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CHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY

Effect of a Novel Vacuolar-H⁺-ATPase Inhibitor on Cell and Tumor Response to Camptothecins

Istituto Nazionale Tumori, Milan, Italy (G.Pe., R.S., G.Pr., L.D.B., M.T., F.Z.); Consiglio Nazionale delle Ricerche, Università di Pavia, Pavia, Italy (A.C.C.); and Nikem Research, Baranzate, Milan, Italy (P.M., P.B., C.F.)

Received February 24, 2006; accepted May 18, 2006.

	Abstract

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The vacuolar-H⁺-ATPase, functionally expressed in cell membranes, is known to play a relevant role in intracellular pH regulatory mechanisms, because it is implicated in pumping protons into the extracellular environment or in sequestrating excess protons into acidic vacuolar compartments. Because tumor cells exist in a hypoxic microenvironment and produce acidic metabolites, this regulatory mechanism is recognized as a protective function. This study was designed to investigate the effect of NiK-12192 [4-(5,6-dichloro-1H-indol-2-yl)-3-ethoxy-N-(2,2,6,6-tetramethyl-piperidin-4-yl)-benzamide], an indole derivative identified as an effective inhibitor of vacuolar-H⁺-ATPase, on the cytotoxic activity of two camptothecins, i.e., topotecan and SN-38 (7-ethyl-10-hydroxycamptothecin, the active metabolite of irinotecan). The cellular studies performed in two pairs of human colon carcinoma cell lines, i.e., LoVo and LoVo/DX (overexpressing P-glycoprotein) and HT29 and HT29/Mit (overexpressing breast cancer resistant protein), indicated an enhancement of the antiproliferative effect of camptothecins by concomitant exposure to subtoxic concentrations of NiK-12192. Studies of subcellular distribution indicated that whereas topotecan alone localized mainly in mitochondria and endoplasmic compartment, the simultaneous presence of NiK-12192 caused a cytoplasmic redistribution. In HT29/Mit cells, NiK-12192 reverted the pattern of acidification induced by topotecan. The potentiation of topotecan efficacy by NiK-12192 was documented by an increased efficacy of the combination in both the HT29 tumor xenografts, being more evident in the topotecan-resistant HT29/Mit tumor. In conclusion, the vacuolar-H⁺-ATPase inhibitor NiK-12192 was able to potentiate the cytotoxic/antitumor effects of camptothecins, either in in vitro or in in vivo systems. Such findings support a potential interest for the use of vacuolar-H⁺-ATPase inhibitors in combination therapy to improve camptothecin efficacy.

Tumor cells often exist in a hypoxic microenvironment and develop high glycolytic activity, which, in turn, produces acidic metabolites. Four major types of pH regulatory mechanisms have been identified in tumor cells, i.e., Na⁺-H⁺ exchangers, bicarbonate transporters, proton-lactate symporters, and proton pumps (Putnam, 2001). Among them, the vacuolar-H⁺-ATPase has been shown as essential (Nishi and Forgac, 2002). At the cell surface, vacuolar-H⁺-ATPase plays a role in maintaining an intracellular environment favorable for growth while producing an acidic extracellular environment favorable for cell invasion (Montcourrier et al., 1994; Sennoune et al., 2004). In the intracellular membranes of organelles, vacuolar-H⁺-ATPase plays a role in drug compartmentation observed in resistant cells (Larsen et al., 2000). The vacuolar-H⁺-ATPase has been shown to be overexpressed in multidrug-resistant cells, and altered cytosolic pH has been implicated in drug resistance (Simon et al., 1994; Raghunand and Gillies, 2000). Thus, vacuolar-H⁺-ATPase may be a potential target to increase the therapeutic efficacy of antitumor agents, by modulating pH-dependent cellular resistance mechanisms (Izumi et al., 2003). The role of vacuolar-H⁺-ATPase inhibitors in tumor therapy is a matter of investigation, and recent studies have shown the ability of proton-pump inhibitors to sensitize tumor cells to various cytotoxic agents (McSheehy et al., 2003; Ouar et al., 2003; Luciani et al., 2004).

Vacuolar-H⁺-ATPase inhibitors have been known for many years, with the natural compound bafilomycin A1 being the most extensively studied (Bowman et al., 1988). However, bafilomycin A1 is not able to distinguish among the various types of vacuolar-H⁺-ATPase; therefore, the in vivo treatment is toxic, due to an inhibition of all of the essential vacuolar-H⁺-ATPases and to systemic alteration of cellular physiology (Farina and Gagliardi, 2002). Starting from bafilomycin A1, extensive structure-activity relationship studies allowed the identification of the key structural requirements for the vacuolar-H⁺-ATPase inhibitory activity (Gagliardi et al., 1999). SB242784, a potent and selective inhibitor of the osteoclast vacuolar-H⁺-ATPase, has been identified as a potent inhibitor of in vitro (Nadler et al., 1998) and in vivo bone resorption (Visentin et al., 2000). Further modification of the SB242784 structure, in which the diene chain was replaced by substituted phenyl ring, led to the identification of new classes of vacuolar-H⁺-ATPase inhibitors (Farina et al., 2001). Among the compounds of the series, NiK-12192, a novel substituted indol-2yl-N-(2,2,6,6-tetramethylpiperidin-4-yl)benzamide (Fig. 1), was selected for development due to its capability to inhibit the enzyme in various biological systems.

View larger version (8K): [in this window] [in a new window]   Fig. 1. Chemical structure of NiK-12192.

	Materials and Methods

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Drugs. Topotecan (Hycamtin; Glaxo SmithKline S.p.A., Verona, Italy), SN-38 (provided by Prof. L. Merlini, University of Milan, Milan, Italy), bafilomycin A1 (Sigma-Aldrich, Milan, Italy), and NiK-12192 (Fig. 1; provided by Nikem Research, Milan, Italy) were used throughout the study.

For in vitro studies, 1 mg/ml stock solutions were stored in dimethyl sulfoxide for NiK-12192 and SN-38 and in ethanol for topotecan, and they were diluted before their use in culture medium. Maximal final concentrations of dimethyl sulfoxide and ethanol in culture medium were 0.6 and 0.1%, respectively, which have been verified to be nontoxic on our cell systems. For in vivo studies, topotecan was dissolved in sterile distilled water, and 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.

Bafilomycin-Sensitive ATPase Assay. Inhibition of bafilomycin-sensitive ATPase activity was assayed by measuring the release of inorganic phosphate, during 30 min of incubation, of the microsomal fraction in 96-well plates at 37°C, as described by Boyd et al. (2001). Procedures for the preparation of membranes have been described previously (Boyd et al., 2001). All of the ATPase assays were performed in the presence of oligomycin and vanadate as inhibitors of F- and P-ATPases, to exclude possible interactions.

Cell and Tumor Lines. The combination of camptothecins and NiK-12192 was investigated in two human colorectal carcinoma cell lines, HT29 and LoVo. The panel included the HT29/Mit subline, selected for resistance to mitoxantrone, overexpressing BCRP, and the LoVo/DX subline, selected for resistance to doxorubicin, overexpressing P-glycoprotein (Grandi et al., 1986; Perego et al., 2001). HT29 and HT29/Mit cell lines were maintained in McCoy's medium (Cambrex Bio Science Verviers S.p.r.l., Verviers, Belgium), and LoVo and LoVo/DX cells were maintained in F-12 medium (BioWhittaker), both with 10% heat-inactivated fetal bovine serum added (Invitrogen, Gaithersburg, MD). All cell lines were maintained in a humidified atmosphere with 5% CO₂ at 37°C. For in vivo studies, tumor lines were obtained by s.c. injection in nude athymic mice of exponentially growing tumor cells, and lines were maintained by successive passages of tumor fragments, as described previously (Pratesi et al., 1989).

Antiproliferative Activity Studies in Cell Cultures. Cells were seeded in 96-well plates. After 24 h, cells were exposed to NiK-12192, to the camptothecins or to both compounds for 72 h. After treatment, cells were fixed in 50% trichloroacetic acid and processed for sulforhodamine B assay (Skehan et al., 1990). Dose-response curves were plotted, and analysis of drug interaction was done by a modified method of Drewinko (Zanchi et al., 2005). Combination index is calculated as the ratio SFa x SFb/SFab, where SFa, SFb, and SFab are the surviving fraction of cells treated with the compound a or b or a and b together, respectively. A combination index >1 indicates greater than additive effects, i.e., synergism (the higher the value, the greater the degree of synergism); a combination index of 1 indicates additivity; and a combination index <1 indicates antagonism.

Subcellular Localization of NiK-12192 and Topotecan. HT29 and HT29/Mit cells were seeded on coverslips and treated, 48 h later, with 22 µM topotecan with or without 1 µM NiK-12192 for 40 min. The fluorescence imaging analysis was performed by means of an Argus VIM 100 processor digital system (Hamamatsu Photonics Deutschland GmbH, Herrsching am Ammersee, Germany), using a high-sensitivity ISIT camera (C2400-09; Hamamatsu Photonics), coupled to a fluorescence microscope (Leica, Wetzlar, Germany). Excitation light was provided by a 100-W mercury lamp and selected by a 366-nm (T% = 40) interferential filter. The images were acquired through a 430-nm-long pass filter, by means of a 95x Leitz objective (iris diaphragm, numerical aperture 1.10–1.33), digitally stored on a magnetic mass memory support, and processed by means of the Argus 100 control program (Hamamatsu Photonics). In cells treated with the combination, the fluorescence of topotecan and Nik-12192 was superimposed.

Determination of Lysosomes Acidity. Cells were treated 24 h after seeding with 1 µM NiK-12192, topotecan (0.1 µM for HT29 or 5 µM for HT29/Mit), or the combination of the two compounds. After 24 h of treatment, trypsinized cells were stained with acridine orange (Sigma-Aldrich, St. Louis, MO) at the final concentration of 1 µg/ml in phosphate-buffered saline for 17 min, washed, and analyzed by FACScan (BD Biosciences) or observed by a fluorescence microscope. In acridine orange-stained cells, the cytoplasm and nucleus fluoresce bright green and dim red, whereas acidic compartments fluoresce bright red. The intensity of the red fluorescence is proportional to the degree of acidity and/or the volume of the cellular acidic compartment (Traganos and Darzynkiewicz, 1994). Therefore, by comparing the mean red/green fluorescence ratio within different cell populations, it is possible to measure a change in the degree of acidity and/or the fractional volume of their cellular acidic compartment. Dot plots of FL1 (green fluorescence) and FL3 (red fluorescence) obtained by FACScan analysis were subdivided into eight regions, and the ratios of the mean values for red and for green fluorescence are reported. Slides were prepared with the same samples and observed by DMRB (Leica) fluorescence microscope equipped with a 100-W lamp. Images were recorded with a Spot Insight digital camera (Delta Sistemi, Rome, Italy). No interference between acridine orange lysosomal staining and topotecan and/or NiK-12192 is expected, because of the different excitation emission wavelengths of acridine orange (excitation at 490 nm and emission in the range of 530–640 nm). Regardless, a quenching effect would result in a decrease of the emission intensity of acridine orange, rather than in a change of the emission color.

Western Blot Analysis. Adherent and floating cells were lysed and prepared for SDS-polyacrylamide gel electrophoresis as described previously (Zuco et al., 2004). After separation on SDS-polyacrylamide gel electrophoresis, proteins were transferred to nitrocellulose filters. Membranes were incubated with anti-BCRP primary antibody (Alexis Corporation, Lausanne, Switzerland) overnight and with peroxidase-conjugated anti-mouse antibody to reveal immunoreactive bands using the enhanced chemiluminescence detection system from GE Healthcare (Little Chalfont, Buckinghamshire, UK). Anti-actin antibody, used as control for loading, was from Sigma-Aldrich.

Antitumor Activity in Vivo. Antitumor activity experiments were performed using 8- to 11-week-old female athymic nude CD-1 mice (Charles River Italia, Calco, Italy). Mice were maintained in laminar flow rooms with constant temperature and humidity. Experimental protocols were approved by the Ethics 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).

Experimental groups included five mice bearing bilateral s.c. tumors (9–10 tumors/group). Tumor growth was followed by biweekly tumor diameter measurements using a Vernier caliper. Tumor weight (TW) was calculated, considering tumor density = 1, according to the formula TW (milligrams) = tumor volume (cubic millimeters) = d² x D/2, where d and D are the shortest and the longest diameter, respectively. Drug treatments started when mean TW was approximately 100 mg. Drugs were delivered orally, by gavage, in a volume of 10 ml/kg body weight according to different treatment schedules and doses. Details are reported under Results. In combination studies, NiK-12192 was delivered starting 1 h after each topotecan treatment. Control mice were treated orally with solvent, in parallel with drug treatments.

Drug efficacy was assessed as 1) tumor weight inhibition percentage (TWI%) = 100 – (mean TW of treated mice/mean TW of control mice x 100), evaluated during and after drug treatment; 2) the log₁₀ cell kill (LCK) achieved by the drug treatment, according to the formula (T–C)/DT x 3.32, where T and C are the mean time (days) required for treated (T) and control (C) tumors, respectively, to reach a determined weight (1 g), and DT is the doubling time of control tumors. To define a synergistic efficacy, in the groups treated with the combination the "expected TWI% value" was calculated by summing TWI% induced by topotecan + TWI% induced by NiK-12192 on the surviving fraction of tumor. The results achieved represent the TWI% due to an additive effect of the two drugs. Synergism is defined when the "observed TWI%" of the combination was superior to the expected value (Berenbaum, 1989).

Drug tolerability was assessed in tumor-bearing mice as 1) lethal toxicity, i.e., any death in treated mice occurring before any death in control mice; 2) body weight loss percentage (BWL%) = 100 – (body weight on day x/body weight on day 1 x 100), where x represents a day after or during the treatment period. Experimental groups were sacrificed by cervical dislocation when mean TW was at a maximum of 1 ± 0.5 g.

Statistical Analysis. Student's t test (two-tailed) was used to compare treated and control samples throughout all of the study. p values lower than 0.05 were considered statistically significant. Analyses were performed with Prism, version 4.0 (GraphPad Software Inc., San Diego, CA).

	Results

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Inhibition of Vacuolar-H⁺-ATPase Activity. The results shown in Table 1 indicate that NiK-12192 was an effective inhibitor of vacuolar-H⁺-ATPase activity in various systems, including bovine chromaffin granules and membranes from human osteoclastoma, human kidney cortex, and human liver.

Antiproliferative Activity. The antiproliferative activity of the tested camptothecins (topotecan and SN-38) or their combination with NiK-12192 was investigated in colon carcinoma cells, including sublines characterized by overexpression of transport systems. Specifically, HT29/Mit cells, selected for resistance to mitoxantrone and characterized by high expression levels of BCRP, exhibited cross-resistance to topotecan and SN-38. LoVo/DX cells, selected for resistance to doxorubicin and characterized by a typical multidrug-resistant phenotype, showed a moderate cross-resistance to SN-38 (Table 2). The IC₅₀ values of NiK-12192 itself were comparable in parental cells and resistant sublines. Using a concomitant 72-h exposure, the combination of topotecan and subtoxic concentrations of NiK-12192 resulted in a sensitization of both the HT29 parental and resistant cells (Fig. 2). A sensitization to SN-38 by NiK-12192 was found in LoVo/DX and their parental cells. The potentiation of camptothecins activity was found with NiK-12192 concentrations not affecting cell growth, whereas at concentrations inhibiting cell proliferation only additive effects were observed (data not shown). An analysis of drug interaction performed with the Drewinko method (Zanchi et al., 2005) also supported the potentiation of camptothecin cytotoxicity. Because the sensitization was more marked in combination with topotecan in HT29 and its mitoxantrone-resistant subline, such cell systems were chosen for further studies.

View larger version (25K): [in this window] [in a new window]   Fig. 2. Synergistic activity of NiK-12192 in combination with camptothecins on HT29 and LoVo parental and resistant-derived cell lines. Cell growth was evaluated by sulforhodamine B assay after 72 h of drug exposure. Numbers indicate the combination index according to Drewinko. topotecan or SN-38 alone; , topotecan or SN-38 + NiK-12192. NiK-12192 was used at subtoxic concentrations, i.e., 0.5 µM in HT29 and HT29/Mit and 1 µM in LoVo and LoVo/DX. Values are the mean ± S.D. of three independent experiments.

Subcellular Localization of Topotecan. To elucidate the cellular bases of the interaction between topotecan and NiK-12192, some aspects of the cellular accumulation and distribution of topotecan with or without NiK-12192 were investigated (Fig. 3). In HT29 and HT29/Mit cell lines, after exposure to topotecan, we observed an emission signal in the perinuclear region with polar cytoplasmic diffusion in some cells and the presence of some round vesicles. These patterns were already reported to indicate a topotecan localization in mitochondria and endoplasmic reticulum, on the basis of the comparative analysis with specific staining for these cell structures (Croce et al., 2004). The concomitant treatment with both compounds resulted in a diffuse distribution of the fluorescence in the cytoplasm, with an intense polar localization in some cells.

View larger version (128K): [in this window] [in a new window]   Fig. 3. Fluorescence images of HT29 and HT29/Mit cells incubated in 22 µM topotecan with or without 1 µM NiK-12192 for 40 min. Images were acquired by means of a processor digital system using a high-sensitivity ISIT camera coupled to a fluorescence microscope under excitation at 366 nm and selected through a 430-nm-long pass filter. Pictures were adjusted to comparable gray levels to allow an easier comparison of the fluorescence patterns.

View larger version (14K): [in this window] [in a new window]   Fig. 4. Fluorescence microscopy of HT29 and HT29/Mit cells exposed to topotecan or NiK-12192 or their combination. Cells were stained with the lysosomotropic agent acridine orange to detect the change of acidification in the acidic compartment.

Drug Effect on BCRP Expression. Because several drugs (in particular, substrates) could modulate the expression of BCRP, the effects of topotecan and NiK-12192 on BCRP protein expression were investigated in HT29 cells under the same conditions of antiproliferative activity experiments. Topotecan caused an appreciable up-regulation of the protein (Fig. 5). This finding is not surprising, because topotecan is a substrate for BCRP (Perego et al., 2001). In contrast NiK-12192 caused a marginal (if any) down-regulation of BCRP. A similar pattern of modulation of BCRP was found in HT29/Mit cells (data not shown).

View larger version (51K): [in this window] [in a new window]   Fig. 5. BCRP expression in HT29 cells. Cells were exposed to IC50 for each drug (topotecan or NiK-12192) for 72 h. The whole-cell lysates then were analyzed by Western blotting with the specific antibody. Actin is shown as control for protein loading. Lane 1, untreated cells; lane 2, cells treated with NiK-12192; and lane 3, cells treated with topotecan.

Antitumor Activity. In all of the in vivo studies, both topotecan and NiK-12192 were delivered p.o., by gavage. In the combination, NiK-12192 was always given daily x three times for repeated cycles, starting the same day of each topotecan treatment (approximately 1 h later). When the combination topotecan + NiK-12192 was tested against HT29/Mit human tumor xenograft, topotecan was given at its maximal tolerated dose (15 mg/kg) every fourth day (q4d) for three times, and NiK-12192 at the dose of 30 mg/kg. As expected for a BCRP-expressing tumor, topotecan was poorly active in inhibiting tumor growth (Table 4). The effect of NiK-12192 itself was negligible. The combination of the two drugs resulted in a clear therapeutic improvement, being the 75% TWI, observed 5 days after treatment end, much higher than the expected value based on the efficacy of each drug alone (58%). Moreover, the observed TWI significantly differed from that of topotecan (p < 0.001). As reflected by the LCK value, the growth inhibition lasted for long time, and 70% TWI was still assessable 10 days later, i.e., 28 days after tumor cells inoculum (data not shown). One mouse treated with the combination died at day 16, and all mice of the group lost weight (20%), indicating some increase of toxicity of the combination in such conditions.

A parallel experiment was designed for mice implanted with the parental HT29 tumor line using a lower dose of NiK-12192, 20 mg/kg (Table 4). Against the parental tumor, topotecan was active, and the combination of the two drugs achieved an improved antitumor effect. Indeed, 5 days after the last treatment, the 88% TWI was significantly higher than that achieved by topotecan alone (p < 0.05) and higher than the expected (77%), considering that NiK-12192 alone exhibited minimal activity. Again, the inhibition of tumor growth lasted for long time, achieving an LCK value of 2.5. However, in spite of the reduced dose of NiK-12192, the combination still resulted in partial toxicity, and a relevant body weight reduction was achieved. Thus, a general toxic effect on the mouse might contribute to the tumor growth inhibition. Growth curves representative of the experiments reported in Table 4 are shown in Fig. 6.

View larger version (16K): [in this window] [in a new window]   Fig. 6. Growth curves of HT29 and HT29/Mit tumor xenografts treated p.o. with solvent (X), 15 mg/kg topotecan (), 20 mg/kg () or 30 mg/kg () NiK-12192, or topotecan + NiK-12192 (). Arrows indicate the days of treatment. Each point reports the mean values ± S.E. of 9 to 10 tumors.

	Discussion

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Vacuolar-H⁺-ATPase has been proposed as a useful target to be exploited for the improvement of antitumor chemotherapy, because modulation of pH is a protective mechanism activated in response to stress conditions, in particular following DNA damage (Paglin et al., 2001; Torigoe et al., 2002). Moreover, vacuolar-H⁺-ATPase has a role as cooperating factor in the multidrug-resistant phenotype mediated by overexpression of ATP-dependent membrane proteins that function as drug efflux pumps (Raghunand and Gillies, 2000). The results reported in this study show that NiK-12192, a novel synthetic vacuolar-H⁺-ATPase inhibitor, was able to sensitize colon carcinoma cells to two camptothecins chosen for their therapeutic interest, i.e., topotecan and SN-38 (the active metabolite of irinotecan). NiK-12192 was effective in sensitizing cells overexpressing P-glycoprotein (LoVo/DX) or BCRP (HT29/Mit) to topotecan and SN-38 cytotoxicity. Other vacuolar-H⁺-ATPase inhibitors derived from natural sources, such as bafilomycin A1 and concanamycin, have been reported to reverse multidrug-resistant phenotype and to potentiate the activity of topoisomerase II inhibitors in cell systems (Ouar et al., 2003). Our findings showed that the NiK-12192 vacuolar-H⁺-ATPase inhibitor was effective in modulating sensitivity of P-glycoprotein-overexpressing cells to SN-38. This finding may be of potential therapeutic interest, because irinotecan is used in clinical treatment of colon carcinoma. In addition, the results indicate the ability of NiK-12192 to increase topotecan effects in cell lines expressing another transport system, the BCRP protein, which is present either in parental HT29 cells, or, at a much higher level, in HT29/Mit cells (Perego et al., 2001; Croce et al., 2004).

The potentiation of the cytotoxic effects of camptothecins by NiK-12192 was reflected in a significant improvement in the antitumor activity in vivo of topotecan in combination with oral NiK-12192. Although NiK-12192 itself was well tolerated at the tested dose, the combination of NiK-12192 with maximal tolerated doses of topotecan may result in an appreciable increase of drug toxicity. Indeed, with the administration sequence investigated in this study, i.e., 3 days of NiK-12192 after each dose of the cytotoxic drug, the schedule of topotecan administration was critical for the toxicity, which was observed when the drug (15 mg/kg) was delivered biweekly, but not when delivered weekly. The increase of toxicity could be expected considering that the target of NiK-12192 is not tumor-specific. Thus, our results indicate that the choice of an appropriate treatment schedule is necessary to avoid undesirable effects. Moreover, preliminary results concerning the pharmacokinetics of NiK-12192 indicated a persistent plasma level, thus suggesting that less frequent administrations might be better tolerated.

The potentiation of antiproliferative/antitumor effects and the modulation of cellular acidification observed with the combined treatment of topotecan and NiK-12192 suggest a linkage between the two events. Indeed, NiK-12192 as well as bafilomycin A1 was effective in modulating the pattern of drug-induced acidification in acidic vesicular compartment, which can be a protective mechanism activated in response to DNA damage, as reported in cells treated with ionizing radiation (Paglin et al., 2001). Our study clearly showed that topotecan was able to enhance acidification in vesicular compartment of resistant cells, and this behavior was consistent with the ability of resistant cells to tolerate stress conditions. When NiK-12192 was present in topotecan-treated cells, the pH reversed to the value of untreated cells, with a pattern comparable with that found in sensitive cells. Although the fluorescence analysis did not allow a quantitation of the intracellular content of topotecan, the available data support a marked change in subcellular drug distribution. Indeed, the concomitant exposure to topotecan and NiK-12192 resulted in an increased distribution of topotecan in nucleus and cytoplasm. A plausible explanation for this behavior is that the acidification of the vesicular compartment influences the subcellular localization of topotecan. It is unlikely that the sensitization of colon carcinoma cells was mediated by interaction of NiK-12192 with the transporters (BCRP or P-glycoprotein), because it was detected in cells expressing either low (parental) or high levels (resistant sublines) of these proteins. However, because changes of acidification could influence the expression of these transport systems (Adams et al., 2006), an indirect effect mediated by protein down-regulation could not be ruled out.

In the present study, NiK-12192 alone exhibited only a marginal activity against the two human tumor xenografts investigated. However, further studies will be necessary to better characterize the pharmacological profile of NiK-12192 and its efficacy when delivered by other routes and schedules. Noteworthy, in combination studies NiK-12192 was effective by oral route, and such a property represents an important preclinical requisite of the compound, in perspective of a possible clinical use in combination with other antitumor agents. In previous studies, different proton pump inhibitors, i.e., pantoprazole and bafilomycin A1, have been reported to show antitumor activity in a human and in a rat tumor xenograft, respectively (McSheehy et al., 2003; Yeo et al., 2004). However, the two studies report different modalities of cell death in the different experimental systems, thus indicating that more investigations are needed to better elucidate the critical factors involved in the antitumor activity of proton pump inhibitors. Other classes of V-ATP-ase inhibitors are known, but results of in vivo antitumor activity are not available (Boyd et al., 2001; Beutler and McKee, 2003; Bowman et al., 2003; Niikura et al., 2004; Huss et al., 2005).

In conclusion, we have shown for the first time that NiK-12192, a small molecule vacuolar-H⁺-ATPase inhibitor, was able to potentiate the antitumor activity of topotecan in pre-clinical tumor models, both in topotecan-sensitive and -resistant tumor xenografts. The synergistic interaction was more evident in the topotecan-resistant BCRP-overexpressing HT29/Mit cell subline, thus supporting the potential interest of the novel inhibitor for the therapy of camptothecin-resistant tumors.

	Acknowledgements

 
We thank Laura Zanesi for editorial assistance.

	Footnotes

 
This work was partially supported by the Associazione Italiana Ricerca sul Cancro, Milan, 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.106.103481.

ABBREVIATIONS: BCRP, breast cancer resistant protein; SN-38, 7-ethyl-10-hydroxycamptothecin; TW, tumor weight; TWI%, tumor weight inhibition percentage; LCK, log₁₀ cell kill; BWL%, body weight loss percentage; R/G, red/green; NiK-12192, [4-(5,6-dichloro-1H-indol-2-yl)-3-ethoxy-N-(2,2,6-tetramethyl-piperidin-4-yl)-benzamide; SB242784, (2Z,4E)-5-(5,6-dichloro-2-indolyl)-2-methoxy-N-(1,2,2,6,6-pentamethylpiperidin-4-yl)-2,4-pentadienamide.

¹ Current affiliation: GlaxoSmithkline, Collegeville, Pennsylvania.

Address correspondence to: Dr. Franco Zunino, Istituto Nazionale Tumori, via Venezian 1, 20133 Milan, Italy. E-mail: franco.zunino{at}istitutotumori.mi.it

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