Department of Physiology, Texas Tech University Health Sciences Center, Lubbock, Texas 79430-6551
Submitted 14 November 2003 ; accepted in final form 21 January 2004
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ABSTRACT |
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metastasis; intracellular pH; migration; sodium ion/hydrogen ion exchanger; bicarbonate transport
Four major types of pHcyt regulatory mechanisms have been identified in tumor cells: Na+/H+ exchangers, bicarbonate (HCO3) transporters, proton-lactate symporters, and proton pumps (11, 38, 42). Recently, the vacuolar H+-ATPase (V-ATPase) has emerged as a novel and important pHcyt regulatory system in some specialized cells, including tumor (25, 26, 28). This proton pump is ubiquitously expressed (33, 35), not only in vacuolar membranes but also in plasma membranes (26, 58) of eukaryotic cells. The V-ATPase is a multi-subunit enzyme complex composed of a membrane sector (V0) and a cytosolic catalytic sector (V1) (35). The integral V0 domain functions in proton translocation, whereas the peripheral V1 domain hydrolyzes ATP. V-ATPase is a member of a family of ATP-driven proton pumps responsible for the acidification of intracellular compartments such as endosomes, lysosomes, Golgi-derived vesicles, and clathrin-coated vesicles (35). In addition to the role of V-ATPase in intracellular compartments, this enzyme is important for plasma membrane functions in various specialized cells (13, 32, 54).
There are several classes of inhibitors of V-ATPase, including the macrocyclic lactones bafilomycin and concanamycin, the benzolactone enamides salicylihalamides and lobotamides, and, more recently, the macrolide lactams chondropsin and poecillastrin (3). Although they exhibit different potencies and selectivity to inhibit V-ATPases from mammals and fungal sources, they all seem to bind to subunit c to exert their effect. Because of the many isoforms, it is possible that mutations may cause resistance to V-ATPase inhibitors. Indeed, mutations in subunit c involved in binding of V-ATPase inhibitors decrease the sensitivity of bafilomycin by 20- to 60-fold and to concanamycin by
3-fold in Neurospora crassa (3).
V-ATPase is functionally expressed in plasma membranes of human tumor cells and may have specialized functions in cell growth, differentiation, angiogenesis, and metastasis (2527). Furthermore, pHcyt is critical for the cytotoxicity of anticancer agents, and V-ATPase has been implicated in the acquisition of the multidrug resistance phenotype (20, 28, 40). Therefore, understanding the mechanisms regulating pHcyt and tumor acidity is important for developing new approaches to cancer chemotherapy, and V-ATPase may represent a potential target for cancer chemotherapy (52). We know that V-ATPases at the cell surface play a role in maintaining an alkaline intracellular environment favorable for growth, while maintaining an acidic extracellular environment favorable for invasion (28, 29). We hypothesize that V-ATPase is important in the acquisition of a more metastatic and invasive phenotype. Because little is known about the mechanisms of pHcyt regulation in breast cancer, we employed human breast cancer cell lines with distinct metastatic potential and determined the distribution and the functional activity of V-ATPase in highly and lowly metastatic cells. For this purpose, we investigated, by immunocytochemistry and confocal laser scanning microscopy, the distribution of V-ATPase in highly and lowly metastatic human breast cancer cells. We determined the enzymatic activity at their plasma membranes, to corroborate the V-ATPase distribution, and in cell homogenates. Then, in living cells, we evaluated the proton fluxes via plasma membrane V-ATPase (pmV-ATPase), as well as via the ubiquitous Na+/H+ exchanger and HCO3-based H+-transporting mechanisms. We also evaluated the kinetics of the migration and invasion of lowly and highly metastatic cells. We used bafilomycin and concanamycin to study V-ATPase activity, proton flux (JH+) via pmV-ATPase, and migration and invasion because they are better known inhibitors.
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MATERIALS AND METHODS |
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Cell culture. Human breast cancer cell lines, MDA-MB-231 (ATCC no. HTB-26; passage 28), MB435s (ATCC no. HTB-129; passage 239), MDA-MB468 (ATCC no. HTB-132; passage 340), and MCF-7 (ATCC no. HTB-22; passage 148) were purchased from American Type Culture Collection (ATCC). The cells were plated in culture dishes and grown as follows. MB231 and MB468 cells were grown in Leibovitz's L-15 (ICN Biomedical, Costa Mesa, CA) containing 2 mM glutamine, 24 mM NaHCO3, 10 mM HEPES, 0.067 g/l penicillin, and 0.143 g/l streptomycin. MB435s cells were grown as MB231 cells, except that the media were supplemented with 10 mg/ml insulin. MCF-7 cells were grown in MEM (nonessential amino acids Earle's balance salt solution) containing 10 mg/ml insulin, 24 mM NaHCO3, 10 mM HEPES, 0.067 g/l penicillin, and 0.143 g/l streptomycin. All cell lines were supplemented with 10% FBS (Gibco, Grand Island, NY) under a 95% air-5% CO2 humidified environment at 37°C. Cells used for experiments were from <10 passages after cryopreservation. For immunocytochemistry, cells were plated on 18-mm-diameter round coverslips. For fluorescence spectroscopy studies of pHcyt with SNARF-1, the cells were grown onto 60-mm petri dishes containing six glass coverslips (9 x 22 mm) at densities of 5 x 105 cells/dish until the cells reached confluency. For kinetic studies of migration, the cells were grown on 12-mm-diameter round coverslips until the cells reached confluency.
Immunocytochemistry. Monoclonal antibodies against V-ATPase subunit A and a were obtained from Molecular Probes. To determine the distribution of V-ATPase, the cells were fixed with 4% paraformaldehyde in PBS (pH 7.4) for 15 min. The cells were rinsed with PBS (2 x 5 min) and then washed with 25 mM glycine in PBS (1 x 5 min), permeabilized with 0.1% Triton X-100 in PBS (1 x 10 min), and blocked with 1% BSA in PBS (1 x 15 min). The cells were incubated with primary antibodies (MAb against A and a subunits) in 1% BSA-PBS for 45 min and then washed extensively with PBS. The cells were then incubated with the secondary antibody (Alexa fluor 568 anti-mouse IgG, Molecular Probes) at the dilution of 1:100 in PBS containing 1% BSA for 45 min at room temperature. After rinsing (3 x 1 min and then 2 x 5 min) in PBS, cells were subsequently incubated with Alexa fluor 488-phalloidin (Molecular Probes) that binds to F-actin to delineate the cell's edges. After rinsing (2 x 5 min) in PBS, cells were mounted in VectaMount solution (Vector Laboratories, Burlingame, CA) and maintained at 4°C overnight. The cells were observed with a confocal laser scanning microscope (LSM 510 META, Zeiss) with a x63 objective (Plan-APOCHROMAT, 1.4 numerical aperture, oil differential interference contrast). Simultaneously acquired images of Alexa fluor 488-phalloidin (actin cytoskeleton, green) and Alexa fluor 568 (V-ATPase, red) fluorescence were collected, and each section was analyzed on a pixel-by-pixel basis utilizing Physiology software version 3.0 (Zeiss) to assess colocalization of actin and V-ATPase.
Isolation of plasma membrane. Plasma membranes of breast cancer cells were obtained as described elsewhere, with modifications (7). Briefly, confluent monolayers from ten 100-mm petri dishes were washed three times with 10 mM Tris·HCl, 1 mM EDTA, and 150 mM NaCl, pH 7.4. Cells were then harvested by scraping with a rubber policeman. Membranes were isolated from the resulting cell suspension after hypotonic lysis and differential centrifugation, followed by treatment with sodium iodide (7). The pellet was resuspended in homogenizer buffer (250 mM sucrose, 1 mM EGTA, and 50 mM Tris·HCl) and then applied to the top of 2040% sucrose gradient and centrifuged at 200,000 g for 1 h at 4°C. The membranes from the interphase 2040% sucrose were diluted in Tris-EDTA buffer and collected by centrifugation for 30 min at 100,000 g. The final pellet was resuspended in homogenizer buffer and stored at 80°C. Electron microscopy analysis of the plasma membrane fractions showed that they were free of mitochondria and other cellular organelles.
V-ATPase enzymatic activity.
The V-ATPase activity in both cell homogenates and isolated plasma membranes from highly and lowly metastatic breast cancer cells was determined from the hydrolysis of radiolabeled ATP, as described (7). Plasma membranes (1 mg/ml) were treated with 0.7 mg/ml deoxycholate for 30 min at 37°C to expose V-ATPase to substrate. Detergent-treated membranes were then diluted 1:10 and incubated at 37°C for 1 h in a total volume of 0.2-ml ATPase assay buffer containing 25 mM Tris·HCl, 4 mM MgCl2, 0.1 mM EGTA, 4 mM [-32P]ATP (Perkin Elmer/NEN Life Sciences), and 50 nM bafilomycin A1, pH 7.0. The V-ATPase activity was estimated by subtracting total ATP hydrolysis minus the bafilomycin-sensitive activity. Protein content was determined by the Lowry method by using BSA as a standard (22).
pHcyt measurements in cell populations. The pHcyt was determined by the fluorescence of SNARF-1 [5 (and 6-)-carboxy-SNARF-1], as described (26, 29). Two coverslips (9 x 22 mm) containing cells at confluency were incubated with 7.5 µM SNARF-1-AM in CPB, at pHex of 7.4 or 8.0, as needed. For experiments containing HCO3 at different pHex values (i.e., 6.8 and 7.4), the HCO3 concentration was estimated as described earlier (11, 12). In these experiments, the pHex was maintained constant by continuously bubbling the CPB containing HCO3 with 5% CO2. Cells were incubated for 45 min at 37°C on a rocker platform (Cole-Parmer, Vernon Hills, IL) followed by 30-min incubation in dye-free buffer to ensure complete ester hydrolysis and leakage of uncleaved dye. The two coverslips were placed back to back in a holder perfusion device and perfused at a rate of 3 ml/min, and the fluorescence of SNARF-1 was monitored with a SLM-8100/DMX spectrofluorometer (Spectronics Instruments, Rochester, NY) equipped for sample perfusion, at 37°C. Fluorescence was monitored in continuous-acquisition mode by using an excitation wavelength of 534 nm and monitoring emissions at 584, 600, and 644 nm, as described (29). The fluorescence emission at 584 nm decreases and that at 644 nm increases, respectively, with increasing pH. The ratio of 644 to 584 nm was used to monitor pH changes. The 600-nm wavelength, which is insensitive to pH, was used to evaluate the efficiency of dye loading, quenching, or other artifacts (26). Fluorescence data were converted to ASCII format for subsequent analyses in SigmaPlot version 8.0 (Jandel Scientific, San Rafael, CA).
Dye calibration. In situ calibration curves were generated, as described previously (29). Briefly, the cells attached to coverslips were perfused with a high-K+ buffer (pH 5.58.0 at 0.2 pH intervals) containing 2 µM valinomycin and 6.8 µM nigericin. The high K+ is used to approximate intracellular K+, and nigericin sets the H+ gradient equal to the K+ gradient, with valinomycin completing the collapse of the K+ gradient. The ratio (644 nm/584 nm) of SNARF-1 was determined at each pH studied during in situ calibrations (29). The following parameters were obtained for SNARF-1 in MB231: acidic dissociation constant (pKa) = 7.934 ± 0.056, Rmin = 0.367 ± 0.003, and Rmax = 5.652 ± 0.429 (n = 39); in MCF-7: pKa= 8.077 ± 0.057, Rmin = 0.429 ± 0.006, and Rmax = 5.442 ± 0.049 (n = 39); in MB435s: pKa = 7.54 ± 0.076, Rmin = 0.435 ± 0.007, and Rmax = 3.167 ± 0.194 (n = 39); in MB468: pKa = 7.822 ± 0.04, Rmin = 0.389 ± 0.001, and Rmax = 3.929 ± 0.186,(n = 39); where Rmin is the ratio observed when the dye is fully protonated, and Rmax represents the ratio of fluorescence obtained when the dye is fully unprotonated. These in situ calibration parameters were used to generate the pHcyt values for each individual experiment, as previously described (12, 26).
Data analysis. The initial rate of recovery from an ammonium chloride-induced acid load is measured as the slope of linear regression curve relating time and pHcyt, as described previously (42). Briefly, cells were perfused with 25 mM NH4Cl in CPB for 5 min to allow entry of NH3 and NH4+ into the cell. Inside of the cell, the NH4+ dissociates into NH3 + H+, thus acid loading the cells. We then removed the NH4Cl and evaluated the pHcyt recovery from this acidification within the first 5 min. The individual pHcyt data points are subtracted from the zenith pHcyt at 5 min and plotted against time to obtain the slope of the linear regression curve relating time and pHcyt. Because the apparent H+ buffering capacity is different in each cell type and could result in distinct JH+, we estimated these parameters as described earlier (42).
Migration kinetics. To measure the migratory ability of highly and lowly metastatic cells, migration was assessed in a wounded monolayer model. Briefly, cells were grown on 12-mm coverslips to confluence and subsequently wounded with a micromanipulator that induces a 250-µm gap. Following this injury, cells migrate to close the wound (46). Cell movement was evaluated under phase-contrast microscopy, and images were captured with a digital camera (Kodak MDS290) every 2 h. Migration was assessed as wound distance at each time point from three randomly selected areas by using Image J software. The experiments were done in the presence and absence of 50 nM bafilomycin A1.
Invasion and migration assays.
At confluence, cells grown in T-25 flasks were loaded with 5 µM calcein-AM for 30 min and then trypsinized, washed, and counted. To evaluate cell invasion in vitro through extracellular matrix proteins, HTS FluoroBlok inserts (8-µm pore size; Becton Dickinson, Franklin Lake, NJ) were coated with matrigel. For cell migration, the HTS FluoroBlock were not coated with matrigel. For cell invasion and migration, cells were seeded at densities of 5 x 104 and incubated at 37°C/5% CO2 for 8 h (MB231) and 24 h (MCF-7) in the presence or absence of 50 nM bafilomycin A1. The inserts were visualized with a x20 objective (UPlan Fl 0.5 Ph1), and images of the bottom and top of the insert were obtained with a real-time confocal imaging system based on a rotating disk (Yokogawa Mod C-10 from McBain Instruments). Cell counts from bottom images compared with counts from top images were used to assess percent invasion and migration. Calcein was excited with the 488-nm line of a 15-mW krypton-argon air-cooled ion laser system (T643-RYB-02 from Melles Griot laser group). The emission signal was collected at 530 nm by using as a detector the Hamamatsu's Orca-100 scan interline cooled (Peltier cooling system) charged-coupled device camera (12 bit). Five images per insert were obtained, and the experiments were done in duplicate. The images were subsequently analyzed, and cells were counted with the assistance of Scion Image software (Scion, Frederick, MD). Percent invasion was corrected for proliferation and calculated by using the following equation (29)
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Statistical analysis. All results are expressed as means ± SE. The significant differences were determined by using a t-test or an ANOVA procedure for multiple comparison of normal distributions. The Mann-Whitney test or the Kruskal-Wallis ANOVA with Dunn's test for multiple comparison was used for nonparametric distributions (SigmaStat v.2.03; Statistical Software, Jandel Scientific). All statistical tests were considered significant at P < 0.05.
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RESULTS |
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These earlier experiments were performed at pHex 8.0 because our laboratory's previous studies in other tumor cell types showed a maximal proton pumping activity at an alkaline pHex (26, 27, 29). To further evaluate the physiological significance of pmV-ATPase, we performed experiments at "cell culture pHex = 7.4" in both highly and lowly metastatic cells. These data show that the JH+ are significantly faster in highly than in lowly metastatic cells (Fig. 3A). Therefore, the JH+ are significantly faster at pHex 7.4 than at 8.0, regardless of the metastatic phenotype (compare Fig. 2). However, studies using magnetic resonance spectroscopy have indicated that the tumor pHex is significantly more acidic than that of normal tissue (10, 15, 40). We, therefore, grew highly and lowly metastatic cells at acidic pHex 6.8, which is consistent with that found in breast cancer cells grown in nude mice (10, 40). We determined that the rates of cell growth at pHex 6.8 were similar to those at pHex 7.4. We evaluated the JH+ in these cells and have determined that the JH+ were significantly faster in cells grown at pHex 6.8 than at 7.4. The JH+ are faster in highly (MB435s = 2.34 ± 0.18 mM H+/min; n = 6) than in lowly (MCF7 = 1.90 ± 0.17 mM H+/min; n = 6, P < 0.05) metastatic cells. These data indicate that pmV-ATPase can be induced by growing cells at acidic pHex. Thus pmV-ATPase expression may offer an adaptive advantage for tumor cells typically exposed to an acidic pHex environment.
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To determine the relative contribution of Na+/H+ exchanger to pHcyt regulation, we determined JH+ in the presence of Na+ and absence of HCO3 and subtracted them from the JH+ in the absence of Na+. These data indicated that lowly metastatic cells exhibit faster JH+ via Na+/H+ exchanger than highly metastatic cells (Fig. 3C). We also determined JH+ in MB435s (0.25 ± 0.25 mM H+/min; n = 6) and MB468 (1.60 ± 0.30 mM H+/min; n = 6) cells and concluded that the JH+ via Na+/H+ exchanger were significantly faster in lowly than in highly metastatic cells (P < 0.05). Altogether, these data indicate that lowly metastatic cells preferentially use the ubiquitous Na+/H+ exchanger and HCO3-based H+-transporting systems, whereas highly metastatic cells preferentially use pmV-ATPase for pHcyt regulation.
Effect of acidic pHex on pHcyt under acute and chronic conditions. We hypothesize that the presence of pmV-ATPase may allow the cells to survive the acidic environment of tumors. We, therefore, evaluated the impact of acute changes in pHex from 7.4 to 6.8 in the presence and absence of HCO3. These experiments indicated that, consistently, the pHcyt is significantly more acidic at an acidic than at an alkaline pHex (Table 1). Furthermore, the presence or absence of HCO3 does not change pHcyt when measured at the same pHex (either acidic or alkaline). The effect of acidic pHex on pHcyt only occurs acutely, i.e., when cells are exposed at acidic pHex for 1 h. When these cells are grown chronically at acidic pHex (i.e., >80 h), the resting pHcyt are significantly more alkaline than in cells grown at pHex 7.4, regardless of their metastatic potential. This is possibly due to overexpression of pmV-ATPases, because the Na+- and HCO3-independent JH+ are significantly faster in cells grown at acidic pHex than in cells grown at pHex 7.4 (compare Table 1).
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DISCUSSION |
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The preferential colocalization of V-ATPase with F-actin at the cell cortex observed in this study suggests that the V-ATPase may distribute to the cell surface by interacting with cytoskeleton elements. Recent studies have shown that V-ATPases bind actin filaments, suggesting that this interaction is an important mechanism controlling transport of V-ATPases from the cytoplasm to the plasma membrane (17, 56). In osteoclasts, it has been shown that the V-ATPases were localized on dotlike organelles associated with the filamentous structures of microtubule extending to the cell surface and resided on the plasma membrane of mature-nuclear osteoclast-like cells (53). Moreover, disruption of the cytoskeleton structure by transfecting highly metastatic melanoma cells with cytokeratin mutants decreases their invasion and V-ATPase expression (27).
The immunocytochemical experiments demonstrated that the V-ATPases are expressed at the cell surface. However, these studies do not address whether V-ATPase is functional. We, therefore, performed subcellular fractionation studies in isolated plasma membranes. The data indicated that a bafilomycin-sensitive ATP hydrolysis system is significantly greater in highly than in lowly metastatic breast cancer cells. Electron microscopy analysis of the isolated plasma membrane indicated that these membranes were free of mitochondria and other organelles. Thus it is unlikely that the V-ATPase activity found at the plasma membrane was due to endosomal contaminants. Furthermore, because the plasma membranes from lowly metastatic cells were isolated with the same protocol and the V-ATPase activity was minor, this supports our contention that a significantly higher number of bafilomycin-sensitive enzymes are present at the plasma membrane of the highly metastatic cells.
To determine whether the presence of V-ATPase at the plasma membrane plays a role in pHcyt regulation, we evaluated whether there were differences in the response to acid loads in highly and lowly metastatic cells. The recovery from acid loads showed that highly metastatic cells exhibited a pHcyt recovery that was faster than in lowly metastatic cells. Importantly, inhibition of V-ATPase with bafilomycin and concanamycin significantly decreased the JH+. These data indicate that the distribution of V-ATPase at the cell surface is responsible for the increased JH+ observed in highly metastatic cells and that V-ATPase is positioned at the cell surface to extrude H+ across the plasma membrane. Early studies from the yeast H+-ATPase-transfected cell model have suggested that overexpression of proton pumps on the cell membrane can increase the pHcyt, trigger cell proliferation, and finally causes tumorigenesis (12, 36). The present study indicates that V-ATPase overexpression at the cell surface increases the JH+ and is responsible for a more metastatic phenotype. Furthermore, we also determined that lowly metastatic cells preferentially used Na+/H+ exchanger and HCO3-based H+-transporting mechanisms, whereas highly metastatic cells preferentially used pmV-ATPase to regulate their pHcyt.
The distribution of V-ATPase at the plasma membrane may induce other effects besides increasing the magnitude of the JH+ in cancer cells. Tumor invasion and metastasis are two hallmarks of the neoplasm malignancy. They are the major causes of the morbidity of the cancer patients. To understand the physiological significance of the V-ATPase expression at the cell surface in invasion and migration, we employed a wounded monolayer model to evaluate the kinetics of migration in the highly and lowly metastatic breast cancer cells. In these experiments, scraping off a 250-µm region in a confluent monolayer of cells resulted in cells migrating toward the wound. When healing was allowed to continue, the wound was completely recovered in 18 and 72 h in highly and lowly metastatic cells, respectively. Bafilomycin A1 treatment decreased wounding behavior in the highly metastatic cells with minor effect on lowly metastatic cells. These data indicate that V-ATPase expression at the cell surface is involved in the faster migratory ability of the highly metastatic cells. We also performed studies to evaluate the ability of the cells to invade through the extracellular matrix. Our data indicated that highly metastatic cells exhibiting pmV-ATPase were more invasive than lowly metastatic cells. Importantly, bafilomycin treatment decreased the migration and invasion in highly metastatic cells. These results are in agreement with a recent study that showed that bafilomycin suppressed cell motility in NIH3T3 A31 mouse fibroblasts, possibly due to alterations of pH gradients in endocytic structures, known to exhibit V-ATPase (51). Furthermore, overexpression of the c subunit of V-ATPase in 10T1/2 fibroblasts has been shown to enhance invasion and the secretion of matrix metalloproteinase (MMP)-2, an enzyme needed for protein degradation during invasion (18). These data suggest that overexpression of V-ATPases is important for invasion.
The precise mechanism of how V-ATPase expression at the cell surface may regulate cell motility and migration is unclear. Many factors are involved in this process, such as Ca2+ (24, 44), chemoattractants (23), collagenases (48), cathepsins (43), MMPs (30), and serine protease (55). No direct evidence for V-ATPase expression at the plasma membrane for tumor invasion has been documented yet, but a relationship between pHcyt and invasion has been suggested. First, all of the proteases mentioned above are pH sensitive. Cathepsins are lysosomal enzymes that have an optimal acidic pH (57). Acidic pH induces the redistribution and release of cathepsin B from a series of metastatic human cell lines (43). Mathematical models have been used to investigate whether altered proteolytic activity at acidic pHex is responsible for the stimulation of a more metastatic phenotype. In these cells, the effect of culture pHex on the secretion and activity of two different classes of proteinases, the MMPs and the cysteine proteinases (such as cathepsin B), has been evaluated (57). The modeling data suggest that changes in MMP activity at acidic pH do not have significant effects on invasive behavior. However, the model predicts that the levels of active cathepsin B are significantly altered by acidic pH. Unfortunately, the theoretical data contradict experimental data that supported a crucial role for MMPs in invasion (29). Nevertheless, these studies suggest a critical role for the cysteine proteinases in tumor progression (57). Subsequent studies have shown that acidic pHex increases the invasive behavior of tumor cells (29). In these studies, the in vitro invasive potential of two human melanoma cell lines, the highly invasive C8161 and lowly invasive A375, were examined. Culturing of either cell line at acidic pHex 6.8 caused dramatic increases in both migration and invasion. These data indicate that culturing of cells at mildly acidic pHex induces them to become more invasive (29). Thus the presence of V-ATPase at the cell surface is a significant contributor to the induction of a more invasive phenotype, because it results in an acidic pHex while maintaining a more alkaline pHcyt needed for cell growth and invasion. Indeed, tumor cells in situ have a lower pHex than normal cells; this is an intrinsic feature of the tumor phenotype, caused by alterations either in acid extrusion from the tumor cells or in clearance of extracellular acid (15, 28, 40). Acidic pHex benefits tumor cells because it promotes invasiveness, whereas an alkaline pHcyt gives them a competitive advantage over normal cells for growth. Furthermore, V-ATPase is anti-apoptotic in tumor cells (49, 59).
The mechanisms involved in the decreased invasion and migration following bafilomycin and concanamycin treatment are unclear. However, it is known that a critical step in directed motility and migration is the asymmetric actin polymerization at the leading edge, to establish cell polarity. Increases in pHcyt promote recruitment and actin binding of cofilin at the leading edge of migratory cells (2). An increase in pHcyt also stimulates the actin-severing activity of cofilin (2). Cofilin localizes at the leading edge in fibroblasts and in cancer cells (8). The complex of actin depolymerizing factor and cofilin tends to bind F and G actin in a pH-dependent fashion (2). Thus disruption of pHcyt regulatory mechanisms may, in turn, affect actin polimerization and thereby cell migration. However, our data indicated that bafilomycin treatment did not affect actin cytoskeleton. Thus inhibition of pmV-ATPase at the leading edge may result in decreased cell migration. Further studies are needed to elucidate if localized pHcyt changes imposed by preferential localization of pmV-ATPase at the leading edge regulates invasion and migration in highly metastatic cells.
The cytoskeleton also contributes to the transport of biosynthetic cargo of vesicles derived from the Golgi apparatus, including endosomes and lysosomes (21), and the actin cytoskeleton is involved in the short transport of secretory vesicles to the plasma membrane (19). Thus inhibition of V-ATPase and pHcyt regulation may lead to a disruption of vesicle trafficking needed for cell movement. A role for cortical actin has been found in neuronal cell line PC-12, in which the motility of secretory vesicles was mediated by actin (19). The role of the endocytic pathway in cell migration is unclear. In migrating neutrophils and other cell types, integrins may recycle from the lagging to the leading through polarized endosomal recycling (5, 6, 37). Because the endosomes and lysosomes are part of the endomembranous compartments with a high turnover and are enriched with V-ATPase, it is possible that disruption of V-ATPase by bafilomycin may alter V-ATPase turnover into the plasma membrane, thus inhibiting the supply to the cell surface with components and proteins needed at the leading edge of migratory cells (31). This, however, requires further investigation.
In conclusion, V-ATPase not only takes part in pHcyt homeostasis, but it is also involved in the acquisition of a transformed phenotype in cancer cells. The preferential expression of V-ATPase at the cell surface is important for the acquisition of invasiveness and metastasis of the tumor cells. Therefore, it appears that V-ATPase is a potential target in cancer therapy and may be an excellent candidate for anticancer drugs.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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