Chemoprevention of Urothelial Cell Carcinoma Growth and Invasion by the Dual COX–LOX Inhibitor Licofelone in UPII-SV40T Transgenic Mice

Published OnlineFirst May 2, 2014; DOI: 10.1158/1940-6207.CAPR-14-0087

Cancer Prevention Research Article Research

Venkateshwar Madka1, Altaf Mohammed1, Qian Li1, Yuting Zhang1, Jagan M.R. Patlolla1, Laura Biddick1, Stan Lightfoot1, Xue-Ru Wu2, Vernon Steele3, Levy Kopelovich3, and Chinthalapally V. Rao1

Abstract Epidemiologic and clinical data suggest that use of anti-inflammatory agents is associated with reduced risk for bladder cancer. We determined the chemopreventive efficacy of licofelone, a dual COX–lipoxygenase (LOX) inhibitor, in a transgenic UPII-SV40T mouse model of urothelial transitional cell carcinoma (TCC). After genotyping, six-week-old UPII-SV40T mice (n ¼ 30/group) were fed control (AIN-76A) or experimental diets containing 150 or 300 ppm licofelone for 34 weeks. At 40 weeks of age, all mice were euthanized, and urinary bladders were collected to determine urothelial tumor weights and to evaluate histopathology. Results showed that bladders of the transgenic mice fed control diet weighed 3 to 5-fold more than did those of the wild-type mice due to urothelial tumor growth. However, treatment of transgenic mice with licofelone led to a significant, dose-dependent inhibition of the urothelial tumor growth (by 68.6%–80.2%, P < 0.0001 in males; by 36.9%–55.3%, P < 0.0001 in females) compared with the control group. The licofelone diet led to the development of significantly fewer invasive tumors in these transgenic mice. Urothelial tumor progression to invasive TCC was inhibited in both male (up to 50%; P < 0.01) and female mice (41%–44%; P < 0.003). Urothelial tumors of the licofelone-fed mice showed an increase in apoptosis (p53, p21, Bax, and caspase3) with a decrease in proliferation, inflammation, and angiogenesis markers (proliferating cell nuclear antigen, COX-2, 5-LOX, prostaglandin E synthase 1, FLAP, and VEGF). These results suggest that licofelone can serve as potential chemopreventive for bladder TCC. Cancer Prev Res; 7(7); 708–16. 2014 AACR.

Introduction pose a very high risk for distant metastases (2). Chemopre- Urinary bladder cancer is one of the common cancers vention has been applied to many different tumor types, worldwide, with the highest incidence rates in industrial- including TCC of the bladder, and has been proposed as a ized countries. In United States, it is the second most management strategy for patients with cancer at risk of frequently diagnosed genitourinary cancer, with an estimat- developing second primary tumors (3). Inflammation is ed 563,640 people currently living with bladder cancer. In a known risk factor for many cancers, particularly epithelial 2014, about 74,690 new cases of bladder cancer are antic- cancers such as those of the urothelium. It plays important ipated (about 56,390 in men and 18,300 in women), and roles at different stages of tumor development, including approximately 15,580 are expected to die of this cancer (1). initiation, promotion, invasion, and metastasis (4). Chron- The majority (90%–95%) of bladder cancers are transition- ic inflammation is associated directly with muscle-invasive al cell carcinomas (TCC). Of these, 70% to 75% are non- bladder cancer, particularly that due to urinary tract infec- invasive, low-grade, papillary TCC, whereas up to 30% of all tion in invasive squamous cell carcinoma (5). diagnosed tumors are classified as invasive TCCs, which The metabolism of arachidonic acid by COX-2 and 5- lipoxygenase (5-LOX) generates prostaglandins and hydro- xyeicosatetraenoic acids, respectively, that mediate a wide Authors' Affiliations: 1Center for Cancer Prevention and Drug Develop- variety of physiologic processes, including cell prolifera- ment, Hem-Onc Section, Department of Medicine, PCS Oklahoma Cancer Center, University of Oklahoma Health Sciences Center, Oklahoma City, tion, immune surveillance, cell differentiation, and inflam- Oklahoma; 2Department of Urology, NYU Medical Center, New York, New mation. Clinically, aberrant expression of the COX-2 and 5- 3 York; and Division of Cancer Prevention, Chemoprevention Agent Devel- LOX enzymes has been observed in urothelial tumors and opment Research Group, National Cancer Institute, Bethesda, Maryland found to be associated with poor prognosis (6, 7). Con- Corresponding Author: Chinthalapally V. Rao, University of Oklahoma Health Sciences Center, 975 NE 10th St., BRC 1203, Oklahoma City, OK centrations of a urinary PGE metabolite (PGE-M) and 73104. Phone: 405-271-3224; Fax: 1-405-271-3225; E-mail: leukotriene E4 (LTE4), biomarkers of the COX-2 and 5-LOX [email protected] pathways, are elevated in smokers (8). In view of the doi: 10.1158/1940-6207.CAPR-14-0087 recognized link between smoking, inflammation, and blad- 2014 American Association for Cancer Research. der cancer, targeting the inflammatory pathways seems to

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be an ideal approach to reduce the risk of urothelial cancer prep Kit (Invitrogen) and PCR. PCR for the SV40T gene development. Several preclinical studies have suggested that wasdoneusingtheprimer50-CTTTGGAGGCTTCTGG- the combination of a selective COX-2 inhibitor and a 5-LOX GATGCAACT-30 (sense) and 50-GCATGACTCAAAAAACT- inhibitor is more effective than either agent alone in pre- TAGCAATTCTG-30 (antisense) and amplifying under the venting cancer cell growth or tumor formation (9, 10). Dual following PCR conditions: denaturation at 95Cfor5 inhibitors that block both COX and 5-Lox may provide minutes, followed by 35 cycles at 95Cfor1minute,58C synergistic anti-inflammatory effects and also decrease gas- for 45 seconds, and 72Cfor45seconds.ThePCR trointestinal side effects associated with NSAIDs(11) and products, when separated on a 2% agarose gel, showed select COX-2 inhibitors. a 550-bp band. Licofelone is one such compound that is currently in phase III clinical trials for treatment of pain and inflam- Bioassay mation associated with osteoarthritis (12, 13) and also Genotyped UPII-SV40T transgenic mice were used in has been shown to prevent colon (14, 15), lung (16), and the efficacy study. The experimental protocol is summa- prostate cancers (17). However, no studies have been rized in Fig. 1A. Five-week-old mice were selected and reported on the chemopreventive effects of licofelone in randomized so that the average body weights in each urothelial cancer. Among the preclinical models for group were equal (n ¼ 30 UPII-SV40T mice per group and urothelial cancer, UPII-SV40T transgenic mice develop n ¼ 12 wild-type mice per group) and were fed a modified invasive urothelial tumors due to the expression of Sim- AIN-76A diet for 1 week. At 6 weeks of age, mice were fed ian virus large T antigen (Tag) driven by the urothelium- control or experimental diets containing 0, 150, or 300 specific uroplakin II (UPII) promoter (18). These mice ppm licofelone (Fig. 1B) until termination of the study. have been used as a model to elucidate the mechanism Mice were checked routinely for signs of weight loss, underlying urothelial tumorigenesis (19, 20) as well as in toxicity, or any abnormalities. Previously, we have estab- chemoprevention studies (21, 22). In the present study, lished maximal tolerated doses/optimal dose of licofe- we determined the chemopreventive efficacy of licofelone lone in mice and found to be >500 ppm fed in the using the UPII-SV40T transgenic mouse model for inva- modified AIN-76A diet (15). The food intake and body sive urothelial TCC. weight of each animal were measured once weekly for the first 6 weeks and then once a month until termination. Materials and Methods After 34 weeks on experimental diets (i.e., 40 weeks age), Animals, diet, and care all mice were euthanized by CO2 asphyxiation and All animal experiments were conducted in accordance necropsied, and urinary bladders were collected and with the guidelines of the Institutional Animal Care and weighed to determine the tumor weight. A portion of Use Committee. Transgenic mice (UPII-SV40T) expres- the urinary bladder was fixed in 10% neutral-buffered sing a Simian Virus 40 large T antigen (SV40T) specifically formalin for histopathologic evaluation, and the rest was in urothelial cells under the control of the Uroplakin II snap frozen in liquid nitrogen for further analysis. (UPII) promoter and reproducibly developing high-grade carcinoma in situ (CIS) and invasive tumors throughout Tissue processing and histologic analysis the urothelium (18) were used. The required numbers of Formalin-fixed, paraffin-embedded tissues were sec- m UPII-SV40T transgenic mice were generated by breeding tioned (4- m) and stained with hematoxylin and eosin as described earlier (22). Animals were housed in venti- (H&E). Sections of each urothelial tumor were evaluated lated cages under standardized conditions (21C, 60% histologically by a pathologist blinded to the experimental humidity, 12-hour light/12-hour dark cycle, 20 air changes groups. Carcinomas were classified into noninvasive CIS, per hour) in the University of Oklahoma Health Sciences invasive carcinomas (lamina propria invasive and muscu- Center rodent barrier facility. Semipurified modified AIN- laris propria invasive) types according to histopathologic 76A diet ingredients were purchased from Bioserv, Inc. criteria as previously described (22). Licofelone was procured from the NCI chemoprevention drug repository. Licofelone (150 and 300 ppm) was RT-PCR premixed with small quantities of casein and then blend- Total RNA from urothelial tumor samples of male mice ed into the diet using a Hobart mixer. Both control and was extracted using the Totally RNA Kit as per the man- experimental diets were prepared weekly and stored in a ufacturer’s instructions. Equal quantities of DNA-free cold room. Mice were allowed ad libitum access to the RNA were used in reverse transcription reactions for respective diets and to automated tap water purified by making cDNA using SuperScript reverse transcriptase reverse osmosis. (Invitrogen). RT-PCR reactions were done for proliferating cell nuclear antigen (PCNA), p53, Bax, caspase 3, Breeding and genotyping Prostaglandin E Synthase 1 (mPGES1), FLAP, VEGF, p16, All mice were bred and genotyped as described earlier and actin using SYBR green and specific primers (Table 1). DD (22). In brief, male UPII-SV40T mice were crossed with Relative gene expression was calculated using the 2 CT wild-type females to generate offspring. Transgenic pups formula (23). All experiments were performed using were confirmed by tail DNA extraction using the mini- replicated tumor samples and at least in triplicate.

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A 0 week 6 weeks 40 weeks B HO 34 weeks O

N CH Control diet/experimental diet CI 3 CH N = 30; 15male + 15female 3 Weaning Necropsy Modified AIN76A with genotyping histopathology Licofelone 0, 150, 300 ppm randomization molecular analysis

C Normal Tumor D Wild type UPII-SV40T

SV40T

PCNA

COX-2

5-LOX

VEGF

ACTIN

Figure 1. A, experimental design for study of the chemopreventive efﬁcacy of licofelone in UPII-SV40T transgenic mice. At 6 weeks of age, groups (30/group UPII-SV40T or 12/group WT) of mice were fed experimental diets containing 0, 150, or 300 ppm licofelone continuously for 34 weeks, and bladders from each mouse were evaluated histopathologically and analyzed for expression of various markers as described in the text. B, structure of licofelone, a dual COX–LOX inhibitor. C, H&E staining showing representative normal urothelium in the wild-type mice and invasive high-grade transitional cell carcinoma in UPII-SV40T transgenic mice. Inset, representative urinary bladders from wild-type and transgenic mice. D, expression of SV40T, PCNA, COX-2, 5-LOX, VEGF, and b-actin as analyzed by RT-PCR.

Immunohistochemistry blocked with 5% nonfat milk (Biorad) in TBS and incubated Modulatory effects of licofelone on the expression of with antibodies for PCNA, p53 cyclin E, and caspase 3 COX-2, 5-LOX, and p21 were evaluated by immunohis- overnight at 4C. Subsequently, membranes were washed tochemistry as described previously (22). Briefly, sections of and incubated with secondary antibody (either goat anti- paraffin-embedded tissues were deparaffinized in xylene, mouse or goat anti-rabbit conjugated with horseradish rehydrated through graded ethanol solutions, and washed peroxidase; 1:10,000 dilution) for 1 hour. Protein was in PBS. Antigen retrieval was carried out by heating the detected on BioMax MR film (Kodak) using chemilumines- sections in 0.01 mol/L citrate buffer (pH 6.0) for 30 minutes cence (Super Signal; Pierce Biotechnology). Equal protein in a boiling water bath. Endogenous peroxidase activity was loading was confirmed by detection of tubulin. Selected quenched by incubation in 3% H2O2 in PBS for 5 minutes. blots were quantified using Image J software. Nonspecific binding sites were blocked using protein block for 20 minutes. Then, sections were incubated overnight at Statistical analysis 4 C with 1:300 dilutions of mAbs against COX-2, 5-LOX, The data are presented as mean SE. Differences in body and p21 (Santa Cruz Biotechnology). After several washes weights were analyzed by ANOVA. Statistical differences with PBS, they were incubated with appropriate secondary between urothelial tumor weights in the control and treated t antibodies for 2 hours, then exposed to avidin–biotin groups were evaluated using unpaired test with Welch complex reagent (Invitrogen). After rinsing with PBS, the correction. Tumor incidences (percentage of mice with slides were incubated with the chromogen 3,30-diamino- urothelial tumors) were analyzed by the Fisher exact test. benzidine for 3 minutes, then counter stained with hema- Differences between control and treatment groups were P < toxylin. Nonimmune rabbit immunoglobulins were substi- considered significant at 0.05. All statistical analysis tuted for primary antibodies as negative controls. Speci- was performed using Graphpad Prism 5.0 Software. mens were observed using an Olympus microscope IX71, Results and digital computer images were recorded with an Olym- pus DP70 camera. General observations All of the transgenic and wild-type mice fed control and Western blotting licofelone-containing modified AIN76A diets were weighed Proteins (60 mg) in lysates from bladders of control and weekly and monitored throughout the study. Gross anato- licofelone-treated mice were separated by SDS-PAGE and my of wild-type and transgenic mice revealed no evidence of transferred to a nitrocellulose membrane. Membranes were any abnormality in organ size, or changes in appearance of

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Table 1. List of primers used for RT-PCR analysis

Gene Forward primer Reverse primer PCNA 50-TAAAGAAGAGGAGGCGGTAA-30 50-TAAGTGTCCCATGTCAGCAA-30 p53 50-TGAAACGCCGACCTATCCTTA-30 50-GGCACAAACACGAACCTCAAA-30 Bax 50-CAGGATGCGTCCACCAAGAA-30 50-GCAAAGTAGAAGAGGGCAACCA-30 p16 50-AGTCCGCTGCAGACAGACTG-30 50- CGGGAGAAGGTAGTGGGGTC-30 Caspase 3 50-AGCAGCTTTGTGTGTGTGATTCTAA-30 50-AGTTTCGGCTTTCCAGTCAGAC-30 Cox-2 50-GCATTCTTTGCCCAGCACTT-30 50-AGACCAGGCACCGACCAAAGA-30 5-LOX 50-GGACCTCAGCATGTGGTATG-30 50-GCTGGGTCAGGGGTACTTTA-30 mPGES1 50-GGAACGACATGGAGACCATCTAC-30 50-TCCAGGCGACAAAAGGGTTA-30 FLAP 50-GCCGGACTGATGTACCTGTT-30 50-GGTGAGCGTCCTTCTCTGTC-30 VEGF 50-GGAGATCCTTCGAGGAGCACTT-30 50-GGCGATTTAGCAGCAGATATAAGAA-30 SV40T 50-GCAGCTAATGGACCTTCTAGG-30 50-GCAATTCTGAAGGAAGGTCCT-30 Actin 50-AGATCTGGCACCACACCTTC-30 50-GGGGTGTTGAAGGTCTCAAA-30

liver, spleen, heart, lung, seminal vesicles, testis, ovaries, or TCC to high-grade invasive TCC. To determine the effect of prostate. Thus, doses applied in the efficacy studies were licofelone on this progression, formalin-fixed bladders expected to be nontoxic. from the control and licofeone-fed transgenic mice were sectioned and stained with H&E and their histopathology Urothelial tumor growth is inhibited by licofelone in was compared. Bladders from the transgenic mice fed transgenic mice control diet had high-grade, muscle-invasive TCC at 40 UPII-SV40T mice spontaneously develop urothelial weeks of age. However, licofelone-fed mice showed signif- tumors, as result of which there is a significant increase icant inhibition of invasive tumors in both male and female in urinary bladder weights compared with wild type. At 40 groups. As a result of licofelone administration, 12% to 50% weeks age, these tumors are histopathologically classified (P < 0.01) of the male mice had tumors that were classified as high-grade tumors invading both lamina propria and as high-grade, noninvasive CIS (Fig. 2E). High-dose licofe- muscularis while wild-type bladders show normal urothe- lone was found to have better effect on tumor invasion lium (Fig. 1C). These tumors showed an overexpression of compared with the lower dose. In the female group, both the PCNA, COX-2, 5-LOX, and VEGF compared with that doses led to prevention of tumor invasion in 41% to 44% of of the normal urothelium from wild-type mice (Fig. 1D). the mice (P < 0.003; Fig. 2F). At the end of the experiment, no significant differences in body weights were observed (Fig. 2A and B). A chemo- Antiproliferative and antiangiogenic effects of preventive effect of dietary licofelone administered at 150 licofelone or 300 ppm was found on urothelial tumor growth. Male Urothelial tumors of the transgenic mice were found to and female UPII-SV40T mice fed control diet had urothe- have significant overexpression of the proliferation and lial tumors that weighed an average of 112.9 9.8 mg and angiogenesis markers PCNA and VEGF (Fig. 1D) compared 19.3 0.8 mg, respectively (Fig. 2C and D). Dietary with those in the normal urothelium of bladders from wild- licofelone at 150 and 300 ppm administered for 34 weeks type mice. To evaluate the antiproliferative and antiangio- significantly inhibited the tumor growth in a dose-depen- genic effects of licofelone on protein and mRNA levels of dant manner that led to reduced urothelial tumor weight these markers, urothelial tumors from the control and in both sexes. Tumors of licofelone-fed male mice weighed licofelone-fed animals were subjected to RT-PCR and West- 65.2% and 82.7% less at the low and high doses, respec- ern blotting. Tumors from mice fed the licofelone diet tively (39.3 9.2 mg; P < 0.0001 and 19.5 8.9 mg; P < showed a significantly decreased expression of PCNA (Fig. 0.0001) compared with tumors of control mice due to 3A and B) and VEGF (Fig. 3A) compared with those in the significant inhibition of tumor growth (Fig. 2C). A similar control group. The tumor suppressor p16 (Fig. 3A) was effect of licofelone was observed in female mice; tumors induced in the licofelone group while Cyclin E protein (Fig. from the transgenic mice fed the experimental diet weighed 3B) was significantly inhibited. 35% to 49% less at both doses, respectively, (12.6 0.8 mg; P < 0.0001 and 6.8 1.1 mg; P < 0.0001) than those of Licofelone induces proapoptotic molecules in the control group (Fig. 2D). urothelium Molecular effects of licofelone were determined by Licofelone prevents progression of urothelial tumor to analyzing expression of mRNA and protein for various invasive TCC proapoptotic/antitumor molecules. RT-PCR analysis of Urothelial tumors in UPII-SV40T mice progress gradually the mRNA showed a significant increase in the levels of from normal-looking epithelium to low-grade noninvasive the tumor suppressor protein p53 in tumors from

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A C E 40 125 100

100 30 80

SEM) 75 P ± 60 < 0.012 20 P < 0.0001 50 40 P < 0.0001 10 (mg; mean 25 20 Urothelial tumor weight % of Mice with invasive TCC Body weight (g; mean ± SEM) 0 0 0 0 150 300 0 150 300 0 150 300 Conc. of licofelone in diet (ppm) Conc. of licofelone in diet (ppm) Conc. of licofelone in diet (ppm) B D F 30 100 20

80 P 15 P < 0.0001 < 0.005 20 P < 0.003 P < 0.0001 60 10 40 10 5

(mg; mean ± SEM) 20 Urothelial tumor weight % of Mice with invasive TCC Body weight (g; mean ± SEM) 0 0 0 0 150 300 0 150 300 0 150 300 Conc. of licofelone in diet (ppm) Conc. of licofelone in diet (ppm) Conc. of licofelone in diet (ppm)

Figure 2. A and B, body weights of the male and female transgenic mice fed control or experimental diets at 40 weeks of age. C and D, effect of dietary licofelone on urothelial tumor weights of male and female transgenic mice, respectively. The weight of tumors from licofelone-fed male mice was signiﬁcantly lower (65.2% and 82.7% in low- and high-dose groups, respectively) compared with control mice (P < 0.0001). Similarly, in the female mice, 35% to 49% (P < 0.0001) inhibition was observed. E and F, effect of dietary licofelone on incidence of invasive TCC in male and female transgenic mice, respectively. Licofelone treatment led to a dose-dependent, signiﬁcant reduction of tumor weight and tumor invasion in both groups.

licofelone-fed mice (Fig. 3A). Similarly, p53 protein was Discussion induced by licofelone in the treated group as determined Clinically, 75% to 85% of patients with bladder cancer by immunoblotting (Fig. 3B). The proapoptotic mole- present with nonmuscle-invasive bladder cancer (NMIBC) cules Bax (Fig. 3A) and caspase 3 (Fig. 3A and B) were also that is confined to the mucosa (stage Ta, CIS) or submucosa induced by the licofelone treatment, suggesting that lico- (stage T1). These patients can be identified based on haema- felone may lead to inhibition of tumor growth by induc- turia, the most common finding in NMIBC and cytology of ing apoptosis. Licofelone treatment also led to an increase urine. If untreated, approximately 54% of patients with CIS in p21 expression (Fig. 4A). progress to muscle-invasive disease; even in treated patients, these tumors often recur and some progress to invasive or Licofelone inhibits expression of proinflammatory metastatic carcinoma, for which the treatment options COX-2 and 5-LOX molecules become limited. Hence, there is a need to develop targeted The effect of licofelone on expression of the proinflam- regimens that will prevent disease progression, improve matory molecules COX-2, 5-LOX, mPGES-1, and FLAP was response to treatment, and also prevent recurrence. Inflam- determined. Urothelial tumors from the transgenic mice fed mation of the bladder due to infectious as well as noninfec- control diet showed a significant overexpression of COX-2 tious etiologies such as medication, radiation, foreign bodies, and 5-LOX (Figs. 1D and 4A) compared with normal urothe- chemicals, and autoimmune responses affects the bladder lium. Licofelone had an inhibitory effect on both of these function directly and also is a potential risk factor for urothe- proinflammatory molecules (Fig. 4A). Similarly, there was lial cancer development. Inflammatory pathways thus have significant downregulation of mPGES1 (Fig. 4B) and FLAP been considered important targets for bladder cancer preven- (Fig. 4B) that can contribute to proinflammatory effects. tion as well as to increase the response to chemotherapy (24).

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A B Control Lic 150 Lic 300

PCNA 1.5 1.5 2.5 ** p53

2.0 Caspase 3 1.0 1.0 Cyclin E 1.5 Tubulin * 1.0 0.5 0.5 ** 1.5 2.5 * * 0.5 * 2.0

Relative mRNA expression (PCNA) 0.0 Relative mRNA expression (VEGF) 0.0 Relative mRNA expression (p16) 0.0 1.0 1.5 Con Con Con Lic 150 Lic 300 Lic 150 Lic 300 Lic 150 Lic 300 1.0 0.5 ** 0.5

8 6 8 0.0 Relative protein expression (p53) 0.0 * *** Relative protein expression (PCNA) *** Con Con Lic 150 Lic 300 Lic 150 Lic 300 6 6 * 4 * 6 1.5 ** *** 4 4 4 1.0 2 2 2

2 0.5 Relative mRNA expression (p53) 0 Relative mRNA expression (Bax) 0 0 ****** Relative mRNA expression (Caspase 3) Con Con Con 0 0.0 Lic 150 Lic 300 Lic 150 Lic 300 Lic 150 Lic 300 Relative protein expression (Cyclin E) Relative protein expression (Caspase 3) Con Con Lic 150 Lic 300 Lic 150 Lic 300 *, P < 0.05; **, P < 0.005; ***, P < 0.0005 *, P < 0.05; **, P < 0.005; ***, P < 0.0005

Figure 3. A, effect of licofelone on expression of PCNA, VEGF, p16, p53, Bax, and caspase 3 markers using replicate tumor samples from each group. RT-PCR analysis was done on cDNA synthesized from mRNA of urothelial tumors of mice fed control and licofelone-containing diets as described in Materials and Methods. Licofelone treatment led to an increase in p53, Bax, caspase 3, and p16, and a decrease in PCNA, VEGF mRNAs. B, effect of licofelone on PCNA, p53, caspase 3, and Cyclin E proteins was determined by Western immunoblotting and quantiﬁed using image J software as described in Materials and Methods. Expression of PCNA and Cyclin E was found to be inhibited, whereas the expression of p53 and caspase 3 proteins was found to be induced.

Prominent expression of COX-2 and 5-LOX enzymes has manner (28). Similarly, targeting 5-LOX using the specific been described in bladder cancers, and expression of these inhibitor AA861 caused a dose-dependent inhibition of proteins correlates with tumor grade and invasion (6, 7, 25– bladder cancer cell growth (25). While targeting either of 27). Overexpression of COX-2 and 5-LOX enzyme meta- these enzymes has shown inhibitory effects, it has been bolites has been reported in smokers, who represent a high- found that COX-2 inhibition includes shunting of arachi- risk population for bladder cancer (8). We found the donic acid into the 5-LOX pathway leading to increased risk urothelial tumors of the UPII-SV40T transgenic mice to for cardiovascular disease (8). The striking interrelationship have elevated levels of these enzymes (COX-2, 5-LOX) of COX-2 and 5-LOX biologic functions suggests that drugs compared with normal tissue (Figs. 1D and 4A). Hence, that are able to block both COX-2 and 5-LOX pathways may this transgenic mouse was an appropriate model to test the provide a better approach to bladder cancer prevention. effect of anti-inflammatory agents targeting these enzymes. Licofelone is a promising novel dual COX–LOX inhibitor COX-2 inhibitors such as nimesulide (28) and celecoxib with proven chemopreventive efficacy in several cancer (29), also traditional NSAIDs such as indomethacin (30) models both in vitro and in vivo. Here, we have shown that and ibuprofen (31), have been demonstrated to prevent licofelone has a significant inhibitory effect on urothelial bladder cancer in in vitro and in vivo models. Sabichi and tumor growth and invasion in both male and female mice in colleagues (32) did not observe a clinical benefit of cele- a dose-dependant manner. Licofelone was able to signifi- coxib in preventing recurrence of NMIBC in a randomized cantly prevent tumor invasion in the female mice even at a controlled trial, although, celecoxib had a marginally sig- lower dose compared with that male. This is particularly nificant effect on reducing metachronous recurrences com- interesting in view of the differences in the risk for urothelial pared with placebo. However, nimesulide was capable of cancer incidence between these two sexes. Also, hormonal preventing bladder tumor recurrences in a dose-dependent factors are known to play an important role in bladder

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AB1.5

Normal Control tumor Treated tumor 1.0 * Figure 4. A, effect of licofelone on p21, COX-2, and 5-LOX 0.5 *** expression in urothelial tumors. Immunohistochemical analysis ﬁ 0.0 was performed with paraf n-

Relative expression mRNA (mPGES1) embedded and microsectioned on C c 300 bladder tissues as described in Lic 150 Li 1.5 Materials and Methods. A signiﬁcant decrease in COX-2 and 5-LOX expression with an 1.0 increased expression of p21 was seen in the treatment groups. B, ** effect of licofelone on 0.5 expression of mPGES1 and FLAP analyzed by RT-PCR. 5-LOX COX-2 p21

Relative mRNA expression (FLAP) expression Relative mRNA 0.0 50 Con ic 300 Lic 1 L

*, P < 0.05; **, P < 0.005; ***, P < 0.0005

cancer, with androgen (20, 33) having significant influence (17) cancer cells. A dose-dependent inhibition of PCNA on tumor growth. The differences in sex hormonal levels expression with an increase in apoptosis also was observed between the sexes may explain some of the excess risk in licofelone-treated lung tumors (16) as well as colon observed in males, kinetics of tumor growth, and also their tumors (15, 36). Similarly, dose-dependent inhibition of response to drugs. From the present study as well as the COX-2 and 5-LOX was observed with licofelone treatment etiological observations, it seems that strong interventions in lung tumors (16). We found that licofelone had a or combinational strategies may result in effective preven- modulatory effect on cell-cycle regulatory proteins like tion of the disease in the males due to their association with Cyclin E and p16. Although expression of oncogenic Cyclin higher risk. We found that licofelone suppressed prolifer- E was inhibited in licofelone-treated tumors, expression of ation markers such as PCNA while inducing proapoptotic the tumor suppressor p16 was induced. Cyclin E is an markers such as p53, Bax, and caspase 3. Licofelone also important regulator of entry into the S phase in the mam- showed inhibitory effects on expression of the inflamma- malian cell cycle and may play an oncogenic role because it tion-related enzymes COX-2 and 5-LOX in the urothelial has been observed to undergo amplification in bladder and tumors. Previous studies have shown that transgenic mice many other cancers (37). The tumor suppressor protein p16 overexpressing COX-2 specifically in the urothelium exhibit is most frequently deleted (38) or inactivated by hyper- a high incidence of transitional cell hyperplasia in the methylation (39) in bladder cancers. Inactivation of the p53 bladder with a proportion of lesions progressing to invasive pathway is observed in bladder cancers and in the SV40T in carcinoma in conjunction with significant amplification of vitro, in vivo models. Here, licofelone-treated urothelial genes associated with the cell cycle and proliferation (34). tissues were found to have higher levels of p53 and p21 Here, we demonstrate that suppression of inflammatory protein compared with the untreated groups. NSAIDs are pathways by licofelone is associated with prevention of this known to protects p53 tumor suppressor function by inhi- progression of urothelial tumors to invasive disease and bition of electrophilic prostaglandin formation (40) and modulation of genes that affect cell proliferation and apo- also exert antitumor effects in COX-independent manner ptosis. Similar in vivo effects of licofelone have been by effect transcriptional factors, cell-cycle regulators like reported in colon and lung tumor studies. Licofelone inhib- cyclins and p21 (41). ited colonic tumors in a mouse colon cancer model by 72% Angiogenesis plays a key role in tumor growth and to 100%, caused a significant reduction in small intestinal metastasis. VEGF, one of the major angiogenic factors, was polyps (15), and provided better efficacy that helped cel- found to be greater in bladder cancer tissue compared with ecoxib in suppressing tumor growth. In a benzo(a)pyrene- normal urothelium (Fig. 1D). It correlated with the histo- induced lung cancer model, licofelone inhibited tumor logic grade and the presence of carcinoma in situ. Patients multiplicity by 26% with a significant decrease in tumor with higher VEGF had a significantly shorter progression- incidence (16). free survival compared with those with lower levels (42). Induction of apoptosis by licofelone has been reported in Elevated levels of VEGF and its receptors were found in various cancer cell lines, including colon (35) and prostate bladder cancer specimens and expression correlated with

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Licofelone Prevents Urothelial Cancer In Vivo

the invasiveness (43). We observed a significant downre- Authors' Contributions gulation of VEGF in the urothelial tumors of licofelone- Conception and design: V. Madka, A. Mohammed, X.-R. Wu, V. Steele, L. Kopelovich, C.V. Rao treated mice (Fig. 3B). COX-2 and VEGF function coordi- Development of methodology: V. Madka, A. Mohammed, J.M.R. Patlolla, nately in induction of angiogenensis (44). Suppression of X.-R. Wu, L. Kopelovich, C.V. Rao COX-2 by aspirin led to inhibition of the VEGF pathway, Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): V. Madka, Q. Li, Y. Zhang, J.M.R. Patlolla, and the inhibition of the COX-2/VEGF-dependent pathway C.V. Rao was effective in reducing tumor-associated angiogenesis, Analysis and interpretation of data (e.g., statistical analysis, biosta- tistics, computational analysis): V. Madka, A. Mohammed, S. Lightfoot, tumor growth, and tumor metastasis (45). In the present L. Kopelovich, C.V. Rao study, we observed inhibition of both COX-2 and 5-LOX Writing, review, and/or revision of the manuscript: V. Madka, along with VEGF in the licofelone-treated urothelial A. Mohammed, V. Steele, L. Kopelovich, C.V. Rao Administrative, technical, or material support (i.e., reporting or orga- tumors. nizing data, constructing databases): L. Biddick, S. Lightfoot, L. Kope- lovich, C.V. Rao Conclusions Study supervision: V. Madka, A. Mohammed, V. Steele, C.V. Rao Targeting of inflammatory pathways is an attractive strategy for prevention of urothelial cancers. In the present Acknowledgments The authors thank the University of Oklahoma Health Sciences Center study, we have shown that licofelone, a dual COX/LOX Rodent Barrier Facility and Dr. Julie Sando for valuable suggestions and inhibitor, inhibits urothelial tumor growth and prevents editorial help. invasion in vivo. The tumor-inhibitory effects of licofelone correlate with effects on inflammatory and tumor biomar- Grant Support kers. Given the proven safety profile of licofelone, it is a This study was funded by NIH/NCI (NCI-CN53300). promising candidate for urothelial cancer prevention and is The costs of publication of this article were defrayed in part by the worth considering for further development for the future payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate clinical trials. this fact.

Disclosure of Potential Conﬂicts of Interest Received March 10, 2014; revised April 21, 2014; accepted April 23, 2014; No potential conﬂicts of interest were disclosed. published OnlineFirst May 2, 2014.

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716 Cancer Prev Res; 7(7) July 2014 Cancer Prevention Research

Chemoprevention of Urothelial Cell Carcinoma Growth and Invasion by the Dual COX−LOX Inhibitor Licofelone in UPII-SV40T Transgenic Mice

Venkateshwar Madka, Altaf Mohammed, Qian Li, et al.

Cancer Prev Res 2014;7:708-716. Published OnlineFirst May 2, 2014.

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