Impact of intravesical instillation of a novel biological response modifier (P-MAPA) on progress of non-muscle invasive bladder cancer treatment in a rat model

This work describes the effects of immunotherapy with Protein Aggregate Magnesium-Ammonium Phospholinoleate-Palmitoleate Anhydride in the treatment of non-muscle invasive bladder cancer in an animal model. NMIBC was induced by treating female Fischer 344 rats with N-methyl-N-nitrosourea. After treatment with MNU, the rats were distributed into four experimental groups: Control (without MNU) group, MNU (cancer) group, MNU-BCG (Bacillus Calmette-Guerin) group, and MNU-P-MAPA group. P-MAPA intravesical treatment was more effective in histopathological recovery from cancer state in relation to BCG treatment. Western blot assays showed an increase in the protein levels of c-Myc, COUP-TFII, and wild-type p53 in P-MAPA-treated rats in relation to BCG-treated rats. In addition, rats treated with P-MAPA intravesical immunotherapy showed the highest BAX protein levels and the lowest proliferation/apoptotic ratio in relation to BCG-treated rats, pointing out a preponderance of apoptosis. P-MAPA intravesical treatment increased the wild-type p53 levels and enhanced c-Myc/COUP-TFII-induced apoptosis mediated by p53. These alterations were fundamental for histopathological recovery from cancer and for suppress abnormal cell proliferation. This action of P-MAPA on apoptotic pathways may represent a new strategy for treating NMIBC.


Introduction
Bladder cancer (BC) among other cancers one of the most common malignancy of the urinary tract and is the second causing of death amid urogenital tumors. Males are 3 to 4 times more likely to develop BC than females [1, The proto-oncogene c-Myc has a pivotal role in oncogenesis in several human tumors. Studies demonstrated that c-Myc plays important regulatory roles in growth control, differentiation, apoptosis, angiogenesis, and metabolism [17,18]. Historically, there is a tendency to classify c-Myc as a gene responsible for cell immortalization [17,18]. Paradoxically, it became clear that, in certain circumstances, c-Myc protein could act by inducing apoptosis [17][18][19]. Many genes involved in programmed cell death, such as p53, p21, and BAX, contain regions responsive to c-Myc in their promoters [17,18]. Possible pro-apoptotic pathways mediated by c-Myc, p19 ARF activation is particularly important since, in cooperation with Ras protein, this protein represses Mdm-2 (negative regulator of p53), thereby inducing p53 release and triggering apoptotic mechanisms [17,20]. In contrast to these studies, the role of c-Myc in mediating apoptosis in NMIBC is still not clear.
In view of the remarkable antitumor activity of P-MAPA against NMIBC and its immunomodulatory action on TLRs and p53, we hypothesized that P-MAPA could trigger apoptosis in NMIBC by increasing p53, COUP-TFII, and c-Myc protein levels. To address this question, we examined the effect of treatment with P-MAPA in an animal model of chemically induced NMIBC and compared the results with those obtained by treating the animals with BCG.

Experimental procedures
Twenty female Fischer 344 rats seven weeks old were obtained from the Multidisciplinary Center for Biological Investigation (CEMIB) at the University of Campinas (UNICAMP). The animal experiments were approved by an institutional Committee for Ethics in Animal Use (CEUA/ UNICAMP, protocol no. 2684-1). Prior to intravesical catheterization with a 22-gauge angiocatheter, the rats were anesthetized with 10% ketamine (60 mg/kg, i.m.; Vibra ® , Roseira, SP, Brazil) and 2% xylazine (5 mg/kg, i.m.; Vibra ® , Roseira, SP, Brazil). The animals remained anesthetized for approximately 45 min after catheterization to prevent spontaneous micturition. Five control rats (Control group) received 0.30 ml of 0.9% physiological saline every other week for 14 weeks. For NMIBC induction, 15 rats received n-methyln-nitrosourea (MNU; 1.5 mg/kg, dissolved in 0.30 ml of 1 M sodium citrate, pH 6.0-Sigma, St. Louis, MO, EUA) intravesical every other week for eight weeks [9,12]. Two weeks after the last dose of MNU, all rats were submitted to ultrasonography to evaluate the occurrence of tumors. The ultrasounds were evaluated using a portable, software-controlled ultrasound system with a 10-5 MHz 38-mm linear array transducer. The animals from Control group showed no Page 3 of 10 24 mass infiltrating the bladder walls (Fig. 1a). The ultrasonography of urinary bladder from MNU group showed a mass (average tumor size 3,5 X 3.9 mm) infiltrating the caudal and dorsal bladder walls (Fig. 1b, c). The color Doppler flow image showed high vascularity in the mass (Fig. 1c).
MNU-treated rats were further divided into three groups (n = 5 per group): the MNU group received 0.30 ml of 0.9% physiological saline, the MNU-BCG group received 10 6 CFU (40 mg) of BCG (Fundação Ataulpho de Paiva, Rio de Janeiro, RJ, Brazil); and the MNU-P-MAPA group received P-MAPA (5 mg/kg, intravesically; Farmabrasilis, Campinas, SP, Brazil). All of the rats were treated with saline, BCG or P-MAPA intravesical every other week for six weeks.
At the end of the treatments, the rats were euthanized and the urinary bladders were collected and processed for histopathological analysis and western blotting.

Histopathological analysis
Samples of urinary bladders (n = 5 per group) were fixed in Bouin solution for 12 h and then washed in 70% ethanol, dehydrated in an increasing series of alcohols, cleared in xylene for 2 h and embedded in plastic polymer (Paraplast Plus, St. Louis, MO, USA). Subsequently, 5-µm thick sections were cut on a rotary microtome (Slee CUT5062 RM 2165; Slee Mainz, Mainz, Germany), stained with hematoxylin-eosin (Sigma, St. Louis, MO, EUA) and photographed with a Leica DM2500 photomicroscope (Leica, Munich, Germany). A senior uro-pathologist analyzed the urinary bladder lesions based on the criteria of the Health/World International Society of Urological Pathology Organization [21].

Determination of the proliferative and apoptotic indexes
The same tissue samples used for histopathological analysis were also used to determine the proliferative and apoptotic indexes. The proliferative index was determined based on Ki-67 immuno-detection. The samples were cut into 6-µm thick sections and antigen was retrieved by boiling the sections in a 10 mM citrate buffer, pH 6.0, three times for 5 min each in a conventional microwave oven. The sections were subsequently incubated in 0.3% H 2 O 2 to block endogenous peroxidase activity and nonspecific binding sites were blocked by incubating the sections in blocking solution at room temperature. The primary mouse monoclonal anti-Ki-67 (RRID: AB_442101; 1:50) was diluted in 1% BSA and applied to the sections overnight at 4 °C. Bound antibody was detected with an Advance™ HRP kit (Dako Cytomation Inc., USA). The sections were lightly counterstained with Harris' hematoxylin and photographed with a photomicroscope (DM2500 Leica). Ten randomly selected fields were examined per rat, resulting in 50 fields per group with a ×40 objective lens. The number of cells stained for Ki-67 in each field was counted and the total number of positive cells was expressed as a percentage of the total number of cells counted.
DNA fragmentation (TUNEL) was evaluated by the terminal deoxy-nucleotidyl transferase (TdT) method using a FragEL™ DNA kit (Calbiochem, La Jolla, CA, USA). Apoptotic nuclei were identified using a diaminobenzidine chromogen mixture. Ten randomly selected microscopic fields were analyzed per sample, resulting in 50 fields per group, using a Leica DM2500 photomicroscope with a × 40 objective. Sections were lightly counterstained with Harris' hematoxylin. The apoptotic index was determined by dividing the number of apoptotic nuclei by the total number of nuclei counted in each field.

Western blotting of p53, c-Myc, COUP-TFII, and BAX
Urinary bladder samples (n = 5 per group) were weighed and homogenized in 50 μL of RIPA lysis buffer (EMD Millipore Corporation, Billerica, MA, USA) per mg of tissue. Aliquots containing 70 μg of protein were separated by SDS-PAGE on 10% or 12% polyacrylamide gels under reducing conditions. After electrophoresis, the proteins were transferred to Hybond-ECL nitrocellulose membranes (Amersham, Pharmacia Biotech, Arlington Heights, IL., USA). The membranes were blocked with TBS-T containing 1% BSA (bovine serum albumin) and incubated overnight at 4 °C with primary mouse monoclonal anti-p53 (RRID: AB_303198; 1:1500), rabbit monoclonal antic-Myc (RRID: AB_731658; 1:5000), mouse monoclonal anti-COUP-TFII (RRID: AB_742211; 1:1000), and rabbit polyclonal anti-BAX (RRID: AB_306191; 1:1000), all diluted in 1% BSA. All primary antibodies were from Abcam (Cambridge, MA, USA). The membranes were then incubated for 2 h with rabbit or mouse secondary HRP-conjugated antibodies (diluted 1:3000 in 1% BSA; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Peroxidase activity was detected by incubation with a diaminobenzidine chromogen (Sigma Chemical Co., St Louis, MO, USA). Western blots were run in duplicate and urinary bladder samples were pooled from five rats per group for each repetition. Semi-quantitative densitometry (IOD-integrated optical density) analysis of the bands was done using ImageJ v.1.47 software (National Institutes of Health, Bethesda, MD, USA; available at: http:// rsb. info. nih. gov/ ij/) followed by statistical analysis. β-Actin was used as an endogenous housekeeping gene for standardization of the intensity of band staining. The results were expressed as the mean ± standard deviation of the ratio of the band intensity relative to that of β-actin [12].

Statistical analyses
Quantitative results were expressed as the mean ± standard deviation whenever possible. Western blotting data, proliferative and apoptotic indexes, and the proliferation/apoptotic ratio (P/A) were compared among groups by one-way analysis of variance (ANOVA) followed by the Tukey test, with the level of significance set at 1% (p < 0.01). Histopathological results were compared with a proportion test. The difference between the two proportions was tested using test of proportion with a type-I error of 1%.

P-MAPA promotes histopathological recovery from cancer state
The control group showed no histological changes in bladder tissue ( Fig. 2a; Table 1). Three different cell types composed the normal bladder urothelium: basal cell layer, intermediate cell layer, and surface cell layer (umbrella cells) (Fig. 2a). In contrast, MNU group showed deep microscopic changes in the urinary bladder tissue such as pT1 (Fig. 2b, c) and pTa in 80% and 20% of the rats, respectively (Table 1). In addition, 80% of the rats from this group showed keratinizing squamous metaplasia.
In the MNU-BCG group, pTa (Fig. 2d), pTis (Fig. 2e), and papillary hyperplasia were the most frequent urothelial lesions found in 40%, 40%, and 20% of the rats, respectively ( Table 1). The bladder urothelium morphology in the MNU-P-MAPA group was very similar to that of the control group (Fig. 2f, g). Normal urothelium was observed in 40% of the rats ( Fig. 2f; Table 1). Flat hyperplasia (Fig. 2g) was the only histopathological change found in the MNU-P-MAPA group (60% of the rats, Table 1), which is not considered a negative report; it might represent a pre-cancerous lesion. P-MAPA-treated rats showed no urinary calculi and macroscopic haematuria. These urinary changes were only identified in the MNU and MNU-BCG groups.

P-MAPA intravesical immunotherapy increases wild-type p53, COUP-TFII, and c-Myc levels
In response to P-MAPA treatment, p53 and COUP-TFII protein levels were significantly increased in relation to MNU-BCG group (Fig. 3a, c). In addition, the lowest p53 and COUP-TFII levels were found in the MNU group (Fig. 3a,  c).
Western blot assays showed an increase in the levels of c-Myc in the MNU group compared to the other groups (Fig. 3b); they were also significantly higher in the P-MAPAtreated rats compared to the BCG-treated rats (Fig. 3b).

High COUP-TFII, c-Myc, and p53 levels decrease proliferation and increase apoptosis after treatment with P-MAPA
The apoptotic and proliferative indexes showed different cell cycle kinetics after each treatment (Fig. 4). A significant decrease in urothelial cell proliferation in the P-MAPAtreated rats was observed compared to BCG-treated rats (Fig. 4).

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The animals treated with P-MAPA intravesical immunotherapy showed significant increase in both apoptotic index (Fig. 4) and BAX levels (Fig. 3d) compared to the BCG-treated animals.
As a consequence of P-MAPA effects in the cell cycle kinetics, the proliferation/apoptotic ratio (P/A) was significantly lower compared to BCG treatment, pointing out a preponderance of apoptosis (Fig. 5).

Discussion and conclusions
The management of NMIBC persists as a challenge due to the high rates of recurrence and progression to muscle invasive stage. BCG use in NMIBC is limited by treatment failure, adverse effects, and intolerance that appear on twothirds of patients [4,22,23]. Metabolic disorders, such as diabetes mellitus type 2, could contribute to the failure of BCG in NMIBC [24]. In cases of BCG treatment failure, radical cystectomy is the only available therapeutic option. However, in patients ineligible for radical cystectomy, the treatment possibilities are limited [4,22,25].
In view of these problems and limitations associated with the use of BCG, novel therapies are needed to treat NMIBC and prevent disease progression while preserving bladder function and ensuring acceptable quality of life for patients. These new therapies would also supply an alternative for those ineligible for radical cystectomy. P-MAPA, a biological response modifier that shows novel therapeutic properties compared to standard treatments, appears to be a valuable candidate drug for the treatment of NMIBC. In previous reports, we described several therapeutic properties of P-MAPA. [9,10,12], Although all the studies performed in animal models to assess the therapeutic potential of P-MAPA on NMIBC compared with BCG does not involve tumoral resection (TURBT) due to experimental restrictions, all of them, used a model of tumoral induction based on intravesical instillations of MNU. As reported in the literature [26] the use of MNU like TURBT, lead to an altered urothelial surface, favoring the implantation of tumoral cells, or in other words, the use of intravesical MNU may result in an altered presentation of urothelial surface similar to those induced by TURBT.
Here, by using the NMU rat model to study NMIBC, we found that treatment with BCG led to histopathological recovery in only 20% of the rats. In contrast, intravesical immunotherapy with P-MAPA provided better histopathological recovery from cancer in relation to BCG treatment (MNU-BCG group).
Few years ago, an important research and strategies for the cancer therapy (e.g., NMIBC), mainly analyzing molecules that bind to and activate TLRs were studied [10,12,27]. Our previous knowledge with animal model on NMIBC, P-MAPA increased TRIF and IRF-3 protein levels, indicating an activation of MyD88-independent pathway [9,12]. P-MAPA induction of MyD88-independent pathway, which is a TRIF-dependent pathway, produced an enhancement of IFN-γ and iNOS (macrophages type 1-M1) protein contents. P-MAPA immunotherapy led to distinct activation of innate immune system of TLRs 2 and 4-mediated, differently to BCG, overcoming in an augmented interferons signaling pathway [12], giving a more efficient effect in the NMIBC treatment.
IFN-γ and IRF-3 induction (interferon signaling pathway) by P-MAPA, when compared proliferation/apoptotic ratio in the animal treatment by P-MAPA to BCG was significantly lower in the animals treated with the former than with BCG treatment, denoting prevalence of the apoptotic pathway [12].
Thereby, P-MAPA on activation of interferon signaling pathway was highly more effective in the enhancement of immunogenic cell death in relation to inflammatory cytokines signaling pathway through BCG. Thus, P-MAPA is considered as a cytotoxic immunotherapy [9,12]. P-MAPA immunotherapy also increased p53 levels [9,12]. The expression of proteins and inflammatory cytokines is altered after p53 activation in cells related with immune system [28]. TLRs expression was altered in cancer cell lines after p53 activation, resulting in increased ligand-mediated expression of cytokines downstream from the corresponding TLR [28,29].
The modulation of TLR expression in tumors associated with immune cells in response to DNA-impairing and/ or p53-inducing agents could be useful in approaches that use TLRs for cancer management [13,20]. Studies demonstrated that wild-type and p53 mutants modulate TLR expression differentially. Shatz et al. [28] proposed that management of normal or mutant p53 responses along with immune challenges that include TLR targets could increase inflammatory and immune type responses. New treatments based on restore wild-type p53 to cancer cells, may stop  or significantly reduce cancer growth [29,30]. These treatments involve medicines that could alter "mutated p53" and make it behave as wild-type p53 [29]. Furthermore, other therapies could block proteins that degrade p53 in cancer cells, resulting in increased cancer cell p53 levels with consequent cancer cell death. We have previously shown that immunomodulation of TLRs 2 and 4 by P-MAPA led to wild-type p53 activation in NMIBC [9,12]. In addition, in  letters (a, b, c, d) indicate significant differences (p < 0.01) between the groups after the Tukey test 24 Page 8 of 10 our study, we have shown that intravesical immunotherapy with P-MAPA remarkably increased wild-type p53 protein levels in a rat model of NMIBC. This effect was not seen with BCG therapy, which suggested that immunotherapy with P-MAPA could be a relevant pharmacological approach for increasing wild-type p53 protein levels, in agreement with Menendez et al. [29]. In mouse fibroblasts studies have shown that induction of p53 in response to activated oncogenes such as E1A, Raf, Ras, β-catenin, v-abl, E2F1, and c-Myc involves the p19 ARF protein that binds and also inhibits Mdm2 [19,31]. At normal conditions, p53 is a short-lived protein because of its fast proteasomal degradation [33][34][35]. This kind of degradation is largely mediated by Mdm2 which is one of p53's own target gene products, that fits as an E3 ubiquitin ligase for p53, although Mdm2-independent pathways for p53 degradation also exist [32,33,[36][37][38].
The inhibition or suppression of apoptosis is considered an important factor in leading tumorigenesis in vivo [18]. Although c-Myc protein can drive apoptosis in various biological levels [18,39] the simple overexpression of c-Myc in pancreatic islets does not lead to carcinogenesis except if apoptosis is blocked, e.g., by p53 loss, p19 ARF knockout, or Bcl-xl overexpression [18,40]. Seoane and Massague [41] demonstrated that depletion of c-Myc in colorectal cancer cell lines reduced the downstream effectors of p53 signaling, resulting in a decrease in apoptosis. These authors concluded that, in the absence of c-Myc, the levels of the anti-apoptotic cell cycle arrest protein p21 were enhanced, while those of pro-apoptotic genes such as BAX were reduced, coming out in cell cycle arrest rather than apoptosis. Phesse et al. [18] elucidated the fundamental function of c-Myc in signaling DNA damage-induced apoptosis in vivo through the control of p53 protein. These authors investigated the apoptotic response to DNA damage after deleting the c-Myc gene in intestinal enterocytes from adult murine intestine and found that c-Myc deletion completely abolished the immediate wave of apoptosis that followed ionizing irradiation and treatment with cisplatin, a situation that mimicked the phenotype of p53 deficiency in the intestine. Since c-Mycdeficient intestinal enterocytes do not upregulate p53, Phesse et al. [18] concluded that these results reflected an upregulation of the E3 ubiquitin ligase Mdm2 that targets p53 for degradation in these cells. In addition, low level overexpression of c-Myc elicited persistent apoptosis in response to DNA damage, indicating that c-Myc acts as a decisive cell survival rheostat after DNA damage.
High E3 ubiquitin ligase Siah-2 (seven in absentia homolog 2) and androgen receptor protein levels play an important role in urothelial carcinogenesis, presumably leading to the escape of urothelial cancer cells from attack by the immune system [10]. The latter authors also demonstrated that intravesical treatment with P-MAPA downregulated Siah-2 protein levels, an essential step for histopathological recovery from cancer. In support of Phesse et al. [18] and Garcia et al. [10], the present study demonstrated that immunotherapy with P-MAPA enhanced c-Myc protein levels in a rat model of NMIBC, resulting in the downregulation of ubiquitin ligase Siah-2 and an increase in wild-type p53 levels. As a result of the increase in wild-type p53 levels, the proliferation/apoptotic ratio was remarkably lower and the BAX protein level was significantly higher in rats treated with P-MAPA, indicating a predominance of apoptosis. We have thus identified an important mechanism of action for P-MAPA, namely the mediation of c-Myc-induced apoptosis by p53; this phenomenon was not observed after treatment with BCG. Few years ago, the role of other factors in regulating p53 activity has been investigated, particularly that of COUP-TFII [14,16]. The importance of COUP-TFII in p53 signaling and its interaction with c-Myc in triggering apoptotic mechanisms in NMIBC is poorly understood. Huang et al. [14] found that COUP-TFII expression induced a distinguished accumulation of p53 protein when contrast with steady-state protein levels in HCT116 cells. This finding suggested that COUP-TFII was a bona fide agonist of the p53 transcriptional network. These authors also demonstrated that expression of COUP-TFII in avian and zebrafish developmental systems activated p53 and produced apoptosis in vivo, resulting in a phenotype like to that of p53 overexpression.
When patients exhibit COUP-TFII-positive colorectal tumors they have a better overall survival rate than with those with tumor with COUP-TFII-negative [42]. Other work also found relatively low expression of COUP-TFII in ovarian tumors in relation to healthy tissue [43]. A high COUP-TFII transcript level was related to an increased survival and its expression inhibited the TGF-β-dependent epithelial-mesenchymal transition and chemoresistance in human breast cancer [44]. Based on the latter results, Xu et al. [16] indicated that COUP-TFII serves either as a tumor promoter and suppressor in different tumor types. Here, we have demonstrated for the first time that COUP-TFII levels were significantly lower in NMIBC rats compared to healthy rats (control group). Intravesical immunotherapy with P-MAPA significantly increased these levels, which were like to those in the control group. In the BCG group, COUP-TFII levels were significantly lower compared to the P-MAPA and control groups. All of these findings indicated that COUP-TFII levels were associated with c-Myc in the activation of wild-type p53, resulting in enhanced apoptosis in NMIBC.
In conclusion, we have identified an important mechanism of action for P-MAPA based on an enhancement in wild-type p53 levels and the mediation of c-Myc/COUP-TFII-induced apoptosis by p53. This overall pathway was fundamental for histopathological recovery from cancer and for suppress abnormal cell proliferation. The action of P-MAPA in these apoptotic pathways may represent a new strategy for treatment of NMIBC.