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CHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY

Antimetastatic Effect of a Small-Molecule Vacuolar H⁺-ATPase Inhibitor in in Vitro and in Vivo Preclinical Studies

Fondazione Istituto di Ricovero e Cura a Carattere Scientifico Istituto Nazionale Tumori, Milan, Italy (R.S., G.Pe., G.Pr., M.T., E.F., L.D.B., P.C., F.Z.); Facoltà Medicina Veterinaria Università degli Studi di Milano, Milan, Italy (E.R.); Consiglio Nazionale delle Ricerche, Università di Pavia, Pavia, Italy (A.C.C., G.B.); and Nikem Research, Baranzate, Milan, Italy (P.M., C.F.)

Received July 12, 2007; accepted September 28, 2007.

	Abstract

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On the basis of the evidence that vacuolar H⁺-ATPase is implicated in the development of the metastatic phenotype, we have explored the possibility to target the enzyme function in an attempt to control the metastatic behavior of tumor cells. In this study, we used an indole derivative, NiK-12192 [4-(5,6-dichloro-1H-indol-2-yl)-3-ethoxy-N-(2,2,6,6-tetramethyl-piperidin-4-yl)-benzamide], recently identified as an effective inhibitor of vacuolar H⁺-ATPase, as a potential antimetastatic agent in the treatment of NSCLC H460 xenograft, which is able to induce lung metastases in mice. Oral administration of NiK-12192 caused a significant inhibition of formation of spontaneous metastases. In contrast, the drug exhibited a negligible effect on the development of artificial metastases (i.e., after i.v. injection of tumor cells), thus supporting that the drug affects the early events of the metastatic process (e.g., migration and invasion). Cellular effects are consistent with this interpretation. In conclusion, the available results show for the first time that a vacuolar H⁺-ATPase inhibitor is effective in modulation of the metastatic behavior of a lung carcinoma, supporting its potential therapeutic interest as a novel treatment approach.

Proton transporters implicated in pH regulatory mechanism include the Na⁺/H⁺-exchanger, Na⁺/K⁺-ATPase pump, vacuolar H⁺-ATPases, H⁺/Cl^– symporter and proton lactate symporters (Putnam, 2001; Harguindey et al., 2005). The vacuolar H⁺-ATPase is expressed in plasma membrane and cytoplasmic organelles and can contribute to the metastatic potential of many tumor cells (Sennoune et al., 2004a,b; Martinez-Zaguilan et al., 1993, 1998). On the basis of its function, it is conceivable that inhibition of vacuolar H⁺-ATPase function/expression may affect the invasive behavior of tumor cells. Indeed, the knocking down of ATP6L, the 16-kDa subunit of vacuolar H⁺-ATPase, in a human hepatocellular carcinoma cell line resulted in inhibition of growth and metastases in nude mice (Lu et al., 2005). In clinical specimens, vacuolar H⁺-ATPase was detected in invasive pancreatic carcinomas (Ohta et al., 1996).

We have recently reported that the vacuolar H⁺-ATPase inhibitor NiK-12192, a lead compound of a novel series of substituted indole derivatives (Farina et al., 2001), is able to potentiate the antitumor activity of the camptothecin topotecan at well tolerated doses (Petrangolini et al., 2006). Although NiK-12192 exhibits a marginal effect on the s.c. growing tumor xenografts, the intra-/extracellular modulation of pH is expected to influence spreading of tumor cells and metastasis development. The present study was based on the hypothesis that inhibition of vacuolar H⁺-ATPase, which has a prominent role in modulating the pH gradient, may influence the metastatic behavior of tumor cells. Therefore, the aim of the study was to investigate the role of NiK-12192 on the growth and the metastatic spreading of a human lung tumor, H460, which is able to form spontaneous lung metastases when implanted s.c. in nude mice (Corti et al., 1996). The results indicated that oral treatment with NiK-12192 resulted in a relevant antimetastatic effect in animal models. Moreover, in vitro studies confirmed the inhibitory activity of the compound in various assays designed to investigate cellular effects relevant to the metastatic behavior.

	Materials and Methods

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Drugs
NiK-12192 and topotecan used in this study are products of Nikem Research (Milan, Italy) and Glaxo SmithKline (Verona, Italy), respectively. Bafilomycin A1 is provided by Sigma-Aldrich (St. Louis, MO).

For in vitro studies, stock solutions (1 mg/ml) were stored in DMSO for NiK-12192 and diluted before use in saline solution. In experiments, the final concentration of DMSO in the medium was 0.1%, which has been verified to be nontoxic in our cell system. For in vivo studies, NiK-12192 was dissolved by adding absolute ethanol and Cremophor ELP (both 5% of the final volume) and diluted (just before use) by adding 0.9% NaCl. Topotecan was dissolved in sterile distilled water.

H460 Human Tumor Model System
The NCI-H460 nonsmall cell lung tumor cell line (hereafter named H460) was used in the in vitro studies. For animal experiments, H460 tumor cells were adapted to grow as ascitis in nude mice and maintained in vivo by i.p. passages (5 x 10⁶ cells/mouse in 0.5 ml of PBS). In brief, cells were collected from the donor mice approximately 7 days after inoculum, and, after washing, cell number and viability were determined by trypan blue exclusion. Cell suspension was injected s.c. or i.v. to achieve spontaneous or experimental metastases (Cassinelli et al., 2006).

In Vitro Studies
Intracellular pH Determination. Cells grown on coverslips were incubated for 40 min with SNARF-4F [5-(and -6) carboxylic acid, acetoxymethyl ester, acetate, stock solution (80 mM in DMSO); Invitrogen, Eugene, OR] at the final concentration of 8 µM. Dilutions were performed directly in the medium deprived of serum to prevent the hydrolysis of the fluorogenic substrate. After incubation, the coverslips were rinsed with PBS and mounted upside down on the microscope slides. SNARF-4F incubation was performed on control (untreated) cells and on cells pretreated with NiK-12192 (2 µM, 24 h) (Marcotte and Brouwer, 2005). Microspectrofluorometric analysis was performed under epi-illumination conditions by means of a Leitz microspectrograph (Wetzlar, Germany) equipped with an Optical Multichannel Analyzer (EG&G, model 1420/512; Princeton Applied Research, Princeton, NJ). Excitation light was provided by a 100-W Hg lamp (Osram, Berlin, Germany), combined with KG1 and BG38 antithermal filters, and selected by a 366-nm interference filter (T% = 25). Fluorescence spectra were recorded from single cells (cytoplasmic area selected by means of a field diaphragm) in the 550- to 800-nm interval. The intracellular pH values were estimated from the blue shift of the emission band centered at approximately 655 nm at pH 7.2, according to the reference spectra reported by the dye producer. Measurements were performed by means of an oil immersion Fluotar Leitz objective (100x NPL, incorporated iris diaphragm; numerical aperture, 0.60–1.32).

Vacuolar H⁺-ATPase Expression and Subcellular Localization. For vacuolar H⁺-ATPase expression, Western blot analysis was performed after 24 h of treatment with Nik-12192 (2 µM), as already reported (Zuco et al., 2004). Antivacuolar H⁺-ATPase D primary antibody (Sc-20945; Santa Cruz Biotechnology, Santa Cruz, CA) was used.

For subcellular localization of the protein, cells were seeded on coverslips in six multiwell plates, treated for 24 h, fixed in 4% paraformaldehyde, and then incubated for 60 min with vacuolar H⁺-ATPase antibody. After washing in PBS containing 1% bovine serum albumin, samples were incubated with the FITC-secondary anti-rabbit antibody (Invitrogen) and mounted with Mowiol (Mowiol 4.88; Calbiochem, San Diego, CA) and observed by fluorescence microscope DMRB (Leitz). Images were taken by a 50x immersion oil objective, with an excitation light provided by a 100-W Hg lamp and an excitation filter at 450 to 490. Images were recorded with a Spot Insight digital camera (Delta Sistemi, Rome, Italy) equipped with a system of image analysis (IAS 2000; Delta Sistemi).

Migration and Invasion Assays. H460 tumor cells were seeded in complete medium and, after 24 h, treated with different concentrations of the drug for 24 h. Then, cells were harvested and transferred in the upper chamber of 24 well Transwell plates (Costar; Corning Incorporated, Corning, NY) in serum-free medium (1.2 x 10⁵ cells/well), whereas conditioned medium was added to the lower chamber. To maintain cells under drug exposure during the assay, the drug was added in the upper and lower compartments of the chambers at the same concentration as in the previous treatment. In migration assay, after 5 h of incubation at 37°C, filters were cleaned on the upper side with a cotton swab, fixed in 99% ethanol, and stained with a solution of 0.4% sulforhodamine B in 1% acetic acid, and migrated cells were counted under an inverted microscope. All experiments were performed three times in duplicate.

In the invasion assay, the procedure was the same as described above, except that the Transwell chamber membranes were coated with 12.5 µg of Matrigel/well (BD Biosciences, San Jose, CA) and that the Transwell plates were processed after 24 h of incubation at 37°C.

Wound Healing Assay. H460 tumor cells were seeded onto 24-well plates at 4.5 x 10⁵ cells/well in growth medium. A single scratch wound was created on confluent monolayers by using a micropipette tip (Liang et al., 2007). Cells were washed with PBS to remove cell debris, supplemented with serum-free medium containing NiK-12192, and monitored. Images were captured by a Spot Insight digital camera (Delta Sistemi) using a 4x contrast phase objective at 0 and 48 h postwounding, and wound distance (micrometers) was measured by system of image analysis (IAS 2000). Three different regions for each wound were considered. The experiment was performed two times.

β3 Expression and Cellular Localization. Cells, seeded on coverslips in six multiwell plates, were treated with NiK-12192 (2 µM). After 24 h of drug exposure, samples were incubated with a 1:400 solution of FITC-conjugated mouse anti-human-vβ3 monoclonal antibody (Mab1976F; Chemicon International, Temecula, CA) for 1 h, mounted with Mowiol, and observed by fluorescence microscope as reported above.

Tubulin and Actin Microscopic Analysis. For confocal experiments, cells were grown on coverslips, treated with NiK-12192 (2 µM) for 24 h, fixed with 4% paraformaldehyde for 30 min, and then washed and added with 70% ethanol for 20 min at –20°C. After washing with PBS containing 1% bovine serum albumin, cells were incubated with phalloidin, fluorescein-isothiocyanate-labeled 1:200 (Sigma-Aldrich), and monoclonal anti-β-tubulin 1:2000 antibody (clone SDL3D10; Sigma-Aldrich) for 1 h at 37°C. Samples were then incubated with Alexa 594-conjugated anti-mouse IgG antibody (Invitrogen) for 30 min, mounted with Prolong Gold Antifade reagent (Invitrogen) and analyzed by confocal microscopy (Microradiance 2000; Bio-Rad Laboratories, Inc., Hercules, CA) equipped with Ar (488 nm) and HeNe (543 nm) lasers. Images (1024 x 1024 pixels) were obtained using a 60x oil immersion lens and analyzed using Lasersharp 2000 software. Reported images represent a single z-section of the samples with 0.5-µm step. The pinhole diameter was regulated according to the value suggested by the acquisition software to obtain the maximum resolution power.

Three-Dimensional Cell Culture. H460 tumor cells (0.8 x 10⁴ cells/plate) were cultured in bacteriological plates (5-cm diameter) in complete culture medium. After 6 days, single spheroids were transferred to 96-well plates in culture medium containing NiK-12192 at different concentrations. Measures of spheroid diameters were taken by a micrometer lens in the inverted microscope at 0, 24, 48, and 72 h. Values are reported as mean percentage of the diameter length over that of time 0 (100%). Six spheroids for each point were considered.

In Vivo Studies
Animals. All experiments were carried out using female athymic nude CD-1 mice, 8 to 11 weeks old (Charles River, Calco, Italy). Mice were maintained in laminar flow rooms with constant temperature and humidity. Experimental protocols were approved by the Ethic Committee for Animal Experimentation of the Istituto Nazionale Tumori (Milan, Italy), according to the United Kingdom Coordinating Committee on Cancer Research Guidelines (Workman et al., 1998).

Antitumor and Antimetastatic Activities. The effects of NiK-12192, topotecan, or combination of them on the growth of primary tumors and spontaneous lung metastases were tested in mice inoculated s.c. in the right flank with H460 ascitic tumor cells (2 x 10⁶/mouse). Each control or drug-treated group included 9 to 11 mice. The growth of the s.c. tumor was followed by biweekly measurements of tumor diameters with a Vernier caliper. Tumor weight (TW) and TW inhibition percentage in treated over control mice were calculated as previously reported (Cassinelli et al., 2006). Drug treatment was delivered p.o., by gavage, daily for 9 weeks (weekends excluded), starting the day after cell injection (day 1). NiK-12192 was delivered at the dose of 30 mg/kg, topotecan at the dose of 1 mg/kg. In the mice treated with the combination, NiK-12192 was delivered approximately 1 h after topotecan. At day 63, tumor-bearing mice were sacrificed by cervical dislocation, and their lungs were removed. Lung lobes were spliced between two glass slides, and the metastatic nodules were macroscopically counted against a bright light. Reading of metastases was performed by two independent observers, unaware of the experimental group, with an interobserver reproducibility >95%. The metastatic nature of these areas was confirmed by histological analysis.

To induce experimental lung metastases, mice (seven to eight per group) were injected i.v. with ascitic H460 cells (2 x 10⁶/mouse) (Cassinelli et al., 2006) and then treated p.o. daily with NiK-12192 (30 mg/kg), and/or topotecan (1 mg/kg) for 7 weeks (weekends excluded), from day 1. At day 83, all mice were sacrificed for lung observation as described above. In the presence of confluent metastatic nodules, lungs were defined as "tumor invaded," and tumor nodules were not counted. The number of 500 metastases was ascribed. For ethical reasons, mice presenting signs of suffering were sacrificed before day 83.

For histological observation, s.c. implanted tumor-bearing mice (three to four mice per group) were treated with NiK-12192 (30 mg/kg) alone or in combination with topotecan (1 mg/kg), daily for 2 weeks, starting at day 1. The day after the last treatment, mice were sacrificed, and their tumors were excised and fixed in 10% buffered formalin for hematoxylin-eosin staining. Angiogenesis was analyzed by detection of microvessel density (MVD). Number of blood vessels was assessed in three fields, at 400x, within non-necrotic areas. Only vascular units containing erythrocytes were considered (Belotti et al., 1996).

Statistical Analysis. The Student's t test was used to compare TW in control and treated mice. The number of metastases and microvessels in control and treated mice was compared by the Mann-Whitney test. All analyses were performed using GraphPad Prism software (GraphPad Prism Inc., San Diego, CA).

	Results

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Modulation of Intracellular pH. NiK-12192 was able to decrease the intracellular pH, as assessed by means of the pH-dependent shift of the SNARF-4F emission band through the microspectrofluorometric analysis. Indeed, as shown in Fig. 1, in NiK-12192-treated cells, the pH probe exhibited a shift to shorter wavelengths consistent with a more acidic pH environment with respect to the control cells. The emission peak position of control cells was constant at approximately 650 nm, whereas those of the cells treated for 24 h with NiK-12192 (2 µM) were always comprised between 640 and 630 nm. The estimation of the intracellular pH value was performed exclusively on the base of the blue shift of the SNARF-4F longer emission band and indicated a decrease from approximately 7.2 for the control to a pH range of approximately 6.2 for NiK-12192-treated cells. The increase in the emission band centered at approximately 560 nm was not considered because it can be due both to the conversion of SNARF-4F emission at acidic pH and to a shoulder of the NiK-12192 emission. Although the level of vacuolar H⁺-ATPase expression was not modified by treatment with the compound (Fig. 2A), NiK-12192 produced changes in intracellular localization (Fig. 2B). In control samples, vacuolar H⁺-ATPase localized in the cytoplasm and was evident as spots in most cells. Treatment with the inhibitor caused a more homogeneous and diffuse distribution of the enzyme.

View larger version (26K): [in this window] [in a new window]   Fig. 1. Intracellular pH modulation by NiK-12192 in H460 cells. SNARF-4F fluorescence spectra were recorded in single cells from NiK-12192-treated and -untreated samples at 366-nm excitation and 550- to 800-nm interval emission. Each spectrum derives from a single cell. Three spectra are reported for treated samples. Only one spectrum is reported for control cells, due to the homogeneous results.

View larger version (54K): [in this window] [in a new window]   Fig. 2. Vacuolar H+-ATPase expression and subcellular localization in H460 cells. A, Western blot analysis performed on nitrocellulose filters incubated with anti-vacuolar H+-ATPase D primary antibody and peroxidase-conjugated anti-rabbit antibodies. Actin was used as control of loading. 1, untreated control; 2, cells treated with NiK-12192 (2 µM) for 24 h. B, subcellular localization as by fluorescence microscopy in H460 cells untreated or treated with NiK-12192 (2 µM) for 24 h and stained with anti-vacuolar H+-ATPase D antibody and with the FITC-secondary anti-rabbit antibody.

View larger version (37K): [in this window] [in a new window]   Fig. 3. Adhesion, migration, and invasion in NiK-12192-treated H460 cells. After 24 h of exposure to NiK-12192, cells were seeded in Transwell plates with membranes coated with (invasion) or without (migration) matrigel, as reported under Materials and Methods. Data indicate the percentages (±S.D.) of adherent, migrated, or invaded cells in comparison with untreated samples; data are derived from two independent experiments performed in duplicate.

View larger version (99K): [in this window] [in a new window]   Fig. 4. Effect of NiK-12192 on wound healing of H460 cell monolayers after scratching. Monolayers of H460 cells were scratched and treated with NiK-12192 (0.5 µM). At 48 h, the distance between the rims of the scratching was measured by an Image Analysis System. Pictures of the same samples at different times after scratching are reported and are representative of three different regions from a single wound.

Modulation of Factors Related to Cell-Cell Interactions. Because the β3 integrin is highly expressed in lung carcinoma cells (Albert et al., 2006) and associations between integrins and vacuolar H⁺-ATPase have been already reported (Lee et al., 2004), the intracellular localization of β3 was investigated in H460 cells after treatment. As shown in Fig. 5, in untreated cells, the integrin was found mainly polarized in the cell cytoplasm, whereas in treated cells, β3 was more homogeneously diffused in the cytoplasm, thus suggesting a different behavior of treated cells in cell-cell and cell-matrix interactions.

View larger version (93K): [in this window] [in a new window]   Fig. 5. β3 Intracellular localization in H460 cells. After 24 h of exposure to NiK-12192 (2 µM), cells were incubated with anti-β3 monoclonal antibody and stained with secondary FITC antibody. Pictures were taken by fluorescence microscope at 500x.

View larger version (93K): [in this window] [in a new window]   Fig. 6. Effect of NiK-12192 on cytoskeleton proteins. Images of H460 cells double-stained with anti-tubulin monoclonal antibody followed by Alexa-594-conjugated anti-mouse (red) and FITC-phalloidin (actin fibers, green) obtained by sequential scanning with confocal laser microscope. The third panel of each row represents the merge of green and red channels; one of 24 frames is shown. Long and regular fibers of tubulin in the cell cytoplasm and filaments of actin forming pseudopodia are visible in control cells (top row). In H460 cells after 24 h of drug exposure (2 µM), actin and tubulin fibers looked unstructured, and spots of aggregation were evident (bottom row). Scale bar, 10 µm.

The drug ability to interfere with three-dimensional growth of H460 cells was examined on agar substratum because this effect may have relevance in the metastatic growth. Spheroids with a diameter of 100 to 150 µm were treated with subtoxic concentrations of the drug, and their size was then determined at various exposure times (Fig. 7). In spheroids treated with NiK-12192, a concentration-dependent inhibition growth was found already at 24 h, also with the low concentration (0.5 µM).

View larger version (16K): [in this window] [in a new window]   Fig. 7. Effect of NiK-12192 on the three-dimensional growth of H460 spheroids. Single spheroids were grown in 96-well plates. Measures of spheroid diameters were taken by a micrometer lens in an inverted microscope at various times. Values are reported as percentage of the diameter length over that of time 0. Each point is referred to as mean value (±S.D.) of six spheroids. , control; , NiK-12192, 0.5 µM; , NiK-12192, 2 µM.

View larger version (29K): [in this window] [in a new window]   Fig. 8. Effect of NiK-12192 on the growth of primary H460 tumor. Mice were implanted s.c. with H460 lung cancer cells (2 x 106 cells/mouse) and treated p.o. with solvent (x), 1 mg/kg topotecan (TPT) (), 30 mg/kg NiK-12192 (), or TPT + NiK-12192 (). Treatments were delivered daily from day 1, 5 days/week, for 9 weeks. TPT was delivered approximately 1 h before NiK-12192. Means of 9 to 11 tumors/group are reported.

To further get insight into the steps of the metastatic process affected by NiK-12192, we examined the drug effects on experimental lung metastases, i.e., induced by i.v. injection of H460 ascitic cells, thus bypassing the first steps of the metastatic process. Mice were treated p.o. with NiK-12192, 30 mg/kg, according to the same schedule as for spontaneous metastasis experiment (for 7 weeks) and sacrificed for lung observation at day 83 (Table 1). At such a time, all mice in the control group presented a large metastatic burden and most of them had tumor-invaded lungs; no relevant differences could be observed in the NiK-12192-treated mice. In contrast, topotecan was able to significantly reduce the number of lung metastases (P < 0.05 versus control mice). Interestingly, the combination of NiK-12192 and topotecan resulted in an even higher inhibition of the metastases number versus control mice (P < 0.01) and in the lack of metastasis in one of seven mice.

Because angiogenesis, in particular the migration of microvascular endothelial cells, is known to be regulated by extracellular pH (Orive et al., 2003; Rojas et al., 2006), the effect of NiK-12912 treatment on tumor neovascularization was investigated in primary s.c. growing H460 tumors treated daily (30 mg/kg) for 2 weeks (Fig. 9). Tumor angiogenesis appeared only marginally reduced by treatment with NiK-12192, because the MVD of treated tumors was lower compared with that of control tumors (30% inhibition, but not significantly different). Topotecan, according to the daily schedule of treatment, showed a higher antiangiogenic activity (42% inhibition, P < 0.01), and the combined treatment achieved a somewhat higher MVD inhibition (52%, P < 0.005 versus control).

View larger version (19K): [in this window] [in a new window]   Fig. 9. MVD in H460 tumor xenografts. Mice implanted s.c. with H460 lung cancer cells (2 x 106 cells/mouse) were treated p.o. with solvent (control) or TPT (1 mg/kg) or NiK-12192 (30 mg/kg) or TPT + NiK-12192. Treatments were delivered daily, from day 1, for 2 weeks. Topotecan was delivered approximately 1 h before NiK-12192. The day after the last treatment, tumors were excised and fixed in 10% buffered formalin for hematoxylin-eosin staining. MVD was assessed in three fields, at 400x, within non-necrotic areas. Means of three to four tumors/group (S.D.) are reported. *, P < 0.01; **, P < 0.005 versus control.

	Discussion

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Despite the significant inhibition of the metastatic potential of s.c. growing H460 tumor, NiK-12192 exhibited a marginal effect on the development of artificial metastases (i.e., after i.v. injection of the same tumor cells). The poor effect shown by NiK-12192 against artificial lung metastases and primary tumor supports the hypothesis that the compound was able to inhibit the early steps of the metastatic process, rather than the tumor cell growth in the target organ.

We have previously shown, either in cellular and in in vivo systems, that NiK-12192 is able to potentiate topotecan efficacy against human colon carcinomas, possibly by a cytoplasmic redistribution of the cytotoxic drug and by modulating the expression of membrane-associated transport systems (Petrangolini et al., 2006). In the present study, we showed that the combination topotecan + NiK-12192 resulted in a significant reduction of spontaneous metastasis incidence over that achieved by each single drug. The mechanisms of antimetastatic effects of NiK-12192 and topotecan are substantially different. The effects of topotecan were probably related to its cytotoxic activity because both spontaneous and artificial metastases were inhibited by the topotecan treatment. However, the antimetastatic effect of topotecan could reflect multiple events including a direct tumor growth inhibition and an early antiangiogenic effect, which could account for the topotecan efficacy in reducing both spontaneous and artificial metastases. The antiangiogenic effects of topotecan in primary tumor could at least in part account for the synergistic inhibition of spontaneous metastasis by the topotecan + NiK-12192 combination. The angiogenic effects of camptothecins have been already described and are more evident when the drugs are delivered by the daily low-dose schedule (Petrangolini et al., 2003). The effect of NiK-12192 on microvessel density, which may be expected on the basis of the knowledge that a significant number of angiogenic factors acts closely related to intracellular H⁺ dynamics (Orive et al., 2003), was marginal compared with that achieved by the cytotoxic drug, suggesting that the antiangiogenic effect is not a primary mechanism of antimetastatic activity.

In the previous study, an increased toxicity was achieved by the combination topotecan + NiK-12192, when the topotecan maximum tolerated dose (every 4th day for three times) was delivered (Petrangolini et al., 2006). Thus, a different regimen was investigated in the present study, i.e., a daily low-dose for long time. Such regimen was well tolerated, and, despite the prolonged time of treatment (up to 9 weeks), no signs of toxicity (nor lethal toxicity, nor body weight loss) were observed in mice treated with topotecan + NiK-12192, thus indicating that the toxicity of the combination can be modulated in this regimen.

The ability of NiK-12192 to inhibit vacuolar H⁺-ATPase activity appears to influence a number of cellular functions, which may be relevant to the observed antimetastatic efficacy in the in vivo studies. Indeed, the results achieved in the in vitro assays support the ability of NiK-12192 to modulate the intracellular pH and to inhibit a series of metastasis-associated events. Our results indicated that in H460 cells, NiK-12192 was able to cause cytoskeleton modifications, including aggregation of actin and tubulin microfilament as well as reduction of cell pseudopodia. In addition, it was able to modulate the subcellular distribution of adhesion molecules such as vβ3 integrin. The reduced cell motility was reflected in the inhibition of cell migration, invasion, and likely in wound healing. Several lines of evidence have indicated a relation among cellular functions involving transport systems, cytoskeleton, and adhesion molecules (Vitavska et al., 2003; Zuo et al., 2006). These functions, which may be modulated by intracellular and extracellular pH, play a critical role in cellular interactions with the extracellular matrix. A lot of evidences suggested important interactions among vacuolar H⁺-ATPase and cytoskeleton; the aminoterminal domain of the vacuolar H⁺-ATPase subunit B has been already demonstrated to contain a filamentous actin binding site (Holliday et al., 2000; Zuo et al., 2006), and the vacuolar H⁺-ATPase subunit C can bind both F- and G-actin (Vitavska et al., 2005) by controlling the dynamics of the actin cytoskeleton. A modification of the cytoskeleton has been in fact observed in our model after a 24-h treatment with NiK-12192, resulting in a lack of regular structure for actin filaments together with the absence of pseudopodia.

Vacuolar H⁺-ATPases are implicated in multiple diseases, including osteoporosis, deafness, and cancer, and their role as drug target is under study (Bowman and Bowman, 2005). A recent study reported the efficacy of another vacuolar H⁺-ATPase inhibitor, FR202126, in reducing osteolysis induced by a metastatic breast cancer in a syngenic mouse model. However, the compound was unable to prevent lung or liver metastasis (Niikura, 2007). It is unclear whether the drug specificity for various vacuolar H⁺-ATPase isoforms expressed in different cells (including osteoclasts) may be responsible for the different effects of these inhibitors. The ability of the natural macrolide vacuolar H⁺-ATPase inhibitor bafilomycin A1 to decrease in vitro both invasion and migration of human breast metastatic cells (Sennoune et al., 2004a) strongly supports the key role of vacuolar H⁺-ATPase in these two fundamental processes of tumor malignancy. The implication of the enzyme in modulation of the metastatic phenotype is supported by the observation that cells with a low metastatic potential are characterized by a widespread distribution of the enzyme in cytoplasm. Relevant to this point is the NiK-12192-induced modulation of intracellular distribution of the enzyme.

In conclusion, we have shown for the first time that a vacuolar H⁺-ATPase inhibitor, NiK-12192, has a strong antimetastatic effect in a preclinical model of spontaneous lung metastases. Moreover, the effect was further increased by the combination with a cytotoxic drug, thus supporting a potential interest for vacuolar H⁺-ATPase inhibitors in the therapy of cancer.

	Footnotes

 
This work was partially supported by the Associazione Italiana Ricerca sul Cancro, Milan, Italy, and by the Ministero della Salute, Rome, Italy.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.128587.

ABBREVIATIONS: NiK-12192, 4-(5,6-dichloro-1H-indol-2-yl)-3-ethoxy-N-(2,2,6,6-tetramethyl-piperidin-4-yl)-benzamide; DMSO, dimethyl sulfoxide; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; TW, tumor weight; MVD, microvessel density; TPT, topotecan; FR202126, (2,6-dichloro-N-[3-methyl-4-(3-methyl-2-oxo-1-imidazolidinyl)-8-quinolinyl]benzamide).

Address correspondence to: Dr. Franco Zunino, Fondazione Istituto di Ricovero e Cura a Carattere Scientifico Istituto Nazionale Tumori, Via Venezian 1, 20133 Milan, Italy. E-mail: franco.zunino{at}istitutotumori.mi.it

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