ORIGINAL ARTICLE

Melatonin prevents the development of hyperplasticurothelium induced by repeated doses of cyclophosphamide

Daša Zupančič & Gaj Vidmar & Kristijan Jezernik

Received: 16 October 2008 /Revised: 14 February 2009 /Accepted: 19 March 2009 /Published online: 21 April 2009# Springer-Verlag 2009

Abstract Repeated cyclophosphamide (CP) chemotherapyincreases the risk of developing bladder cancer, whichcould be due to the extremely rapid proliferation ofurothelial cells observed in hyperplastic urothelium inducedby CP treatment. We investigated the effect of melatonin onthe development of urothelial hyperplasia induced byrepeated CP treatment. Male ICR mice were injected withCP (150 mg/kg) or melatonin (10 mg/kg) with CP once aweek for 3, 4 and 5 weeks. Transmission and scanningelectron microscopy, immunohistochemistry and Westernblot analysis were used to study the ultrastructure,apoptosis, proliferation and differentiation of urothelialcells. Repeated doses of CP caused the development ofhyperplastic urothelium with up to ten cell layers andincreased proliferation and apoptotic indices regardingKi-67 and active caspase-3 immunohistochemistry, respec-tively. Scanning electron microscopy observations, cytoker-atin and asymmetrical unit membrane immunohistochemistryand Western blot analysis showed a lower differentiationstate of superficial urothelial cells. Melatonin co-treatmentprevented the development of hyperplastic urothelium,statistically significantly decreased proliferation and apo-ptotic indices after four and five doses of CP and causedhigher differentiation state of superficial urothelial cells.

Keywords Urothelium . Cyclophosphamide . Hyperplasia .

Melatonin

Introduction

The three-layered mammalian urinary bladder urothelium isa normally very stable and highly differentiated epithelium[1]. Nevertheless, it shows a great capacity for regenerationafter a mechanically or chemically induced damage [1–3].Urothelial regeneration usually gives rise to hyperplasia andcan contribute to a wide range of neoplasms [2]. Hicks andChowaniec [2] and our unpublished data showed thatrepeated intraperitoneal doses of cyclophosphamide (CP)cause persistent urothelial hyperplasia. Cohen et al. havedeveloped a model of carcinogenesis which incorporatesseveral basic principles: cancer results from genetic errorswithin normal cells; carcinogenesis is a multi-stage process,requiring more than one genetic event; and a chance of agenetic event occurring per cell division is a finite quantityand, therefore, that increased cell division increases thechance for a genetic event [4]. Based on this model, theythen postulate that changes which lead to increased cellproliferation in the urothelium should be carefully evaluat-ed as a possible contributing factor to carcinogenesis [5–7].CP has long been known to produce severe hyperplasiafollowing cytotoxic damage to the urothelium [8, 9].However, it is also a weak complete carcinogen and afterprolonged application will produce cancer of the urothe-lium, but its hyperplastic action is probably associated withits late-stage carcinogenic activity rather than with its first-stage initiating activity [10]. In a lifelong experiment of thecarcinogen action of CP, low-dose oral application of CPled to the induction of bladder cancer in 30% to 43% of rats[11]. However, in the last few months of the experiments,

Virchows Arch (2009) 454:657–666DOI 10.1007/s00428-009-0765-3

D. Zupančič (*) :K. JezernikInstitute of Cell Biology, Faculty of Medicine,Lipičeva 2,1000 Ljubljana, Sloveniae-mail: dasa.zupancic@mf.uni-lj.si

G. VidmarInstitute for Rehabilitation,Linhartova cesta 51,1000 Ljubljana, Slovenia

repeated intraperitoneal injections of CP were given to ratsand mice and no bladder tumours were observed [2, 12].Nevertheless, it is known that patients treated withcyclophosphamide have up to a ninefold increased risk ofdeveloping bladder cancer [13, 14].

Urothelial carcinogenesis in the rat and mouse proceedsthrough a sequence of morphologic changes beginning assimple hyperplasia [15]. In the rat, it then progresses tonodular and papillary hyperplasia, while in the mouse nodularhyperplasia is considerably more common than papillaryproliferations [4]. The rat model then progress to papillomasand strongly resembles low-grade papillary urothelial neopla-sia in humans, whereas the mouse model resembles the high-grade, highly malignant disease of humans [15].

A single-dose CP treatment causes a destruction of thebladder urothelium [8, 9, 16, 17], followed by rapidproliferation of urothelial cells resulting in reversible hyper-plasia and gradual restoration of normal three-layeredurothelium [6, 18–20]. It is documented that reversiblehyperplasia is accompanied not only by an increasedproliferation activity of urothelial cells but also by anincreased number of apoptotic cells [20] and lower differen-tiation state of superficial urothelial cells [16, 21, 22]. In thehyperplastic urothelium, most of the urothelial cells undergoapoptosis within the superficial layer and are exfoliated intothe lumen of the urinary bladder [20] and subsequentlyremoved from the body with urine and lost for detection.Thus, it is important to identify apoptotic cells before classicalfeatures of apoptosis are present, which can be achieved byimmunohistochemical detection of active caspase-3 [19, 23,24]. Specific antisera allow a reliable detection of apoptoticcells without overlap with necrotic cell population [25, 26].Caspase-3 is an effector caspase and when activated byinitiator caspases, such as caspase-8, it cleaves varioussubstrates, leading to the apoptotic cell death [24, 27].

The terminally differentiated superficial urothelial cells–umbrella cells have two unique characteristics: a subapicaltrajectorial network of cytokeratins [22] and membrane–protein plaques, which cover the apical surface of theumbrella cells, forming a rigid-looking asymmetrical unitmembrane (AUM) [28–31]. Immunostaining against AUMproteins may serve as a well-defined marker of celldifferentiation, since it shows no labelling signal in theundifferentiated cells with microvilli, whereas stronglabelling in the umbrella cells with rigid-looking micro-ridges can be observed [21, 22]. Anti-AUM antibody ismade against highly purified bovine AUM and reactsstrongly with uroplakin III (47 kDa) [32]. Differentiationof urothelial cells can also be characterised by changes inthe cytokeratin (CK) expression patterns. For example, CK7 is expressed in differentiated and undifferentiated cells,whereas CK 20 is present exclusively in terminallydifferentiated umbrella cells [33–37].

In the last decade, research has specifically addressed therole of melatonin, the main secretory product of the pinealgland, in controlling the proliferation of neoplastic cells[38]. Both in vitro and on different animal models oftumorigenesis, melatonin has been found to inhibit cellproliferation and to delay tumour progression [39, 40].Most studies exploring the relationship between melatoninand oncogenesis have focused on melanoma and breastcancer, but more recently melatonin has been shown toinfluence neoplastic growth in many other cancers includ-ing carcinomas of the urinary bladder [41]. Furthermore,increasing evidence indicates that melatonin may exert itsanticancer effects not only via inhibition of cell prolifera-tion but also by stimulation of differentiation and apoptosis[39]. Moreover, it is documented that melatonin has aprotective effect against single-dose CP-induced urothelialdamage [42, 43] and a favourable impact on regenerationand restoration of normal urothelium, since it reduces thenumber of apoptotic and proliferating urothelial cells andresults in their earlier differentiation [43].

Repeated doses of CP cause the development of urothelialhyperplasia, which could lead to bladder cancer development[6]. In different studies, melatonin has been found to inhibitcell proliferation and also influence differentiation andapoptosis. The aim of this study was therefore to examinethe effect of melatonin on the development of hyperplasticurothelium and on the proliferation, apoptosis and differen-tiation of urothelial cells after repeated doses of CP.

Materials and methods

Animals and treatment

The animal experiments were approved by the Slovenianveterinary administration of the Ministry of Agriculture,Forestry and Food according to the Animal HealthProtection Act and Introductions for Granting Permits forAnimal Experimentation for Scientific Purposes. Fortyalbino ICR (CD-1) adult male mice were divided into tengroups by a simple random sampling method. They werehoused in plastic cages at 23±2°C and 50% to 60% relativehumidity. Basal diet and water were available ad libitum.

Group 1 (control) animals were injected with saline oncea week for 3 weeks and they served as controls, whereasgroup 2 (Mel) received 15 mg/kg of body weight ofmelatonin once a week for 3 weeks and served as controlfor melatonin administration only. All the animals fromgroups 3 to 10 were injected with CP (Sigma) at a dose of150 mg/kg of body weight once a week for 3, 4 and5 weeks and half of them (groups 7 to 10) also received15 mg/kg of body weight of melatonin with CP, before andthe day after CP injection. All the drugs were administered

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intraperitoneally (i.p.). Animals were sacrificed after three(3×), four (4×) and five (5×) doses of CP or melatonin andCP co-treatment (Mel+CP) and 1 month after five doses(1 m) of CP or melatonin and CP co-treatment (Mel+CP).

From each group, the bladders were processed forelectron microscopy, for active caspase-3, Ki-67, CK 7,CK 20 and AUM immunohistochemistry and for Westernblot analysis.

Electron microscopy

The tissue was fixed in 4.5% paraformaldehyde and 2%glutaraldehyde for 3 h. For transmission electron micros-copy, the samples were then postfixed in osmium tetroxide,dehydrated in ethanol and embedded in Epon. Ultrathinsections were stained with lead citrate and uranyl acetateand examined with a Jeol 100 CX electron microscope(Jeol Ltd., Tokyo, Japan). For scanning electron microsco-py, the samples were postfixed in osmium tetroxide andcritical-point dried. After sputter-coating with gold, theywere examined at 15 kV with a Jeol JSM 840 A scanningelectron microscope (Jeol Ltd., Tokyo, Japan).

Immunohistochemical detection of active caspase-3, Ki-67and AUM

The tissue was fixed in 4% paraformaldehyde overnightand embedded in paraffin. After deparaffinisation, theendogenous peroxidase activity was blocked by 3% H2O2

in methanol. For detection of Ki-67, sections weremicrowave-heated. Non-specific labelling was blocked by2.5% bovine serum albumin (Sigma, Taufkirchen, Ger-many) for Ki-67 or by 20% normal swine serum for activecaspase-3 and AUM. Sections were incubated with primarymonoclonal anti-Ki-67 (clone MIB-5, Dako, Glostrup,Denmark; 1:50 dil.), polyclonal anti-active-caspase-3(R&D Systems, Wiesbaden, Germany; 1:1,500 dil.) orpolyclonal rabbit antibodies raised against highly purifiedbovine AUM (a kind gift from Prof. T.-T. Sun, Departmentof Dermatology, New York University Medical School,New York, NY 10016; 1:10,000 dil.) overnight at 4°C. Fornegative controls, the incubation with primary antibody wasomitted or the specific primary antibody was replaced by anon-relevant antibody. For secondary antibodies, biotiny-lated rabbit anti-mouse (Dako, Glostrup, Denmark; 1:100dil.) or swine anti-rabbit immunoglobulins (Dako, Glostrup,Denmark; 1:400 dil.) were applied for 1 h at roomtemperature, followed by ABC/HRP complex (Dako,Glostrup, Denmark) incubation. After the standard DAB(Sigma, Taufkirchen, Germany) development procedure,sections were counterstained with haematoxylin and exam-ined with a Nikon Eclipse TE300 fluorescence microscope(Nikon Corporation, Tokyo, Japan).

Immunohistochemical detection of CK 7 and CK 20

The tissue was frozen and cut into 7-µm-thick cryosectionsand fixed in absolute ethanol (−20°C) for 20 min. Afterblocking with 2% BSA in PBS, incubation with primarymonoclonal anti-CK 7 (clone OV/TL 12/30, Dako, Glostrup,Denmark; 1:20 dil.) and anti-CK 20 (clone KS20.8, Dako,Glostrup, Denmark; 1:100 dil.) was performed overnight at4°C. For negative controls, the incubation with primaryantibody was omitted or the specific primary antibody wasreplaced by a non-relevant antibody. Adequate secondaryantibodies conjugated with TRITC (Sigma, Taufkirchen,Germany) were incubated for 1 h at 37°C. After washing,the sections were mounted in mounting medium Vectashieldwith DAPI (Vector Laboratories, Burlingame, CA, USA) andexamined with a Nikon Eclipse TE300 fluorescence micro-scope (Nikon Corporation, Tokyo, Japan).

Western blot analysis

Urothelial cells were scraped from the wall of the urinarybladder and homogenised in ice-cold buffer (0.8 M Tris–HCl,7.5% SDS, 1 mM phenylmethylsulfonyl fluoride). The lysatewas centrifuged and the protein concentration in the superna-tant was determined by using a BCA™ protein assay kit(Pierce, Rockford, IL, USA). Sample proteins (30 µg/lane)were size-fractionated on 12% SDS–polyacrylamide gels andthen transferred to Hybond ECL nitrocellulose membranes(Amersham Biosciences, Little Chalfont, UK) by electro-blotting. After blocking overnight at 4°C in 5% skim milk inPBS-Tween, membranes were incubated for 2 h at roomtemperature with primary polyclonal rabbit antibodies raisedagainst highly purified bovine AUM (a kind gift fromProf. T.-T. Sun, Department of Dermatology, New YorkUniversity Medical School, New York, NY 10016, USA;1:10,000 dil.) or monoclonal anti-CK 7 (Santa Cruz Biotech-nology, Inc., Santa Cruz, CA, USA; 1:200 dil.). After washingin PBS-Tween, membranes were incubated for 1 h withadequate secondary antibodies horseradish peroxidase-conjugated goat anti-rabbit (Sigma, Taufkirchen, Germany),diluted 1:1,000, or anti-mouse (Dako, Glostrup, Denmark),diluted 1:1,000. Membranes were finally probed with enhancedchemoluminescence reagent (Amersham Biosciences, LittleChalfont, UK) and exposed to X-ray films. Rabbit anti-actinantibodies, diluted 1:1,000 (Sigma, Taufkirchen, Germany),were used to confirm an equal loading of protein in each lane.

Methods and counting for quantitative analysis

Four animals of each group were used for immunohisto-chemical detection of active caspase-3 and detection ofKi-67 on paraffin sections. Urinary bladders were cut intoserial tissue sections and three sections of each urinary

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bladder were examined. Cells with a positive immunohis-tochemical reaction against active caspase-3 and Ki-67were accepted as labelled and used for scoring of apoptoticand proliferation indices, respectively. All urothelial cells pertissue section were counted and the indices for each tissuesection were defined as a percentage ratio of labelledurothelial cells to the total number of counted cells.

Statistical analysis

For each animal, the mean of the three slices was used forfurther analysis of apoptotic index and of proliferationindex. First, the distributions of both indices were plotted

for each group using boxplots. Because of departures fromnormality, and because of non-homogeneous variances, thedata were analysed using two-way analysis of variance onranks instead of on original observations, which is a robustyet efficient solution [44].

Since the effect of different doses of CP was found to behighly statistically significant, post-hoc comparisons wereperformed between different doses of CP using the Scheffeprocedure on the ranked data (it should be noted that othermethods, such as Sidak's, or Dunnet's method for plannedcomparisons with baseline, lead to equivalent conclusions).Since the effect of group was also highly statisticallysignificant, while the (non-)significance of the interaction

Fig. 1 TEM micrograph. a Necrotic cells with swollen cytoplasm and nucleus in the superficial layer of the urothelium (arrows) after five dosesof CP. b Normal urothelium after five doses of melatonin and CP co-treatment. Scale bar 5 μm

Fig. 2 Boxplot of apoptotic index distribution (thick line denotesmedian, box denotes 1st and 3rd quartile and whiskers denote thesmallest and the largest observation within 1.5 interquartile range fromthe 1st and the 3rd quartile, respectively)

Fig. 3 Boxplot of proliferation index distribution (thick line denotesmedian, box denotes 1st and 3rd quartile and whiskers denote thesmallest and the largest observation within 1.5 interquartile rangesfrom the 1st and the 3rd quartile, respectively)

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effect should be treated with caution in ANOVA on rankeddata [45], the groups with or without melatonin co-treatment were compared after different doses of CP usingthe exact Mann–Whitney test.

For morphological data (urothelial differentiationmarkers and hyperplasia appearance), which were in theform of ratings of four-point scale (from 1 = least present to4 = maximum presence), the median value of the threesections was calculated and analysed in the same way asthat for apoptotic and proliferation indices.

Results

In control and melatonin-treated animals, the urotheliumwas composed of three cell layers and was ultrastructurallynormal. Repeated CP treatment at 3, 4 and 5 weeks causedthe development of hyperplastic urothelium with up to tencell layers. After three, four and five doses of CP,prominent dilatations in the intercellular spaces were

observed and some necrotic cells were perceived in thesuperficial layer of the urothelium (Fig. 1a). Melatonin co-treatment resulted in the normal appearance of theurothelium (Fig. 1b). One month after five doses of CPand 1 month after five doses of melatonin and CP co-treatment, the urothelium was composed of three cell layersand was ultrastructurally similar to normal urothelium.

Active caspase-3-positive and Ki-67-positive cells incontrol and melatonin-treated animals were extremely rareand the mean for all indices was zero (Figs. 2 and 3). Afterrepeated doses of CP (3×, 4× and 5×), substantial elevationof apoptotic and proliferation index was detected, whilemelatonin co-treatment resulted in much less apoptotic andproliferation index increase (Figs. 2 and 3). An activecaspase-3 and Ki-67 reaction positive signal was observedmainly in the cytoplasm of the superficial urothelial cells andin the nuclei of the basal cell layer, respectively (Figs. 4 and 5).One month after five doses of CP and 1 month after fivedoses of melatonin and CP co-treatment, the apoptotic andproliferation indices returned to near the initial zero level.

Fig. 4 Active caspase-3 immunohistochemistry. a Active caspase-3-positive cell (brown) in the urothelium after four doses of CP and b afterfour doses of melatonin and CP co-treatment. Scale bar 10 μm

Fig. 5 Ki-67 immunohistochemistry. a Ki-67-positive cells (brown nuclei) are most abundant in the basal layer of the urothelium after four dosesof CP and b after four doses of melatonin and CP co-treatment. Scale bar 10 μm

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Statistical analyses of urothelial differentiation markersand hyperplasia appearance are summarised in Table 1.They show that CK 7 was present in the urothelium of allgroups of animals. CK 7 labelling was observed in all celllayers of hyperplastic urothelium and normal three-layeredurothelium after repeated doses of CP and after repeatedmelatonin and CP co-treatment, respectively (Fig. 6a, b).Western blot analysis confirmed the equal presence of CK7 in all samples (Fig. 7). All superficial urothelial cells ofcontrol, melatonin-treated and 1 month after five doses ofCP or melatonin- and CP-treated animals were CK 20positive and AUM positive (Table 1). After repeated dosesof CP, CK 20 labelling of superficial cells was completelynegative (Fig. 6c), while some superficial cells were CK20 positive after melatonin and CP co-treatment (Fig. 6d).Labelling with antibodies against AUM exhibited a similarpattern to CK 20 labelling. Weak anti-AUM staining wasapparent after repeated doses of CP (Fig. 6e), whereasmelatonin and CP co-treatment resulted in stronger AUMstaining of the superficial urothelial cells (Fig. 6f). Theresults of Western blot analysis proved the higher

expression of UP III (detected with anti-AUM antibody)in urothelial cells after four and five doses of melatoninand CP co-treatment than after four and five doses of CP(Fig. 7). Scanning electron microscopy revealed that cellswith microvilli and ropy ridges were present on theluminal surface of the urothelium after repeated doses ofCP (Fig. 6g), while cells with microridges were rare. Co-treatment with melatonin diminished the appearance ofmicrovilli on the luminal surface and resulted in prevailingof cells with ropy ridges and microridges (Fig. 6h).

Table 1 Summary of the statistical analyses of differentiation markers and hyperplasia appearance in urothelial cells after repeated doses of CPand after repeated melatonin and CP co-treatment

Mean (range) p values for effects ofdose, group andinteraction

Summary of post-hoccomparisons regard-ing CP doseaControl/

Mel3× 4× 5× 1m

CK 7 CP 4.0 (4–4) 4.0 (4–4) 4.0 (4–4) 4.0 (4–4) 4.0 (4–4) NA All CP doses equalMel +CP

4.0 (4–4) 4.0 (4–4) 4.0 (4–4) 4.0 (4–4) 4.0 (4–4)

CK 20 CP 4.0 (4–4) 1.0 (1–1)b 1.0 (1–1)b 1.0 (1–1)b 4.0 (4–4) <0.001, <0.001, <0.001 4×, 5×, 3× < Control/Mel, 1 mMel +

CP4.0 (4–4) 2.2 (2–3)b 2.0 (2–2)b 2.0 (2–2)b 4.0 (4–4)

AUM CP 4.0 (4–4) 2.2 (2–3) 2.0 (2–2)b 2.0 (2–2)b 4.0 (4–4) <0.001, <0.001, <0.001 3×, 4×, 5× < Control/Mel, 1 mMel +

CP4.0 (4–4) 2.2 (2–3) 3.0 (3–3)b 4.0 (3–3)b 4.0 (4–4)

Microvilli CP 1.0 (1–1) 3.5 (3–4)b 2.0 (2–2) 2.0 (2–2) 1.0 (1–1) <0.001, 0.001, 0.005 Control/Mel, 1m<5×, 4× < 3×Mel+

CP1.0 (1–1) 2.2 (2–3)b 1.8 (1–2) 1.5 (1–2) 1.0 (1–1)

Ropyridges

CP 1.0 (1–1) 2.8 (2–3) 2.5 (2–3) 3.0 (3–3) 1.0 (1–1) <0.001, 0.009, 0.039 Control/Mel, 1m<3×, 4×, 5×Mel +

CP1.0 (1–1) 2.0 (2–2) 2.5 (2–3) 2.2 (2–3) 1.0 (1–1)

Microridges CP 4.0 (4–4) 1.0 (1–1)b 1.5 (1–2)b 2.0 (2–2)b 4.0 (4–4) <0.001, <0.001, <0.001 3×, 4×, 5× < Control/Mel, 1 mMel +

CP4.0 (4–4) 3.5 (3–4)b 3.2 (3–4)b 3.5 (3–4)b 4.0 (4–4)

Hyperplasia CP 1.0 (1–1) 3.0 (3–3)b 3.0 (3–3)b 3.2 (3–4)b 1.0 (1–1) <0.001, <0.001, <0.001 Control/Mel, 1m<5×, 4×, 3×Mel+

CP1.0 (1––1) 1.8 (1–2)b 1.2 (1–2)b 1.0 (1–1)b 1.0 (1–1)

Because the sample size is four in all cells, the distribution of scores is completely captured by mean and range

NA not applicablea Post-hoc comparisons regarding dose are summarised using homogeneous subsets, which are separated by the < (less than) symbol, wherebyordering within homogeneous subsets corresponds to observed means across the two groups. For example, for CK 20, scores were significantlyhigher at baseline and after 1 month then after all three CP doses, whereby observed scores were higher on average at 1 month than at baseline(though not significantly), while the average score was the lowest after 3× dose, somewhat higher after 4× dose, followed by 5× dose (with thosedifferences not appearing to be significant)b Significant difference between groups for the given dose

Fig. 6 Differentiation markers. a CK 7 labelling in hyperplasticurothelium after five doses of CP. b CK 7 labelling in normal three-layer urothelium after five doses of melatonin and CP co-treatment. cNo CK 20 labelling in urothelium after three doses of CP. d Positive CK20 labelling in superficial cells of urothelium after three doses ofmelatonin and CP co-treatment. e Weak AUM labelling of superficialurothelial cells after four doses of CP. f Strong AUM labelling of allsuperficial cells after four doses of melatonin and CP co-treatment. gAfter five doses of CP, urothelial cells are covered by microvilli (arrow)and ropy ridges (arrowhead). h After five doses of melatonin and CPco-treatment, cells are covered by microridges. Scale bar 10 μm

b

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The distributions of apoptotic index and proliferationindex are depicted in Figs. 2 and 3, respectively.

For apoptotic index and for proliferation index, the maineffects of different doses of CP and of CP only or melatoninand CP co-treatment were statistically significant (p<0.001from two-way ANOVA on ranked data), while theinteraction effect was not (p=0.161 and p=0.053 forapoptotic and proliferation indices, respectively).

Post-hoc comparisons between different doses of CPindicate that, for both apoptotic and proliferation index, theaverage does not differ statistically significantly betweencontrol/Mel and 1-m groups (p=0.605 and p=0.175 forapoptosis and proliferation index, respectively), while thatlevel differs significantly from the 3×, 4× and 5× doses ofCP (p<0.05), which, in turn, do not differ significantlyamong themselves (p>0.05).

Comparisons between the CP and Mel+CP groups afterdifferent doses showed that the groups differed mostnotably at 4× and 5× (p=0.029 for apoptosis andproliferation index at both doses). They differed less at 3×without reaching statistical significance (p=0.029 forapoptosis and proliferation index), they were practicallyequal at 1 m (p=0.686 and p=0.200 for apoptosis andproliferation index, respectively), and they were identical atthe onset of the study as expected (p=1.000 for bothindices). It should be noted that the p values forcomparisons between groups after different doses of CPmight have been adjusted for multiple tests, but they areconservative enough because of data aggregation (and thusa reduced sample size, which is very small).

Discussion

Hicks and Chowaniec [2] and our previous unpublishedexperiments showed that repeated doses of CP causeurothelial hyperplasia. In the present study, we demonstrat-ed that melatonin prevents the development of hyperplasticurothelium induced by repeated CP treatment.

Cohen and co-workers [4–7, 46, 47] proposed a modelof carcinogenesis which takes into account the proliferativeeffects of agents in carcinogenesis. In the current experi-ment, we demonstrated that urothelial hyperplasia inducedby repeated-dose CP is accompanied by increased prolifer-ation activity of urothelial cells. Cohen et al. assumed that,every time a cell undergoes a cell division, there is a lowprobability, but not zero, of a genetic error occurring [4, 6].It is, in general, accepted that at least two irreversiblegenetic events must occur for tumour formation. Thus, theextremely rapid proliferation of urothelial cells seenfollowing repeated CP treatment greatly increased thelikelihood of at least one genetic error occurring (initiation).If the increased cell proliferation would be continued for along period of time, the likelihood of a second promotingevent would increase sufficiently that a significant inci-dence of tumours would occur. Persistent high-proliferationindices observed after repeated CP doses could increase theprobability of bladder carcinogenesis and therefore explainthe fact that patients treated with repeated CP chemotherapyhave up to ninefold increased risk of developing bladdercancer [13, 14].

In numerous experiments in vitro and in different animalmodels of tumorigenesis, melatonin has been found toinhibit proliferation and neoplastic growth [38, 40, 48].Some studies also documented that melatonin reduced thecell proliferation in neoplastically unchanged cells [49–51].It is therefore not surprising that, in the present experiment,melatonin significantly decreased the proliferation activityof urothelial cells and therefore prevented hyperplasia fromdeveloping. Since the rapid proliferation of urothelial cellsseen after repeated doses of CP could increase thelikelihood of bladder carcinogenesis [4–7], melatonin mightprevent the development of bladder tumours by decreasingthe proliferation activity of urothelial cells.

Experimentally induced urothelial hyperplasia is accom-panied by an increased number of apoptotic cells [20, 52–54]. It happened similarly in the present experiment, sincenot only increased proliferation but also increased apoptotic

Fig. 7 Western blot analysis of CK 7 and AUM. CK 7 (54 kDa) isequally present in urothelial cells after repeated doses of CP and afterrepeated doses of melatonin and CP co-treatment. After four and fivedoses of melatonin and CP co-treatment, the expression of UP III

(47 kDa; detected with anti-AUM antibody) is higher than after fourand five doses of CP. The immunoblot experiments were performedtwice with similar results. MW molecular weight standards

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indices were found in the hyperplastic urothelium afterrepeated doses of CP. Melatonin co-treatment significantlydecreased not only the proliferation but also the apoptosisof urothelial cells. Numerous reports documented the roleof melatonin in inhibiting apoptosis in different cells afterischemia [55–57], hyperoxia [58] or oxidative stress [59].Moreover, melatonin also decreased the apoptosis ofurothelial cells after a single-dose CP treatment [43]. Allthese experiments investigated the acute injury in whichmelatonin diminished cell death, while in the present studythe anti-apoptotic effect of melatonin was observed inchronic hyperplastic state. It is possible that since melatonininhibited the proliferation activity of urothelial cells andprevented the development of hyperplastic urothelium,decreased apoptosis of urothelial cells appeared per se andnot as a consequence of direct melatonin action.

The scanning electron microscopy observations andexpression of urothelial key differentiation markers, the CK7, CK 20 and AUM, showed that the differentiation state ofsuperficial urothelial cells of hyperplastic urothelium, inducedby repeated doses of CP, was lower than in control andresembled the differentiation of superficial urothelial cellsduring reversible hyperplasia following the destruction of theurothelium after a single dose of CP [16, 21, 22, 60].Melatonin co-treatment resulted in the higher differentiationstate of superficial urothelial cells as indicated by theappearance of urothelial apical surface and confirmed bythe expression of analysed differentiation markers. Theseresults are similar to a variety of in vivo and in vitroexperimental models of neoplasia, in which evidence wasshown that melatonin influences the differentiation of tumourcells [39, 61]. Although it is documented that melatonin iscapable of promoting differentiation of neoplastically un-changed osteoblasts and ovarian cells [49, 62], we do notknow whether melatonin influences urothelial cell differen-tiation directly or through its preventive effect against CP-induced hyperplasia. Further studies will be needed to gain acomplete insight into this question.

In conclusion, our results document that repeated dosesof CP cause the development of hyperplastic urotheliumwith rapid proliferation of urothelial cells, which could leadto an increased risk of bladder cancer. Melatonin co-treatment prevents the development of hyperplastic urothe-lium, statistically significantly decreases proliferation andapoptotic indices and causes higher differentiation state ofsuperficial urothelial cells. Despite the lack of clinicalstudies testing its clinical relevance, the present resultssuggest that supplementing patients treated with repeatedCP chemotherapy with adjuvant melatonin therapy maydecrease their risk of developing bladder cancer.

Acknowledgement The authors would like to thank Prof. T.-T. Sunfor providing the anti-AUM antibody used in this study.

Conflict of interest statement We declare that we have no conflictof interest.

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