR analysis ( Figure SB). Survivin and p65 protein expression was detected in different time points after TNF-α treatment by western blot. Figure 3E,F suggested that p65/RelA expression was remarkably inhibited by siRNA transfection, accordingly, survivin expression revealed a relative downregulation, furthermore, there was no significant upregulation of the two proteins after TNF-α stimulation. Taken together, these results suggest that the activation of the NF-κ B signaling pathway significantly contributes to the upregulation of survivin expression, whereas deactivation of NF-κ B downregulates survivin expression.
NF-κB overexpression stimulates the luciferase activity of the survivin promoter. Multiple transcription enhancer-binding sites within the 5′ flanking region of the survivin gene have been identified, including four NF-κ B sites 30,31 . In B-cell lymphomas 26 , colorectal cancer 27 and T-cell leukemia 31 , NF-κ B has been reported to bind to the promoter region of the survivin gene and enhance transcription. Hence, we further validated whether NF-κ B induces survivin expression by binding to the promoter region in bladder cancer cells. A luciferase reporter plasmid containing the survivin promoter region was constructed ( Figure SA), and a dual luciferase reporter assay was performed to measure the interaction. After transfection with the luciferase plasmid, the cells were treated with TNF-α (50 ng/ml) alone or pretreated with BAY 11-7082 (2 μ M) for 2 h followed by the same TNF-α treatment. As shown in Fig. 3G, TNF-α significantly stimulated the luciferase activity of the survivin (E,F) One normal bladder mucosa tissue and four bladder cancer tissues which were pathologically diagnosed with bladder urothelial carcinoma staging from pT1 to pT4, were examined by western blotting analysis. The gels were run under the same experimental conditions. The band intensities were calculated by AlphaEase FC software. β -Tubulin was used as an internal control for total protein measurement, and lamin B1 was used as a nucleoprotein reference. The ratio of target gene to β -Tubulin/lamin B1 was used to conduct the statistical analysis. *P < 0.05 and **P < 0.01, as determined by Student's T-test.
promoter luciferase construct, whereas BAY 11-7082 pretreatment significantly inhibited the luciferase activity, which was otherwise stimulated by TNF-α alone. Furthermore, we transfected the cells with negative control (NC) oligo or siRNA against p65, after 24 hours the cells were separately treated with control/TNF-α , and the cell luciferase activity was measured after another 24 hours. Figure 3H showed that TNF-α remarkably stimulated the luciferase activity of the cells received transfection of NCs, in contrast, survivin promoter luciferase activity of p65 knockdown cells could no longer be upregulated by TNF-α treatment.

YM-155 significantly decreases survivin expression and inhibits bladder cancer cell proliferation.
Next, we investigated the carcinogenic function of the NF-κ B/survivin pathway in bladder cancer cells. The   The activation of NF-κ B signaling significantly enhanced survivin promoter luciferase activity. In contrast, pretreatment with BAY 11-7082 (2 μ M for 2 h) effectively hampered the increase in luciferase activity induced by TNF-α (50 ng/ml). (H) p65 knockdown significantly decreased the luciferase activity which was otherwise stimulated by TNF-α . The gels were run under the same experimental conditions. The band intensities were calculated by AlphaEase FC software. β -Tubulin was used as an internal control for total protein measurement, and lamin B1 was used as a nucleoprotein reference. The ratio of target gene to β -Tubulin/lamin B1 was used to conduct the statistical analysis. *P < 0.05 and **P < 0.01, as determined by Student's T-test.
imidazolium-based agent Sepantronium bromide (YM-155) was used to inhibit survivin expression. YM-155 has been confirmed to specifically decrease survivin expression and to induce apoptosis in many cancers 32,33 . Moreover, recent phase II studies have revealed encouraging anti-tumor activities of YM-155 combined with other anti-neoplastic agents 34,35 . However, few studies have reported the effects of YM-155 in bladder cancer. Therefore, it is necessary to determine its function in bladder cancer cell lines. We treated 5637 and T24 cells with YM-155 and found that the agent significantly decreased survivin expression in a dose-dependent manner (Fig. 4A). Next, we treated the cells with YM-155 (100 μ M) for 24 h, and cell proliferation was examined by CCK8 and colony formation assays (Fig. 4B,C). Meanwhile, the cell cycle-and apoptosis-related proteins p-Rb, cyclin A, cyclin D, cleaved-caspase 3 and cleaved-caspase 9 were also detected by western blotting (Fig. 4D). Similar to findings of previous studies, YM-155 specifically inhibited proliferation and induced apoptosis and the cycle arrest of bladder cancer cells.

NF-κB suppresses apoptosis and increases the proliferation of bladder cancer cells by targeting
survivin. NF-κ B activation is a common phenomenon in cancers, which results in the aberrant expression of distinct sets of NF-κ B target genes, leading to malignant transformation, metastatic dissemination, abnormal cell proliferation or resistance to cell death 36 . In this context, we investigated whether NF-κ B promotes cell survival by upregulating survivin expression in bladder cancer. Apoptosis was induced by YM-155 treatment. Next, the p65/RelA expression plasmid was transfected to activate NF-κ B. CCK8 and colony formation assays (Fig. 5A,B) revealed that YM-155 remarkably reduced the proliferation of the tested cells, whereas NF-κ B overexpression reverted the impaired cell proliferation, which was otherwise inhibited by YM-155 alone. Flow cytometric analysis further demonstrated the alterations in the cell cycle and apoptosis. As shown in Fig. 5C, YM-155 alone significantly induced apoptosis, and NF-κ B overexpression combined with YM-155 reduced apoptosis compared with YM-155 treatment alone. Similarly, a cell cycle analysis (Fig. 5D) suggested that YM-155 alone significantly impaired cell cycle progression at the G0/G1 phase, whereas NF-κ B overexpression combined with YM-155 treatment promoted cell cycle progression to the G2/M phase compared with YM-155 treatment alone. Next, we measured survivin and p65/RelA expression as well as that of the cell cycle-and apoptosis-related proteins p-Rb, cyclin A, cyclin D, cleaved-caspase 3 and cleaved-caspase 9 within each group by western blotting (Fig. 5E). Our in vitro results suggested that YM-155 potently induced apoptosis and inhibited the proliferation of bladder cancer cell lines by downregulating survivin expression. In contrast, the recovery of survivin expression induced by NF-κ B overexpression significantly reduced the anti-tumor effects of YM-155. These findings indicate that NF-κ B suppresses apoptosis and increases the proliferation of bladder cancer cells by upregulating survivin.

NF-κB promotes tumor growth by upregulating survivin expression in vivo.
We next investigated the carcinogenic function of the NF-κ B/survivin pathway in bladder cancer using a xenograft mouse model. For NF-κ B stable overexpression, we used recombinant lentiviruses encoding the CDS sequence of p65/RelA (purchased from GenePharma, Shanghai, China) to infect 5637 cells; a negative control lentiviral vector encoding a nonsense sequence was used as a vehicle control. The stably transduced 5637 cells were selected using puromycin. To assess the efficiency of lentiviral gene delivery, p65/RelA expression was assessed by western blotting at 20 days post-infection ( Figure SC). Lv-NC-5637 and Lv-RelA-5637 cells were separately injected into the flanks of athymic nude mice to establish xenograft tumors. When tumor sizes reached approximately 20 mm 3 , the mice were divided into four groups (2 groups received Lv-NC-5637 injection, and 2 groups received Lv-RelA-5637 injection; 3 mice/group). The groups of mice with tumors established by Lv-NC-5637 or Lv-RelA-5637 were continuously infused with YM-155 at a dose of 5 mg/kg/day for 7 days; the other two groups of mice were infused with the vehicle control. Tumor growth was monitored throughout the survival period, and the mice were euthanized and tumors were excised at 15 days after infusion ( Figure SE). The tumor growth curve (Fig. 6A) showed that the tumors in the YM-155 plus Lv-NC-5637-treated mice grew significantly slower than those in the vehicle control plus Lv-NC-5637-treated mice. In contrast, in the NF-κ B-overexpressing (Lv-RelA-5637) groups, the tumor growth inhibition resulting from YM-155 was significantly reduced, and the tumors in the mice treated with vehicle control plus Lv-RelA-5637 revealed the fastest growth rate among all of the groups. Accordingly, the same was observed in terms of tumor size and weight measurements (Fig. 6A,B).
To further determine whether NF-κ B induces survivin expression in xenograft tumors, the tumor tissues were dissected and subjected to IHC staining. Our results (Fig. 6C,D) demonstrated that YM-155 significantly inhibited survivin expression compared with the vehicle control, whereas NF-κ B overexpression reverted the survivin expression that was otherwise inhibited by YM-155; NF-κ B overexpression without YM-155 treatment significantly upregulated survivin expression. Ki67 expression was also detected, and in accordance with the tumor growth, NF-κ B overexpression without YM-155 treatment revealed the highest expression level of ki67, whereas YM-155 with vector significantly decreased ki67 expression and tumor growth. The data detailed above suggest that in vivo, NF-κ B enhances the proliferation and resistance to apoptosis of bladder cancer cell lines by upregulating survivin expression.

Discussion
In this study, we have shown that survivin serves as a downstream mediator of NF-κ B signaling in bladder cancer malignant progression. We have provided clear evidence that in bladder cancer, NF-κ B activation enhances the expression of survivin both in vitro and in vivo. Accordingly, with survivin overexpression, cell proliferation and resistance to apoptosis is significantly stimulated, which further leads to increased xenograft tumor growth. Moreover, the YM-155 agent potently suppresses the expression of survivin in bladder cancer cells, inducing apoptosis and cell cycle arrest, consequently suppressing xenograft tumor growth. Our study further demonstrates that the NF-κ B/survivin pathway plays a tumorigenic function in bladder cancer cell lines, and for the first time, we document the mechanism of YM-155 in bladder cancer tumor growth in a nude mouse model. The major issues underlying poor clinical outcome and even metastasis-related death in bladder cancer patients have been identified in tumors prone to progression, recurrence and chemoradiation resistance 37,38 . To overcome these issues, a number of molecular markers have been studied and are suggested to be involved in the biological process of malignant transformation of bladder cells (e.g., the cell cycle regulators p53, pRb and cyclin A, as well as the apoptosis mediators IAP and Bcl-2 family proteins 19,[38][39][40] ). NF-κ B is a family of nuclear transcription regulators, which affect cellular responses to multiple extracellular stimuli 41 . Moreover, constitutive NF-κ B activation, which commonly occurs in malignant tissues, results in the strong transactivation of a wide variety of reporter genes, resulting in perturbations in cell cycle progression, resistance to apoptosis and for the treatment of cells was 100 nM. The gels were run under the same experimental conditions. The band intensities were calculated by AlphaEase FC software. β -Tubulin was used as an internal control for total protein measurement, and lamin B1 was used as a nucleoprotein reference. The ratio of target gene to β -Tubulin/lamin B1 was used to conduct the statistical analysis. *P < 0.05 and **P < 0.01, as determined by Student's T-test. enhanced angiogenesis 36 . Several studies have demonstrated that NF-κ B activation, as indicated by nuclear p65/ RelA subunit expression, is positively associated with tumor histological grade, T-category and chemoradiation resistance, particularly in muscle-invasive bladder cancers 42,43 . In this study, we detected a significant increase in nuclear p65/RelA levels in bladder cancer tissues compared to that of adjacent normal tissues, and p65/RelA expression was also correlated with pathological progression of bladder cancer. Moreover, our xenograft tumor model revealed increased tumor growth in the mice receiving injections of stable NF-κ B-overexpressing 5637 cells. These results are consistent with previous studies.
The inhibitor of apoptosis (IAP) protein family, which was originally defined as a class of endogenous caspase inhibitors, has garnered increasing attention for its disparate biological functions in survival, mitosis and intracellular signaling 11 . The Baculovirus IAP Repeat (BIR) domain is the defining structural characteristic of IAP molecules, which mediate protein recognition and protein-to-protein interactions 10 . Several IAPs also contain additional structural domains, such as the carboxyl-terminus RING, an ubiquitin-associated domain, that facilitates the ubiquitination process 10 . Survivin is a unique member of the IAP family, a well-known cancer-specific molecule, and a molecular marker for poor clinical outcome of tumors, including bladder cancer 44,45 . Indeed, it has been widely reported that survivin is a target of NF-κ B signaling and promotes cancer progression as well as drug resistance in many cancers. However, to date, no reports have been made to confirm the NF-κ B/survivin pathway in bladder cancer using a stable NF-κ B-overexpressing xenograft tumor model. Here, using a xenograft tumor assay, we not only demonstrated the NF-κ B/survivin axis both in vitro and in vivo, but we also tested the anti-cancer function of YM-155.
YM-155, a small molecule that selectively suppresses survivin mRNA expression, has been investigated in preclinical cell lines, xenograft models and phase I/II studies [33][34][35]46 . YM-155 has been shown to be safe and well tolerated at an ideal dose of approximately 5 mg/kg/day by continuous infusion for 7 days, according to phase I and II studies 47,48 . In a study concerning the anti-tumor activities of YM-155 in a wide variety of human cancer cell lines and xenograft models, YM-155 was reported to elicit significant anti-tumor activity in a bladder cancer (UM-UC-3) xenograft model 49 . In our study, we inoculated nude mice with the bladder cancer cell line 5637, and YM-155 or vehicle control was continuously administered at 5 mg/kg/day for 7 days. Our results showed that YM-155 potently suppresses xenograft tumor growth, as well as the expression of the survivin gene. Importantly, it also significantly hampered the tumor growth induced by NF-κ B activation.
In bladder cancer, survivin mediates resistance to apoptosis and cell proliferation induced by NF-κ B both in vitro and in vivo. Finally, YM-155 is a promising agent for bladder cancer management. The relative levels of expression were quantified and analyzed using LightCycler TM 480 software 1.5.1.6.2 (Roche, Basel, Switzerland). The real-time value for each sample was averaged and compared using the Ct method. The relative expression level (defined as a fold change) of each target gene (2 −ΔΔCt ) was normalized to the endogenous β -actin reference (Δ Ct) and related to the amount of target gene in the control sample, which was defined as the calibrator at 1.0. Three independent experiments were performed to analyze the relative gene expression, and each sample was tested in triplicate.

Methods
Patients and tissue specimens. Four pairs of bladder urothelial carcinoma tissues and adjacent normal mucosa tissues (located > 3 cm from the tumor) were freshly collected from four patients. Another one non-tumor bladder mucosa tissue specimen and four bladder urothelial carcinoma tissue specimens were freshly collected from the other four patients who were pathologically diagnosed with bladder urothelial carcinoma staging from pT1 to pT4. The patients were hospitalized, pathologically diagnosed with bladder urothelial carcinoma and underwent transurethral bladder tumor resection or radical cystectomy at the Urology Department at the First Hospital of China Medical University (Shenyang, China). The study was conducted according to an institutional review board-approved protocol (2012-33) by Medical Ethics Committee of the First Affiliated Hospital of China Medical University, and written informed consent was obtained from each patient for surgery and research purposes. All of the cases were classified according to the 1997 UICC TNM classification for the stage and according to OMS 2004 for the grade. The pathological sections of five non-tumor bladder mucosa tissue specimens and forty bladder cancer tissue specimens were provided by Department of Pathology at the First hospital of China Medical University, and the pathological diagnosis and analysis of immunochemistry staining result in this study were made in collaboration with Department of Pathology.
Small interfering RNA, plasmid construction and retroviral infection. Three pairs of siRNAs against p65/RelA were synthesized by GenePharm (GenePharma Corporation, Shanghai, China). The Sequences of siRNAs were as following: The pGL3-survivin-promoter luciferase plasmid was purchased from GenePharm (GenePharma Corporation, Shanghai, China). The recombinant lentivirus vector pGLV-NF-κ B and the negative control vector (pGLV-NC) containing a nonsense sequence were purchased from GenePharm (GenePharma Corporation, Shanghai, China). We transfected 5637 cells with pGLV-NF-κ B or pGLV-NC for 3 days. Then, the stably transduced 5637 cells were selected using puromycin for 20 days by adding the minimum concentration of puromycin required to kill untransduced 5637 cells. The efficiency of retroviral infection was measured by western blotting.
Cell proliferation assay. Cell proliferation was determined using Cell Counting Kit-8 (CCK-8) (Dojindo, Tokyo, Japan) and a cell colony formation assay, according to the manufacturer's protocol. Cells in the logarithmic phase of growth were seeded into 96-well culture plates at 3 × 10 3 cells per well for 24 h. Next, the cells were differentially treated with TNF-α , BAY 11-7082 or YM-155. At the indicated times, the assays were performed by directly adding the CCK-8 reagent into the medium and incubating in 37 °C for 1 h. The absorbance value was measured at 450 nm to determine cell viability using a 96-well plate reader. (model 680, Bio-Rad, Hertfordshire, UK).
For cell colony formation assay, the cells were plated in 24-well plates (300 cells per well) and incubated for 7 days in complete medium. Colonies were fixed with 10% formaldehyde for 10 min and stained with 1.0% crystal violet for 5 min. The number of colonies, defined as > 50 cells/colony, was counted.
Protein extraction and western blotting. Cells were harvested in RIPA lysis buffer (Beyotime, Shanghai, China) and boiled for 10 min at 90 °C. Protein concentrations were measured using the BCA assay. Approximately, 50 μ g of protein extract from cultured cells or 100 μ g from fresh surgical bladder tissues were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The gels were then electrotransferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA), which were then incubated with the indicated primary antibodies in 5% nonfat milk in TBS-T overnight at 4 °C. Next, the membranes were washed for 15 min each and immediately incubated with anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibodies for 1 h at 37 °C. The immunobands were visualized using ECL detection reagents (Transgen Biotechnology, Beijing, China) on a MicroChemi TM Chemiluminescent Imaging System (DNR Bio-Imaging Systems, Mahale HaHamisha, Jerusalem, Israel). Antibodies against p65/RelA and survivin were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against Cyclin A, Cyclin D, p-Rb, cleaved caspase-3 and cleaved caspase-9 were purchased from Abcam (Cambridge, MA, USA). The housekeeping protein β -Tubulin (Sigma-Aldrich, St. Louis, MO, USA) was used as an internal control for total protein measurement, and lamin B1 (Sigma-Aldrich, St. Louis, MO, USA) was used as a nucleoprotein reference. The densitometric values were calculated by AlphaEase TM FC 6.0 software (Alpha Innotech, Santa Clara, CA, USA), and the ratios of target protein to β -tubulin were used to conduct the statistical analysis.
Nuclear/cytoplasmic fractionation. A Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Shanghai, China) was used to extract the nuclear and cytoplasmic proteins from cultured cells and tissues, according to the manufacturer's protocol. Briefly, cells were washed with cold PBS, resuspended in buffer containing 1 mM DTT and 1 mM PMSF, and incubated on ice for 15 min. Detergent was added, and the cells were vortexed for 30 s at the highest speed. The nuclei and supernatant (cytoplasm) were separated by centrifugation at 4 °C. The nuclei were resuspended in buffer containing 1 mM DTT and 1 mM PMSF, incubated on ice for 30 min, and vortexed with interruptions. Nuclear extracts were collected by centrifugation at 14,000 × g for 10 min at 4 °C. For nuclear protein extraction of tissues, 60 mg of frozen bladder tissues were excised, immediately suspended in buffer containing 1 mM DTT and 1 mM PMSF, homogenized on ice, and then incubated for 15 min. The subsequent procedure was the same as that for cell nuclear and cytoplasmic protein extraction. (pCMV4-RelA-GFP) or the empty vector for 24 h. Then, pGL3 luciferase reporter plasmids, containing the promoter sequence of the survivin/BIRC5 gene, were transfected into the cells for another 48 h. Luciferase and Renilla signals were measured using a Dual Luciferase Reporter Assay Kit (Promega, Madison, WI, USA) according to the manufacturer's protocol.
Xenograft tumor model. BALB/c nude mice (4-6 weeks old, 14-16 g) were purchased from Beijing Vital River Experimental Animal Technology Co. Ltd. The mice were housed in barrier facilities on a 12-h light/dark cycle. The study was approved by Medical Laboratory Animal Welfare and Ethics Committee of China Medical University and the methods were carried out in accordance with the approved guidelines. Lv-RelA-5637 and Lv-NC-5637 cells were separately injected into the flanks of athymic nude mice to establish a xenograft tumor. When tumor sizes reached approximately 20 mm 3 , the mice were divided into four groups (two groups were established by Lv-RelA-5637 and the other two by Lv-NC-5637; 3 mice/group). Each of the mice with xenograft tumors expressing Lv-RelA-5637 or Lv-NC-5637 were continuously infused with YM-155 at 5 mg/kg/ day for 7 days; the other two groups of mice were infused with vehicle control. Tumors were examined every 3 days; the lengths and widths were measured using calipers, and tumor volumes were calculated using the equation (length × width 2 )/2. At 15 days after infusion, the mice were euthanized, and the tumors were excised and weighed. Next, xenograft tumors were lysed in RIPA lysis buffer (Beyotime, Shanghai, China) for western blotting or fixed in 10% (v/v) buffered formalin and paraffin-embedded for IHC staining.
Statistical analysis. The data are shown as the mean values ± sd. All statistical analyses were performed using SPSS (Statistical Package for the Social Sciences) 21.0 statistical software (SPSS Inc., Chicago, IL, USA). A two-tailed Student's t-test was used to assess significant differences between two groups of data in all pertinent experiments. Pearson's correlation coefficient analysis was used to determine the correlation between gene expressions. A P-value ≤ 0.05 was considered significant.