Background
Bladder cancer is one of the most aggressive epithelial tumors and is characterized by a high rate of early systemic dissemination. Patients with metastatic bladder cancer are routinely treated with cisplatin (CDDP)-based systemic chemotherapy, such as M-VAC (methotrexate, vinblastine, doxorubicin, CDDP) or GC (gemcitabine, CDDP) regimens. Such CDDP-based regimens have generally produced a complete or partial response in approximately 50-70% of patients [
1,
2]. However, tumors treated with CDDP finally acquire platinum resistance, and no standard of care exists when tumors develop after CDDP-treatments. These disappointing results have prompted an ongoing search for novel agents and multidrug combinations in this area.
NF-κB, a heterodimer consisting mainly of p65 and p50 proteins, functions as a transcription factor that induces inflammatory cytokines and antiapoptotic proteins. A growing body of evidence indicates that the activation of NF-κB is associated with resistance to apoptosis, expression of angiogenic proteins, and carcinogenesis due to its fundamental effects on cellular dedifferentiation and proliferation in malignancies [
3,
4]. DHMEQ, is a novel and potent NF-κB inhibitor [
5] that binds to a Cys residue of p65 and acts at the level of nuclear translocation [
6]. The mechanism by which DHMEQ inhibits activation of NF-κB is unique because DHMEQ inhibits NF-κB translocation from the cytoplasm to the nucleus [
7]. Using this agent, we previously showed that inhibition of the NF-κB pathway led to a potent induction of apoptosis in renal cell cancer, bladder cancer, and prostate cancer cells [
8-
10], suggesting that the regulation of NF-κB may be a potent therapeutic target for urogenital cancer.
The aim of the present study was to investigate the efficacy of NF-κB blockade as a new modality for treating platinum-resistant advanced bladder cancers. Also, we evaluated the efficacy of other chemotherapeutic agents such as gemcitabine, paclitaxel and carboplatin as second line chemotherapy for CDDP-resistant bladder tumor cell lines. To the best of our knowledge, no study has ever addressed the impact of NF-κB blockade when bladder tumors develop acquired resistance toward CDDP-based treatments. Also, few studies have described the changes in NF-κB expression in such tumors. We believe that these results may highlight the importance of NF-κB regulation as well as the clinical potency of DHMEQ in the treatment of metastatic bladder cancer.
Methods
Cell lines and agents
Two human invasive bladder cancer cell lines, T24 and T24PR, were used. T24 cells were obtained from the American Type Culture Collection (Rockville, MD, USA). T24PR cells were established in our laboratory as an acquired platinum resistant cell line [
11]. Briefly, T24 cells were grown and passaged upon reaching confluence in medium containing CDDP over a 6-month period to develop platinum resistance. All cells were routinely maintained in RPMI-1640 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Dainippon Pharmaceutical, Tokyo, Japan), at 37°C in a humidified 5% CO
2 atmosphere. DHMEQ, synthesized as described previously [
10,
12], was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mg/ml and stored at −20°C. This stock solution was diluted in culture medium to a final concentration of <0.1%. CDDP and paclitaxel were kindly supplied by Nippon Kayaku Co. (Tokyo, Japan). Gemcitabine and carboplatin were obtained from Wako Pure Chemical Industries (Osaka, Japan).
Cell extracts and western blotting analysis
Proteins were extracted from the cytoplasm and nucleus separately using NE-PER nuclear and cytoplasmic extraction reagents (Pierce Biotechnology, Rockford, IL, USA) according to the manufacturer’s protocol. The extracted nucleus protein (20 μg) and cytoplasmic protein (20 μg) with sample buffer containing 2-mercaptoethanol was separated on 12.5% SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA) and then incubated with 5% skim milk overnight. The membrane was then incubated overnight with primary antibodies against NF-κB p65 (Cell Signaling Technology, Beverly, MA, USA), Lamin A⁄C (Santa Cruz Biotechnology, Dallas, TX, USA), beta-actin (Sigma-Aldrich, St. Louis, MO, USA), Bcl-2 (Santa Cruz Biotechnology, Dallas, TX, USA) and survivin (Santa Cruz Biotechnology, Dallas, TX, USA). After incubation with appropriate secondary antibodies, signals were visualized using an ECL Western blotting system (Amersham, Piscataway, NJ, USA).
Electrophoretic mobility shift analysis
A nuclear extraction kit (Affymetrix, Santa Clara, CA, USA) was used to prepare nuclear extracts and an EMSA “Gel Shift” kit (Affymetrix, Santa Clara, CA, USA) was used in gel shift assay. The binding reaction mixture contained 2 μL of nuclear extract (at a concentration of 2 μg/μL), 1 μL of poly (dI–dC), and biotin-labeled p65 probe in binding buffer (75 mM NaCl, 1.5 mM EDTA, 1.5 mM DTT, 7.5% glycerol, 1.5% NP-40, 15 mM Tris–HCl; pH 7.0). Samples were incubated for 30 min at 15°C in this mixture. DNA/protein complexes were separated from free DNA on a 6% non-denaturing polyacrylamide gel in 0.25 mM TBE buffer. The gel was transferred to a nylon membrane and detected using streptavidin-HRP and chemiluminescent substrate. The following sequence was used as a p65 probe (Affymetrix, Santa Clara, CA, USA): 5′-CATCGGAAATTTCCGGAAATTTCCGGAAATTTCCGGC-3′.
Cell growth assay
All cell lines were seeded at a density of 5 × 103 cells per well into 96-well culture plates. Following 24 hour incubation in RPMI 1640 medium with 10% fetal bovine serum, the cells were incubated for 48 hours with various concentrations of DHMEQ. To evaluate the changes of sensitivity to anticancer agents, the cells were incubated for 48 hours with various concentrations of anticancer agents (cisplatin, gemcitabine, paclitaxel and carboplatin) in each cell line. In combination analysis, cells were incubated for 48 hours with 3 μg/ml of DHMEQ and various concentrations of chemotherapeutic agents in a similar way. Cells treated with the same concentration of DMSO were served as controls. At the end of the incubation period, cell viability was determined using a Premix WST-1 Cell Proliferation Assay System (Takara Bio Inc, Shiga, Japan) and microplate spectrophotometer (Bio-Rad Laboratories, Inc, Tokyo, Japan). The absorbance value of each well was determined at 450 nm with a 655 nm reference beam in a microplate reader (Bio-Rad, Tokyo, Japan).
Resistance factor (RF) analysis also provided a qualitative measure of the extent of acquired resistance against anticancer agents, and was calculated as the half maximal (50%) inhibitory concentration (IC
50) of resistant line/IC
50 of parent line. IC
50 values were determined in three independent experiments. Combination index (CI) analysis provided a qualitative measure of the extent of drug interaction. A CI of less than 1, equal to 1 and more than 1 indicates synergy, additive and antagonism, respectively [
13,
14].
Apoptosis assay
Flow cytometric analysis was performed using transferase-mediated nick-end labelling (TUNEL) assay to detect apoptosis. TUNEL assay was performed using ApopTag kits (Sigma Chemical, Atlanta, GA, USA). The cells (1×106 cells) were seeded in 100 mm dishes and incubated for 24 hours in RPMI 1640 medium with 10% fetal bovine serum. Following 48 hour incubation in medium containing 10 μg/ml of DHMEQ, apoptosis was detected by flow cytometry, and subsequent analysis was carried out according to the manufacturer’s protocol.
Murine xenograft bladder cancer model
All animal procedures were carried out in accordance with ARRIVE guidelines. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Keio University {Permit Number: 10228-(1)}. Six-week-old athymic nude mice (BALB/c) with an average body weight of 20 g were obtained from Sankyo Lab Service Co. (Tokyo, Japan). T24PR cells (2 × 106 cells), suspended in 100 μl of matrigel (Becton Dickinson Labware, Lincoln Park, NJ, USA), were implanted subcutaneously into the flank of each mouse. In the first set of in vivo experiments, the mice were randomly assigned to 2 groups, each consisting of 10 animals. On day 7 after cancer cell implantation, the mice were injected intraperitoneally with 2 mg/kg DHMEQ daily. The control group was administered vehicle DMSO solution.
In the second set of in vivo experiments, the mice were assigned to 3 groups (control, paclitaxel alone, or combined paclitaxel and DHMEQ), each consisting of 10 animals. Paclitaxel (10 mg/kg) was administered intraperitoneally on day 14 and day 21 after cancer cell implantation, while DHMEQ was injected intraperitoneally at 2 mg/kg from day 7 after cancer cell implantation. The mice were carefully monitored and tumor size was measured every three days. Tumor volume (V) was calculated according to the formula V = length × width × height × 0.52. Four weeks after implantation, the mice were sacrificed and the tumors were evaluated histologically.
Immunostaining for Ki-67 and apoptosis
Formalin-fixed, paraffin-embedded tissue sections (4 μm) were stained with hematoxylin and eosin (H&E) for tumor pathology. These sections were deparaffinized, rehydrated, and washed in phosphate-buffered saline. Endogenous peroxidase was quenched. A blocking step was included using 1% bovine serum albumin together with avidin and biotin blocking solutions. To determine the proliferative activity, Ki-67 immunostaining was performed using an anti-Ki-67 monoclonal antibody (MIB-1; Dako, Carpinteria, CA, USA). Apoptosis was measured by TUNEL assay using a commercially available apoptosis in situ detection kit (Wako Pure Chemical, Osaka, Japan). Visualization of the immunoreaction was performed with 0.06% 3,3′-diaminobenzidine (DAB) (Sigma Chemical, Atlanta, GA, USA). A dark accumulation of DAB in the nuclei was judged to indicate a positive reaction for TUNEL and Ki-67. The apoptotic index was calculated as the average number of TUNEL-positive cells in 10 areas at high power field (×400). The proliferation index was calculated as the average number of cancer cells with nuclei stained for Ki-67 in 10 areas at high power field (×400).
Statistical analysis
All data are presented as the mean ± SE. Comparisons of two different groups were performed using the Mann–Whitney U-test. P-values <0.05 were accepted as being statistically significant.
Statistical analyses were performed with R Statistical Language version 2.9 and SPSS version 18.0 statistical software package.
Discussion
Bladder cancer is one of the most aggressive epithelial tumors and is characterized by a high rate of early systemic dissemination. The prognosis for patients with advanced or metastatic bladder cancer remains poor [
15]. The vast majority of patients treated with CDDP-based regimens develop progressive disease within 8 months of treatment, and the median survival is reported to be only 13–15 months [
2,
16]. Furthermore, there is still no approved treatment option for patients who develop disease recurrence or progression after CDDP-based regimens [
17].
In the present study, we have demonstrated the cytotoxic effect of DHMEQ, a potent NF-κB inhibitor, after the development of acquired platinum-resistant bladder cancer cells. Strong nuclear localization of NF-κB was observed in T24PR cells whereas relatively weak nuclear expression of NF-κB was observed in T24 cells. DHMEQ reversibly inhibited the DNA-binding activity of NF-κB and consequently induced a significant dose-dependent decrease in cell viability due to apoptosis in T24PR cells. We further examined the efficacy of DHMEQ using mouse xenograft tumors, and observed a significant inhibitory effect on tumor growth especially in DHMEQ-treated tumors of T24PR cells. These data suggest that DHMEQ may be useful even in acquired platinum-resistant tumors and shed light on the impact of NF-κB inhibition as a new modality when tumors develop acquired resistance toward CDDP-treatments.
The DNA-binding activity of p65 was significantly inhibited after 2–6 hours of 10 μg/ml of DHMEQ and then gradually recovered. DHMEQ covalently binds to the cysteine residue to induce irreversible inhibition [
18,
19]. However, after a long incubation period, possible newly formed NF-κB appears. We think inhibition of NF-κB for several hours would be sufficient to increase the drug sensitivity. So we believe that DHMEQ has a long-lived efficacy without continuing inhibition of NF-κB translocation to the nucleus. In fact, we have examined and reported similar cytotoxic results for DHMEQ in various types of cancer cells, even though the NF-κB inhibition is short-lived [
9,
12,
20]. Furthermore, we have shown that Bcl-2 and survivin were suppressed by DHMEQ in a dose-dependent manner. It is likely that the increase of drug sensitivity is due to the decrease of anti-apoptosis protein expression in our present study.
NF-κB activation has been found to be involved in many types of cancer including genitourinary cancer such as prostate cancer and renal cell cancer [
21-
23]. In bladder tumors as well, the impact of NF-κB activation on tumorigenesis has been described [
24] and our previous work focused on the efficacy of NF-κB blockade by DHMEQ in a mouse xenograft model of invasive bladder cancer KU-19-19 cells [
10]. Also, several researchers attempted to examine the association between the status of NF-κB expression and resistance to chemotherapy in bladder tumors. Using immunohistochemical analysis from 116 bladder cancer patients, Levidou et al. reported a close association between the aggressiveness of bladder tumors and nuclear NF-κB expression, and suggested NF-κB expression has an impact as an independent indicator for prognosis in bladder UC patients [
24]. Wang et al. also reported that NF-κB activity and sensitivity to chemotherapy are inversely correlated in cancer treatments [
25]. Inhibition of NF-κB not only leads to enhanced apoptosis but also to increased sensitivity to radiation or chemotherapy in several tumor cells such as fibrosarcoma and colorectal cancer cell lines as well as xenograft models or pancreatic carcinoma cells [
25-
27]. With regard to the association between NF-κB activation/expression and chemoresistance, Antoon et al. reported that the breast cancer chemo-resistance cell line MCF-7TN-R overexpressed NF-κB. Furthermore, inhibition of the NF-κB cascade with a sphingosine kinase-2 inhibitor decreased NF-κB activation as well as tumor growth
in vitro and
in vivo [
26].
Paclitaxel, which is one typical taxane agent, has been used in patients with advanced urothelial carcinoma who were refractory to prior CDDP based chemotherapy. Paclitaxel alone yields a 42% response rate against urothelial carcinoma when used as a first-line treatment [
27], but yields only a 10% response rate in patients who were treated previously [
28]. Considering the low response rate of paclitaxel when used alone as a second-line treatment, its combinations with gemcitabine, cisplatin, carboplatin, and ifosfamide have been investigated, and the response rate was found to increase to 15–40% [
28-
31]. In the present study, examining further the clinical potency of DHMEQ, we investigated the efficacy of combination therapy with paclitaxel and DHMEQ in platinum-resistant tumors. Indeed, the results showed a significant difference in tumor growth between the DHMEQ-only group and combination-treated group. As shown in Figure
10, paclitaxel inhibited the growth of T24PR tumors, however, its combination with DHMEQ had a stronger antitumor effect. The reduction of tumor volume in tumors treated with the combination treatment and paclitaxel alone treatment as compared to vehicle control was 66.9% and 17.0%, respectively. Therefore, we propose that combination therapy consisting of taxane agents and DHMEQ may be an effective choice for patients with CDDP refractory bladder tumor.
Acknowledgements
This study was supported in part by Grants-in-Aid for Scientific Research (No. 22791495 to Tanaka N, No. 20591866 to Kikuchi E and No. 21390445 to Oya M) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
YI carried out all studies, performed the statistical analysis, and wrote manuscripts. EK, NT, KU, and MO conceived of the study, and participated in its design and coordination and helped to draft the manuscript. ES, TS, and AM helped to carried out the molecular genetic studies.TK and RM helped to carried out immunoassays. All authors read and approved the final manuscript.