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cancers Article Intravesical Instillation of Azacitidine Suppresses Tumor Formation through TNF-R1 and TRAIL-R2 Signaling in Genotoxic Carcinogen-Induced Bladder Cancer Shao-Chuan Wang 1,2,3, Ya-Chuan Chang 2, Min-You Wu 2, Chia-Ying Yu 2, Sung-Lang Chen 1,2,3 and Wen-Wei Sung 1,2,3,* 1 Department of Urology, Chung Shan Medical University Hospital, Taichung 40201, Taiwan; [email protected] (S.-C.W.); [email protected] (S.-L.C.) 2 School of Medicine, Chung Shan Medical University, Taichung 40201, Taiwan; [email protected] (Y.-C.C.); [email protected] (M.-Y.W.); [email protected] (C.-Y.Y.) 3 Institute of Medicine, Chung Shan Medical University, Taichung 40201, Taiwan * Correspondence: [email protected] or fl[email protected]; Tel.: +886-4-2473-9595 Simple Summary: Approximately 70% of all bladder cancer is diagnosed as non-muscle invasive bladder cancer and can be treated by transurethral resection of the bladder tumor, followed by intravesical instillation chemotherapy. Bacille Calmette-Guérin (BCG) is the first-line agent for intravesical instillation, but its accessibility has been limited for years due to a BCG shortage. Here, our aim was to evaluate the therapeutic role of intravesical instillation of azacitidine, a DNA methyltransferase inhibitor, in bladder cancer. Cell model experiments showed that azacitidine inhibited TNFR1 downstream pathways to downregulate HIF-1α, claspin, and survivin. Concomitant Citation: Wang, S.-C.; Chang, Y.-C.; upregulation of the TRAIL R2 pathway by azacitidine ultimately drove the tumor cells to apoptosis. Wu, M.-Y.; Yu, C.-Y.; Chen, S.-L.; Sung, Rats with genotoxic carcinogen-induced bladder cancer showed a significantly reduced in vivo tumor W.-W. Intravesical Instillation of burden after intravesical instillation of azacitidine. These findings might support further clinical Azacitidine Suppresses Tumor trials of azacitidine in bladder cancer. Formation through TNF-R1 and TRAIL-R2 Signaling in Genotoxic Abstract: Azacitidine, an inhibitor of DNA methylation, shows therapeutic effects against several Carcinogen-Induced Bladder Cancer. malignancies by inducing apoptosis and inhibiting tumor cell proliferation. However, the anti- Cancers 2021, 13, 3933. https:// tumor effects of azacitidine on urinary bladder urothelial carcinoma (UBUC), especially following doi.org/10.3390/cancers13163933 intravesical instillation (IVI), are not established. Here, UBUC cell lines were used to analyze the Academic Editor: Andrea Morrione in vitro therapeutic effects of azacitidine. Potential signaling pathways were investigated by antibody arrays and Western blotting. The N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced rat UBUC Received: 18 June 2021 model was used for in vivo quantitative analysis of tumor burden. Azacitidine significantly inhibited Accepted: 2 August 2021 DNMT expression in UBUC cell lines and reduced cell viability and clonogenic activity, as determined Published: 4 August 2021 by MTT and colony formation assays, while also inducing significant cytotoxic effects in the form of increased sub-G1 and Annexin V-PI populations (all p < 0.05). Antibody arrays confirmed the in vitro Publisher’s Note: MDPI stays neutral suppression of TNF-R1 and the induction of TRAIL-R2 and their downstream signaling molecules. with regard to jurisdictional claims in TNF-R1 suppression reduced claspin and survivin expression, while TRAIL-R2 activation induced published maps and institutional affil- cytochrome C and caspase 3 expression. Rats with BBN-induced bladder cancer had a significantly iations. reduced tumor burden and Ki67 index following IVI of azacitidine (p < 0.01). Our study provides evidence for a reduction in BBN-induced bladder cancer by IVI of azacitidine through alterations in the TRAIL-R2 and TNF-R1 signaling pathways. These findings might provide new insights for further clinical trials. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. Keywords: bladder cancer; azacitidine; intravesical instillation; TNF-R1; TRAIL-R2 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Cancers 2021, 13, 3933. https://doi.org/10.3390/cancers13163933 https://www.mdpi.com/journal/cancers Cancers 2021, 13, 3933 2 of 13 1. Introduction Urinary bladder urothelial carcinoma (UBUC), also known as bladder cancer, is a highly prevalent malignancy worldwide. In 2020, the estimated new cases and deaths were 81,400 and 17,980, respectively, in the United States [1]. Non-muscle invasive bladder cancer (NMIBC) accounts for 70% of all bladder cancer diagnoses [2], and the management of NMIBC consists mostly of tumor excision via transurethral resection of the bladder tumor (TURBT). Subsequent intravesical instillation (IVI) of chemotherapeutic agents is performed within 24 h after surgery to eliminate residual cancer cells [3]. IVI therapy directly instills chemotherapy or immunotherapy agents, such as mit- omycin C and Bacille Calmette-Guérin (BCG), into the bladder [2]. BCG is the first-line treatment for NMIBC, and it significantly reduces the recurrence of malignancy [4]. BCG has several disadvantages, such as side effects that include a localized inflammatory re- sponse and BCG infection; however, a shortage of BCG agent currently limits accessibility to this treatment [5,6]. In addition, the tumor recurrence rate remains at about 40% fol- lowing chemotherapy instillation [7], indicating that any reduction in the rate of UBUC recurrence will require the exploration of alternative agents or agent combinations for IVI bladder cancer therapy. The progression of many cancers involves epigenetic abnormalities, such as aberrant DNA or histone methylation, which result in disturbances of gene expression [8,9]. Epige- netic therapy, therefore, plays an essential role in cancer treatment, and the vast majority of epigenetic drugs are histone deacetylase inhibitors (HDACi) and DNA methyltrans- ferase inhibitors (DNMTi) [10]. Ongoing or completed clinical trials of epigenetic drugs for treating bladder malignancy have mainly involved HDACi treatments, with only about 20% investigating DNMTi efficacy [11]. Similarly, very few studies have explored the intravesical instillation of DNMTi as adjuvant therapy. One promising DNMTi inhibitor is azacitidine, a type of hypomethylating agent that incorporates into DNA and RNA. It also binds to DNMT1 as an irreversible inhibitor [12], thereby preventing DNA methylation and reactivating tumor suppressor genes [13]. Azaci- tidine has been used as therapy for myelodysplastic syndromes and it improves outcomes for elderly patients diagnosed with acute myeloid leukemia [14,15]. Azacitidine also has therapeutic effects on solid tumors [16]. Currently, most clinical trials have focused on azacitidine as therapy for leukemia, but a few studies have examined the potential of intravenous injection as therapy for urologic malignancies, such as castration-resistant prostate cancer [17]. Azacitidine promotes the expression of tumor suppressor genes and inhibits the pro- liferation of bladder cancer cell lines, suggesting a potential therapeutic role in UBUC [18]. Azacitidine can also increase the sensitivity of drug-resistant bladder urothelial carcinoma cells to chemotherapy agents [19]. We hypothesized that azacitidine might show an anti- tumor effect and reduce the tumor recurrence rate in UBUC. The aim of the present study was to examine the anti-tumor effects of azacitidine on UBUC cell lines and to conduct in vivo experiments to assess the feasibility of using IVI of azacitidine as a treatment for UBUC. 2. Materials and Methods 2.1. Cell Culture Two UBUC cell lines, T24 and UMUC3, were purchased from The American Type Culture Collection (Manassas, VA, USA) and stored according to the supplier’s instructions. Cells were grown in RPMI medium containing 10% fetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin, 2 g/mL NaHCO3, 1 mM sodium pyruvate, and 0.1 mM non- ◦ essential amino acids. All cell lines were cultured at 37 C and supplied with 5% CO2. 2.2. MTT Assays The MTT assay was performed to detect the cytotoxicity of azacitidine. Briefly,2 × 104 cells were seeded into 96-well plates, incubated overnight, and exposed to azacitidine (0, 1, and Cancers 2021, 13, 3933 3 of 13 10 µM) for 1 or 24 h and harvested on the 72nd hour. MTT solution (0.5 mg/mL) was added to the wells and incubated for 3 h at 37 ◦C. The reaction was ended by the removal of the supernatant, followed by dissolving the formazan product in DMSO. Optical density was measured with an ELISA reader using a 570 nm filter. Each experiment was performed in triplicate and the cell viability of each group was calculated and analyzed compared to the control. 2.3. Clonogenic Assay T24 (250 cells) and UMUC3 (500 cells) treated with azacitidine (0, 1, and 10 µM) were seeded into each well of a 6-well plate and incubated for 3 or 10 days. The resulting colonies were fixed with 95% ethanol for 20 min and stained with 20% Giemsa solution for 30 min at room temperature, as previously described [20]. Each experiment was performed in triplicate and colony counts were calculated for each group. 2.4. Flow Cytometry Analysis Flow cytometry was conducted with a FACSCanto™ II Cell Analyzer (BD Biosciences, Franklin Lakes, NJ, USA) to determine the cell cycle distribution and percentage of apop- totic cells in each group, as previously described [21]. T24 and UMUC3 cells were seeded in 6-well plates and treated with azacitidine (0, 1, and 10 µM) for 72 h. For cell cycle distribution analysis, cells were fixed in pre-chilled 70% ethanol. The fixed cells were then re-suspended in PBS containing 0.4 µg/mL PI and 0.5 mg/mL RNase, and analyzed by flow cytometry. Apoptosis was analyzed using an Annexin V-FITC apoptosis detection kit (Strong Biotech Corporation, Taipei, Taiwan) according to the recommended protocol. Cells were re-suspended in binding buffer (100 µL), and 2 µL Annexin V-FITC and 2 µL PI were added. After a 15 min reaction in the dark, the cell apoptosis rate was evaluated by flow cytometry.

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