Chemoprevention of urothelial cell carcinoma growth and invasion by the dual COX-LOX inhibitor licofelone in UPII-SV40T transgenic mice

Abstract

Epidemiological 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 cyclooxygenase (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=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 3-5-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 females mice (41-44%; p<0.003). Urothelial tumors of the licofelone-fed mice showed an increase in apoptosis (p53, p21, Bax, Caspase3) with a decrease in proliferation, inflammation and angiogenesis markers (proliferating cell nuclear antigen (PCNA), COX2, 5LOX, prostaglandin E synthase 1 (mPGES1), FLAP, and vascular endothelial growth factor (VEGF). These results suggest that licofelone can serve as potential chemopreventive for bladder TCC.

Introduction

Urinary bladder cancer is one of the common cancers worldwide, with the highest incidence rates in industrialized countries. In United States, it is the second most frequently diagnosed genito-urinary cancer, with an estimated 563,640 people currently living with bladder cancer. In 2014, about 74,690 new cases of bladder cancer are anticipated (about 56,390 in men and 18,300 in women) and approximately 15,580 are expected to die of this cancer (1). The majority (90-95 %) of bladder cancers are transitional cell carcinomas (TCC). Of these, 70-75% are non-invasive, low-grade, papillary TCC; while up to 30% of all diagnosed tumors are classified as invasive TCC, which pose a very high risk for distant metastases (2). Chemoprevention has been applied to many different tumor types, including TCC of the bladder, and has been proposed as a management strategy for cancer patients at risk of developing second primary tumors (3). Inflammation is a known risk factor for many cancers, particularly epithelial cancers such as those of the urothelium. It plays important roles at different stages of tumor development, including initiation, promotion, invasion and metastasis (4). Chronic inflammation is associated directly with muscle-invasive bladder cancer, particularly that due to urinary tract infection in invasive squamous cell carcinoma (5).

The metabolism of arachidonic acid (AA) by cycloxygenases-2 (COX2) and 5-lipoxygenase (5-LOX) generates prostaglandins (PGs) and hydroxyeicosatetraenoic acids (HETEs), respectively, that mediate a wide variety of physiological processes including cell proliferation, immune surveillance, cell differentiation, and inflammation. Clinically, aberrant expression of the COX-2 and 5-LOX enzymes has been observed in urothelial tumors and found to be associated with poor prognosis (6, 7). Concentrations of a urinary PGE metabolite (PGE-M) and leukotriene E₄ (LTE₄), biomarkers of the COX-2 and 5-LOX pathways, are elevated in smokers (8). In view of the recognized link between smoking, inflammation and bladder cancer, targeting the inflammatory pathways appears to be an ideal approach to reduce the risk of urothelial cancer development. Several preclinical studies have suggested that the combination of a selective COX-2 inhibitor and a 5-LOX inhibitor is more effective than either agent alone in preventing cancer cell growth or tumor formation (9, 10). Dual inhibitors that block both COX and 5-Lox may provide synergistic anti-inflammatory effects and also decrease gastrointestinal side effects associated with non-steroidal anti-inflammatory drugs (NSAIDs) (11) and select COX-2 inhibitors.

Licofelone is one such compound that is currently in phase III clinical trials for treatment of pain and inflammation associated with osteoarthritis (12, 13) and also has been shown to prevent colon (14, 15), lung (16) and prostate cancers (17). However no studies have been reported on the chemopreventive effects of licofelone in urothelial cancer. Among the preclinical models for urothelial cancer, UPII-SV40T transgenic mice develop invasive urothelial tumors due to the expression of Simian virus large T antigen (Tag) driven by the urothelium-specific uroplakin II (UPKII) promoter (18). These mice have been used as a model to elucidate the mechanism underlying urothelial tumorogensis (19, 20) as well as in chemoprevention studies (21, 22). In the present study, we determined the chemopreventive efficacy of licofelone using the UPII-SV40T transgenic mouse model for invasive urothelial TCC.

Material and methods

Animals, diet and care

All animal experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC). Transgenic mice (UPII-SV40T) expressing a Simian Virus 40 large T antigen (SV40T) specifically in urothelial cells under the control of the Uroplakin II (UPII) promoter and reproducibly developing high-grade carcinoma in situ (CIS) and invasive tumors throughout the urothelium (18) were used. The required number of UPII-SV40T transgenic mice were generated by breeding as described earlier (22). Animals were housed in ventilated cages under standardized conditions (21°C, 60% humidity, 12-h light/12-dark cycle, 20 air changes per hour) in the University of Oklahoma Health Sciences Center rodent barrier facility. Semi-purified modified AIN-76A diet ingredients were purchased from Bioserv, Inc. Licofelone was procured from the National Cancer Institute chemoprevention drug repository. Licofelone (150 and 300 ppm) was premixed with small quantities of casein and then blended into the diet using a Hobart mixer. Both control and experimental diets were prepared weekly and stored in a cold room. Mice were allowed ad libitum access to the respective diets and to automated tap water purified by reverse osmosis.

Breeding and Genotyping

All mice were bred and genotyped as described earlier (22). In brief, male UPII-SV40T mice were crossed with wild-type females to generate offspring. Transgenic pups were confirmed by tail DNA extraction using the mini-prep kit (Invitrogen) and polymerase chain reaction (PCR). PCR for the SV40T gene was done using the primer 5 ′-CTTTGGAGGCTTCTGGGATGCAACT-3 (sense) and 5′-GCATGACTCAAAAAACTTAGCAATTCTG-3′ (antisense) and amplifying under the following PCR conditions: denaturation at 95°C for 5 minutes, followed by 35 cycles at 95°C for 1 minute, 58°C for 45 sec, and 72°C for 45 sec. The PCR products, when separated on a 2% agarose gel, showed a 550 bp band.

Bioassay

Genotyped UPII-SV40T transgenic mice were used in the efficacy study. The experimental protocol is summarized in Fig. 1A. Five-week-old mice were selected and randomized so that the average body weights in each group were equal (n=30 UPII-SV40T mice per group and n=12 wild-type mice per group) and were fed a modified AIN-76A diet for 1 week. At 6 weeks of age, mice were fed control or experimental diets containing 0, 150, or 300 ppm licofelone (Fig 1B.) until termination of the study. Mice were checked routinely for signs of weight loss, toxicity or any abnormalities. Previously, we have established maximal tolerated doses/ optimal dose of licofelone in mice and found to be >500 ppm fed in the modified AIN-76A diet (15) The food intake and body weight of each animal were measured once weekly for the first 6-weeks and then once a month until termination. After 34 weeks on experimental diets (i.e., 40 weeks age), all mice were euthanized by CO₂ asphyxiation and necropsied, and urinary bladders were collected and weighed to determine the tumor weight. A portion of the urinary bladder was fixed in 10% neutral-buffered formalin for histopathological evaluation and the rest was snap frozen in liquid nitrogen for further analysis.

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 β-Actin as analyzed by RT-PCR.

Tissue processing and Histological analysis

Formalin-fixed, paraffin-embedded tissues were sectioned (4-μm) and stained with hematoxylin and eosin (H&E). Sections of each urothelial tumor were evaluated histologically by a pathologist blinded to the experimental groups. Carcinomas were classified into non-invasive carcinoma in situ (CIS), invasive carcinomas (lamina propria invasive and muscularis propria invasive) types according to histopathological criteria as previously described (22).

Realtime PCR

Total RNA from urothelial tumor samples of male mice was extracted using the Totally RNA Kit as per manufacturer's instructions. Equal quantities of DNA-free RNA were used in reverse transcription reactions for making cDNA using SuperScript reverse transcriptase (Invitrogen). Real time PCR reactions were done for proliferating cell nuclear antigen (PCNA), p53, Bax, Caspase 3, Prostaglandin E Synthase 1 (mPGES1), FLAP, vascular endothelial growth factor (VEGF), p16 and Actin using SYBR green and specific primers (Table 1). Relative gene expression was calculated using the 2^−ΔΔCT formula (23). All experiments were performed using replicated tumor samples and at least in triplicate.

Table 1.

List of primers used for real-time PCR analysis

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

Immunohistochemistry (IHC)

Modulatory effects of licofelone on the expression of COX-2, 5-LOX and p21 were evaluated by immunohistochemistry as described previously (22). Briefly, sections of paraffin-embedded tissues were deparaffinized in xylene, rehydrated through graded ethanol solutions, and washed in phosphate-buffered saline (PBS). Antigen retrieval was carried out by heating the sections in 0.01 mol/L citrate buffer (pH 6.0) for 30 minutes in a boiling water bath. Endogenous peroxidase activity was quenched by incubation in 3% H₂O₂ in PBS for 5 minutes. Nonspecific binding sites were blocked using Protein Block for 20 minutes. Then, sections were incubated overnight at 4°C with 1:300 dilutions of monoclonal antibodies against COX2, 5-LOX and p21 (Santa Cruz Biotechnology). After several washes with PBS, they were incubated with appropriate secondary antibodies for 2 hours, then exposed to avidin-biotin complex reagent (Invitrogen). After rinsing with PBS, the slides were incubated with the chromogen 3,3 ′-diaminobenzidine for 3 minutes, then counter stained with hematoxylin. Non-immune rabbit immunoglobulins were substituted for primary antibodies as negative controls. Specimens were observed using an Olympus microscope IX71, and digital computer images were recorded with an Olympus DP70 camera.

Western Blotting

Proteins (60 ug) in lysates from bladders of control and licofelone-treated mice were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Membranes were blocked with 5% nonfat milk (Biorad) in Tris-buffered saline (TBS) and incubated with antibodies for PCNA, p53 cyclin E and Caspase 3 overnight at 4^oC. Subsequently, membranes were washed and incubated with secondary antibody (either goat anti-mouse or goat anti-rabbit conjugated with horseradish peroxidase; 1:10,000 dilution) for 1 h. Protein was detected on BioMax MR film (Kodak) using chemiluminescence (Super Signal, Pierce Biotechnology). Equal protein loading was confirmed by detection of tubulin. Selected blots were quantified using Image J software.

Statistical Analysis

The data are presented as means ± standard errors (SE). Differences in body weights were analyzed by Analysis of Variance. Statistical differences between urothleial tumor weights in the control and treated groups were evaluated using unpaired t-test with Welch's correction. Tumor incidences (percentage of mice with urothelial tumors) were analyzed by Fisher's exact test. Differences between control and treatment groups were considered significant at p <0.05. All statistical analysis was performed using Graphpad Prism 5.0 Software.

Results

General observations

All of the transgenic and wild type mice fed control and licofelone-containing modified AIN76A diets were weighed weekly and monitored throughout the study. Gross anatomy of wild-type and transgenic mice revealed no evidence of any abnormality in organ size, or changes in appearance of liver, spleen, heart, lung, seminal vesicles, testis, ovaries or prostate. Thus, doses applied in the efficacy studies were expected to be nontoxic.

Urothelial tumor growth is inhibited by licofelone in transgenic mice

UPII-SV40T mice spontaneously develop urothelial tumors, as result of which there is a significant increase in urinary bladder weights compared with wild type. At 40 weeks age, these tumors are histopathologically classified as high-grade tumors invading both lamina propria and muscularis while wild type bladders show normal urothelium (Fig 1C). These tumors showed an over-expression of the PCNA, COX-2, 5-LOX and VEGF compared to that of the normal urothelium from wild type mice (Fig 1D). At the end of the experiment, no significant differences in body weights were observed (Fig. 2A & 2B). A chemopreventive effect of dietary licofelone administered at 150 or 300 ppm was found on urothelial tumor growth. Male and female UPII-SV40T mice fed control diet had urothelial tumors that weighed an average of 112.9 ± 9.8 mg and 19.3 ± 0.8 mg, respectively (Fig. 2C and 2D). Dietary licofelone at 150 and 300 ppm administered for 34 weeks significantly inhibited the tumor growth in a dose-dependant manner that led to reduced urothelial tumor weight in both sexes. Tumors of licofelone-fed male mice weighed 65.2 % and 82.7% less at the low and high doses, respectively (39.3 ± 9.2 mg; p<0.0001 and 19.5 ± 8.9mg; p<0.0001) compared with tumors of control mice due to significant inhibition of tumor growth (Fig 2C). A similar effect of licofelone was observed in female mice; tumors from the transgenic mice fed the experimental diet weighed 35% - 49% less at both doses respectively, (12.6 ± 0.8mg; p<0.0001 and 6.8 ± 1.1mg; p<0.0001) than those of the control group (Fig 2D).

Figure 2.

A & B) Body weights of the male and female transgenic mice fed control or experimental diets at 40 weeks of age.

C & 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 to control mice (p<0.0001) similarly in the female mice 35-49% (p<0.0001) inhibition was observed.

E & 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 prevents progression of urothelial tumor to invasive TCC

Urothelial tumors in UPII-SV40T mice progress gradually from normal-looking epithelium to low grade noninvasive TCC to high grade invasive TCC. To determine the effect of licofelone on this progression, formalin-fixed bladders from the control and licofeone-fed transgenic mice were sectioned and stained with H&E and their histopathology was compared. Bladders from the transgenic mice fed control diet had high-grade, muscle-invasive TCC at 40 weeks of age. However, licofelone-fed mice showed significant inhibition of invasive tumors in both male and female groups. As a result of licofelone administration, 12-50% (p<0.01) of the male mice had tumors that were classified as high-grade, non-invasive CIS (Fig 2E). High dose licofelone was found to have better effect on tumor invasion compared with the lower dose. In the female group, both doses led to prevention of tumor invasion in 41% - 44% of the mice (p<0.003) (Fig 2F).

Anti-proliferative and Anti-angiogenic effects of licofelone

Urothelial tumors of the transgenic mice were found to have significant over-expression of the proliferation and angiogenesis markers PCNA and VEGF (Fig 1D) compared with those in the normal urothelium of bladders from wild type mice. To evaluate the anti-proliferative and anti-angiogenic effects of licofelone on protein and mRNA levels of these markers, urothelial tumors from the control and licofelone-fed animals were subjected to real time PCR and Western blotting. Tumors from mice fed the licofelone diet showed a significantly decreased expression of PCNA (Fig 3A and 3B) and VEGF (Fig 3A) compared with those in the control group. The tumor suppressor p16 (Fig 3A) was induced in the licofelone group while Cyclin E protein (Fig 3B) was significantly inhibited.

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. Real time PCR analysis was done on cDNA synthesised 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 Methods. Expression of PCNA and Cyclin E was found to be inhibited while the expression of p53 and caspase 3 proteins was found to be induced.

Licofelone induces pro-apoptotic molecules in urothelium

Molecular effects of licofelone were determined by analyzing expression of mRNA and protein for various proapoptotic/anti-tumor molecules. Real time PCR analysis of the mRNA showed a significant increase in the levels of the tumor suppressor protein p53 in tumors from licofelone-fed mice (Fig. 3A). Similarly, p53 protein was induced by licofelone in the treated group as determined by immunoblotting (Fig 3B). The pro-apoptotic molecules Bax (Fig. 3A) and Caspase 3 (Fig. 3A and 3B) also were induced by the licofelone treatment, suggesting that licofelone may lead to inhibition of tumor growth by inducing apoptosis. Licofelone treatment also led to an increase in p21 expression (Fig 4A).

Figure 4.

A) Effect of Licofelone on p21, COX-2 and 5-LOX expression in urothelial tumors. Immuno-histochemical analysis was performed with paraffin-embedded and microsectioned bladder tissues as described in Materials and Methods. A significant decrease in COX-2 and 5-LOX expression with an increased expression of p21 was seen in the treatment groups.

B) Effect of licofelone on expression of mPGES1 and FLAP analyzed by real-time PCR.

Licofelone inhibits expression of pro-inflammatory COX-2 and 5-LOX molecules

The effect of licofelone on expression of the pro-inflammatory molecules COX-2, 5-LOX, mPGES-1 and FLAP was determined. Urothelial tumors from the transgenic mice fed control diet fed showed a significant over-expression of COX-2 and 5-LOX (Fig. 1D and 4A) compared with normal urothelium. Licofelone had an inhibitory effect on both of these pro-inflammatory molecules (Fig. 4A). Similarly, there was significant down-regulation of mPGES1 (Fig. 4B) and FLAP (Fig. 4B) that can contribute to pro-inflammatory effects.

Discussion

Clinically, 75-85% of patients with bladder cancer present with non-muscle-invasive bladder cancer (NMIBC) that is confined to the mucosa (stage Ta, CIS) or submucosa (stage T1). These patients can be identified based on haematuria, the most common finding in NMIBC and cytology of urine. If Untreated, approximately 54% of patients with CIS progress to muscle-invasive disease; even in treated patients, these tumors often recur and some progress to invasive or metastatic carcinoma, for which the treatment options become limited. Hence, there is a need to develop targeted regimens that will prevent disease progression, improve response to treatment and also prevent recurrence. Inflammation of the bladder due to infectious as well as to noninfectious etiologies such as medication, radiation, foreign bodies, chemicals, and autoimmune responses affects the bladder function directly and also is a potential risk factor for urothelial cancer development. Inflammatory pathways thus have been considered important targets for bladder cancer prevention as well as to increase the response to chemotherapy (24).

Prominent expression of COX-2 and 5-LOX enzymes has been described in bladder cancers and expression of these proteins correlates with tumor grade and invasion (6, 7, 25, 26, 27). Over-expression of COX-2 and 5-LOX enzyme metabolites has been reported in smokers, who represent a high-risk population for bladder cancer (8). We found the urothelial tumors of the UPII-SV40T transgenic mice to have elevated levels of these enzymes (COX-2, 5-LOX) compared with normal tissue (Fig 1D and 4A). Hence, this transgenic mouse was an appropriate model to test the effect of anti-inflammatory agents targeting these enzymes. COX-2 inhibitors such as nimesulide (28), and Celecoxib (29); also traditional NSAIDs such as indomethacin (30), ibuprofen (31) have been demonstrated to prevent bladder cancer in in vitro and in vivo models. Sabichi et al. (32) did not observe a clinical benefit of celecoxib in preventing recurrence of non-muscle-invasive bladder cancer in a randomized controlled trial, although, celecoxib had a marginally significant effect on reducing metachronous recurrences compared with placebo. However, nimesulide was capable of preventing bladder tumor recurrences in a dose-dependent manner (28). Similarly, targeting 5-LOX using the specific inhibitor AA861 caused a dose-dependent inhibition of bladder cancer cell growth (25). While targeting either of these enzymes has shown inhibitory effects, it has been found that COX-2 inhibition includes shunting of arachidonic acid (AA) into the 5-LOX pathway leading to increased risk for cardiovascular disease (8). The striking interrelationship of COX-2 and 5-LOX biological functions suggests that drugs that are able to block both COX-2 and 5-LOX pathways may provide a better approach to bladder cancer prevention.

Licofelone is a promising novel dual COX-LOX inhibitor with proven chemopreventive efficacy in several cancer models both in vitro and in vivo. Here we have shown that licofelone has a significant inhibitory effect on urothelial tumor growth and invasion in both male and female mice in a dose dependant manner. Licofelone was able to significantly prevent tumor invasion in the female mice even at a lower dose compared to that male. This is particularly interesting in view of the differences in the risk for urothelial cancer incidence between these two sexes. Also hormonal factors are known play an important role in bladder cancer, with androgen (33, 34) having significant influence on tumor growth. The differences in sex hormonal levels between the sexes may explain some of the excess risk observed in males, kinetics of tumor growth and also their response to drugs. From the present study as well as the etiological observations it appears that strong interventions or combinational strategies may result in effective prevention of the disease in the males due to their association with higher risk. We found that licofelone suppressed proliferation markers such as PCNA while inducing pro-apoptotic markers such as p53, Bax and caspase 3. Licofelone also showed inhibitory effects on expression of the inflammation-related enzymes COX-2 and 5-LOX in the urothelial tumors. Previous studies have shown that transgenic mice over-expressing COX-2 specifically in the urothelium exhibit a high incidence of transitional cell hyperplasia in the bladder with a proportion of lesions progressing to invasive carcinoma in conjunction with significant amplification of genes associated with the cell cycle and proliferation (35). Here we demonstrate that suppression of inflammatory pathways by licofelone is associated with prevention of this progression of urothelial tumors to invasive disease and modulation of genes that affect cell proliferation and apoptosis. Similar in vivo effects of licofelone have been reported in colon and lung tumor studies. Licofelone inhibited colonic tumors in a mouse colon cancer model by 72-100%, caused a significant reduction in small intestinal polyps (15), and provided better efficacy that did celecoxib in suppressing tumor growth. In a benzo(a)pyrene-induced lung cancer model, licofelone inhibited tumor multiplicity by 26% with a significant decrease in tumor incidence (16).

Induction of apoptosis by licofelone has been reported in various cancer cell lines including colon (36) and prostate (17) cancer cells. A dose-dependent inhibition of PCNA expression with an increase in apoptosis also was observed in licofelone-treated lung tumors (16) as well as colon tumors (15, 37). Similarly, dose-dependent inhibition of COX-2 and 5-LOX was observed with licofelone treatment in lung tumors (16). We found that licofelone had a modulatory effect on cell-cycle regulatory proteins like Cyclin E and p16. While expression of oncogenic Cyclin E was inhibited in licofelone-treated tumors, expression of the tumor suppressor p16 was induced. Cyclin E is an important regulator of entry into the S phase in the mammalian cell cycle and may play an oncogenic role since it has been observed to undergo amplification in bladder and many other cancers (38). The tumor suppressor protein p16 is most frequently deleted (39) or inactivated by hypermethylation (40) in bladder cancers. Inactivation of the p53 pathway is observed in bladder cancers and in the SV40T in-vitro, invivo models. Here licofelone treated urothelial tissue were found to have higher levels of p53 and p21 protein compared to the untreated groups. NSAIDS are known to protects p53 tumor suppressor function by inhibition of electrophilic PG formation (41) and also exert anti-tumor effects in COX-independent manner by effect transcriptional factors, cell cycle regulators like cyclins and p21 (42).

Angiogenesis plays a key role in tumor growth and metastasis. VEGF, one of the major angiogenic factors, was found to be greater in bladder cancer tissue compared with normal urothelium (Fig. 1D). It correlated with the histologic grade and the presence of carcinoma in situ. Patients with higher VEGF had a significantly shorter progression-free survival compared with those with lower levels (43). Elevated levels of VEGF and its receptors were found in bladder cancer specimens and expression correlated with the invasiveness (44). We observed a significant down-regulation of VEGF in the urothelial tumors of licofelone-treated mice (Fig. 3B). COX-2 and VEGF function coordinately in induction of angiogenensis (45). Suppression of COX-2 by aspirin led to inhibition of the VEGF pathway, and the inhibition of the COX-2/VEGF-dependent pathway was effective in reducing tumor-associated angiogenesis, tumor growth, and tumor metastasis (46). In the present study, we observed inhibition of both COX-2 and 5-LOX along with VEGF in the licofelone-treated urothelial tumors.

Conclusions

Targeting of Inflammatory pathways is an attractive strategy for prevention of urothelial cancers. In the present study we have shown that licofelone, a dual COX/LOX inhibitor, inhibits urothelial tumor growth and prevents invasion in vivo. The tumor inhibitory effects of licofelone correlate with effects on inflammatory and tumor biomarkers. Given the proven safety profile of licofelone, it is a promising candidate for urothelial cancer prevention and is worth considering for further development for the future clinical trials.