Keywords: platinum anti-cancer drugs, pharmacokinetics, Wistar rat

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Petr Sova 1 , Adolf Mistr 1 , Ales Kroutil 1 , Martin Semerád 1 , Hana Chlubnová 1 , Veronika Hrusková 1 , Jirina Chládková 2 , Jaroslav Chládek* 3 (1) R&D, PLIVA-Lachema a.s., Brno, Czech Republic, (2) Departments of Pediatrics, (3) Pharmacology and Biochemistry, Charles University in Prague, Faculty of Medicine in Hradec Kralove, Czech Republic Introduction Platinum compounds are very effective anticancer drugs used against solid tumours such as testicular, ovarian, lung, head and neck cancers [START_REF] Lebwohl | Clinical development of platinum complexes in cancer therapy: an historical perspective and an update[END_REF]. Research has focused on new platinum drugs with improved efficacy, which can overcome resistance to currently used drugs.

Another objective is to develop platinum compounds suitable for oral administration which could further increase the therapeutic use of platinum anti-cancer drugs due to flexible and convenient dosing [START_REF] Kelland | The resurgence of platinum-based cancer chemotherapy[END_REF]. Satraplatin [(OC-6-43)-bis(acetato)amminedichloro(cyclohexylamine) platinum(IV)] (also known as JM216), is the first orally administered platinum complex with documented efficacy and acceptable safety in patients with hormone-refractory prostate cancer and small-cell lung cancer [START_REF] Choy | Current status and future prospects for satraplatin, an oral platinum analogue[END_REF].

LA-12 (Figure 1), [(OC-6-43)-bis(acetato)(1-adamantylamine)amminedichloroplatinum(IV)], is a novel platinum(IV) complex with a bulky adamantylamine ligand which has demonstrated higher cytotoxicity in vitro than that of cisplatin and no cross-resistance [START_REF] Zak | Satraplatin activation by haemoglobin, cytochrome C and liver microsomes in vitro[END_REF]5] and also higher antitumor efficacy than satraplatin on human tumor xenografts [6].

Importantly, LA-12 proved role in apoptosis which is not fully dependent on protein p53. The results confirm its therapeutic potential as a very promising anticancer agent targeting both p53+ and p53-cells [7]. Nowadays, LA-12 undergoes clinical testing.

The aim of the present study was to compare the pharmacokinetics, urinary excretion and tissue distribution of platinum in Wistar rats after single oral doses of LA-12 or satraplatin.

Both platinum derivatives were administered to male Wistar rats as suspensions in methylcellulose at four equimolar doses within the range of 37.5 mg to 300 mg LA-12 /kg body weight.

Materials and methods

Che micals and solutions

LA-12 and satraplatin (Fig. 1) were prepared in PLIVA-Lachema a.s. using methods described previously [START_REF] Zak | Satraplatin activation by haemoglobin, cytochrome C and liver microsomes in vitro[END_REF]8]. Nitric acid and phosphoric acid were Suprapur grade from Merck (Darmstadt, Germany). Magnesium nitrate was obtained from Lachema (Brno, The Czech Republic). Triton X-100 was purchased from Sigma-Aldrich (Prague, The Czech Republic). Double deionized water of 18 M cm-1 specific resistivity, obtained in a Milli-Q Plus Millipore system, was used to prepare all the solutions. A 10 mg/mL primary standard solution of platinum was Titrisol Merck. A stock standard solution of 0.1 mg/mL was prepared by dilution of the primary standard solution with 100 mL water after the addition of 0.1 mL nitric acid. Modification solution contained 2 mL nitric acid, 2 mL phosphoric acid, 3 mL Triton X-100 and 5 g magnesium nitrate in 1 L of water. Diluted modification solution was a mixture of 100 mL of the modification solution and 100 mL water.

Animals and dosing

Male albino Wistar-Hahn rats (6-8 weeks) weighing (235-268) g were kept under a 12 h light/dark cycle with free access to water and the pelletized standard diet for 13 days prior to experiments for acclimatization. All animal protocols were approved by the Institute's Animal Experimentation Ethics Committée and animals were treated according to OECD guidelines.

In the morning after overnight fast, the platinum compounds under study were administered by a gastric gavage in a volume of 1 ml/kg of body weight as suspensions in a 0.6 % solution of methylcellulose in water. Rats were given four doses of LA-12 from the range of 37.5 -300 mg LA-12 /kg body weight or four equimolar doses of satraplatin.

Sampling for pharmacokinetics

Rats were randomly assigned to eight dosing groups of 18 rats each and, furthermore, to two of 12 blood sampling intervals (pre-dose and at 15 min, 0.5 h, 1 h, 1.5 h, 2 h, 3 h, 4 h, 8 h, 24 h, 48 h and 72 h). Moreover, rats from six additional groups of four animals each were kept at metabolic cages and urine was collected until 72 h. Two blood samples of 2 mL each were taken from retroorbital plexus under ether anesthesia. Blood was collected into polypropylene test-tubes containing 0.75% K 3 EDTA (20 μL/mL of blood) K 3 EDTA and cooled in a water bath (8 ºC) for 5 min. The samples were then centrifuged in a cooled centrifuge (8 ºC) at 3000 g for 10 min. The first aliquot (0.1 mL) of the supernatant was immediately frozen at -18 °C.

The remaining volume was transferred into an ultrafiltrate filter (Ultrafree-CL, 30 kD, Millipore, Prague, Czech Republic) and centrifuged in a cooled centrifuge (8 ºC) at 2000 g for 30 min. Ultrafiltrate was pipetted into a polypropylene test tube and immediately frozen at -18 °C. After the second blood sampling interval, rats were killed by withdrawing whole blood from the abdominal artery. Liver and kidney were rinsed with 0.9% NaCl, blotted on filter paper, weighed, and immediately frozen at -18 °C.

Platinum analysis

Platinum concentration was determined by a validated method based on electrothermal atomic absorption analysis with Zeeman background correction. An AAnalyst 800 spectrometer (Perkin-Elmer, Norwalk, CT, USA) with longitudinal AC Zeeman-effect background correction with transversely heated graphite tube (THGA™) and autosampler AS 800 was used. The tubes were coated with pyrolytic graphite and equipped with an integrated L'vov platform. The graphite furnace temperature program for platinum was as follows: 110 °C for 30s, 130 °C for 30 s, 1300 °C for 20 s, 2 200 °C for 6 s (peak height absorbance reading), and 2 450 °C for 3s.

Samples of biological fluids (plasma, ultrafiltered plasma, urine) were diluted with 0.15% modification solution prior analysis. Solid samples (kidney, liver) were homogenized and weighted into PTFE vessel. Then, nitric acid (6 mL), hydrochloric acid (2 mL), hydrogen peroxide (1 mL) and hydrofluoric acid were added. Vessels were fixed into high pressure rotor bodies and digested by temperature program: 10 minutes increasing up to 200 ºC, 20 minutes holding at 200 ºC. After cooling down, solutions were transferred into volumetric flasks and diluted with water.

The linearity of the calibration lines for biological fluids was confirmed (r 2 >0.999) over the range of 0.01-0.50 mg/L. Based on the assayed concentrations of spiked quality control samples (0.06, 1.0, 5.0 mg/L diluted to linear calibration range of 0.02, 0.20, 0.20 mg/L), the inter-day accuracy and precission were adequate. The relative errors were between -2.4 and 16.7 % and the coefficients of variation were less than 6.4 %. No effect was obsered of the storage of plasma, ultrafiltrate and urine at -18 oC for up to 3 months and of four freeze/thaw cycles on the accurate determination of platinum (relative diferencess less than ±15%). Similarly, storage of biological fluids for 3 h at room temperature resulted in acceptable differences from -0.37 % to -6.1 %. Calibration standards of platinum tissue concentrations were prepared using standard additio n method. The assay was linear in the range of 0.02-0.50 mg/L. Accuracy of liver platinum determination at three concentrations (0.05, 0.075 and 0.10 mg/L) was characterized by the relative errors from -10.4% to -2.2%. Repeated analyses of liver samples spiked with 0.05 mg/L platinum resulted in the coefficient of variation less than 2%.

Data analysis

Pharmacokinetic modelling was performed using the Kinetica software, version 4.0 (InnaPhase Corporation, Thermo Fisher Scientific Inc.Waltham, MA, USA). In the first step, population values for parameters of compartmental models were estimated using plasma platinum (ultrafiltrate platinum) concentrations of all animals. In the second step, individual estimates of pharmacokinetic parameters were obtained according to the maximum a posteriori Bayesian fitting method. Non-compartmental pharmacokinetic analysis (NCA) was performed also. For each compound and dose level, data from 18 animals were combined into a concentration-time profile using naive data pooling (3 animals per sampling interval).

Maximum concentration in plasma (C max ) and time to maximum concentration (T max ) were determined directly from the observed data. The area under the plasma (ultrafiltrate) platinum concentration-time curve from zero up to the last concentration above the quantification limit (AUC 0-t ) was calculated by the linear trapezoidal method. The area under the plasma (ultrafiltrate) platinum concentration-time curve from zero up to infinity (AUC 0-∞ ) was determined as the sum of the AUC 0-t and of the extrapolated part AUC extra , i.e. the ratio of the concentration predicted at the time interval t and the terminal rate constant λ z . The terminal rate constant was estimated by linear regression analysis of ln C vs. time data using the last three concentrations above the lower limit of quantification. Oral clearance (CL/F) was calculated by dividing the dose of platinum with the AUC 0-∞ . Apparent volume of distribution at equilibrium after oral administration (V ss /F) was calculated using the formula: V ss /F = (dose.AUC)/AUMC, where AUMC is the area under the first moment of the concentrationtime curve from zero up to infinity. Apparent volume of distribution during terminal phase after oral administration (V Z /F) was obtained as follows: V Z /F = dose/(AUC 0-∞ . λ z ).

Statistical analysis was performed using GraphPad Prism, version 5.0 (GraphPad Software, San Diego, California). Platinum concentrations and all pharmacokinetic characteristics were log transformed before testing. Dose-related changes in pharmacokinetic characteristics and their comparison between LA-12 and satraplatin were evaluated using two-way analysis of variance with comound, dose and compound×dose as fixed effects. Tissue concentrations afer corresponding doses were compared between compounds using analysis of variance for repeated measures. When a significant effect was found, the Tukey test was used to compare the means. For all statistical procedures, P values < 0.05 were taken as significant.

Results

The pharmacokinetics of plas ma platinum and ultrafiltrate platinum

The mean concentration-time profiles of plasma platinum and ultrafiltrate platinum are shown in Fig. 2. Platinum was detected in all plasma samples 15 min after oral administration of both compounds. The concentration-time curves were flat and peaked between 1.5 and 4 h for both compounds after all doses with the exception of 75 mg/kg satraplatin (8 h). After 72h, plasma platinum decreased to a value in the range of 10 to 20 % of the maximum concentration.

Maximum concentrations of ultrafiltrate platinum were observed between 1-3 h.

Ultrafilterable platinum decreased more rapidly than plasma platinum. Within 8 h, its concentration dropped below 10% of the maximum concentration or it was less than the lower limit of quantification (0.01 mg/L) (Fig. 2).

Based on the statistical evaluation and graphical inspection of goodness-of-fit plots, plasma platinum concentrations were best described with an open two-compartment model with first-order absorption and elimination from the central compartment. Individual predicted concentrations of both compounds agreed well with assayed values (data not shown).

Pharmacokinetic parameters of plasma platinum obtained by population modelling and posthoc Bayesian estimation are summarized in Table 1. Ultrafiltrate platinum concentrations were well described with an open one-compartment model with first-order absorption and elimination from the central compartment (data not shown). The pharmacokinetic characteristics are listed in Table 2. Pharmacokinetic parameters estimated using noncompartmental analysis of the mean concentration-time profiles of plasma and ultrafiltrate platinum are given in Table 3.

After administration of both compounds, concentrations of plasma platinum and derived characteristics of systemic platinum exposure (C max and AUC) increased less than in proportion to the 8-fold increase of the dose (Tables 1 and3). Similar for LA-12 and satraplatin, the pharmacokinetic parameters CL/F and V d raised with the dose (P<0.001, one-way ANOVA). In the whole range of doses, the AUC of plasma platinum was higher after satraplatin (P<0.001). The values of ultrafiltrate platinum CL/F and V d showed a similar dose-dependency as those of plasma platinum (Table 2 and3). However, administration of LA-12 as compared to satraplatin resulted in significantly higher AUC values of ultrafiltrate platinum after the doses of 150 mg and 300 mg/kg, respectively. The C max values were significantly higher starting from the the dose of 75 mg/kg LA-12 and upward (Fig. 3).

Urinary excretion of platinum

Excretion of platinum in urine is shown in Fig. 4. After all doses, highest amounts of platinum were found in the first of the three 24-h intervals of collection. The mean 72-h urinary recovery of platinum in urine was less than 5% regardless of the dose and compound. After administration of satraplatin, the 72-h urinary recovery of platinum decreased with the dose (P<0.01). For LA-12, a numerical decrease was observed that did not reach statistica l significance (P=0.41) (Fig. 4). Renal clearance of platinum showed no dose-dependency and was comparable (P>0.05) for both derivatives (Table 2).

Platinum in the kidney and liver

After the doses less than 150 mg/kg, platinum in the kidney and tissue homogenates peaked between 2 and 4 h and than decreased slowly. After higher doses, tissue distribution of platinum was delayed and secondary peaks appeared on the concentration-time curves at 24 h or 72 h (Fig. 5). Kidney tissue concentrations of platinum and derived characteristics AUC 0- 72h and C max (Fig. 6) were comparable between LA-12 and satraplatin after doses of 37.5 -150 mg/kg but the highest dose of LA-12 resulted in markedly higher concentrations. Platinum concentrations and AUC 0-72h and C max values in the liver homogenate were approximately two-fold higher after LA-12 accross all four doses. In both organs, the characterstics of platinum exposure increased in proportion to dose only after the doses of 37.5 mg/kg and 75 mg/kg, respectively. With further dose escalation, they augmented less (Fig. 6).

Discussion

This first direct comparison of LA-12 to satraplatin shows that in the range of doses under study (37.5 mg/kg -300 mg/kg), the pharmacokinetics of plasma and ultrafiltrate platinum, urinary excretion and tissue distribution of platinum are similar after a single oral administration of both compounds to Wistar rats. For both platinum derivatives, the characteristics of platinum exposure (C max and AUC) evaluated in the plasma, plasma ultrafiltrate and tissues increased less than in proportion to dose. Urinary recovery of platinum expressed as a fraction of the dose decreased with increasing dose of satraplatin. For LA-12, it displayed a decreasing trend but the differences did not reach statistical significance.

Moreover, the pharmacokinetic characteristics V/F and CL/F of plasma and ultrafiltrate platinum displayed similar dose-dependent increases. These findings are consistent with a dose-related decrease in the extent of oral bioavailability (F) of LA-12 and satraplatin most likely due to saturable intestinal absorption. Nonlinear pharmacokinetics of plasma and ultrafiltrate platinum has been previously reported in mice for oral satraplatin after doses ranging from 9.5 to 200 mg/kg and in a phase I study after doses from 60 to 700 mg/m 2 , respectively [9, 10].

LA-12 molecule was designed with the purpose of its enhanced penetration and higher accumulation in cancer cells due to increased lipophilicity caused by incorporation of bulky hydrophobic ligand -adamantylamine. This presumption was confirmed by the higher cytotoxicity and increased platinum levels in cancer cells in comparison with other platinum based antitumor drugs [5,11,12]. The range of doses investigated is therapeutically relevant as shown in the study directly comparing anti-tumour activities of LA-12 and satraplatin in vivo in mice using the human carcinoma xenografts of colon HCT116, prostate PC3, and ovarian A2780 and A2780/cisR (resistant to cisplatin). The dailyx5 repeated dose regimen of equimolar doses of LA-12 and satraplatin, administered in two cycles, was selected for this evaluation. The highest effect was reached with LA-12 at a dose of 60 mg/kg. LA-12 was able to overcome resistance to cisplatin of the ovarian carcinoma A2780 subline. The activities of LA-12 in all doses and all used tumor xenografts were higher than equimolar doses of satraplatin [6]. In another study, 135 mg/kg satraplatin p.o. (q7dx4 schedule) exhibited comparable activity to that observed for i.v. cisplatin and carboplatin across four human ovarian carcinoma mice xenografts of widely differing sensitivity to cisplatin and carboplatin [13].

Platinum anticancer drugs rapidly form a variety of reactive intermediates, which bind irreversibly to constituents of blood and plasma as well as in tissues. Investigating the pharmacokinetics of the parent compound and its metabolites is, therefore, technically difficult due to problems concerning sample handling and sensitivity and specificity of analytical methods. Efficient hyphenated bioanalytical techniques such as high performance liquid chromatography combined with inductively coupled plasma mass spectrometry (HPLC-ICPMS) and HPLC-tandem mass spectrometery (HPLC-MS/MS) are increasingly used in speciation of platinum for assessing stability, biotransformation, and protein and red blood cell binding of platinum drugs both in vitro and in vivo [14,15]. The analysis of samples from in vivo and in vitro studies with satraplatin with the help of high performance liquid chromatography coupled with mass spectrometry (HPLC-MS) and HPLC-off-line AAS demonstrated that satraplatin underwents rapid and extensive biotransformations by reduction to the platinum(II) compound (JM118), and by hydrolysis to other metabolites (JM383, JM518, JM559). Using these techniques, very low (in mice) or undetectable (in patients) concentrations of the parent drug were described in the plasma ultrafiltrate after oral administration of satraplatin [16,17]. According to results of HPLC-ICPMS analyses, satraplatin concentrations decrease rapidly with the initial half-life of 6.3 min during in vitro incubation at 37 o C in human blood. Satraplatin-derived Pt becomes rapidly and irreversibly bound to the red blood cell membrane [18]. After oral administration of satraplatin, these processess most likely proceed to a significant extent before blood sampling. The parent drug is reduced by haemoglobin and possibly other heme proteins and its reduction products attack proteins and low-molecular-weight compounds [19]. The stability of satraplatin and JM118 is significantly higher during incubation at 37 o C in human plasma. The compounds disappear from plasma with half-lives of 2.5 h and 1.1 h, and the rate of irreversible protein binding of platinum is characterized with the half-life of 5.2 h and 1.1 h, respectively [START_REF] Bell | Co mparative protein binding, stability and degradation of satraplatin, JM118 and cisplatin in human plasma in vitro[END_REF].

In the present study, measurements of platinum in biological fluids and tissues were performed using flameless AAS. This strategy is generally accepted for the investigation of body exposure to platinum after administration of platinum complexes [START_REF] Lebwohl | Clinical development of platinum complexes in cancer therapy: an historical perspective and an update[END_REF] and the same approach was used in several in vivo studies with satraplatin [9,10, [START_REF] Raynaud | Metabolism, protein binding and in vivo activity of the oral platinum drug JM216 and its biotransformation products[END_REF][START_REF] Vouillamoz-Lorenz | Pharmacokinetics of satraplatin (JM216), an oral platinum (IV) complex under daily oral administration for 5 or 14 days[END_REF][START_REF] Kurata | Pharmacokinetic and pharmacodynamic analysis of bis-acetato-ammine-dichlorocyclohexylamine-platinum(IV) (JM216) administered once a day for five consecutive days: a phase I study[END_REF]. The concentration of plasma platinum is a sum of ultrafilterable platinum and platinum irreversibly bound to plasma proteins and other macromolecules. Plasma ultrafiltrate platinum comprises the parent drug, its biotransformation products and platinum bound to small molecules such as glutathione, methionine and cysteine. The pharmacokinetics of plasma ultrafiltrate platinum were approximately linear within the range of 37.5 to 150 mg/kg of LA-12 while no such relationship was observed after satraplatin. Higher doses of LA-12 (> 75 mg/kg) resulted in a higher bioavailability of plasma ultrafiltrate platinum compared to satraplatin. Since it is generally accepted that plasma ultrafiltrate platinum is a better predictor of efficacy and/or toxicity, it potentially implies that higher and more predictable effects may be attainable after LA-12 compared to satraplatin. Hanada and coworkers have shown that cisplatin and its ultrafilterable metabolites (i.e. "mobile platinum") distribute rapidly into tissues and organs after i.v. administration to rats and are transformed to platinum bound to macromolecules (fixed platinum) which persist in tissues over long time [START_REF] Hanada | Pharmacokinetics and toxicodynamics of cisplatin and its metabolites in rats: relationship betwee n renal handling and nephrotoxicity of cisplatin[END_REF]. In agreement with this view, the concentrations of platinum in the kidney and liver tissue increased less than proportionally to dose after the doses exceeding 75 mg/kg. Concentrations of platinum in the kidney homogenate were approximately two-fold higher after 150 and 300 mg LA-12 compared to equimolar doses of satraplatin. The same difference was observed in the liver after all four doses. The characteristics of tissue exposure to platinum (C max and AUC 0-72h ) document extensive platinum distribution into the kidneys and liver after administration of both compounds. The pattern of tissue distribution of platinum after oral administration of LA-12 to Wistar rats has been described previously. The relative organ concentrations at 24 h postdosing were liver > kidneys > > spleen > lungs > heart = skin > muscle. Platinum was undetectable in the brain [START_REF] Sova | Pharmacokinetics and tissue distribution of platinum in rats following single and multiple oral doses of LA-12 [(OC-6-43)-bis(acetato)(1-adamantylamine)amminedichloroplatinum (IV)][END_REF].

The limitation of this study is that direct comparison of LA-12 and satraplatin with respect to the in vitro kinetics of platinum plasma protein binding and RBC uptake was not performed. Given the instability of satraplatin, LA-12 and their metabolites, the differences observed between the concentrations of plasma and plasma ultrafiltrate platinum after administration of both compounds could from a part be ascribed to changes occuring in vitro during sample processing and storage. To limit this possibility, the experiments with LA-12 and satraplatin ran in parallel, blood samples were immediately cooled in ice-water bath and processed at 8 o C within a fixed time frame of 45 min: plasma was separated within 15 min and ultrafiltrate was collected within additional 30 min. In vitro investigations of platinum distribution in the blood for cisplatin and oxaliplatin found no significant changes in plasma and ultrafiltrate platinum after cooling and processing samples within an hour [START_REF] Johnsson | Sample handling for determination of free platinum in blood after cisplatin exposure[END_REF][START_REF] Morrison | Validation of a highly sensitive ICP-MS method for the determination of platinum in biofluids: application to clinical pharmacokinetic studies with oxaliplatin[END_REF].

Moreover, data on platinum urinary excretion obtained for both derivatives argue against the existence of arteficial differences in concentrations of plasma ultrafiltrate platinum generated after sampling (i.e. ex vivo). The renal clearance of free platinum was similar regardless of the dose and compound administered. This indicates that the amount of platinum excreted in urine was directly proportional to the AUC of plasma ultrafiltrate platinum.

In conclusion, this study shows that platinum concentrations and characteristics C max and AUC evaluated in the plasma, plasma ultrafiltrate and kidney and liver tissues increase less than in proportion to dose following a single-dose administration of 37.5-300 mg/kg of LA-12 and satraplatin to Wistar rats. The dose-related increases in the pharmacokinetic characteristics V/F and CL/F of platinum and ultrafiltrate platinum as well as a drop in platinum urinary recovery are consistent with a dose-related decrease in the extent of oral bioavailability of both compounds most likely due to saturable intestinal absorption.

Regarding the extent of platinum exposure, plasma platinum concentrations were higher after satraplatin while higher doses of LA-12 (150 and 300 mg/kg) resulted in higher plasma ultrafiltrate and kidney platinum concentrations and approximately two-fold higher concentrations of liver platinum were found after LA-12 across the whole range of doses. adamantylamine as non-leaving amine group: Synthesis, characterization, and in vitro antitumour activity against the panel of cisplatin resistant cancer cell lines. J Med Chem 47:761-763 5. Kozubik A, Horvath V, Svihalkova-Sindlerova L, Soucek, K, Hofmanova J, Sova P, et al

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  comparative study of pharmacokinetics, urinary excretion and tissue distribution of platinum in rats following a single-dose oral administration of two platinum(IV) complexes LA-12 (-6-43)-bis(acetato)(1adamantylamine)amminedichloroplatinum(IV) and satraplatin (-6-43)bis(acetato)amminedichloro(cyclohexylamine)platinum(IV) Petr Sova, Adolf Mistr, Ales Kroutil, Martin Semerád, Hana Chlubnová, Veronika Hrusková, Jirina Chládková, Jaroslav Chládek To cite this version: Petr Sova, Adolf Mistr, Ales Kroutil, Martin Semerád, Hana Chlubnová, et al.. A comparative study of pharmacokinetics, urinary excretion and tissue distribution of platinum in rats following a single-dose oral administration of two platinum(IV) complexes LA-12 (-6-43)-bis(acetato)(1-adamantylamine)amminedichloroplatinum(IV) and satraplatin (-6-43)bis(acetato)amminedichloro(cyclohexylamine)platinum(IV). Cancer Chemotherapy and Pharmacology, 2010, 67 (6), pp.1247-1256. 10.1007/s00280-010-1411-0. hal-00615337 A comparative study of pharmacokinetics, urinary excretion and tissue distribution of platinum in rats following a single dose oral administration of two platinum(IV) complexes LA-12 (OC-6-43)-bis(acetato)(1-adamantylamine)amminedichloroplatinum(IV) and satraplatin (OC-6-43)bis(acetato)amminedichloro(cyclohexylamine)platinum(IV).

( 2005 )Fig. 1 Fig. 2 Fig. 3

 2005123 Fig.1Chemical structure of (OC-6-43)-bis(acetato)(1-

Fig. 4

 4 Fig. 4 Mean ( SD) urinary recovery of the platinum dose (%) after a single dose oral

Fig. 5 Fig. 6

 56 Fig. 5 Mean (SEM) platinum concentration versus time curves in the kidney and liver

Table 1 .

 1 Pharmacokinetic parameters of plasma platinum obtained by population modelling and post-hoc Bayesian estimation following single oral doses of LA-12 and Satraplatin.

	Compound	Dose (mg/kg)	k a (1/h)	k e (1/h)	t 1/2 (h)	CL/F (mL/min/kg)	AUC (mg.h/L)	k 12 (1/h)	k 21 (1/h)	t 1/2 (h)	t 1/2 (h)	V/F (L/kg)
	LA-12	37.5	0.58±0.05	0.12±0.008	5.6±0.37	12.1±2.33	19.0±4.2	0.48±0.03 0.092±0.003 1.0±0.09	41±1.7	5.8±1.1
		75	0.56±0.03	0.12±0.01	5.6±0.45	16.2±4.82	29.4±8.6	0.49±0.04 0.096±0.004 1.0±0.08	40±1.5	7.8±2.2
		150	0.50±0.06 0.095±0.009 7.3±0.69	12.5±2.77 74.1±17.3 0.52±0.03	0.13±0.01 0.99±0.06	39±3.0	8.6±2.6
		300	0.72±0.12 0.048±0.008	14±2.4	21.4±3.70 84.8±16.5 0.27±0.05 0.17±0.008 1.5±0.28	45±2.2	27±0.70
	P-value		0.30	< 0.0001	< 0.0001	< 0.0001	< 0.0001	< 0.0001	< 0.0001	< 0.0001 < 0.0001	< 0.0001
	Satraplatin 37.5	0.76±0.03 0.065±0.003	11±0.33	6.63±0.60	33.4±3.1	0.15±0.04 0.10±0.007 2.1±0.51 35.8±3.8 6.2±0.49
		75	0.40±0.02 0.070±0.004 9.9±0.57	8.16±0.79	54.5±6.3	0.17±0.02 0.16±0.008 1.9±0.22 22.9±1.2 7.0±0.39
		150	0.32±0.05 0.064±0.003	11±0.34	9.36±0.65	92.8±6.4	0.26±0.03 0.086±0.003 1.7±0.20 50.2±1.8 8.7±0.63
		300	0.17±0.03 0.078±0.004 8.9±0.34	12.0±0.71	147±8.9	0.26±0.02 0.052±0.003 1.9±0.31	120±6.5	9.2±0.53
	P-value		< 0.0001	< 0.0001	< 0.0001	< 0.0001	< 0.0001	< 0.0001	< 0.0001	0.0076	< 0.0001	< 0.0001
	Data are means±SD of 18 animals. P-values refer to the effect of the dose.

Table 2 .

 2 Pharmacokinetic parameters of plasma ultrafiltrate platinum obtained by population modelling and post-hoc Bayesian estimation following single oral doses of LA-12 and Satraplatin.

	Compound	Dose (mg/kg)	k a (1/h)	C ma x (mg/L)	T ma x (h)	AUC (mg.h/L)	k e (1/h)	t 1/2 (h)	CL/F (mL/min/kg)	CL R a (mL/min/kg)	V/F (L/kg)
	LA-12	37.5	1.10 b	0.175±0.061 1.2±0.03 0.565±0.187	0.67±0.05	1.0±0.06	423±116	14.0±5.2	38.1±10.0
		75	1.2±0.31 0.325±0.063 1.1±0.23 1.05 ±0.200	0.65±0.12	1.1±0.16	440±103	14.7±2.8	40.5±3.70
		150	0.79±0.18 0.433±0.080 1.4±0.23	1.60±0.366	0.69±0.14	1.1±0.23	576±118	11.6±4.2	52.1±1.63
		300	0.54±0.19 0.545±0.195 1.5±0.25	2.22±0.454	0.86±0.08	0.81±0.11	821±140	17.6±5.9	56.8±6.89
	P-value		-	< 0.0001	< 0.0001	< 0.0001	< 0.0001	< 0.0001	< 0.0001	0.48	< 0.0001
	Satraplatin 37.5	1.10 b	0.146±0.023 1.5±0.08 0.736±0.209	0.34±0.05	2.0±0.28	315±59.6	12.4±3.8	53.6±5.40
		75	0.68±0.10 0.213±0.030 1.7±0.20 0.982±0.158	0.53±0.07	1.3±0.13	460±74.4	14.9±1.7	51.7±1.68
		150	0.43±0.05 0.240±0.040 1.9±0.27	1.26±0.357	0.67±0.12	1.1±0.27	710±140	15.8±5.3	65.5±4.29
		300	0.31±0.07 0.288±0.056 1.9±0.20	1.69±0.109	0.81±0.03	0.85±0.10 1040±60.8	15.8±7.3	77.4±3.31
	P-value		-	< 0.0001	< 0.0001	< 0.0001	< 0.0001	< 0.0001	< 0.0001	0.73	< 0.0001
	Data are means±SD of 18 animals; a…CL

R was evaluated in four animals in each group; b…the interindividual variability was impossible to estimate; P-values refer to the effect of the dose.

Table 3 .

 3 Non-compartmental pharmacokinetic analysis of the mean concentration versus time profiles of plasma platinum and ultrafiltrate platinum following single oral doses of LA-12 and Satraplatin. interval was 72 h, ultrafiltrate platinum was below the quantification in all samples collected at 10 h

	Compound Sample	Dose (mg/kg)	C ma x (mg/L)	T ma x (h)	AUC 0-t (mg.h/L) a	AUC (mg.h/L)	z (1/h)	t 1/2 (h)	CL/F (mL/min/kg)	V z /F (L/kg)	V ss /F (L/kg)
	LA-12	plasma	37.5	0.844	4.0	17.4	20.2	0.027	25.8	10.9	24.4	22.8
			75	1.15	3.0	29.3	33.2	0.029	24.1	13.3	27.6	26.3
			150	2.30	3.0	67.7	75.9	0.033	21.1	11.6	21.2	23.5
			300	2.53	1.5	60.7	67.1	0.032	21.4	26.3	48.9	51.4
	Satraplatin plasma	37.5	1.79	4.0	25.3	29.4	0.024	28.4	7.52	18.4	15.3
			75	2.08	8.0	53.3	60.5	0.027	25.5	7.28	16.0	14.2
			150	2.60	3.0	66.4	85.5	0.020	35.2	10.3	31.4	29.4
			300	3.20	4.0	81.5	99.3	0.025	27.7	17.8	43.8	44.7
	LA-12	ultrafiltrate	37.5	0.162	1.0	0.427	0.625	0.30	2.3	352	70.1	73.4
			75	0.292	1.0	1.15	1.41	0.22	3.1	313	84.7	87.8
			150	0.465	3.0	1.72	1.91	0.30	2.3	462	91.4	99.2
			300	0.778	1.5	1.48	1.78	0.57	1.2	995	104	154
	Satraplatin ultrafiltrate	37.5	0.171	2.0	0.658	0.720	0.33	2.1	303	55.0	67.2
			75	0.200	1.5	0.644	1.27	0.20	3.4	347	102	113
			150	0.292	3.0	1.16	1.21	0.51	1.4	730	85.9	139
			300	0.286	2.0	1.53	1.71	0.33	2.1	1030	191	249
	a…the last sampling

LA-12 Satraplatin 0 6 12 18 24 30 36 42 48 54 60 66 72


Sumito Sato

Charlotte Kopitz

Bettina Grismayer

Nathalie Beaufort

Ute Reuning

Manfred Schmitt

Thomas Luther

Matthias Kotzsch

Achim Krüger

PhD Viktor Magdolen
email: viktor.magdolen@lrz.tum.de

Overexpression of the urokinase receptor mRNA splice variant uPAR-del4/5 affects tumor-associated processes of breast cancer cells in vitro and in vivo

Keywords: breast cancer, splice variant, adhesion, invasion, tumor xenograft, uPAR

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Introduction

The serine protease urokinase-type plasminogen activator (uPA), its serpin-type inhibitor PAI-1, and its cell surface receptor uPAR (CD87), drastically influence tumor cell proliferation, migration, invasion, and metastasis [START_REF] Blasi | The urokinase receptor: Focused cell surface proteolysis, cell adhesion and signaling[END_REF][START_REF] Blasi | uPAR: a versatile signalling orchestrator[END_REF][START_REF] Reuning | Multifunctional potential of the plasminogen activation system in tumor invasion and metastasis[END_REF]. Accordingly, these factors represent important biomarkers in a number of malignancies, including breast cancer [START_REF] Harbeck | Urokinase-type plasminogen activator (uPA) and its inhibitor PAI-I: novel tumorderived factors with a high prognostic and predictive impact in breast cancer[END_REF][START_REF] Høyer-Hansen | Urokinase receptor variants in tissue and body fluids[END_REF][START_REF] Harris | American Society of Clinical Oncology 2007 update of recommendations for the use of tumor markers in breast cancer[END_REF].

uPAR is an ubiquitous 45-60 kDa glycoprotein, which is composed of three homologous domains (DI, DII, and DIII, from the amino -to the carboxy terminus). It is anchored to the plasma membrane by a glycosyl phosphatidyl inositol (GPI) moiety [START_REF] Ploug | Structure-function relationships in the interaction between the urokinase-type plasminogen activator and its receptor[END_REF]. uPAR exhibits features related to proteolysis as well as cell adhesion: on one hand, upon binding of its canonical ligand uPA, uPAR focuses the extracellular proteolytic network including plasmin and matrix metalloproteinases to the cell surface [START_REF] Blasi | uPAR: a versatile signalling orchestrator[END_REF][START_REF] Ploug | Structure-function relationships in the interaction between the urokinase-type plasminogen activator and its receptor[END_REF][START_REF] Smith | Regulation of cell signalling by uPAR[END_REF], on the other, it interacts with the adhesive extracellular matrix (ECM) protein vitronectin as well as with various integrins, thereby regulating cell adhesion and signaling [START_REF] Blasi | uPAR: a versatile signalling orchestrator[END_REF][START_REF] Smith | Regulation of cell signalling by uPAR[END_REF]9]. For these reasons, uPAR is a major regulator of ECM remodeling and cell adhesion/migration [START_REF] Blasi | uPAR: a versatile signalling orchestrator[END_REF][START_REF] Smith | Regulation of cell signalling by uPAR[END_REF].

Recently, crystal structures of complexes between soluble uPAR (lacking the GPI anchor) and an antagonist peptide (interacting with the uPA binding site of uPAR) or the N-terminal fragment of uPA (ATF; harboring the uPAR binding site) have been determined [START_REF] Llinas | Crystal structure of the human urokinase plasminogen activator receptor bound to an antagonist peptide[END_REF][START_REF] Huai | Structure of human urokinase plasminogen activator in complex with its receptor[END_REF]. These studies revealed that the three domains in uPAR are packed closely and form a cone -shaped central cavity which is wide open and displays a significant depth. The major contact areas between uPAR and these ligands are formed by DI and DII residues, respectively. Binding of uPA to uPAR dramatically increases the affinity of uPAR to vitronectin [START_REF] Gårdsvoll | Mapping of the vitronectin-binding site on the urokinase receptor: involvement of a coherent receptor interface consisting of residues from both domain I and the flanking interdomain linker region[END_REF]. The crystal structure of the ternary complex of soluble uPAR, ATF and the uPAR-binding domain of vitronectin, SMB, showed that there is no direct contact between SMB and ATF: whereas the uPAR binding site of uPA occupies the central cavity of the receptor, SMB binds to the outer side of DI and the DI-DII linker region [START_REF] Huai | Crystal structures of two human vitronectin, urokinase and urokinase receptor complexes[END_REF]. Thus, uPA/uPAR-interaction may stabilize an active conformation of uPAR, leading to its high affinity binding to vitronectin.

Wild-type uPAR (uPAR-WT) is encoded by seven exons. Several mRNA splice variants of uPAR have been identified and their expression analyzed in human cells and tissues [START_REF] Stewart | Characterisation of urokinase plasminogen activator receptor variants in human airway and peripheral cells[END_REF][START_REF] Luther | Identification of a novel urokinase receptor splice variant and its prognostic relevance in breast cancer[END_REF][START_REF] Pyke | An alternatively spliced variant of mRNA for the human receptor for urokinase plasminogen activator[END_REF].

One of these splice variants, uPAR-del4/5, lacking exons 4 and 5, and thus encoding a uPAR form lacking domain DII, was found to be specifically overexpressed in breast and ovarian cancer cells [START_REF] Luther | Identification of a novel urokinase receptor splice variant and its prognostic relevance in breast cancer[END_REF]. Besides, high uPAR-del4/5 mRNA expression levels were found to be significantly associated with short disease-free survival of breast cancer patients [START_REF] Luther | Identification of a novel urokinase receptor splice variant and its prognostic relevance in breast cancer[END_REF][START_REF] Kotzsch | Prognostic relevance of uPAR-del4/5 and TIMP-3 mRNA expression levels in breast cancer[END_REF][START_REF] Kotzsch | Urokinase receptor splice variant uPAR-del4/5-associated gene expression in breast cancer: identification of rab31 as an independent prognostic factor[END_REF]. Moreover, uPAR-del4/5 mRNA is a highly sensitive, statistically independent prognostic factor for distant metastasis-free survival in untreated node-negative breast cancer patients [START_REF] Kotzsch | Urokinase receptor splice variant uPAR-del4/5-associated gene expression in breast cancer: identification of rab31 as an independent prognostic factor[END_REF].

In order to investigate the tumor biological effects of uPAR-del4/5 splice variant expression, we stably transfected MDA-MB-231 breast cancer cells with expression plasmids encoding either this variant or wild-type uPAR. By proliferation, adhesion, and invasion assays, we first analyzed the phenotype of the cell transfectants in vitro, and then monitored the impact of uPAR-del4/5 overexpression on experimental metastasis in a xenograft tumor model in mice in vivo.

Material and methods

Cell culture and cell transfection

The human breast adenocarcinoma cell line MDA-MB-231 (American Type Culture Collection [ATCC], Manassas, VA) was cultured in DMEM (Gibco BRL, Eggenstein, Germany) supplemented with 10 % (v/v) fetal calf serum (FCS), 10 mM HEPES, 0.55 mM Larginine and 0.272 mM L-asparagine (Sigma-Aldrich, Saint-Louis, MO). In some experiments, MDA-MB-435, CAMA-1, MCF-7 breast cancer cells (ATCC), and OV-MZ-6 ovarian cancer cells [START_REF] Möbus | Morphological, immunohistochemical and biochemical characterization of 6 newly established human ovarian carcinoma cell lines[END_REF], respectively, were also used. Cells were routinely checked to be free of mycoplasma.

Cloning of pRcRSV-derived expression plasmids encoding uPAR(-variants) has been reported elsewhere [START_REF] Luther | Identification of a novel urokinase receptor splice variant and its prognostic relevance in breast cancer[END_REF]. Cells were transfected using Lipofectin ® (Invitrogen, Karlsruhe, Germany). Cell transfectants were selected by addition of 1 mg/ml G418 to the cell culture medium (Gibco BRL). In an independent transfection experiment, clones of MDA-MB-231-uPAR-del4/5 cells were isolated by limited dilution.

ELISA

Cells were washed with phosphate-buffered saline (PBS), pH 7.4, then lysed for 60 min at 4 °C in Tris-buffered saline (TBS), pH 7.4 containing 1 % (v/v) Triton-X 100 and a protease inhibitor cocktail ("complete + EDTA"; Roche Diagnostics, Mannheim, Germany). Cell lysates were centrifuged at 13,000 x g for 15 min at 4 °C. Protein content was determined using the Micro BCA TM protein assay reagent kit (Pierce, Stonehouse, UK). uPAR antigen was determined applying the IMUBIND uPAR ELISA kit #893 (American Diagnostica Inc., Stamford, CT). uPAR antigen levels in cell lysates are expressed as ng per mg of total protein.

Western blot

Proteins, separated by electrophoresis on 12 % (w/v) polyacrylamide gels (SDS-PAGE), were transferred to polyvinylidene fluoride membranes (Millipore Corporation, Bedford, MA) in a semi-dry transfer device (Biometra, Göttingen, Germany). Membranes were incubated for 60 min in PBS, pH 7.4, containing 0.1 % (v/v) Tween-20 (PBS-T) and 5 % (w/v) dried skimmed milk, followed by an overnight incubation with the primary monoclonal antibodies (mAb) IIIF10 or IID7, both directed to uPAR, diluted in PBS-T supplemented with 1 % (w/v) dried skimmed milk. The epitope-mapped mAb IIIF10 is directed against DI, mAb IID7 against DII of uPAR [START_REF] Luther | Epitope-mapped monoclonal antibodies as tools for functional and morphological analyses of the human urokinase receptor in tumor tissue[END_REF]. After washes in PBS-T, binding of the antibodies were visualized by incubation of the membranes with a horseradish peroxidase-conjugated secondary goat Ab against mouse Ig (Jackson ImmunoResearch Lab, West Grove, PA), followed by chemoluminescence reaction using ECL (Amersham Biosciences, Little Chalfont, UK).

Immunofluorescence

Cells were seeded in human fibronectin-coated (10 µg/ml, Becton Dickinson, Heidelberg, Germany) 8-chamber glass slides (Permanox-type Lab-Tek slides; Nunc, Roskilde, Denmark) and cultured overnight. Cell monolayers were fixed in 4 % (w/v) paraformaldehyde in PBS, pH 7.4, for 30 min at room temperature (RT). After several washes in PBS, cells were incubated for 1 h at RT in PBS containing 2 % (w/v) bovine serum albumin (BSA), and then probed with mAb IIIF10 diluted in PBS, 1 % (w/v) BSA for 1 h at RT. Cells were washed and incubated for 45 min at RT in the dark with the secondary Alexa488-labeled goat-anti-mouse IgG (Sigma-Aldrich) diluted in the same buffer. After final washes with PBS, cells were mounted in PBS and fluorescence signal intensity evaluated by confocal laser scanning microscopy (CLSM).

Solid-phase uPA binding assay

The solid-phase uPA ligand-binding assay was performed in a similar manner as described previously [START_REF] Goretzki | Quantitative assessment of interaction of urokinase-type plasminogen activator and its receptor (CD87) by use of a solid-phase uPA-ligand binding assay[END_REF]. Briefly, wells of microtiter plates (Maxisorb; Nunc) were coated overnight at 4 o C with 100 µl of rec-uPAR 1-277 (1 µg/ml). After blocking, 100 µl of sample containing the aminoterminal fragment (ATF) of uPA (250 ng/ml) and 50 µl of cell culture supernatants were added to the wells for 1 h at RT. Cell culture supernatants were derived from vector-control Chinese hamster ovary (CHO) cells or from transfected CHO cells overexpressing soluble forms of uPAR-WT or uPAR-del4/5 [START_REF] Luther | Identification of a novel urokinase receptor splice variant and its prognostic relevance in breast cancer[END_REF]. After four washes, plates were incubated with biotinylated murine mAb #377 directed to the kringle domain of human uPA (American Diagnostica Inc.) for 1 h at RT. After washes in PBS, avidin-coupled peroxidase was added for 1 h at RT. Finally, wells were washed and binding of avidin-peroxidase detected by addition of 3,3',5,5'-tetramethlybenzidine (TMB; 1 mg/ml) and 0.003 % (v/v) H 2 O 2 in 0.1 M sodium acetate buffer, pH 6.0. After termination of the reaction with 0.5 M H 2 SO 4 , absorbance was measured at 450 nm.

Cell-based in vitro assays

Proliferation assays. Transfected cells were seeded in a 24-well plate at a density of 15,000 cells per well, and incubated for 48 to 96 h at 37 °C. After washes in PBS, cells were detached using PBS, 0.05 % (w/v) EDTA, and living cells counted in a hemocytometer upon Trypan blue exclusion under a light microscope.

Cell adhesion assays. 96-well plates were coated overnight at 4 °C with vitronectin (1 µg/well), fibronectin (1 µg/well), collagen type I or IV (1 µg/well), all diluted in PBS. Cells were resuspended in culture medium containing 0.5 % (w/v) BSA, seeded at a density of 40,000 cells/well, and allowed to adhere for 2 h at 37 o C. Non-adherent cells were removed by washing with PBS and number of adherent cells quantified by a hexosaminidase activity assay.

For this, cells were incubated with p-nitrophenyl-N-acetyl-β-D-glucosaminide (Sigma-Aldrich) diluted to 15 mM in a 100 mM sodium citrate buffer, pH 5.0, 0.5 % (v/v) Triton X-100, for 90 min at 37 °C. The reaction was terminated by the addition of stop buffer (0.2 M NaOH, 5 mM EDTA) and the optical density recorded at 405 nm.

Cell invasion assays. Invasion assays were performed using Transwell inserts (8 µm pore size; Becton Dickinson). Basement membrane complex growth factor reduced Matrigel TM (11.3 mg/ml) was diluted 1:24 in cold PBS and applied to the upper side of the insert. After drying for 24 h in a laminar hood, inserts were rehydrated with FCS-free DMEM, 0.1 % (w/v) BSA.

Cells were resuspended in culture medium and seeded in the upper chamber of the device at a density of 50,000 cells/chamber. In some experiments, 2 g of proteolytically active highmolecular-weight uPA (ProSpec-Tany TechnoGene, Rehovot, Israel) were added to the upper chamber. The lower chambers were filled with 750 µl DMEM supplemented with 10 % (v/v) FCS as a chemoattractant. After a 24 to 48 h incubation at 37 °C, Matrigel TM and non-invaded cells, located on the upper side of the insert, were removed with tissue paper, whereas invaded cells on the lower side of the insert were fixed, stained using Diff-Quick (Dade Behring AG, Switzerland), and counted under a light microscope.

Experimental animal model

MDA-MB-231 cells are tumorigenic and invasive cells, which upon intravenous injection in mice colonize to the lungs [START_REF] Krüger | Hydroxamate-type matrix metalloproteinase inhibitor batimastat promotes liver metastasis[END_REF]. Transfected MDA-MB-231 cells were genetically tagged with the lacZ gene, allowing X-Gal staining, as reported previously [START_REF] Kopitz | Reduction of experimental human fibrosarcoma lung metastasis in mice by adenovirus-mediated cystatin C overexpression in the host[END_REF]. Pathogen-free female athymic (nu/nu) mice (4-6 weeks old) were obtained from Charles River Laboratories (Sulzfeld, Germany). Mice were allocated to four groups and intravenously inoculated (tail vein) with 1 x 10 6 (a) control MDA-MB-231-vector cells (n = 11), (b) batch-transfected MDA-MB-231-uPAR-del4/5 cells (n = 10), (c) cloned MDA-MB-231-uPAR-del4/5 cells (n = 10), or (d) batch-transfected MDA-MB-231-uPAR-WT cells (n = 9). At day 35 post-injection, animals were sacrificed, lungs isolated and the lacZ-tagged tumor cells stained with X-Gal (Roche Diagnostics) [START_REF] Krüger | The bacterial lacZ gene: an important tool for metastasis research and evaluation of new cancer therapies[END_REF]. Thereafter, blue stained metastases foci were counted and their size measured, considering nodules with diameter > 0.18 mm as macrometastases.

Statistical analyses

Data are expressed as mean ± SE (standard error) of the indicated number of experiments.

Group differences and p-values were calculated by employing the Mann-Whitney test. A pvalue < 0.05 was considered statistically significant.

Results

Generation of uPAR overexpressing breast cancer cell lines

MDA-MB-231 breast cancer cells endogenously express uPAR, as seen by Western blot analysis and immunofluorescence (Fig. 1, see MDA-MB-231 vector cells). Transfection of these cells with a plasmid encoding uPAR-WT resulted in a distinct overexpression of uPAR, which reacts with both mAbs IIIF10 and IID7, directed to domains DI and DII, respectively (Fig. 1a). The observed uPAR reaction pattern was comparable to that reported fo r other cells (see e.g. [START_REF] Beaufort | The human airway trypsin-like protease modulates the urokinase receptor (uPAR, CD87) structure and functions[END_REF]). Transfection of cells with uPAR-del4/5 expression plasmid led to the occurrence of a uPAR species that -as expected -migrated faster than uPAR-WT in SDS-PAGE and reacted with mAb IIIF10 directed to domain DI, but not with mAb IID7 directed to domain DII (Fig. 1a). Importantly, uPAR-del4/5 overexpression in MDA-MB-231 cells did not affect mRNA expression levels of endogenous uPAR-WT mRNA, as assayed by quantitative RT-PCR (data not shown). The uPAR antigen content, measured by a commercially available ELISA, was distinctly increased in uPAR-WT and uPAR-del4/5 overexpressing cells as compared to the vector control cells (MDA-MB-231 vector control cells: 6.5 ng/mg total protein; uPAR-WT cells: 51.7 ng/mg; uPAR-del4/5 cells: 12.0 ng/mg). It is of note that using this ELISA, we are not able to discriminate between endogenous and overexpressed protein and not between different forms of uPAR. Immunofluorescence further confirmed uPAR overexpression (Fig. 1b). The staining pattern was found to be rather heterogeneous within the transfected cell populations, as expected for batch-transfected cells.

Moreover, both uPAR-WT and uPAR-del4/5 displayed an enhanced membrane staining pattern (Fig. 1b), strongly suggesting correct integration of the respective uPAR species into the cell membranes.

In vitro phenotype of uPAR-del4/5 overexpressing breast cancer cells

As compared to vector-transfected cells, neither uPAR-WT nor uPAR-del4/5 overexpressing MDA-MB-231 cells displayed significant differences with respect to cell morphology and cell proliferation (data not shown). In contrast, MDA-MB-231-uPAR-del4/5 cells showed a moderate (i.e., 14 to 20 %) but significant (p < 0.05) reduction of their capacity to adhere to certain adhesive ECM proteins, such as vitronectin, fibronectin, and type I collagen (Fig. 2 and Table 1). Interestingly, this reduced adhesive capacity of uPAR-del4/5 overexpressing cells was not restricted to the MDA-MB-231 cell line, since transfection of other human breast (i.e., MDA-MB-435, CAMA-1, MCF-7) or ovarian (i.e., OV-MZ-6) cancer cell lines, reduced cell adhesion to various ECM components in the same fashion (Table 1). It is of note that uPAR-WT is an acknowledged pro-adhesive factor, for instance by engagement with its ligand vitronectin, in a number of cell types, including human breast and ovarian cancer cells [START_REF] Wei | Regulation of integrin function by the urokinase receptor[END_REF][START_REF] Xue | Urokinase-type plasminogen activator receptors associate with beta1 and beta3 integrins of fibrosarcoma cells: dependence on extracellular matrix components[END_REF].

Next, uPAR-WT and uPAR-del4/5 overexpressing cells were investigated and compared to vector-control cells in a Matrigel TM invasion assay. As expected, overexpression of uPAR-WT was associated with a distinct, about 3-fold enhanced cell invasive capacity, which was further about 2-fold enhanced in the presence of exogenously added uPA (Fig. 3a, lower panel).

Strikingly, MDA-MB-231-uPAR-del4/5 cells displayed a strongly impaired invasion (i.e., about 40 % reduction), which was not affected by the presence of uPA (Fig. 3a, upper panel).

In fact, this latter observation was in agreement with solid-phase ligand-binding assays indicating efficient uPA binding to recombinant uPAR-WT, but not to uPAR-del4/5 protein (Fig. 4). Again, reduced invasion upon uPAR-del4/5 transfection was confirmed in another cancer cell model, namely in human ovarian OV-MZ-6 cancer cells (Fig. 3b).

In vivo phenotype of uPAR-del4/5 overexpressing breast cancer cells

Lung is a major site for metastatic spread of cancer cells during breast cancer progression [START_REF] Krüger | Reduction of breast carcinoma tumor growth and lung colonization by overexpression of the soluble urokinase-type plasminogen activator receptor (CD87)[END_REF][START_REF] Setyono-Han | Suppression of rat breast cancer metastasis and reduction of primary tumour growth by the small synthetic urokinase inhibitor WX-UK1[END_REF]. In order to explore the impact of uPAR-del4/5 overexpression in vivo, we intravenously injected mice with MDA-MB-231-vector, -uPAR-WT, or -uPAR-del4/5 cells and analyzed incidence, number, and size of nodules of experimental lung metastases. Most mice of the vector and uPAR-WT groups (10/11 and 7/9, respectively; Fig. 5a) had numerous lung metastases (on the average 57 and 37, respectively; Fig. 5b). Moreover, 16.6 to 28.0 % of the nodules were classified as macrometastases (size over 0.18 mm; Fig. 5c). Remarkably, in the group of mice which had received MDA-MB-231-uPAR-del4/5 cells, 5 out of 10 mice had no detectable lung metastasis (Fig. 5b). Similarly, the number of metastases in colonized lungs (on the average from 57 to 4; Fig. 5b) as well as the percentage of macrometastases (on the average from 16.6 to 0.8 %, Fig. 5c) was considerably reduced in the uPAR-del4/5 group, as compared to the vector control group. Similar results were observed upon injection of an independently generated MDA-MB-231-uPAR-del4/5 clone (Fig. 5), displaying a comparable antigen level, measured by ELISA (batch-transfected uPAR-del4/5 cells: 12.0 ng/mg; uPAR-del4/5 clone: 10.2 ng/mg). These observations suggest that overexpression of uPAR-del4/5 in vivo not only reduced adhesion, but also impaired growth of breast cancer cell-derived lung metastases in vivo.

Discussion

Aberrant mRNA splicing is a common feature of malignant disorders. Whether this process results from a general dysregulation of tumor cell functions, and thus represents a by-product of cellular transformation, or whether increased aberrant splicing contributes to the malignant phenotype of cancer cells, is a not yet conclusively answered question [START_REF] Ward | The pathobiology of splicing[END_REF][START_REF] Fackenthal | Aberrant RNA splicing and its functional consequences in cancer cells[END_REF]. Still, a growing number of evidence demonstrates that tumor-associated mRNA splicing may lead to cancer cell-specific production of new proteins displaying unique functions, which are crucially affecting cancer development. Mis-spliced mRNAs, which escape the so-called nonsense-mediated mRNA degradation, normally lead to synthesis of truncated, mutated, misfolded, and/or unstable proteins. However, by alternative splicing, it is also possible that certain protein domains are deleted from the encoded protein, which may result in loss or gain of function [START_REF] Fackenthal | Aberrant RNA splicing and its functional consequences in cancer cells[END_REF]. In a recent study on splice variants of the human Adhesion family of G protein-coupled receptors, more than half of the identified splice variants appeared to code for functional proteins, lacking one or more extracellular domains, which take part in proteinprotein interactions, without affecting the seven transmembrane region. Thus, alternative splicing apparently can influence interaction of these receptors with other proteins [START_REF] Bjarnadóttir | Identification of novel splice variants of Adhesion G protein-coupled receptors[END_REF].

The recently identified, tumor-associated variant of uPAR, uPAR-del4/5, displays the specific deletion of the complete DII of three-domain uPAR [START_REF] Luther | Identification of a novel urokinase receptor splice variant and its prognostic relevance in breast cancer[END_REF]. In fact, uPAR-del4/5 protein, consisting of domains DI+DIII fused together, exhibits unique characteristics significantly affecting the behavior of malignant cells: upon analyses of the impact of uPAR-del4/5 overexpression in human MDA-MB-231 breast cancer cells, we observed a significantly impaired cell adhesion and invasion in vitro, along with a distinctly decreased colonization of lungs (in terms of metastatic nodule occurrence, number, and size) in an experimental metastasis mouse model system.

Binding of uPA to its cell surface receptor uPAR involves all three domains of uPAR [START_REF] Blasi | uPAR: a versatile signalling orchestrator[END_REF][START_REF] Llinas | Crystal structure of the human urokinase plasminogen activator receptor bound to an antagonist peptide[END_REF].

As demonstrated by a sensitive solid-phase ligand-binding assay (Fig. 4) and by an uPA/uPAR-complex ELISA (data not shown), uPAR-del4/5 does not interact with uPA. Consequently, we observed that addition of uPA to uPAR-del4/5 overexpressing cells -in contrast to uPAR-WT overexpressing cells -had no effect on cell invasion through Matrigel TM (Fig. 3). uPA/uPAR interaction does not only focus proteolytic activity to the tumor cell surface, but is also a prerequisite for the binding of another uPAR ligand, the ECM protein vitronectin [START_REF] Gårdsvoll | Mapping of the vitronectin-binding site on the urokinase receptor: involvement of a coherent receptor interface consisting of residues from both domain I and the flanking interdomain linker region[END_REF]. Furthermore, interaction of uPA with uPAR is a major determinant of physical and functional interactions of uPAR with a number of integrins [START_REF] Blasi | uPAR: a versatile signalling orchestrator[END_REF]9] and is also involved in other tumorassociated functions, e.g. regarding tumor cell survival and angiogenesis [START_REF] Blasi | uPAR: a versatile signalling orchestrator[END_REF][START_REF] Alfano | The urokinase plasminogen activator and its receptor: role in cell growth and apoptosis[END_REF]. Obviously, uPAR-del4/5 is not capable of modulating these uPA-dependent, tumor-associated functions of uPAR-WT by competing for uPA binding. Still, overexpression of uPAR-del4/5 resulted in a moderate, significantly decreased cell adhesion to an array of adhesive ECM proteins (Fig. 2, Table 1). uPAR-del4/5 also strongly affected cellular invasion in vitro (Fig. 3), a process that depends on both extracellular proteolysis and cell adhesion/detachment capacities. In addition, overexpression of the uPAR-del4/5 splice variant led to a major reduction of experimental metastasis in vivo, compared to both vector control and uPAR-WT overexpressing cells (Fig. 5). It is tempting to speculate that such a phenotype might reflect the capacity of uPAR-del4/5 to behave as a dominant-negative receptor, e.g. via competition with endogenous uPAR-WT for binding to certain integrins such as 5 ß 1 or v ß 3 and/or via an additional uPAR-WT independent process. uPAR-del4/5 mRNA is a statistically independent prognostic factor for distant metastasis-free survival in breast cancer patients. High levels of uPAR-del4/5 mRNA are associated with a poor prognosis of patients [START_REF] Luther | Identification of a novel urokinase receptor splice variant and its prognostic relevance in breast cancer[END_REF][START_REF] Kotzsch | Prognostic relevance of uPAR-del4/5 and TIMP-3 mRNA expression levels in breast cancer[END_REF][START_REF] Kotzsch | Urokinase receptor splice variant uPAR-del4/5-associated gene expression in breast cancer: identification of rab31 as an independent prognostic factor[END_REF]. However, the results of the present paper, especially regarding the reduced invasive capacity of uPAR-del4/5 overexpressing cells in vitro (Fig. 3) and the reduction in lung colonization of these cells in the in vivo experimental metastasis animal model (Fig. 5) rather advert to the conclusion that the uPAR-del4/5 protein acts as a forceful tumor suppressor. On the one hand, high expression of uPARdel4/5 mRNA might reflect the malignant status of the cancer disease, rather than the functions arising thereof at the protein level. O n the other hand, as documented for the adverse effects of different PAI-1 concentrations on tumor angiogenesis [START_REF] Durand | Plasminogen activator inhibitor-I and tumour growth, invasion, and metastasis[END_REF], uPAR-del4/5 protein, depending on the height of its expression level, may display both agonistic and antagonistic properties. Therefore, further extensive studies using clones expressing uPAR-del4/5 protein to a different extent (low vs. high expression) and/or variations of uPAR-del4/5 : uPAR-WT protein ratios may help to explore this latter possibility.

The metastatic process requires a complex sequence of events [START_REF] Steeg | Metastasis suppressors alter the signal transduction of cancer cells[END_REF]. Invasive tumor cells, originating from an in situ cancer surrounded by an intact basement membrane, degrade ECM proteins, induce reversible changes in cell-cell and cell-ECM adherence, and migrate through the ECM. The metastasizing cells then enter circulation, extravasate and disseminate to eventually colonize at a distant site, finally leading to micrometastases and angiogenic metastases. The experimental animal metastasis model used in the present study focuses on the later events of the metastatic process, i.e. metastatic seeding and growth. Here, uPAR-del4/5 overexpression did not only reduce occurrence and number of metastatic nodules, but also the percentage of macrometastases (Fig. 5), which indeed indicates an impact o f the uPAR splice variant on extravasation of circulating tumor cells and/or tissue colonization, as well as on metastatic tumor growth.

In conclusion, although several uPAR mRNA splice variants have been described, some being clearly associated to disease [START_REF] Stewart | Characterisation of urokinase plasminogen activator receptor variants in human airway and peripheral cells[END_REF][START_REF] Luther | Identification of a novel urokinase receptor splice variant and its prognostic relevance in breast cancer[END_REF][START_REF] Pyke | An alternatively spliced variant of mRNA for the human receptor for urokinase plasminogen activator[END_REF], no experimental analysis of their cellular properties was reported so far. The present study, combining in vitro and in vivo experimental approaches, clearly demonstrates tumor biologically relevant effects mediated by uPAR-del4/5 in human breast cancer and also ovarian cancer cells. Thus, production of uPAR-del4/5 might represent a striking example for tumor-associated alternative splicing leading to modified protein species strongly modulating tumor biological processes. hamster ovary (CHO) cell lines [START_REF] Luther | Identification of a novel urokinase receptor splice variant and its prognostic relevance in breast cancer[END_REF] were added to ATF in the ligand binding assay. Bars represent the amount of ATF bound to immobilized rec-uPAR. Whereas conditioned medium of vector-transfected control cells as well as of cells overexpressing a soluble form of uPAR-del4/5 did not compete for binding of ATF to rec-uPAR, a significant reduction of rec-uPAR/ATF interaction was observed in the presence of conditioned medium from cells overexpressing uPAR-WT. Purified, soluble rec-uPAR, interfering with interaction of ATF to immobilized rec-uPAR, was used as a positive control in the assay. Table 1. uPAR-del4/5 overexpression reduces the adhesive capacity of human breast and ovarian cancer cells in vitro.

Figure legends

Tables

Cell adhesion assays were performed as described in the legend to Fig. 2. Results were normalized to the number of adherent vector-transfected cells to uncoated wells and are expressed as the mean +/-SE. At least four independent experiments were performed in triplicates. The asterisk indicates a statistically significant difference between uPAR-del4/5 overexpressing calls and vector-control cells (p < 0.05).

VN

Fig. 1 .

 1 Fig. 1. Expression of uPAR-WT and uPAR-del4/5 in stably transfected MDA-MB-231 cells. (a) Lysates from uPAR-WT-, uPAR-del4/5-, or vector-transfected MDA-MB-231 cells (50 g total protein) were analyzed for uPAR-WT / uPAR-del4/5 expression by Western blot analysis applying a mAb directed to uPAR domain DI (mAb IIIF10, left-hand panel) or to DII (mAb IID7, right-hand panel) of uPAR. (b) Analysis of MDA-MB-231 cell transfectants for uPAR-WT / uPAR-del4/5-expression by immunofluorescence using mAb IIIF10 directed to domain DI of uPAR. Typical fluorescent images (lower panel) together with the corresponding differential interference contrast images (upper panel) are depicted. In order to convert fluorescence staining intensity into colors of a glow scale, the look-up table glowOv/Un LUT provided with the CLSM scanning software was applied: low intensity (red), medium intensity (yellow), and high intensity (white).

Fig. 2 .

 2 Fig. 2. uPAR-del4/5 overexpression reduces the adhesive capacity of human breast cancer cells in vitro. MDA-MB-231 cells were passed to cell culture plates which were pre-treated with the extracellular matrix proteins vitronectin (VN), fibronectin (FN), and type I (Col I) or type IV (Col IV) collagen. After 2 h of cell cultivation, the number of adherent cells was monitored by the hexosaminidase activity assay. Four independent experiments were performed in triplicates each. The results are given in %, normalized to the number of adherent vectortransfected cells onto uncoated wells. Whisker box plots indicate the 25 th and 75 th percentile, the vertical bars indicate the 10 th and 90 th percentile. The mean value is indicated by a bar within the box. Statistically significant differences (p < 0.05) are indicated by an asterisk.

Fig. 3 .

 3 Fig. 3. uPAR-del4/5 overexpression reduces the invasive capacity of human breast and ovarian cancer cells in vitro. (a) Stably transfected MDA-MB-231 breast cancer cells were seeded into the upper compartments of Matrigel TM -coated invasion chambers in the absence or presence of 2 g of proteolytically active uPA. After 96 h of incubation, invaded cells were fixed, stained, and counted. Four independent experiments were performed in duplicates each. The results are given in %, normalized to the number of invaded vector-transfected cells, in the absence of uPA. (b) Stably transfected human ovarian cancer OV-MZ-6 cells were analyzed (in the absence of uPA) for their invasive capacity in a similar manner as described above for the MDA-MB-231 cells. Four independent experiments were performed in duplicates each. The results are given in %, normalized to the number of invaded vector-transfected cells. Whisker box plots indicate the 25 th and 75 th percentile, the vertical bars indicate the 10 th and 90 th percentile. The mean value is indicated by a bar within the box. Statistically significant differences (p < 0.05) are indicated by an asterisk.

Fig. 4 .

 4 Fig. 4. uPAR-WT, but not uPAR-del4/5 interacts with uPA. (a) Schematic representation of the solid-phase uPA-ligand-binding assay ([21]; for details see also Materials and methods). (b) Cell culture supernatants from stably transfected Chinese

Fig. 5 .

 5 Fig. 5. Overexpression of uPAR-del4/5 affects lung colonization and metastatic growth of human breast cancer cells. Stably transfected, lacZ-tagged, MDA-MB-231 cells were inoculated into nude mice via tail vein injection. Mice received either vector control cells (white columns), uPAR-del4/5 cells (gray columns) or uPAR-WT cells (black columns). In addition, an independently generated MDA-MB-231-uPAR-del4/5 transfected cell clone, displaying a comparable uPAR antigen level as the batch-transfected uPAR-del4/5 cell line (hatched gray columns), was analyzed. Animals were sacrificed at day 35 after injection, lungs were collected and stained with X-Gal. (a) The number of mice presenting lung metastasis is expressed as percentage of total mouse number. (b) Metastases were counted in lungs of the mice. Results are expressed as the mean number of metastases +SE counted in lungs colonized by cancer cells. (c) Metastases with a diameter ≥ 0.18 mm were considered as macrometastases. Histograms illustrate the mean percentage + SE of macro-versus total metastases in mice lungs. Statistically significant differences (p < 0.05) to the vector control group are indicated by an asterisk.

Acknowledgements

This study was supported in part by grants provided by the Deutsche Krebshilfe e.V., Germany (Grant No. 106 162) to MK and VM, and by the Framework Programme 7 project HEALTH-2007-201279, Microenvimet to AK. We are grateful to Sabine Creutzburg, Katja Richter, and Antje Zobjack for excellent technical assistance.

Competing interests

The authors declare that they have no competing interests.