Selective Susceptibility of Human Bladder Transitional Cell Carcinoma T24 Cells towards NBD Peptide

Department of Biotechnology, Faculty of Biomedical Sciences, Technology and Research, Sri Ramachandra Institute of Higher Education and Research (Deemed to be University), Chennai, India Department of Medical Biochemistry, Dr. ALM PG Institute of Basic Medical Sciences, University of Madras, Chennai, India Department of Integrative Biology, School of Biosciences and Technology, VIT University, Vellore, India Department of Bioinformatics, Faculty of Biomedical Sciences, Technology and Research, Sri Ramachandra Institute of Higher Education and Research (Deemed to be University), Chennai, India Department of Biotechnology, School of Life Sciences, Pondicherry University, Pondicherry, India Faculty of Allied Health Sciences, Sri Ramachandra Institute of Higher Education and Research (Deemed to be University), Chennai, India Department of Pathology, SRMC&RI, Sri Ramachandra Institute of Higher Education and Research, (Deemed to be University), Chennai, India


Introduction
Bladder Cancer (BC) is the second most common malignancy of the genitourinary system in industrialized countries (Antoni et al., 2017). It is a heterogeneous disease which makes it difficult for accurate molecular analysis of the tumor, identification of the correct biomarkers for assessment and choice of treatment 185 modality (Jakobsson et al., 2018). BC incidence is based on genetic predisposition, lifestyle or environmental factors (Burger et al., 2012). BC is predominant in older male patients ≥ 65 years of age at the time of diagnosis. Also, recurrence and chemoresistance are prevalent in BC (Lu et al., 2017;Wang et al., 2018;Woolbright et al., 2018). There are two major types of BC including the superficial disease and the invasive disease. Bacillus Calmette-Guerin (BCG) immunotherapy is the treatment offered after Transurethral Resection of Bladder Tumor (TURBT) for superficial BCs. Many investigations report BCG as effective in preventing BC recurrence (Pettenati and Ingersoll, 2018;Guallar-Garrido and Julián, 2020). However, it is still controversial when considering its ability to prevent BC progression (Kaufman et al., 2009;Buchwald and Efstathiou, 2015). Treatments offered for invasive BC are surgery with neoadjuvant-, adjuvant-, chemo-and radiation therapy which lacks in-depth analysis of the underlying mechanism due to the lack in the number of randomised Control Trials (RCT) performed for BC (Soloway, 2013). Thus it becomes important to identify the inherent heterogeneity and personalize treatment in BC by understanding the key players in its pathogenesis.
Nuclear Factor kappa B (NF-κB) signaling cascade plays an important role in cell survival (Zhang et al., 2017). A number of molecules can block NF-κB pathway and thus the critical node either directly or indirectly through its signaling pathway members to suppress cancer. Drugs that utilize this strategy are effective in reducing tumor but have poor clinical efficacy (Ianaro et al., 2009;Karin et al., 2002). Proteosome inhibitors are the only exception from this trait which is used for the treatment of some haematological malignancies that demonstrate the key role of NF-κB during their pathogenesis (Richardson et al., 2004). There are many reports stating the involvement of NF-κB in BC (Durairajan et al., 2018;Levidou et al., 2008;Mukherjee et al., 2015;Yeh et al., 2010). However, there are limited reports that have considered the NF-κB signaling cascade and BC in a therapeutic setting. This study is the first of its kind to the best of our knowledge to consider NF-κB associated peptide as a treatment option for BC. NF-κB Essential Modulator (NEMO) is a regulatory protein that belongs to the NF-κB pathway. It allows catalytic units (IKKα, IKKβ) to bind in a region called NEMO Binding Domain (NBD) (May et al., 2000). This domain is termed NBD and is of significance since the core peptides are capable of eliciting NF-κB inhibitory effects. NBD has shown antiinflammatory activity (Baima et al., 2010) in murine models against colitis (Hong et al., 2012), rheumatoid arthritis/osteoclastogenesis (Jimi et al., 2004), kernicterus (Li et al., 2015) and pancreatitis in rats (Long et al., 2009) and also against canine refractory diffuse large B-cell lymphoma (Gaurnier-Hausser et al., 2011). The inherent heterogeneity of BC and the success rate behind BCG treatment establish the inflammatory milieu prevalence. Thus this preliminary in vitro study analyzes the potential held by NBD peptide as an antineoplastic agent in BC.

Cell Culture and Treatment
T24 cell line (Human transitional cell carcinoma of the urinary bladder), NIH-3T3-L1 (mice fibroblasts), CHO (Chinese Hamster Ovary) and Vero (African green monkey kidney) were obtained from the National Centre for Cell Science (NCCS), Pune, India. T24 cells are resistant to cisplatin and were cultured as monolayers in McCoy's 5A medium. NIH-3T3-L1, CHO and Vero were cultured in DMEM. Each of the medium used for maintenance was supplemented with 10% FBS, L-Glutamine and 3 mM sodium bicarbonate/ 20 mM HEPES. A combination of antibiotics (penicillin, streptomycin, kanamycin) and an antimycotic (amphotericin B) was used. The cells were maintained at 37°C with 5% CO2 and 85-95% humidity. The maintained cultures were subcultured for assays and drug treated at optimal growth (Abdel-Mageed, 2003;Chong et al., 2000).
NBD was prepared in serum free McCoy's 5A medium and used for treatment. TAT is a cell 186 penetrating peptide (Trans-Activator of Transcription protein derived from human immunodeficiency virus) tagged to NBD peptide to ensure cell entry. It was employed as a vehicle control at a final concentration not exceeding 100 µM in the cell culture experiments. The standard drug, cisplatin was prepared in 0.9% sodium chloride to avoid isomerisation, kept protected from light and used at a final concentration of 50 µM (Tian et al., 2008). Gemcitabine was prepared in serum free McCoy's 5A medium and used at a final concentration of 50 nM in the cell culture experiments (Fechner et al., 2003). Based on the half maximal inhibitory concentration (IC50) of NBD peptide, all treatments were carried out and incubated for 48 h along with select concentrations: 12.5, 25, 50 and/or 100 μM in every experiment, except for confocal microscopy where 20 μM FITC-NBD was used.

Confocal Microscopy for Localization
T24 cells were seeded on a coverslip placed inside a 35 mm Petri dish at a concentration of 1×10 4 cells.
McCoy's 5A medium was added and incubated to attain 80% confluency. FITC tagged NBD peptide (20 μM) was treated for different time periods (10 min, 45 min, 4 h and 24 h). The plates were incubated in the dark at 37°C for the designated durations and counterstained with 10 μL PI (1 mg/mL) for 1 min. The coverslips were briefly rinsed with PBS and mounted onto another coverslip for documentation according to the previous report with slight modifications (Choi et al., 2003).

Cell Proliferation and Viability Assessment by MTT Assay
T24, NIH-3T3-L1, CHO and Vero cells were seeded at a concentration of 1×10 4 cells/well on a 96 well plate with 10% medium. At about 80% confluency, different concentrations of the NBD peptide (12.5, 25 and 50 μM), TAT peptide (100 μM) as vehicle control and standard drugs (Cisplatin, 50 μM and Gemcitabine, 50 nM) were treated and the volume was made up to 100 μL. The drug treated plates were incubated for 48 h at 37°C for cell proliferation and viability assessment. Finally, 20 μL of MTT reagent (1 mg/mL) was added and incubated for 4 h at 37°C for quantification. The purple formazan crystals formed were dissolved using 100% DMSO and read at 570 nm in BMG Fluostar Optima multimode reader (Wang et al., 2012). The IC50 value for NBD peptide on T24 cells was determined from the MTT experiment. Synergistic action of IC50 NBD peptide was tested with a combination of standard drugs. The tested combinations are IC50 NBD and cisplatin; IC50 NBD and gemcitabine; IC50 NBD, cisplatin and gemcitabine.

Cell Cycle Analysis Using Flow Cytometry
T24 cells were seeded at a concentration of 1×10 5 cells/ well on a 12 well plate and allowed to attain confluency. Different concentrations of the NBD peptide: 12.5, 18.5 (IC50), 25 μM, TAT and standard drugs were treated. Combinations of NBD peptide along with the standard drugs were also treated. The plates were incubated at 48 h at 37°C, harvested by trypsinization, rinsed with PBS and centrifuged at 1500 rpm for 10 min. Cells were fixed overnight at 4°C in ice-cold alcohol (70%). The fixed cells were washed with PBS and centrifuged at 1500 rpm for 10 min before analysis. Triton X-100 (0.5%) and RNase (0.1 mg/mL) was added and incubated for an hour. Propidium iodide (40 μg/mL) was added to the mix and incubated for 45 min in the dark. Cell cycle analysis was carried out on FACS Calibur (BD Biosciences) (Ma et al., 2009;Wang et al., 2013).

Assessing Cell Death by Acridine Orange/Ethidium Bromide (AO/ EtBr) Fluorescence Staining Assay
T24 cells were seeded at a concentration of 1×10 5 cells/well on a 6-well plate added with 10% medium and allowed to attain confluency. NBD (12.5, 18.5 and 25 μM), TAT peptide, standard drugs and the combination were treated. The plates were incubated at 48 h at 37°C followed by addition of 10 μL AO/EtBr (1 mg/mL) to each well for morphological documentation (Ishaq et al., 2014). The fluorescence screen was applied and the cells were photographed under a microscope (Nikon Eclipse Ti-S, USA).

Caspase-3 Activity
Quantification of caspase-3 release was carried out according to the EnzChek® Caspase-3 Assay Kit #2 (Molecular Probes: MP 13184) protocol. Briefly, T24 cells at 1×10 4 cells per well were resuspended in 50 μL of the 1× cell lysis buffer. Cells were incubated on ice for about 30 min to ensure optimal lysis. After incubation, the cells were centrifuged at 5000 rpm for 5 min in a centrifuge. Then 50 μL of the supernatant from each sample was transferred to individual microplate wells. 1× cell lysis buffer (50 μL) was used as a "no-enzyme control" to determine the background fluorescence. Then 50 μL of 2× substrate working solution was added to each sample (NBD: 100 µM, NBD and standard drug combination) and untreated control. The microplate was covered and incubated at room temperature for 30 min. Fluorescence was measured on a microplate reader (BMG FLUOSTAR OPTIMA multimode reader) using excitation/emission wavelengths of 492/520 nm (Cerón et al., 2010).

Cellular Damage Detection Using Lactate Dehydrogenase (LDH) Release
T24 cells were seeded on 48-well plates at a density of 2.5×10 4 cells/well and allowed to become confluent overnight. At >80% confluency, the cells were treated with NBD peptide for 48 h along with standard drugs and vehicle control. After treatment, the conditioned media was used to assess the LDH released by the damaged cells. Buffered substrate (1 mL) and conditioned media (0.1 mL) was added and incubated at 37°C (in a water bath). To this mix, 0.2 mL of NAD+ solution was added, mixed gently and incubated at 37°C for 15 min. DNPH reagent (1 mL) was added and incubated further for 15 min. Finally, 10 mL of sodium hydroxide (0.4 N) was added and the absorbance was read at 440 nm. Standards were also run parallelly and treated as for assays with sodium pyruvate. The amount of color developed was directly proportional to the number of lysed cells (Elumalai et al., 2012).

Detection of Apoptosis by DAPI Staining
T24 cells were seeded on 12-well plates at a density of 5×10 4 cells/well and allowed to become confluent overnight. For the nuclear changes, the monolayer of cells was washed with PBS, fixed with 3% paraformaldehyde (10 min) and were permeabilized with 0.2% Triton X-100 in PBS (10 min) at ambient temperature. The cells were finally incubated with 0.5 µg/mL of DAPI for 5 min to identify the apoptotic nuclei which were intensely stained or fragmented or had condensed chromatin. The changes were observed and recorded under a fluorescent microscope (ex 359 nm; em 461 nm) (Tajmohammadi et al., 2019).

Wound Healing Assay
T24 cells were seeded at a concentration of 1×10 5 cells/well on a 6-well plate and allowed to attain confluency before treatment. Confluent monolayers were wounded by physical shearing across the well using a sterile micropipette tip. The diameter of the wound inflicted was measured immediately using a phase contrast microscope (Nikon Eclipse Ti-S) with ImagePro software. NBD (18.5 and 100 μM), TAT peptide, standard drugs and the combination were treated. The plates were incubated for 60 h at 37°C, observed for wound closure and documented (Gao et al., 2006).

Statistical Analysis
All the experiments were repeated thrice for accuracy and each sample was assessed in triplicates. The data are presented as the mean ± SD. Statistical analysis was performed with IBM SPSS Statistics 23v and differences between groups were examined using one-way Analysis of Variance (ANOVA) followed by Tukey's multiple comparison test wherever applicable to prove statistical significance. P value of ≤ 0.05 was considered statistically significant.

NBD Peptide Localization and Selective Inhibition of T24 Cancer Cells
The ability of the NBD peptide to gain entry into the T24 cells and their stability inside the cells was determined using confocal microscopy after incubation of cells with the peptide for 10 min, 45 min, 4 h and 24 h. The NBD peptide tagged with FITC fluorochrome emits fluorescence upon intracellular localization and is not affected by pH changes in the endolysosomal compartments. The minimum time taken for NBD peptide cell penetration was ≤ 10 min where it was able to localize the cytosol and nucleus with detectable fluorescence. At 45 min of treatment, there were prominent signals from the NBD peptide in the cytoplasm and nucleus retaining cell morphology until 4 h of treatment. The T24 cells started to show aggregation at 24 h of treatment, however, the fluorescence signal was stable with detectable emissions from the peptide. Figure 1 shows that the NBD peptide was stable for up to 24 h of treatment and was capable of acting on the cell's internal machinery. The cytotoxicity was tested on NIH-3T3-L1, CHO and Vero (non-cancerous) cells along with T24 cancer cell line. NIH-3T3-L1, CHO and Vero cells were resistant to NBD peptide treatment (P≤ 0.05). However, T24 BC cells were susceptible to NBD peptide treatment (25, 50, 100 µM) at 48 h duration (P≤ 0.05). The susceptibility was compared with the untreated control cells and standard drugs like cisplatin and gemcitabine (Fig. 2). The inhibition of T24 cell growth in MTT assay allowed us to determine the IC50 value as 18.5 µM and this concentration was used to test the adjuvant capability of NBD peptide. A combination of NBD peptide along with the standard chemotherapeutic drugs was tested on T24 cells. The results showed higher susceptibility of T24 cells to the synergistic action of NBD: Cisplatin: Gemcitabine combination at 48 h (P≤ 0.05) than the individual agents (Fig. 3).

NBD Peptide Elicits Cell Cycle Inhibition
Flow cytometry was performed on T24 cells treated for 48 h with NBD peptide (12.5, 18.5 and 25 µM), standard chemotherapeutic drugs and the combination of NBD with standard chemotherapeutic drugs. Control represents the normal cell cycle progression in T24 cells. Treatment with the NBD peptide increases the accumulation of cells in the sub G0-G1 phase with an overall reduction in the number of events recorded. The 188 standard drugs cisplatin shows an increase in the cells at sub G0-G1 and disruption of G2-M phase; gemcitabine shows an increase in the cells at sub G0-G1 phase; dual action of cisplatin and gemcitabine shows accumulation of cells in the S phase indicating a disruption of G2-M phase. The combination of NBD along with standard drugs indicates the effects of the individual agents combined. The NBD & gemcitabine combination shows G2-M phase disruption which was absent in their individual effect and has increased the accumulation of cells in sub G0-G1 phase. The NBD & standard drugs combination panel shows an overall reduction in the number of events and an increase of cells in sub G0-G1 phase. The histograms of flow cytometry (Fig. 4) and the number of events after gating are tabulated (Table 1).

NBD Peptide Induces Caspase-3 Independent Cell Death of T24 Cells and Inhibits Cell Migration
The accumulation of sub G0-G1 population in cell cycle after NBD treatment led us to use acridine orange and ethidium bromide staining to reveal the mode of death induced by NBD peptide. The early or late apoptosis marked by the orange-yellow coloration was scanty in the standard drug panel and negligible in the combination treatment panel (Fig. 5). To confirm that there was no caspase activity we quantified the cysteine-aspartate proteases that are released when the cells undergo apoptosis. We tested the highest concentration of NBD peptide (100 µM) on T24 cells for 48 h. Negligible effect was observed with the combination of NBD and standard chemotherapeutic drugs when compared with untreated cells (Fig. 6). Similarly, the LDH release was quantified to assess cellular damage as it is a stable cytosolic enzyme, released into the culture medium upon cell lysis. Oxidation of lactate to pyruvate (reversible) is catalysed by LDH,  forming NAD+ from NADH and thus the determination of LDH is based on the detection of NADH in the reaction. Thus Fig. 7 indicates T24 cell death by necrosis (resultant of ruptured plasma membrane) upon treatment with NBD and its combination with cisplatin and gemcitabine. Further confirmation for the non-apoptotic mode of death was by DAPI staining where there was no chromatin condensation at the nuclear membrane periphery except in combinations that contained cisplatin: NBD and cisplatin; NBD, cisplatin and gemcitabine (Fig. 8).   Cancer metastasis is mimicked by the process of wound healing on the monolayer of cells as they require the same phenomenon of proliferation and migration. In our study, the ability of T24 cells to convalesce to their viable state after NBD treatment was demonstrated using cell migration technique. The T24 cells after treatment with NBD peptide, standard drugs and the combination showed significant cell migration inhibition in wound closure when compared to the untreated cells. NBD peptide, when used at the IC50 concentrations of 18.5 µM was unable to restrict the migration of T24 cells and hence the highest concentration of NBD peptide (100 µM) was used in the wound closure assay. The highest concentration of NBD peptide combination along with standard drugs had better inhibition of cell migration than the individual agents (Fig. 9).

Discussion
BC is a recurrent disease and lacks thorough investigations (Mukherjee et al., 2015). The tissue microenvironment is a niche that comprises various signaling factors that are involved in several regulatory functions of a cell. BC has highly active NF-κB along with other transcription factors (Degoricija et al., 2014). During BC initiation presence of aberrant NF-κB in the tumor milieu has been reported (Levidou et al., 2008;Zhu et al., 2017;Mukherjee et al., 2017). Targeted therapy is sought after in the treatment of BC (Jones et al., 2016) overexpression of NF-κB heterodimers can be used to differentiate cancer cells from normal cells. Previously, we were able to show that the NF-κB RelA preferably binds to other dimers of the family other than p50 during BC progression (Durairajan et al., 2018). Since the involvement of the NF-κB heterodimers in BC progression was evident, we utilized this to explore the treatment potential by targeting this pathway.

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NBD peptide was identified to have antiinflammatory properties by acting as indirect NF-κB inhibitor (Gilmore, 2006;Gilmore and Garbati, 2011). NF-κB inhibition either directly or indirectly will affect the basal NF-κB activity required for cell survival and upon its inhibition will lead to toxicity (Davé et al., 2007). Few studies investigating NBD peptide have eradicated this concern where prolonged NBD peptide administration ameliorated inflammatory responses in inflammatory animal models effectively without side effects (Jimi et al., 2004;di Meglio et al., 2005) while retaining the basal level activity of the NF-κB heterodimer to perform the regulatory functions (Jana et al., 2017;May et al., 2000). NBD peptide was able to reduce skeletal muscle atrophy and tumor induced muscle wasting dosedependently with the ability to decrease the tumor burden (Madonna et al., 2012;Wysong et al., 2011).
NBD peptide core amino acids determine its activity and any changes in the core region disrupt its antiinflammatory activity, rendering it inactive (Dai et al., 2004). Analysis of binding energy hot spots between those on NEMO (Nuclear Factor kappa B (NF-κB) essential modulator) and IKKβ (IκB kinase subunit β) interacting interface identified an NBD (NEMO Binding Domain) region on IKKβ to contain the highest concentration of hot-spot residues (W739, W741 and L742). The residing region of these residues creates a druggable binding site on NEMO (Golden et al., 2013). In the present study, NBD peptide was fused with HIV coat protein fragment, TAT to achieve cell penetration. This peptide fragment, NBD inhibits the growth of T24 BC cells. According to our hypothesis, we observed dose-dependent inhibition of the T24 cells with half maximal inhibition at 48 h, while the non-cancerous (NIH-3T3-L1 cells, CHO and Vero) cells did not show any cytotoxicity (Fig. 2). The specificity of NBD peptide towards cancer cells makes it evident that it targets a specific molecule in T24 BC cells that is uncommon in the non-cancerous cell lines. The viability assay confirmed dose-dependent cell death and the NBD peptide's ability to interrupt cellular processes to bring about cell death. The unaffected growth of the noncancerous cells after NBD treatment was high when compared with the untreated control group. The statistical significance of the T24 cancer cell line (P≤ 0.05) is due to the decrease in the number of viable cells that are compared with the untreated control group.
NBD peptide localization in the nucleus was suggestive of the interference to cell cycle. Accumulation of the cells in Sub G0-G1phase indicated necrosis of cells caused by NBD peptide treatment. Combining NBD peptide with standard drugs showed decrease in the overall number of events in all cell cycle phases. Further, differential staining of T24 cells with acridine orange and ethidium bromide revealed majority of cells emitting red fluorescence post NBD peptide treatment suggesting necrosis. Ethidium bromide is known to shift its fluorescence upon binding to intracellular DNA after entering cells with damaged membranes in necrotic cells (Liu et al., 2015;Vethakanraj et al., 2015). This was confirmed by the negligible caspase activity at the highest concentration of the NBD peptide tested compared to the untreated control cells (Fig. 6). Thus acridine orange/ethidium bromide staining, caspase activity, LDH release, DAPI staining confirmed that there was no apoptosis in T24 cells after NBD peptide treatment and also inhibited cell migration in vitro. To the best of our knowledge, this is the first report on NBD peptide to have necrotic activity in T24 bladder cancer cell line. Our observations in this study was in accordance with other studies that state different cell lines do respond differently to a treatment based on their cancer phenotype (McConnell et al., 2015); the factors that cause a peptide to act differently in a tumor microenvironment (Pan et al., 2014); the amino acid difference from the originally reported NBD peptide (May et al., 2000;Dai et al., 2004;May et al., 2002) and the different cell penetrating peptide tags that allow them to act unlike their original counterparts (Shaheen et al., 2018).
Currently, peptide based trials have been predicted to be clinically successful in urological cancers (Obara et al., 2018) and the ability of NBD peptide to sensitize cancer cells to chemotherapy have been reported in pancreatic cancer (Holcomb et al., 2008). Our study was in conformity with another study that combined NBD peptide with other chemotherapeutics like cisplatin and gemcitabine producing higher rates of proliferation inhibition than the peptide or drug alone (Ndikuyeze et al., 2014). Definitive use of NBD peptide determines its function to act as a cell protective agent with minimal adverse effects (Rehman et al., 2003) or to act as an antiinflammatory/anti-cancer agent. This dynamic change in action for NBD could be due to more than just inhibition of activated NF-κB (Cerón et al., 2010). Our study suggests that the mechanism of NBD peptide action varied from each cell type analyzed. This only emphasizes the need for more mechanistic studies that allows characterization of the physiological and immunological responses with NBD treatment in BC.

Conclusion
We investigated NBD peptide in BC since the available chemotherapeutics lack specificity with adverse side effects. In our study, we have shown that NBD peptide selectively reduces the proliferation of BC cells in vitro by necrosis after localization. NBD peptide lacked cytotoxicity towards non-cancerous cell lines across all tested doses. The combination treatment of 195 NBD peptide along with cisplatin and gemcitabine showed stronger inhibition than single drug treatment. Though we need further investigations to understand NBD peptide's mechanism of action, its use as an anticancer agent in the recurrent and often chemoresistant BC seems promising.