Int J Clin Exp Pathol 2017;10(10):10197-10204www.ijcep.com /ISSN:1936-2625/IJCEP0030152

Original Article Rosiglitazone induces apoptosis on human bladder cancer 5637 and T24 cell lines

Xiaoyuan Xu1, Jianjun Wang2, He Jiang1, Lirong Meng3, Bin Lang3

1Key Laboratory of System Bio-medicine of Jiangxi Province, Jiujiang University, Jiujiang, China; 2Department of Orthopedic Surgery, People Hospital of Zhuhai, The Third Affiliated Hospital of Jinan University, Zhuhai, China; 3School of Health Sciences, Macao Polytechnic Institute, Macao, China

Received April 8, 2016; Accepted June 13, 2016; Epub October 1, 2017; Published October 15, 2017

Abstract: Rosiglitazone is a synthetic ligand of peroxisome proliferator-activated receptor γ (PPARγ), and it can in-duce apoptosis and autophagy in a variety of cancer cells. In the present study, we aimed to investigate the influ-ence of rosiglitazone on the proliferation and apoptosis of the 5637 and T24 human bladder cancer cell lines. The results demonstrated that the level of growth inhibition rate was gradually increased by treating the 5637 and T24 cells with higher doses of rosiglitazone and longer incubation time. Rosiglitazone exerted a potent inhibiting effect on migration of the 5637 and T24 cell lines. Moreover, rosiglitazone exerted a antineoplastic activity by inducing apoptosis and cell cycle arrest. Furthermore, treatment with rosiglitazone led to decrease the anti-apoptotic protein Bcl-2 level and increase the pro-apoptotic protein caspase 3 level in 5637 and T24 cells. Importantly, the protein expression of PPAR γ was significantly increased in the present of rosiglitazone in 5637 and T24 cells as compared to control group. In conclusion, the present study demonstrates that rosiglitazone has a potential antineoplastic activity in human bladder cancer cell lines, and the underlying mechanism was mediated, at least partially, through regulation of apoptosis-related protein and PPAR γ expression.

Keywords: Rosiglitazone, bladder cancer, peroxisome proliferator-activated receptor γ (PPAR γ)

Introduction

Bladder cancer is one of the most commonly diagnosed malignancy and is a major cause of morbidity and mortality in the worldwide [1]. In United States, more than 74,000 newly diag-nosed cases and 16,000 deaths are confirmed in 2015 [2]. In China, with an expected 80,500 newly diagnosed cases and 32,900 deaths are predicted in 2015 [3]. In clinical treatment, che-motherapy is still the standard of care, howev-er, patient outcomes is not effective or is poorly tolerated [4]. In fact, there have been no major advances for the treatment of bladder cancer in the last few decades [4, 5].

Rosiglitazone is known to activate peroxisome proliferator activated receptor γ (PPAR γ) and is used in the treatment of type II diabetes [6]. PPAR γ is a ligand-activated transcription factor that regulates growth and differentiation within normal and cancer cell [7]. A growing number of studies have demonstrated that rosiglitazone

has been used to suppress cancer cell growth and induce cell apoptosis, including renal can-cer cell [8], adrenocortical cancer cell [7, 9], non small cell lung cancer cell [10] and colorec-tal cancer cell [11, 12]. These results suggest that the activation of PPAR γ is a potential can-cer therapeutic method in various cancers. In contrast to that rosiglitazone promotes bladder cancer cell migration and invasion through the activation of PPAR γ [13]. Endogenous PPAR γ ligands include unsaturated fatty acids and sev-eral prostanoids [14]. Synthetic ligands com-prise the insulin-sensitizing thiazolindinedione (TZD) class (troglitazone, pioglitazone and rosi-glitazone) [12, 15]. Although extrinsic PPAR γ ligands mediating cancer cell apoptosis have been studied extensively, the detailed mecha-nisms of rosiglitazone in human bladder cancer 5637 and T24 cell apoptosis remains to be clarified.

In the present study, we investigated the effect of the PPAR γ ligand, rosiglitazone, on the prolif-

Rosiglitazone induces apoptosis in bladder cancer cells

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eration and apoptosis of the 5637 and T24 human bladder cancer cell lines, and the under-lying molecular mechanisms were involved.

Materials and methods

Cell culture

5637 and T24 cell lines were obtained from the Cell Resource Center, Shanghai Institutes for Biological Sciences (SIBS, China) and were maintained in RPMI-1640 (Invitrogen, USA) supplemented with 10% FBS (Invitrogen, USA) at 37°C in a humidified incubator (Thermo, USA), 5% CO2, 95% air atmosphere.

Cell viability detection by MTT

5637 and T24 cells proliferation were moni-tored by a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-dip- henyltetrazolium bromide (MTT) Cell Pro- liferation/Viability Assay kit (R&D SYSTEMS) in according to the guidelines. The concentration of original rosiglitazone was 10 mM, which was diluted 200, 500, 1000, 2000 and 5000 times with final concentrations of rosiglitazone of 50 μM, 20 μM, 10 μM, 5 μM and 2 μM, respe- ctively.

Transwell Migration assay

The cells were pretreated, trypsinized, and resuspended in 5% FBS medium to achieve a density of 1 × 105 cells/mL. Transwell inserts (8-mm pore size, Corning, Corning, NY) were placed in wells containing media with 10% FBS. A volume of 200 mL of cell suspension medium with 5% FBS was added to the upper chamber and incubated at 37°C with 5% carbon dioxide for indicated times. After incubation for an appropriate period, the upper side of the mem-brane was washed and wiped off using cotton swabs, and the cells on the lower membrane surface were fixed with methanol for 10 min-utes and stained with 1% toluidine blue (wt/vol, prepared in phosphate-buffered saline), for 5 minutes, and then washed with phosphate-buffered saline twice. After the dye had dried, 100 mL 10% acetic acid was added to the upper chamber and vortexed for 10 minutes, then transferred to 96-well plates and the OD570 values were measured with BioTex SynergeMX.

Wound healing assay

5637 and T24 cells were trypsinized and count-ed. 1 × 105 cells were reseeded in each well of

a new 6-well plate. With incubation overnight, the confluent cells monolayers were scratched with a 10 μL sterile pipette tip. Then the non-adherent cells were washed off with sterilized PBS and serum-free medium was added into the wells. The gap area caused by the scratch was monitored by the inverted microscope (Olympus, Japan). Three random non-overlap-ping areas in each well were pictured at 0 h, 4 h and 8 h post-scratch. Scratch width between the two linear regions was quantitated for assessing capacity of cells migration.

Flow cytometry for the detection of cell cycle progression

5637 and T24 cells were collected after diges-tion and were washed twice with PBS and cen-trifuged at 1,000 rpm for 5 min. The superna-tant was discarded, and the cells were then resuspended and fixed in ice-cold 75% ethanol and stored at 4°C. After two washes in PBS, the cells were stained with propidium iodide (PI) and subjected to flow cytometric analysis of the percentage of cells in G0/G1, S and G2/M phases (BD Biosciences, USA). Treatment with each drug concentration was conducted in trip-licate. The data were processed by Cell Quest Software (BD Biosciences, USA).

Western blotting

5637 and T24 cells were harvested and washed with cold phosphate-buffered saline and lysed in RIPA buffer (50 mM Tris-HCl, pH 7.6; 150 mM NaCl; 1% Triton-X-100, 1% sodium deoxycho-late, 0.1% sodium dodecyl sulfate) supplement-ed with complete miniprotease inhibitor cock-tail tablets. The protein concentration was esti-mated using the Bio-Rad protein assay (Bio-Rad, Marnes-la-Coquette, France). Samples containing 30 μg of protein were separated on 10% SDS-PAGE gel, transferred to nitrocellu-lose membranes (Bio-Rad Laboratories, Her- cules, CA, USA). After saturation with 5% (w/v) non-fat dry milk in TBS and 0.1% (w/v) Tween 20 (TBST), the membranes were incubated with the following antibodies: Bcl-2, caspase-3 and PPAR γ (1:1000, Santa Cruz Biotechnoogy, CA, USA). After three washes with TBST, The membranes were next incubated with the appropriate HRP (horseradish peroxidase)-con-jugated antibody visualized with chemilumines-cence (Thermo, USA).

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Statistical analysis

The data from these experi-ments were reported as mean ± standard deviation (SD) for each group. All statistical an- alyses were performed by using PRISM version 4.0 (Graph Pad). Inter-group dif-ferences were analyzed by one-way ANOVA, and followed by Tukey’s multiple compari-son test as a post test to com-pare the group means if over-all P < 0.05. Differences with P value of < 0.05 were consid-ered statistically significant.

Results

Rosiglitazone inhibits 5637 and T24 cells proliferation

We used the MTT assay to monitor the cell growth inhibi-

Figure 1. 5637 (A) and T24 (B) cells were incubated with rosiglitazone in different concentration for 24 h, 48 h and 72 h, and the cell viability was examined by MTT assay. Values were expressed as mean ± SD, n = 3 in each group. *P < 0.05 versus control group.

tion rate, and the results demonstrated that growth inhibition of 5637 (Figure 1A) and T24 (Figure 1B) cells were verified in the present of rosiglitazone. The level of inhibition rate was gradually increased by treating the 5637 (Figure 1A) and T24 (Figure 1B) cells with high-er doses of rosiglitazone and longer incubation time.

Rosiglitazone inhibits bladder cancer 5637 and T24 cells migration

We examined the potential roles of rosigli-tazone on migration capacities of bladder can-cer cell lines. After 24 h incubation, the tran-swell assay was conducted to assess cells migration capability in vitro. Compared with control, rosiglitazone exerted a potent inhibit-ing effect on migration of the 5637 (Figure 2A) and T24 (Figure 2B) cell lines under the condi-tion of higher concentration. Subsequently, the scratch closed assay was performed to mea-sure the cell migration inhibition of rosigli-tazone, and cells were reseeded and then scratches were made 24 h later. Incubation of rosiglitazone led to retarded wound closing

Figure 2. Migration activity of 5637 (A) and T24 (B) cells exposure to rosiglitazone in different concen-

tration for 24 h was measured by transwell assay. Values were expressed as mean ± SD, n = 3 in each group. *P < 0.05 versus control group.

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ld significantly increase the population in G0/G1 phase and decrease the population in S phase (Figure 5C and 5D). Furthermore, we examined rosiglitazone-induced apoptosis in human bladder cancer 5637 and T24 cell lines through an apoptotic mechanism. Triphosphate nick-end labeling (TUNEL) staining were mea-sured when 5637 and T24 cells were exposed to rosiglitazone for 24 h. As shown in Figure 6A and 6B, rosiglitazone administration showed a significant cell apoptosis in a concentration-dependent manner. The apoptosis-related markers protein expression were measured by western bltting. Treatment with rosiglitazone led to decrease the anti-apoptotic protein Bcl-2 level and increase the pro-apoptotic protein caspase 3 level in 5637 (Figure 6C) and T24 (Figure 6D) cells. Importantly, the protein expression of PPAR γ was significantly increas- ed in the present of rosiglitazone in 5637 (Figure 6E) and T24 (Figure 6F) cells as com-pared to control group.

Figure 3. Cell scratch assay was used to detect the migration of 5637 (A) and T24 (B) cells exposure to rosiglitazone in different concentration for 0 h, 4 h and 8 h.

compared with control group in T24 cell lines (Figures 3B and 4B). However, the wound closing had no obvious differ-ent between the rosiglitazo- ne-treated groups and con- trol group in 5637 cell line (Figures 3A and 4A).

Rosiglitazone regulates bladder cancer 5637 and T24 cells cycle progression and apoptosis by regulating apoptosis-related protein and PPAR γ

Next, we performed flow cy- tometry to measure cell cycle distribution. In bladder cancer 5637 cell lines, incubation with rosiglitazone resulted in a significant decrease in the G0/G1 population and a cor-responding increase in the S phase population compared with control group (Figure 5A and 5B). In T24 cell lines, rosi-glitazone administration cou-

Figure 4. The migration inhibition of scratch assay was transformed to the percentage of the initial dis-

tance between the two edges for 5637 (A) and T24 (B) cells. Values were expressed as mean ± SD, n = 3 in each group. *P < 0.05 ver-sus control group.

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Discussion

Our data suggested that rosiglitazone could inhibit human bladder cancer 5637 and T24 cells proliferation and migration and induce cell apoptosis in vitro. Simultaneously, rosiglitazone administration resulted in a significant decre- ase in anti-apoptotic protein expression and increase in pro-apoptotic protein expression. Moreover, rosiglitazone administration can induce cell cycle arrest in human bladder can-cer 5637 and T24 cell lines. These results sug-gest that rosiglitazone may be a potential ther-apeutic drug for human bladder cancer.

In this paper we demonstrated that rosigli-tazone strongly inhibited human bladder can-cer cell proliferation when the concentration of rosiglitazone was more than 10 μM. In HCT-15 human colorectal cancer cell line [11], the IC50 values at 24 and 48 h were 48.84 μmol/L and

33.33 μmol/L, respectively. Interestingly, the similar results in CaCo-2, HT29 and SW480 human colon cancer cells show that the cells viability is markedly suppressed when the con-centration of rosiglitazone is more than 10 μM [12]. These results obtained about the inhibi-tion of cell proliferation by rosiglitazone support the hypothesis that PPAR γ ligands can sup-press cancer cell proliferation.

PPAR γ, a ligand-activated intracellular tran-scription factor, belongs to the nuclear receptor superfamily, and PPAR γ agonists can induce antineoplastic signalling pathways in different cancer cell lines, animal models and clinical tri-als [16]. In HT-29 human colon cancer cells, rosiglitazone induces caveolin-1 by PPAR γ-de- pendent signaling to improve cancer cell drug resistance [17]. PPAR γ agonists, troglitazone and 15-deoxy-Delta (12, 14)-prostaglandin J2 (15d-PGJ2), show dose-dependent inhibitory

Figure 5. Representative photographs of cell cycle analysis for 5637 (A and B) and T24 (C and D) cells were exposed to rosiglitazone in different concentration for 24 h. Values were expressed as mean ± SD, n = 3 in each group. *P < 0.05 versus control group.

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effects on the proliferation of the gastric can-cer cells [18]. PPAR γ agonists have potent in vitro cytotoxicity, the possible mechanism of which is through induction of apoptosis and cell cycle arrest [19]. Increasing evidence indicates that PPAR γ agonists in combination with other drugs may be the more successful strategies for cancer therapy [11, 20]. Notably, there is evidence to show the potential linkage between the development of bladder cancer and long-term, high doses of oral pioglitazone for the treatment of type 2 diabetes [21]. The combi-nation of 15d-PGJ2 and survivin inhibition play a potentially role in the therapeutical manipula-tion of bladder cancer by inducing the produc-tion of reactive oxygen species (ROS) [22]. Moreover, PPARγ agonist DIM-C can be an excellent alternative to bladder tumors resis-tant to EGFR inhibition, which constitutes ratio-nal for combination therapy with EGFR inhibitor in bladder cancer [23].

Previous studies indicate that PPAR γ is widely expressed in bladder cancer cell lines and

and T24 cell lines through inhibition of proli- feration, induction of apoptosis and cell cycle arrest.

Acknowledgements

Our research was supported by the Science and Technology Development Fund of Macao (Grant No. 064/2012/A).

Disclosure of conflict of interest

None.

Address correspondence to: Bin Lang, School of Health Sciences, Macao Polytechnic Institute, Macao, China. Tel: 853-66708612; E-mail: blang@ipm.edu.mo

References

[1] Wang C, Ge Q, Zhang Q, Chen Z, Hu J, Li F and Ye Z. Targeted p53 activation by saRNA sup-presses human bladder cancer cells growth

Figure 6. 5637 (A) and T24 (B) cells exposure to rosiglitazone in different concentration for 24 h, triphosphate nick-end labeling (TUNEL) staining was measured by flow cytometry. The protein expression of Bcl-2 and caspase 3 was measured by western blotting in 5637 (C) and T24 (D) cells. The protein expression of PPAR γ was measured by western blotting in 5637 (E) and T24 (F) cells. Values were expressed as mean ± SD, n = 3 in each group. *P < 0.05 versus control group.

tumor tissues, and its expres-sion has been correlated with tumor grade and stage [24-26]. Bladder cancer cells with different PPAR γ expression have different capacity of migration and invasion, and there was a positive correla-tion between the increase in PPAR γ expression and migra-tion and invasion capacity [13]. These results demon-strate that high expression of PPAR γ and PPAR γ agonist play an important role in blad-der cancer cell migration and invasion [13]. In contrast, our study indicated that rosigli-tazone could induce apopto-sis and inhibit migration in human bladder cancer with a time- and dose-dependent manner. The PPAR γ agonist troglitazone induces autopha-gy, apoptosis and necroptosis in bladder cancer cells [27].

In conclusion, the present study demonstrates that rosi-glitazone is able to exert a antineoplastic activity in hu- man bladder cancer 5637

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10203 Int J Clin Exp Pathol 2017;10(10):10197-10204

and metastasis. J Exp Clin Cancer Res 2016; 35: 53.

[2] Siegel RL, Miller KD and Jemal A. Cancer sta-tistics, 2015. CA Cancer J Clin 2015; 65: 5-29.

[3] Chen W, Zheng R, Baade PD, Zhang S, Zeng H, Bray F, Jemal A, Yu XQ and He J. Cancer statis-tics in China, 2015. CA Cancer J Clin 2016; 66: 115-132.

[4] Powles T, Eder JP, Fine GD, Braiteh FS, Loriot Y, Cruz C, Bellmunt J, Burris HA, Petrylak DP, Teng SL, Shen X, Boyd Z, Hegde PS, Chen DS and Vogelzang NJ. MPDL3280A (anti-PD-L1) treat-ment leads to clinical activity in metastatic bladder cancer. Nature 2014; 515: 558-562.

[5] Li J, Zhuang C, Liu Y, Chen M, Zhou Q, Chen Z, He A, Zhao G, Guo Y, Wu H, Cai Z and Huang W. shRNA targeting long non-coding RNA CCAT2 controlled by tetracycline-inducible system in-hibits progression of bladder cancer cells. Oncotarget 2016; [Epub ahead of print].

[6] Gerstein HC, Yusuf S, Bosch J, Pogue J, Sheridan P, Dinccag N, Hanefeld M, Hoogwerf B, Laakso M, Mohan V, Shaw J, Zinman B and Holman RR. Effect of rosiglitazone on the fre-quency of diabetes in patients with impaired glucose tolerance or impaired fasting glucose: a randomised controlled trial. Lancet 2006; 368: 1096-1105.

[7] Cerquetti L, Sampaoli C, Amendola D, Bucci B, Masuelli L, Marchese R, Misiti S, De Venanzi A, Poggi M, Toscano V and Stigliano A. Rosigli- tazone induces autophagy in H295R and cell cycle deregulation in SW13 adrenocortical cancer cells. Exp Cell Res 2011; 317: 1397-1410.

[8] Taub M. Cancer drug troglitazone stimulates the growth and response of renal cells to hy-poxia inducible factors. Biochem Biophys Res Commun 2016; 471: 342-347.

[9] Cantini G, Lombardi A, Piscitelli E, Poli G, Ceni E, Marchiani S, Ercolino T, Galli A, Serio M, Mannelli M and Luconi M. Rosiglitazone inhib-its adrenocortical cancer cell proliferation by interfering with the IGF-IR intracellular signal-ing. PPAR Res 2008; 2008: 904041.

[10] Hazra S, Batra RK, Tai HH, Sharma S, Cui X and Dubinett SM. Pioglitazone and rosiglitazone decrease prostaglandin E2 in non-small-cell lung cancer cells by up-regulating 15-hydroxy-prostaglandin dehydrogenase. Mol Pharmacol 2007; 71: 1715-1720.

[11] Miao R, Xu T, Liu L, Wang M, Jiang Y, Li J and Guo R. Rosiglitazone and retinoic acid inhibit proliferation and induce apoptosis in the HCT-15 human colorectal cancer cell line. Exp Ther Med 2011; 2: 413-417.

[12] Cerbone A, Toaldo C, Minelli R, Ciamporcero E, Pizzimenti S, Pettazzoni P, Roma G, Dianzani MU, Ullio C, Ferretti C, Dianzani C and Barrera

G. Rosiglitazone and AS601245 decrease cell adhesion and migration through modulation of specific gene expression in human colon can-cer cells. PLoS One 2012; 7: e40149.

[13] Yang DR, Lin SJ, Ding XF, Miyamoto H, Messing E, Li LQ, Wang N and Chang C. Higher expres-sion of peroxisome proliferator-activated re-ceptor gamma or its activation by agonist thia-zolidinedione-rosiglitazone promotes bladder cancer cell migration and invasion. Urology 2013; 81: 1109, e1101-1106.

[14] Forman BM, Chen J and Evans RM. Hypoli- pidemic drugs, polyunsaturated fatty acids, and eicosanoids are ligands for peroxisome proliferator-activated receptors alpha and del-ta. Proc Natl Acad Sci U S A 1997; 94: 4312-4317.

[15] Lehmann JM, Moore LB, Smith-Oliver TA, Wilkison WO, Willson TM and Kliewer SA. An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated re-ceptor gamma (PPAR gamma). J Biol Chem 1995; 270: 12953-12956.

[16] Grommes C, Landreth GE and Heneka MT. Antineoplastic effects of peroxisome prolifera-tor-activated receptor gamma agonists. Lancet Oncol 2004; 5: 419-429.

[17] Tencer L, Burgermeister E, Ebert MP and Liscovitch M. Rosiglitazone induces caveolin-1 by PPARgamma-dependent and PPRE-in- dependent mechanisms: the role of EGF re-ceptor signaling and its effect on cancer cell drug resistance. Anticancer Res 2008; 28: 895-906.

[18] Sato H, Ishihara S, Kawashima K, Moriyama N, Suetsugu H, Kazumori H, Okuyama T, Rumi MA, Fukuda R, Nagasue N and Kinoshita Y. Expression of peroxisome proliferator-activat-ed receptor (PPAR) gamma in gastric cancer and inhibitory effects of PPAR gamma ago-nists. Br J Cancer 2000; 83: 1394-1400.

[19] Xiong X, Ye Y, Fu L, Dai B, Liu J, Jia J, Tang J, Li L, Wang L, Shen J and Mei C. Antitumor activity of a novel series of alpha-aryloxy-alpha-methyl-hydrocinnamic acid derivatives as PPAR gam-ma agonists against a panel of human cancer cell lines. Invest New Drugs 2009; 27: 223-232.

[20] Dicitore A, Caraglia M, Colao A, Zappavigna S, Mari D, Hofland LJ, Persani L and Vitale G. Combined treatment with PPAR-gamma ago-nists in pancreatic cancer: a glimmer of hope for cancer therapy? Curr Cancer Drug Targets 2013; 13: 460-471.

[21] Hillaire-Buys D, Faillie JL and Montastruc JL. Pioglitazone and bladder cancer. Lancet 2011; 378: 1543-1544; author reply 1544-1545.

[22] Wang Y, Tan H, Xu D, Ma A, Zhang L, Sun J, Yang Z, Liu Y and Shi G. The combinatory ef-

Rosiglitazone induces apoptosis in bladder cancer cells

10204 Int J Clin Exp Pathol 2017;10(10):10197-10204

fects of PPAR-gamma agonist and survivin in-hibition on the cancer stem-like phenotype and cell proliferation in bladder cancer cells. Int J Mol Med 2014; 34: 262-268.

[23] Mansure JJ, Nassim R, Chevalier S, Szymanski K, Rocha J, Aldousari S and Kassouf W. A novel mechanism of PPAR gamma induction via EGFR signalling constitutes rational for combi-nation therapy in bladder cancer. PLoS One 2013; 8: e55997.

[24] Yoshimura R, Matsuyama M, Segawa Y, Hase T, Mitsuhashi M, Tsuchida K, Wada S, Kawahito Y, Sano H and Nakatani T. Expression of peroxi-some proliferator-activated receptors (PPARs) in human urinary bladder carcinoma and growth inhibition by its agonists. Int J Cancer 2003; 104: 597-602.

[25] Choi W, Czerniak B, Ochoa A, Su X, Siefker-Radtke A, Dinney C and McConkey DJ. Intrinsic basal and luminal subtypes of muscle-invasive bladder cancer. Nat Rev Urol 2014; 11: 400-410.

[26] Sandes EO, Lodillinsky C, Langle Y, Belgorosky D, Marino L, Gimenez L, Casabe AR and Eijan AM. Inducible nitric oxide synthase and PPAR gamma are involved in bladder cancer pro-gression. J Urol 2012; 188: 967-973.

[27] Yan S, Yang X, Chen T, Xi Z and Jiang X. The PPARgamma agonist Troglitazone induces au-tophagy, apoptosis and necroptosis in bladder cancer cells. Cancer Gene Ther 2014; 21: 188-193.