Higher antitumor activity of vinflunine than vinorelbine against an orthotopic murine model of transitional cell carcinoma of the bladder

Urologic Oncology 7 (2002) 159–166

Original article

Higher antitumor activity of vinflunine than vinorelbine against an orthotopic murine model of transitional cell carcinoma of the bladder R. Daniel Bonfil, Ph.D.a,*, Daniela M. Russo, M.S.a, M. Mercedes Binda, M.S.a, François M. Delgado, M.D.b, Marc Vincenti, M.D.b a

Laboratory of Fundación de Investigación del Cáncer at CEFYBO, Serrano 669, (C1414DEM) Buenos Aires, Argentina b Pierre Fabre Oncologie, 45, place Abel-Gance, 92654 Boulogne Cedex, France Accepted 2 February 2002

Abstract The aim of this report was to investigate the feasibility of systemic treatment of transitional cell carcinoma of the bladder with vinflunine (VFL), and to compare its activity in respect to vinorelbine (VRL). Exposure of MB49 murine bladder cancer cells to both drugs showed a higher chemosensitivity of the cells to VRL than to VFL (IC50 values of 60 nM and 400 nM, respectively). Pretreatment of MB49 cells with non-cytotoxic drug concentrations revealed an inhibition of control in vitro invasiveness of 40 to 70% (1–25 nM VRL) and 22 to 80% (1–100 nM VFL) (P0.0001, ANOVA). The intraperitoneal administration of the drugs twice a week for 4 weeks in C57Bl/6 female mice revealed that VFL was very well tolerated, with a 8-fold increase in the maximum tolerated dose in respect to VRL (40 mg/kg and 4.8 mg/kg, respectively). The administration schedule was evaluated in C57Bl/6 female mice inoculated transurethraly with 5104 MB49 cells. Intravesical tumor incidence on day 21 was 0% and 17% in mice treated intraperitoneally with 20 and 10 mg/kg VFL respectively (P0.0017 and P0.0001, Fischer’s Exact Test), contrasting with 75–83% obtained in all VRL-treated groups and Controls. All mice treated with 20 mg/kg VFL were still alive 60 days after intravesical MB49 tumor implantation, as well as 50% of those treated with 10 mg/kg VFL, while most of the remaining mice (Control and VRL-treated) died before day 32. These studies clearly demonstrate the activity of VFL against a murine bladder cancer model, with a favorable toxicity profile. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Bladder neoplasms; Chemotherapy; Vinca lkaloids

1. Introduction Therapy for superficial bladder cancer generally involves frequent transurethral tumor resections, intravesical chemotherapy with various agents such as adryamicin, thiotepa and mytomicin, or immunotherapy with BCG [1]. However, these treatments are usually not curative. In fact, in the year 2001, the expected number of individuals that would succumb due to bladder cancer was 12,400 [2]. Recently, in an attempt to find a more effective intravesical treatment for transitional cell carcinoma (TCC) of the bladder, we carried out preclinical studies with vinorelbine (VRL), the latest clinically approved semisynthetic Vinca alkaloid. Inhibitory effects of VRL on in vitro cell proliferation and invasion of the murine TCC of the bladder cell line MB49 [3], as well as on its orthotopic tumor development in syngeneic mice [4] were observed.

* Corresponding author. Current address: Departments of Urology and Pathology, Wayne State University School of Medicine, 540 E. Canfield, Room 9105, Detroit, MI 48201. Fax 313-577-0057.

Intravesical therapy has the advantage that high drug concentrations can be delivered directly to the bladder tumor, thus minimizing the toxicity generally observed with most drugs when administered systemically [1]. Conversely, this route can induce local irritative symptoms such as moderate to severe urgency or chemical cystitis, as consequence of traumatic catheterization [1]. Vinflunine (VFL), 20,20-difluoro-3,4-dihydrovinorelbine, a derivative of VRL, showed very good systemic tolerability with lower toxicity than its precursor in animal models [5,6]. Moreover, VFL demonstrated in vitro antiproliferative effects in several murine and human tumor cell lines [7], and important in vivo antitumor activities against subcutaneous tumor grafts [5,6]. In general, mice treated systemically with VFL showed significant lower tumor growth and longer survival than those treated with VRL [5,6]. Recently, VFL demonstrated in vitro synergistic cytotoxic effect, when combined with other drugs, against human lung cancer and leukemic cells [8], and suggested possible antivascular activity when assayed in a murine transplantable colon adenocarcinoma tumor [9].

1078-1439/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S1078-1439(02)00 1 8 4 - 9

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Based on these features, VFL seems to be a good candidate for systemic treatment of various tumors, including bladder cancer, where difficulties due to intravesical therapy could be avoided. In this study our aim is to compare the effects of VFL and VRL on in vitro cell proliferation and invasion of a murine cell line derived from a TCC of the bladder, as well as the activity of both drugs when administered systemically against tumor cells orthotopically transplanted. 2. Material and methods 2.1. Cell line MB49, derived from a TCC of the bladder induced in a C57BL/6J mouse by 7,12-dimethylbenzanthracene [10], was used. It was grown as a monolayer in RPMI-1640 culture medium (Sigma, St. Louis, Missouri, USA) with 10% fetal bovine serum (GEN, Buenos Aires, Argentina) in a 5% CO2 humidified atmosphere at 37C. 2.2. Drugs VRL (MW 1079) and VFL (MW 1116) were provided by Pierre Fabre Medicament (Boulogne-sur-Seine, France). The drugs were maintained at 8C and diluted to appropriate concentrations just before use. 2.3. Chemosensitivity assay MB49 cells in exponential growth were harvested, resuspended in complete medium, and seeded in 96-well tissue culture plates at a concentration of 2.5103 cells/100 l per well. The cells were allowed to attach to the surface for 24 h and then exposed for 3 h to VRL or VFL prepared in complete medium, in final concentrations ranging from 104 to 103 M. Cells were washed with phosphate buffered saline (PBS) and then allowed to grow in drug-free complete culture medium for 48 h. At that time a modified colorimetric MTT assay [11] was used as a chemosensitivity test. Briefly, the medium of each well was aspirated and replaced with 100 l of culture medium, and then 10 l of MTT (5 mg/ml) were added. Tissue culture plates were incubated in the dark at 37C for 3 h. The medium was aspirated and 150 l ethanol 96 were added per well to dissolve the water insoluble MTT-formazan crystals formed. Plates were shaken in an ELISA reader (BioRad, USA) and the absorbance measured at 595 nm. The inhibitory concentration fifty (IC50) of both drugs was determined as the drug concentration that reduced the absorbance obtained to one half that of untreated cells (control). All experimental measurements were run in quadruplicate. 2.4. Invasion and chemotaxis assays MB49 cells treated for 3 h with non-cytotoxic concentrations of VRL or VFL, were washed and resuspended in RPMI-1640 containing 0.1% fatty acid free bovine serum albumin, BSA (Sigma). The effect of the drugs on the inva-

sive and chemotactic abilities of the tumor cells was assayed as previously described [3]. Briefly, Transwell® (Corning Costar Corporation, Cambridge, Massachusetts, USA) polyvinyl-pyrrolidone-free polycarbonate filters 8 m pore size were coated with Matrigel® (Collaborative Biomedical Products, Bedford, Maryland, USA) 25 g/filter for the chemoinvasion assay or non-coated for the chemotaxis assay. In both cases the inserts were placed in wells containing NIH.3T3 conditioned medium as chemoattractant. Treated MB49 cells resuspended in culture medium with 0.1% BSA were inoculated in the upper compartment at 3105 per well. The Transwell® chambers were incubated overnight at 37C in a humidified atmosphere with 5% CO2. After aspiration of the remaining fluid of the upper compartment, cells that were able to traverse the filter toward the chemoattractant in the lower chamber, were detached from the underneath surface with trypsin, and counted in a Neubauer chamber. All the assays were carried out in triplicate and results were expressed as the percentage of invasiveness or chemotaxis achieved by non-treated cells (control). 2.5. Animals Six to eight weeks-old female C57Bl/6 mice microbiologically tested for pathogenic murine virus and bacteria, were obtained from the Animal Care Facilities of the School of Veterinary Medicine, University of La Plata (La Plata, Argentina), or the National Academy of Medicine (Buenos Aires, Argentina). Mice were fed and watered ad libitum and housed in cabinets with controlled 12 h light/dark cycle and temperature maintained at 24C . 2.6. Determination of MTD and election of drug administration schedule VRL ditartrate and VFL ditartrate were dissolved in sterile saline (0.85% sodium chloride in distilled water) to obtain four different concentrations in each case. C57Bl/6 female mice (mean body weight of 25 g) were inoculated intraperitoneally (i.p.) with 0.2 ml of VRL dilutions of 0.6, 0.3, 0.15 and 0.075 mg/ml, achieving doses of 4.8, 2.4, 1.2 and 0.6 mg/kg body weight, respectively. Other groups of C57Bl/6 female mice received 0.2 ml inoculations i.p. of VFL dilutions of 5, 2.5, 1.25 and 0.625 mg/ml, achieving 40, 20, 10 and 5 mg/kg body weight, respectively. Two different schemes of administration were assayed during 28 days: twice a week (Mondays and Thursdays) and three times a week (Mondays, Wednesdays and Fridays). Control mice were injected with saline. Body weights and deaths were recorded throughout the study. Body weight reductions larger than 15% were considered indicative of toxicity. The administration schedule that led to 100% survival rate in at least three doses for both drugs, was chosen for experimental chemotherapy. Maximum tolerated doses (MTDs) were also included in the studies of therapeutic efficacy. The animals were randomized and each group consisted of 5 mice.

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2.7. Orthotopic tumor implantation For general anesthesia, C57Bl/6 mice were injected i.p. with 0.012 ml sodium pentobarbital (5 mg/ml) (Sigma) per gram of body weight. The animals were placed on a ground plate with their backs, previously shaved, in contact with it using an electrocardiogram conducting gel (Rigógel®, Laboratorio Rigó, Córdoba, Argentina). A 24-gauge teflon angiocatheter (Jelco®, Johnson & Johnson, Arlington, Virginia, USA) was inserted transurethrally into the bladder, and a soft-tipped cautery wire introduced through it until it reached the organ wall. Electrocauterization was achieved by connection of the guide wire to the electroscalpel unit (Model Small 970, Weros®, Argentina), applying a 5 s monopolar coagulation at approximately 20 W. Instillation of tumor cells (5104 MB49 cells/0.1 ml, in complete culture medium) was accomplished through the catheter after removing the steel wire electrode. The syringes used to inoculate the cells were not detached from the catheters and were left in place until the mice awakened (approximately time 2 h). A total number of 162 mice were electrocauterized and intravesically instilled with tumor cells. 2.8. Evaluation of antitumor activity VRL and VFL administration i.p. began one day after the MB49 tumor implantation occurred or seven days later, when the tumor is considered to be already neovascularized and to have an incipient growth. In the first case, mice were sacrificed on day 21 to compare tumor incidence (number of mice bearing intravesical tumors/number of mice transurethrally implanted with MB49 cells) and tumor volumes in all groups (12 animals each). Other groups of mice were sacrificed when considered agonizing for survival analyses (6 mice per group). Tumor volume was calculated as 4/3. . r 3, where r is half the largest tumor diameter, as measured using dial calipers. Treatment of mice in which the drug administration began seven days after tumor cells instillation, to evaluate the effect of drugs on incipient tumor growth, continued until day 60. In these groups (5 mice each) the days at which death occurred in each case were recorded to generate survival curves. 2.9. Histopathological studies Mice in which treatment was initiated on day 1, were sacrificed on day 2. Their bladders were removed, bisected and analyzed using a dissecting microscope to detect intravesical tumor growth and measure the largest and smallest diameters using dial calipers. Four bladders per group were used for histologic studies. Regional lymph nodes, lungs, kidneys and spleens were also removed to determine metastatic dissemination. Tissues were fixed in 10% neutral buffer formaldehyde, embedded in paraffin and 5–6 mthick sections placed on positively charged slides (Probe-On Plus, Fisher Scientific Co., Pittsburgh, Pennsylvania, USA), and processed for routine hematoxylin-eosin. Blind studies carried out by a pathologist, established diagnoses, extent of

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invasion of vesical muscle, if present, and mitotic index. For this purpose, at least 10 fields at 400 were analyzed to count the number of mitotic figures in “hot spot” areas. 2.10. TUNEL assay for the detection of apoptosis The TUNEL (terminal deoxynucleotidyl transferasemediated nick end labeling) assay was performed using a modification of the method originally described by Gavrieli [12,13], using the ApopTag Plus in situ detection kit (Oncor, Gaithersburg, Maryland, USA). Briefly, paraffinembedded tissue sections (four per experimental group) were deparaffinized, rehydrated and then incubated with 20 g/ml of proteinase K (Promega, Madison, Wisconsin, USA) in PBS at pH 7.4 for 15 min at room temperature, followed by microwave treatment (cycles of 10 s every 10 min during 30 min) with 0.01 M citrate buffer, pH 3. Quenching of endogenous peroxide activity was achieved with incubation with 2% hydrogen peroxide in PBS. Specific binding of digoxigenin-labeled UTP to 3-OH ends of fragmented DNA was catalyzed by terminal-deoxynucleotidyl transferase enzyme. After stopping the reaction, the slides were incubated at 4C overnight with 1:750 dilution of biotin-conjugated mouse monoclonal anti-digoxigenin antibody (Sigma), instead of the peroxidase-conjugated anti-digoxigenin provided in the kit. After washing, the sections were incubated for 45 min at room temperature with 1:100 biotinylated rabbit antimouse IgG (Dako, Carpinteria, California, USA). Sections were washed again, and incubated for 45 min at room temperature with 1:100 peroxidase-labeled streptavidin (Dako). Diaminobenzidine tetrahydrochloride (0.5 mg/ml)/ H2O2 (0.01%) was used as chromogen substrate. Methyl green was employed to obtain a light counterstain. The apoptotic index was calculated as the percentage of positive nuclei in an average of 500 cells counted per case, at 400 magnification. 2.11. Statistical analysis Fischer’s Exact Test, ANOVA, and Kruskal-Wallis nonparametric ANOVA and Dunn’s multiple comparisons tests were performed when appropriate. The Kaplan.Meier method and log-rank test were used to compare survival of treated and control animals. All of the analyses were performed using the statistics software package GraphPad Prism®, version 3.00 for Windows® (GraphPad Software, Inc., San Diego, California, USA). Differences were considered statistically significant when P0.05. 3. Results 3.1. Chemosensitivity assays To evaluate the effects of VFL and VRL on MB49 tumor cells, different concentrations of both drugs were added to exponentially growing cultures during 3 h. Two days later, the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) colorimetric assay was carried out and the

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absorbancies obtained at 595 nm were plotted as a percentage of the absorbance shown by non-treated cells (control). A dose-dependent inhibitory effect was shown by both drugs, with IC50 values of 60 and 400 nM for VRL and VFL respectively (Fig. 1A and B), revealing that VFL is approximately 7 times less cytotoxic than VRL when tested for 3 h on MB49 cells. 3.2. Effects of VRL and VFL on invasive and chemotactic activities Non-toxic concentrations of VRL and VFL, as determined by the chemosensitivity assays, were used to evaluate the effect of the drugs on the in vitro invasive ability of MB49 cells. The concentrations utilized to treat the cells were of 1, 10 and 25 nM for VRL, and 1, 10, 50 and 100 nM for VFL. Cells that were able to traverse the Matrigelcoated 8m pore polycarbonate membrane towards chemoattractant factors, were counted and expressed as a percentage of the invasive ability shown by non-treated cells (control). The results obtained in the chemoinvasion assay were plotted (Fig. 2A) and analyzed using ANOVA, showing a significantly different variation between groups (P0.0001). The Tukey-Kramer Multiple Comparisons Test revealed that all concentrations of VRL inhibited significantly the invasive capacity of MB49 cells, related to the Controls (P

Fig. 1. Effects of VRL (A) and VFL (B) on MB49 cell growth. The viable cell number was determined by MTT assay and expressed as a percentage of the absorbance shown by cells treated with vehicle, as described in “Material and Methods”. Each data point represents the mean SE of quadruplicate determinations.

0.001). Similar results were obtained with VFL, although the level of significance obtained with VRL (P0.001) was only showed by the higher doses of VFL used (50 and 100 nM). The lower doses of VFL inhibited in vitro invasion with p0.05. Experiments under similar conditions, were carried out to analyze the effects of non-toxic concentrations of both drugs on MB49 cell motility towards a chemoattractant (Fig. 2B). The chemotaxis assays were carried out similarly to the chemoinvasion assays but without coating the porous polycarbonate filter. The statistical analysis of data by ANOVA reveals that the inhibition of chemotaxis shown by treated cells with respect to the control is extremely significant (P0.0001). The comparison between groups (Tukey-Kramer Multiple Comparisons Test) shows a significant inhibition of chemotaxis demonstrated by VRL when compared to control (P0.01 for 1 nM, and P0.001 for 10 and 25 nM), although only the higher doses of VFL significantly inhibited motility (P0.001 for 50 and 100 nM). 3.3. Selection of drug doses to be used in vivo for evaluations of antitumor activity Two different schedules were designed for i.p. drug administration: twice a week and three times a week. In both

Fig. 2. Effects of drugs on in vitro chemoinvasion (A) and chemotaxis (B) of MB49 cells. After exposure to non-cytotoxic concentrations of VRL and VFL, the ability of cells to traverse Matrigel®-coated (chemoinvasion) or non-coated (chemotaxis) 8 m pore size filters towards a chemoattractant, was measured. Data are mean SE of triplicate determinations expressed as percentage of invasiveness or chemotaxis achieved by non-treated cells (control).

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Table 1 Toxicity study of two different administration schedules of VRL and VFL given i.p. in C57Bl/6 female mice Administration schedule

Duration of treatment

Drug

Twice a week (Mondays and Thursdays)

4 weeks

VRL

VFL

Dose (mg/Kg) 0.6 1.2 2.4 4.8 5 10 20 40

Largest body weight reductiona (%)

0 0 0 20 (day 26) 0 0 0 60 (days 8-9) 0

1.6 (day 6) 5.5 (day 6)

14.2 (day 14)

Saline Three times a week (Mondays, Wednesdays and Fridays)

4 weeks

VRL

VFL

0.6 1.2 2.4 4.8 5 10 20 40

Death rate (%)

0 0 0 100 (days 11, 15, 25, 23, 27) 0 0 20 (day 10) 100 (days 6, 8, 11, 13) 0

3.55 (day 8) 7.14 (day 22)

9.9 (day 14) 11.4 (day 6)

Saline a

Weight changes reported are the maximal losses in body weight in respect to the initial observed, expressed as percentage. Decrease in body weight greater than 15% was considered as a toxicity sign.

cases the same doses were used. The mice were routinely checked for signs of toxicity such as: decrease in body weight greater than 15%, diarrhea, erection of hair, difficulty in moving, and finally mortality. Table 1 summarizes the observations obtained for both administration schedules using four different doses for each of the drugs assayed. Note that the signs of toxicity included in the table (body weight reduction and death rate) were the only ones found. The doses selected were similar to those previously used by Kruczynski et al. [5]. Our aim was to test them in our animals, since differences can be found even in inbred mice of the same strain coming from different facilities. The administration of VFL three times a week resulted in different grades of toxicity at 20 and 40 mg/kg, as demonstrated by an increased mortality rate. For VRL, only the highest dose assayed (4.8 mg/kg) resulted in lethality. In the schema of twice a week only the highest doses assayed of VFL and VRL proved lethal (60% and 20% of mice, respectively). It must be emphasized that, in our hands, VFL revealed a 8- and 4-fold increase in the MTD with respect to VRL using the twice and three times administration schedules, respectively. 3.4. Evaluation of antitumor activity Based on the results obtained in the toxicities studies it was decided to use the twice a week schema, using the three non-toxic doses defined for each of the drugs under study. Initially, we investigated the effects of the drugs on tumor incidence and volume on a fixed day. For that purpose, mice orthotopically implanted with MB49 bladder tumor cells on day 0 were treated i.p. twice a week between days 1 and 21. The results obtained are shown in Table 2. The sta-

tistical analysis of tumor volumes included in Table 2 using Kruskal-Wallis nonparametric ANOVA did not show any significant differences among the groups. However, it must be emphasized that only mice in which intravesical tumors could be measured were included in the analysis. Thus, the antitumor effect produced by i.p. treatment with 10 and 20 mg/kg VFL, where only 16.6% and 0% tumor growth was observed, was not detected by this analysis. Conversely, the Fischer’s Exact Test confirmed the very significantly lower tumor take of those groups with respect to the controls. A second experiment aimed at investigating the effects of the drugs on the survival of mice orthotopically implanted on day 0 with MB49 bladder tumor cells, that were treated i.p. twice a week starting on day 1. The difference

Table 2 Antitumor effects of VRL and VFL against intravesical implanted MB49 TCC of the bladder cells Tumor incidencea (%)

Treatment Control VRL

VFL

0.6 mg/kg 1.2 mg/kg 2.4 mg/kg 5 mg/kg 10 mg/kg 20 mg/kg

10/12 (83.3) 9/12 (75) 9/12 (75) 10/12 (83.3) 10/12 (83.3) 2/12 (16.6)* 0/16 (0)**

Tumor volumeb, mm3 (median [range]) 179.5 [14.2–1436] 267.9 [14.2–972.1] 179.5 [4.2–904.3] 223.7 [4.2–523.3] 146.3 [4.2–381.6] 96.5 [14.2–179.5]

a Number of mice bearing intravesical tumors on day 21/ Number of mice transurethrally implanted with MB49 cells. b Tumor volume was calculated as 4/3. . r 2, where r is half the largest tumor diameter, as measured using dial calipers. * P0.0017, **P0.0001, Fischer’s Exact Test.

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from the previous study was that survival was used as the endpoint. The results obtained are shown in Fig. 3. The median survival for mice receiving no treatment was 21 days, and did not differ significantly from that for mice treated with VNR at doses 0.6, 1.2 or 2.4 mg/kg (21, 22 and 22 days, respectively) or with VFL 5 mg/kg (23 days). Conversely, the median survival for animals intravesically inoculated with MB49 tumor cells receiving VFL at doses 10 and 20 mg/kg was 45 and beyond 60 days, respectively. Odd ratios differed significantly from the group VFL 20 mg/kg for all other groups (P0.005, log-rank test), except for the group VFL 10 mg/kg. The latter had significantly longer mean survival than the remaining groups (P0.02, log-rank test), except for VNR 1.2 mg/kg and, as stated before, VFL 20 mg/kg. Finally, both survival and tumor incidence were analyzed in mice that were intravesically implanted with MB49 cells on day 0, and began their i.p. treatment on day 7. The results obtained (Table 3) showed no significant differences in tumor incidence (Fischer’s Exact Test), although a lower tumor take was observed in mice treated with the highest VFL dose. Conversely, mice bearing incipient bladder tumors that were treated with VFL 10 or 20 mg/kg showed significantly longer mean survival than the rest of the groups (P0.002, log-rank test). In all cases, tumor-bearing mice died as a consequence of the effects of extensive local tumor burden that can cause ureteric obstruction leading to renal failure.

Table 3 Effects of i.p. treatment with VRL and VFL on tumor incidence and survival of C57Bl/6 mice bearing incipient bladder tumors Treatment Control VRL

VFL

0.6 mg/kg 1.2 mg/kg 2.4 mg/kg 5 mg/kg 10 mg/kg 20 mg/kg

Tumor incidence (%)

Survival, days (mean SEM)

5/5 (100) 5/5 (100) 5/5 (100) 4/5 (80) 5/5 (100) 4/5 (80) 2/5 (40)

22.0 0.9 21.8 0.7 21.4 0.7 29.4 8.0 22.2 0.6 36.6 6.2* 51.8 5.6*

*P0.002 when compared to the rest of the groups (log-rank test).

3.5. Histopathological studies The results of histologic analysis confirmed the macroscopic observations. Representative histological aspects of cross sections of bladders obtained from treated and nontreated mice transurethrally inoculated with MB49 tumor cells 21 days earlier, can be seen in Fig. 4A–C. The mitotic and apoptotic indexes were calculated in the biopsies obtained. As can be seen in Table 4, the number of mitotic figures in biopsies obtained from mice treated with VFL was significantly lower than in controls. This was not observed with VRL at the doses assayed. With respect to apoptosis, no statistically significant alteration was observed in either of the groups. However, a tendency to a higher apoptotic index can be seen as the doses of each drug increased. No metastatic foci were detected in lungs, kidneys, spleens or regional lymph nodes of treated or non-treated tumor-bearing hosts. 4. Discussion

Fig. 3. Kaplan-Meier survival curves obtained from C57Bl/6 mice intravesically implanted with MB49 cells that were i.p. treated with:VRL 0.6 mg/kg (––), VRL 1.2 mg/kg (––), VRL 2.4 mg/kg (––), VFL 5 mg/ kg (––), VFL 10 mg/kg (––), VFL 20 mg/kg () or saline (----). Mice treated with VFL 20 mg/kg showed significantly longer survival than all other groups (p0.005, log-rank test), except for the group VFL 10 mg/kg. The median survival time for mice treated with VFL 10 mg/kg was significantly longer than that of the remaining groups (p0.02, log-rank test), except for VNR 1.2 mg/kg. Note that all the mice treated with VFL 20 mg/ kg were still alive by the end of the experiment. There were six animals per group.

In this study, we have compared the effects of VRL and VFL on in vitro growth and invasiveness of the murine TCC of the bladder MB49. Systemic (i.p.) administration of both drugs was also utilized to compare their effects on the orthotopic growth of tumors generated by transurethral inoculation of MB49 tumor cells in syngeneic hosts. Studies aimed at analyzing the survival of treated and non-treated mice intravesically implanted with MB49, were also carried out. Our results revealed an in vitro antiproliferative effect of both VRL and VFL on MB49 cells, with IC50 values of 60 and 400 nM, respectively. This 7-fold larger IC50 value for VFL suggests a lower cytotoxic activity of this drug than VRL on the cell line analyzed. These results agree generally with those obtained by Kruczynski et al. [5], which tested the same drugs on a panel of tumor cell lines including two human bladder TCCs (J82 and T24). In our study both drugs seemed to be very active against the MB-49 cell line used. Although the drug exposure time used by us was only of 3 h, the IC50 values were not excessively high. Non-toxic doses of VRL and VFL were used to treat MB49 cells and to study their effects on in vitro tumor inva-

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Table 4 Effects of VRL and VFL i.p. treatment on mitotic and apoptotic indexes of intravesical TCC tumors Treatment Control VRL

VFL

0.6 mg/kg 1.2 mg/kg 2.4 mg/kg 5 mg/kg 10 mg/kg 20 mg/kg

Mitotic indexa (mean SEM)

Apoptotic indexb (median [range)]

5.0 0.40 4.8 0.5 3.8 0.3 3.8 0.5 2.3 0.35* 2.0 0 *

3.6 (2.0–-9.3) 5.4 (2.4–16.5) 8.5 (4.6–17.0) 9.2 (6.3–13.5) 10.1 (5.3–15.5) 17.0 (15.1–18.5)

Mitotic figures were counted in “hot spots” in at least 10 fields at  400. The apoptotic index was calculated as the percentage of positive nuclei in an average of 500 cells counted per case, at  400 magnification. *Compared to Control P0.01, Tukey-Kramer Multiple Comparisons Test (after P0.0003, ANOVA for all groups). a

b

Fig. 4. (A) Section of a bladder from a control mouse showing an intravesical high grade TCC with total invasion of muscularis propria (400). (B) intravesical high grade TCC with focal invasion of muscularis propria in a mouse treated with 2.4 mg/kg VRL (400). (C) section of a bladder from a mouse treated with 20 mg/kg VFL, showing a normal bladder epithelium with mild dysplasia (400).

siveness. These revealed a dose-response inhibitory action in both cases. The comparison at equivalent drug concentrations showed that VRL appears to have a higher anti-invasive activity in vitro than VFL. Although this inhibitory effect could be explained, at least in part, on the basis of the

reduced cell motility generated in MB49 cells after treatment with the drugs, other mechanisms involved in tumor invasion, such as proteolytic enzymes, could also be affected. Two different administration schedules using four doses of each drug were studied in C57Bl/6 mice, to define the experimental therapy to study. In all cases the i.p. injection was the route used for systemic administration of the drugs. Based on the results obtained, we decided to use a schema in which VRL and VFL were inoculated twice a week i.p. in three doses between 0.6 and 2.4 mg/kg and 5 and 20 mg/kg, respectively. The host tolerance to VFL proved superior to that of VRL, as demonstrated by a 4- to 8-fold increase in MTD. To evaluate the effects of systemic administration of VRL and VFL on bladder cancer, we decided to implant cells from a murine TCC of the bladder transurethrally, instead of subcutaneously. We consider that the intravesical growth of the bladder neoplasm depicts in a better way the development in situ of an autochthonous bladder cancer, and can show more accurately the response of the tumor to the experimental therapy. Although the intravesical median tumor volume shown by mice treated i.p. with VFL was not significantly lower than that of control mice, their tumor incidence was inhibited in a dose-dependent manner and their survival increase was highly significant. These effects were not achieved by i.p. administration of VRL at doses tolerated by the mice. Conversely, animals that were transurethrally implanted with MB49 cells and one day later the i.p. treatment with the highest VFL dose (20 mg/kg) was initiated, had a dramatic diminution in their tumor incidence, showing a complete absence of intravesical tumor (confirmed through histopathological studies) and, as a consequence, 100% survival that continued for at least 60 days. These results, though with less power and significance, were corroborated in mice in which VFL treatment began on day 7, once tumors are already neovascularized and their growth has been initiated. In a previous publication of Hill et al. [6] VFL was shown to have antitumor activity in BXF1299, a human bladder tumor cell line, according to NCI standards (activity defined as 42% T/C ratio) [14]. However, whilst we used a

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syngeneic orthotopic tumor model, their BXF1299 tumor was a subcutaneously xenografted tumor model. Because we observed a reduction of in vitro invasiveness of MB49 cells treated with subcytotoxic doses of VFL, it would have been interesting to evaluate the effect of the drug on metastasis formation. Unfortunately, despite the observation of Gunther using the same tumor model [15], the macroscopic and microscopic analysis of all the organs studied did not reveal the presence of metastases. In conclusion, the preclinical results obtained using this tumor model indicate a clear antitumor activity of VFL against this superficial bladder cancer. The activity obtained by i.p. VFL administration was superior to that revealed previously by us with a single intravesical treatment with VRL [4]. The good overall tolerance of mice to VFL could serve to enhance the antitumor effects observed, since these cannot be obtained with i.p. treatment with VRL at similar doses, due to its toxicity. Based on our results, we believe that VFL could be a good candidate for systemic treatment of bladder cancer and this aspect deserves further investigation. Acknowledgments This work was supported by Pierre Fabre Oncologie, France. The authors greatly appreciate the help of Dr. Daniel Siguelboim in the pathological evaluation of the cancer specimens. We thank Dr. Bridget Hill for critical reading of the manuscript and helpful comments. We acknowledge Claudia Verónica Martínez for her secretarial assistance and Norma Rizzo for her technical help. R. D. Bonfil is member from the Scientific Career of CONICET (National Council of Scientific and Technical Research, Argentina). D. M. Russo and M. M. Binda are recipient s of fellowships granted by CONICET. References [1] Linehan WM, Zbar B, Leacg F, Cordon-Cardo C, Isaacs W. Cancer of the genitourinary system. In: De Vita VT, Hellman S, Rosenberg

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