Int. J. Cancer: 93, 869 – 874 (2001) © 2001 Wiley-Liss, Inc.
Publication of the International Union Against Cancer
ELEVATED EXPRESSION OF VACUOLAR PROTON PUMP GENES AND CELLULAR PH IN CISPLATIN RESISTANCE Tadashi MURAKAMI1,2, Izumi SHIBUYA3, Tomoko ISE1, Zhe-Sheng CHEN4, Shin-ichi AKIYAMA4, Masayuki NAKAGAWA5, Hiroto IZUMI1, Toshitaka NAKAMURA2, Ken-ichi MATSUO6, Yuji YAMADA6 and Kimitoshi KOHNO1* 1 Department of Molecular Biology, University of Occupational and Environmental Health, Yahatanishi-ku Kitakyushu, Japan 2 Department of Orthopedic Surgery, University of Occupational and Environmental Health, Yahatanishi-ku Kitakyushu, Japan 3 Department of Physiology I, University of Occupational and Environmental Health, Yahatanishi-ku Kitakyushu, Japan 4 Department of Cancer Chemotherapy, Institute for Cancer Research, Kagoshima University, Kagoshima, Japan 5 Department of Urology, Faculty of Medicine, Kagoshima University, Kagoshima, Japan 6 Hanno Research Center, Taiho Pharmaceutical Co., Saitama, Japan V-ATPases are proton-translocating enzymes, which are found not only in numerous intracellular organelles but also in the plasma membranes of many eukaryotic cells. Using differential display, we have identified one of the proton pump subunit genes, ATP6C, as a cisplatin-inducible gene. Northern blot analysis demonstrated that expression of other members of the subunit is inducible by cisplatin treatment. Proton pump gene expression is also upregulated in 3 independent cisplatin-resistant cell lines but not in vincristine- or etoposide-resistant cell lines. Cellular pH was significantly higher in cisplatin-resistant cells than in sensitive parental cells. In vitro DNA-binding activity of cisplatin was markedly increased in acidic conditions, suggesting that the cytotoxicity of cisplatin is modulated by cellular pH. Furthermore, the proton pump inhibitor bafilomycin can synergistically potentiate the cytotoxicity of cisplatin but not of etoposide or camptothecin. These results indicate that cellular pH is one of the critical parameters for effective cancer chemotherapy with cisplatin. © 2001 Wiley-Liss, Inc. Key words: V-ATPase; cisplatin; drug resistance; intracellular pH; vacuolar proton pump gene
Cisplatin is a widely used anticancer agent.1,2 Development of resistance to cisplatin is a major obstacle in the clinical treatment of solid tumors.3 Understanding the molecular basis for cisplatin resistance could therefore be important for clinical protocols. In the present study, differential display was used to compare gene expression between parental and cisplatin-treated cells, to identify novel mechanisms involved in the development of cisplatin resistance. Cisplatin resistance is thought to involve several mechanisms, such as increased drug efflux and cellular thiols4 – 6 and increased DNA-repair activity.7,8 Decreased cisplatin accumulation has been manifested in some cisplatin-resistant cells.4,9 The magnitude of this decrease varied with the concentration of extracellular platinum and was greatest at low concentrations, where platinum levels in resistant cells were 2-fold lower than those in parental cells. However, mechanisms of resistance remain undetermined.10 One of the cisplatin-inducible cDNA clones was identified as a V-ATPase, an ATP-dependent proton pump that is commonly upregulated in cisplatin-resistant cell lines. The V-ATPases are multisubunit, ubiquitous components of eukaryotic organisms that function as electrogenic proton pumps.11–13 Proton pumps are responsible for the acidification of intracellular compartments; such acidification is important in a wide variety of cellular events, e.g., release of internalized ligand from receptors, viral infection, degradation of macromolecules and transport of small molecules into vesicle lumens. In addition, V-ATPases are important for such plasma membrane activities as renal acidification, pHi homeostasis and bone resorption. The influence of pHi has been studied with respect to cell growth,14 cell motility,15 tumorigenesis,16 metastasis17 and apoptosis18 in cancer cells. It has been previously reported that reduced pHi is associated with increased cisplatin sensitivity;19 however, a molecular basis has not been specifically addressed.20 In this report, we propose a novel molecular mecha-
nism for cisplatin resistance with respect to the expression of V-ATPases. MATERIAL AND METHODS
Cell culture and cell lines The cisplatin-resistant cell line KB/CP4, the etoposide-resistant cell line KB/VP2 and the vincristine-resistant cell line KB/VJ300 were derived from human epidermoid cancer KB cells.4,21,22 The cisplatin-resistant cell line P/CDP5 was derived from human prostate cancer PC3 cells.23 These cell lines were cultured in Eagle’s minimal essential medium (Nissui Seiyaku, Tokyo, Japan) containing 10% FBS. The cisplatin-resistant cell line A2780/E80, derived from human ovarian cancer A2780 cells,24 was cultured in RPMI-1640 (Nissui Seiyaku) containing 10% FBS. KB/CP4, P/CDP5 and A2780/E80 cells are 63-, 23- and 92-fold resistant to cisplatin compared to parental cells, respectively.4,9,23 KB/VP2 cells are 51-fold resistant to etoposide, and KB/VJ300 cells are 394-fold resistant to vincristine compared to their parental cells.21,22 Drugs BCECF and nigericin were obtained from Molecular Probes (Eugene, OR). Cisplatin was obtained from Sigma (St. Louis, MO), etoposide from Nihon Kayaku (Tokyo, Japan), and camptothecin from Daiichi Seiyaku (Tokyo, Japan). Bafilomycin was obtained from Wako (Osaka, Japan). Differential display Total RNA was isolated by a method described previously.25 The differential display method was performed using the differential display kit from Takara Shuzo (Kyoto, Japan). In brief, Abbreviations: BCECF, 2⬘,7⬘-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein; ECL, enhanced chemiluminescence; HBS, HEPES-buffered solution; LTC4, leukotriene C4; MAb, monoclonal antibody; MDR, multidrug resistance; MRP, multidrug resistance–associated protein; pHi, intracellular pH; SRB, sulforhodamine B; TCA, trichloroacetic acid; VATPase, vacuolar-type H⫹-ATPase. Grant sponsor: Ministry of Education, Culture, Sports, Science and Technology of Japan; Grant sponsor: Princess Takamatsu Cancer Research Fund; Grant number: 99-23106; Grant sponsor: Japan Medical Association; Grant sponsor: Fukuoka Anticancer Research Fund. *Correspondence to: Department of Molecular Biology, Faculty of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu, Fukuoka 807-8555, Japan. Fax:⫹81-93-692-2766. E-mail:
[email protected] Received 16 October 2000; Revised 23 February, 21 March, 18 April 2001; Accepted 8 May 2001 Published online 19 July 2001
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reverse transcription was carried out using 9 anchored primers. Twenty-four primers of 10 mer were used in a PCR with the appropriate anchored primer. KB and cisplatin-treated KB cells were analyzed simultaneously. Gels were dried and autoradiographed for 1–2 days. DNA was eluted from gel by boiling and reamplified by PCR. cDNA fragments were then cloned into pGEM-Teasy (Promega, Madison WI) and sequenced. Northern blot analysis Northern blot analysis was carried out on total RNA extracted from cisplatin-treated cells and various drug-resistant cell lines, as well as cisplatin-resistant cell lines. One of the cDNA clones was identical to that of ATP6C. The cDNA clones of other subunits for V-ATPase (ATP6A1, ATP6B2, ATP6D, ATP6E and ATP6F) were cloned by RT-PCR using total RNA of KB cells with the following primer pairs: ATP6A1, 5⬘-ATGTATGAGCTGGTGGAGGTGGGCC-3⬘ (5A) and 5⬘-TTGACGTGCAGGCCATACTTGCACC-3⬘ (3A); ATP6B2, 5⬘AGGAGACAAGATGGCGCTGC-3⬘ and 5⬘-TCGGCCAGTACAACAGGACC-3⬘; ATP6D, 5⬘-CTCGTTTGACACCTTCCTGG-3⬘ and 5⬘-CATCAGCCATGTATTGAGC-3⬘; ATP6E, 5⬘-GAGCCTGCTGTTCACCGGCC-3⬘ and 5⬘-TGGTACAAACCTGGAGAACC-3⬘; ATP6F, 5⬘-TGGGACGCGTTTGTAGCTCC-3⬘ and 5⬘-TGGCACTGAAAGGCTCAGCC-3⬘. cDNA clones were labeled with random primer using the Megaprime DNA labeling kit (Amersham, Aylesbury, UK), and hybridization was performed at 42°C in Hybrisol I (Oncor, Gaithersburg, MD). Signal intensities were quantified using a bioimaging analyzer (BAS2000; Fuji, Tokyo, Japan). pHi measurements The standard medium used for measurements of pHi was HBS containing 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES and 11.1 mM glucose (pH 7.4). Cultured cells were incubated in HBS with the acetoxymethyl esters of the pH indicator BCECF (BCECF/AM, 1 mM) at room temperature for 1 hr. Cells were then washed twice with dye-free HBS and kept at room temperature until used. Fluorescence was measured as described previously.26 In brief, fluorescence was measured from BCECF-loaded cells in a culture dish, which had a thin glass coverslip bottom and was positioned on the stage of an inverted microscope (IX-50; Olympus, Tokyo, Japan), using a fluorescence-imaging system equipped with a 12-bit digital CCD camera (Quanticell/900; Applied Imaging, Sunderland, UK). Once cells were selected in the optical field with the aid of the microscope, fluorescence intensities at 540 nm (⫾30 nm bandwidth) with excitation at 440 and 490 nm were recorded at an interval of 5 sec. pHi values in individual cells were calculated from the ratio (R) of the fluorescence images measured with excitation at 490 nm to those at 440 nm using the following equation:
Cytotoxicity assays Cells were seeded into 96-well tissue culture plates at concentrations of 5 ⫻ 103 KB cells or 20 ⫻ 103 KB/CP4 cells. On the following day, drugs at increasing concentrations were added to the medium. After 24 hr with drugs, surviving cells were assayed as described previously.29 In brief, surviving cells were fixed with 10% TCA at 4°C for 1 hr and then washed with water. After plates were air-dried, TCA-fixed cells were stained for 30 min with 0.4% (wt/vol) SRB dissolved in 1% acetic acid. At the end of the staining period, SRB was removed and culture plates were quickly rinsed with 1% acetic acid. After plates were air-dried, bound dye was solubilized with 10 mM unbuffered Tris base (pH 10.5) and OD was measured at 564 nm. The quantitative relationship between the SRB staining assay performed at 0 hr after drug treatment and eventual loss of reproductive viability is not known. Isobologram analysis The effect of bafilomycin in combination with cisplatin or other agents at the point of ID50 was analyzed using an isobologram method.30,31 Three isoeffect curves were drawn as follows. Mode I line. When the dose of drug A is chosen, there remains an increment in effect to be produced by drug B. If both drugs are to act independently, the addition is performed by taking the increment in doses, starting from 0, that give log survivals which sum to the ID50. Mode IIa line. When the dose of drug A is chosen, an isoeffect curve can be calculated by taking the dose increment of drug B that in sum gives the total effect, in this case ID50. Mode IIb line. As for IIa, when the dose of drug B is chosen, an isoeffect curve can be calculated by taking the dose increment of drug A that gives the required contribution to ID50. When the dose-response curve of drug A follows first-order kinetics, the mode IIb line will be identical to the mode I line. When both drugs follow first-order kinetics, all 3 isoeffect lines will converge to make a straight line connecting at 1.0 of the ordinate and abscissa. The total area enclosed by these 3 lines represents an additive response. With combinations of graded doses of drug A and the chosen dose of drug B, a single dose-response curve can be drawn. When the experimental ID50 concentration in this combination falls to the left of the envelope, the 2 drugs have supra-additive interaction. When the experimental data point is within the envelope, this combination is noninteractive (additive). When this point is to the right of the envelope but within the square produced by 1.0 of the ordinate and abscissa, the drugs have subadditive interaction. When the point is outside the square, both drugs are considered to be mutually protective. RESULTS
pHi ⫽ pK ⫹ log共R ⫺ R min 兲Ⲑ共R max ⫺ R兲,
where pK, Rmin and Rmax represent the apparent pK for BCECF and the minimum and maximum R values, respectively. The 3 values were estimated to be 6.84, 1.88 and 6.02 by in vivo calibration using HBS of 6 different pH values (6.2, 6.6, 7.0, 7.4, 7.8 and 8.2) and the K⫹/H⫹ exchanger, nigericin, as described elsewhere.27 All pHi measurements were performed at room temperature (about 23°C). DNA platination Salmon sperm DNA was incubated with 10 mM cisplatin in a buffer containing 50 mM Tris (pH 6.8, 7.5, 8.0 or 8.8) and 3 mM NaCl for up to 4 hr at 37°C in the dark. DNA was then slot-blotted using a slot-blot apparatus (GIBCO BRL, Bethesda, MD). The level of platination was visualized by ECL using the CPT2 MAb, which recognizes cisplatin-DNA adducts.28 Data were quantitated by densitometry using the NIH image program (NIH, Bethesda, MD).
Identification by differential display of the cDNA encoding the proton pump upregulated in cisplatin-treated cells To isolate cisplatin-inducible genes, differential display was performed on total RNA from paired KB and cisplatin-treated KB cells. After PCR reamplification, cDNAs were cloned into pGEMTeasy. Sequence analysis showed that dd 6cDNA was identical to a V-ATPase (ATP6C), one of the subunits of the proton pump (data not shown). Since the complex V-ATPase holoenzymes consist of several subunits, we isolated cDNA clones for the other 5 subunits to examine whether proton pump gene expression is inducible by cisplatin treatment. Northern blot analysis demonstrated that all of the subunit genes were inducible (Fig. 1a) by cisplatin at 12 hr and at levels of 1.6- and 11.2-fold. We also examined the expression of these genes in cisplatin-resistant cell lines relative to their drug-sensitive counterparts (Fig. 1b). All 3 cisplatin-resistant cell lines tended to overexpress proton pump genes. Four to 6 subunit genes were upregulated to more than 1.5-fold in the cisplatin-resistant cell lines, where as only 1 subunit gene was upregulated in non-cisplatin-resistant cell lines. In 2 of 3
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FIGURE 2 – pHi of cisplatin-resistant cells. (a) pHi for 25 cells of each cell line was measured. Each dot represents a single cell. The mean for each cell line is shown (horizontal line). (b) Pseudocolor pH images of KB (left) and KB/CP4 (right). In all measurements, extracellular pH was 7.4. Marked intracellular alkalinization was observed in cisplatin-resistant cells. pH values are indicated in the horizontal color bar at the lower right.
sion at steady state, making it unnecessary to increase expression of all subunits in cisplatin-resistant cell lines.
FIGURE 1 – Northern blot analysis of proton pump subunits. (a) Expression of proton pump subunit mRNA in cisplatin-treated KB cells. RNA from KB cells treated with 10 M cisplatin for 0 and 12 hr was separated on a 1% formaldehyde-agarose gel, transferred to a Hybond N⫹ membrane and hybridized with each subunit cDNA probe. Gel staining is also shown (bottom panel). Lane 1, 0 hr; lane 2, 12 hr. (b) Expression of proton pump subunit mRNA in parental and cisplatin-resistant cells. RNA from each cancer cell line was hybridized with each subunit cDNA probe. Gel staining is shown. mRNA size is indicated at the right. A ⬎1.5-fold increase is indicated in bold.
resistant lines, at least 1 of the proton pump subunits was not upregulated. The reason all subunits are not expressed equivalently is unclear. One explanation may be differences in steady-state expression levels of each subunit protein. For example, in PC3 and A2780, subunit B2 (ATP6B2) may already have adequate expres-
Elevation of cellular pH in cisplatin-resistant cell lines To assess the role of overexpression of proton pump genes in controlling pHi, we measured the cellular pH of cisplatin-resistant cells (Fig. 2a). In KB cells, mean cellular pH was 6.76 ⫾ 0.18 (mean ⫾ SD). However, the cellular pH of cisplatin-resistant KB/CP4 cells was significantly increased to 7.49 ⫾ 0.30. The cellular pH of cisplatin-resistant prostate (PC3 6.68 ⫾ 0.11, P/CDP5 7.04 ⫾0.15) and ovarian (A2780 6.65 ⫾ 0.13, A2780/E80 7.49 ⫾ 0.42) cancer cell lines was also elevated when compared to that of parental cisplatin-sensitive cells, suggesting that high cellular pH is common in cisplatin-resistant cell lines (Fig. 2a). In contrast, no substantial differences could be measured in the mean values of cellular pH of KB/VJ300 and KB/VP2 cells (data not shown). The representative fluorescence image shown in Figure 2b was consistent with the results in Figure 2a. Effect of pH on DNA platination The cytotoxicity of cisplatin is believed to result from cisplatinDNA adducts. To assess whether the level of DNA platination was affected by differences in pH, the extent of DNA platination was
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FIGURE 4 – Sensitivity of KB ( , sensitive) and KB/CP4 ( , resistant) cells to bafilomycin, cisplatin, camptothecin and etoposide. Cells were cultured with various concentrations of drugs for 24 hr, and viable cells were then measured. Cisplatin doses were 0.5, 1, 2, 3, 5, 7.5, 10, 15 and 20 M (ID50 3.0 M) for KB and 10, 50, 100, 125, 150, 175, 200 and 250 M (ID50 95 M) for KB/CP4. Bafilomycin doses were 1, 2, 4, 5, 7.5, 10, 15, 20, 50 and 500 nM (ID50 11.2 nM for KB, 7.0 nM for KB/CP4); camptothecin doses were 5, 10, 25, 50, 75, 100, 150, 200 and 400 M (ID50 5.9 M for KB, 8.5 M for KB/CP4); and etoposide doses were 0.5, 1, 2.5, 5, 7.5, 10, 25, 50 and 100 M (ID50 9.1 M for KB, 6.0 M for KB/CP4).
FIGURE 3 – Effect of pH on DNA-cisplatin adduct formation. (a) Salmon sperm DNA (10 g) was incubated at 37°C for up to 4 hr with 10 M cisplatin at the indicated pH and transferred to Hybond N⫹ membranes using a slot blot apparatus. Antibody (CPT2) binding was measured by ECL. (b) Quantitative analysis of the results.
quantitated using the CPT2 MAb, which is specific for DNA platination to cisplatin. Salmon sperm DNA was incubated with cisplatin at varied pH in Tris buffer at 37°C. As shown in Figure 3, DNA platination was increased in a time-dependent manner. We observed decreased DNA platination as a function of increasing pH. Proton pump inhibitors potentiate the cytotoxicity of cisplatin in resistant cells To determine whether overexpression of proton pumps alters the sensitivity to proton pump inhibitor,32,33 cells were exposed to various concentrations of bafilomycin. Cisplatin-resistant cells developed slight collateral sensitivity to bafilomycin at a low concentration (⬍10 nM). However, about 25% of cells survived even under high concentrations (⬎50 nM). The resistance of KB/CP4 cells to cisplatin was about 30-fold, about 1.3-fold to camptothecin and about 0.7-fold to etoposide compared with KB drug-sensitive cells (Fig. 4). Isobolograms at ID50, based on these dose-response curves in combination, were made (Fig. 5), and the cytotoxic activity of anticancer agents in combination with a proton pump inhibitor was then determined. For KB/CP4 cells, for simultaneous
FIGURE 5 – Isobologram analysis of bafilomycin in combination with cisplatin, camptothecin or etoposide. Data from dose-response curves generated in Figure 4 were used to determine the concentrations for combination treatment. Each point is the mean of 3 separate experiments, each performed in triplicate: (a– c) KB cells, (d–f) KB/ CP4 cells, (a,d) bafilomycin with cisplatin, (b,e) bafilomycin with camptothecin, (c,f) bafilomycin with etoposide. Mode I ( ) indicates independent interaction, mode IIa ( ) additive and mode 30 IIb ( ) supra-additive.
and continuous exposure to bafilomycin and cisplatin, the combined data points fell on the left side of the envelope (Fig. 5d). This observation was interpreted as showing that bafilomycin and cisplatin produced a supra-additive effect on KB/CP4 cells. In KB cells, the combination of bafilomycin with cisplatin caused data points to fall on the left side of the envelope and within the envelope of additivity (Fig. 5a). For the combination of bafilomycin with camptothecin or etoposide, the data points fell in the envelope of additivity or with subadditivity (Fig. 5b,c). Similar interactions were observed for KB/CP4 cells with the combination
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of bafilomycin and camptothecin or etoposide (Fig. 5e,f). There was no synergistic effect of bafilomycin with camptothecin and etoposide. However, when both KB and KB/CP4 cells were treated with the combination of bafilomycin and cisplatin, significant potentiation of cytotoxicity was achieved. DISCUSSION
Cisplatin binds preferentially to the N7 atom of guanine residues, especially in regions of 2 or more consecutive guanines. It has been postulated that DNA platination is the key to the cytotoxicity of cisplatin. Cisplatin is effective for a wide variety of solid tumors.10 However, the emergence of cisplatin resistance is a major cause of treatment failure.3 Cisplatin resistance may be caused by increased drug efflux,4,5 an increase in the production of cellular thiols such as glutathione and an increase in the repair rate of cisplatin-DNA adducts.10 These mechanisms result from chronic exposure to anticancer agents. To evaluate this complex phenomenon, immediate early responses against anticancer agents are thought to be an important step in the development of drug resistance. This prompted us to isolate cisplatin-inducible genes using differential display. We found that the genes for proton pump subunits are inducible by cisplatin treatment of human cancer KB cells (Fig. 1a). Proton pump induction might be beneficial for cancer cells, to avoid intracellular acidification, which is thought to be a trigger of apoptosis. In addition, the proton pump is upregulated in cisplatinresistant cell lines (Fig. 1b). Overexpression of the proton pump is closely associated with the development of cisplatin resistance because expression of proton pumps was specifically increased in cisplatin-resistant cell lines but not in other drug-resistant cell lines. We measured pHi to investigate whether proton pumps are functionally expressed in the plasma membrane. V-ATPases not only acidify intracellular compartments but also maintain cytosolic pH.11–13 Cellular pH of tumor tissues is the same or more basic than that of normal tissue using 31P-magnetic resonance spectroscopy. However, electrode-measured pH values in tumors were lower than those observed in normal tissues.34 As shown in Figure 2, overexpression of the proton pump results in intracellular alkalinization. pHi was significantly elevated in 3 cisplatin-resistant cell lines (Fig. 2). Furthermore, DNA-cisplatin crosslinks formed
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well under acidic conditions (Fig. 3). This is probably the reason that elevation of pHi confers cell resistance to cisplatin. Intracellular acidification increased the susceptibility of tumor cells to killing by heat or anticancer agents,2 suggesting that intracellular alkalinization leads to protection against apoptosis induced by anticancer agents. It has also been shown that pHi regulation is related to cisplatin resistance19,20 and that intracellular alkalinization may be caused by the Na⫹/H⫹ exchanger20 or V-ATPase (our results). We did not investigate Na⫹/H⫹ exchanger expression, but we did show overexpression of V-ATPases in cisplatin-resistant cell lines. More detailed examinations are required to address the molecular mechanisms of intracellular alkalinization. As shown in Figure 5, there was a significant synergistic effect of bafilomycin with cisplatin, especially in cisplatin-resistant cells. We also observed a difference in sensitivity to bafilomycin between parental and cisplatin-resistant cells. Cisplatin-resistant KB/ CP4 cells showed collateral sensitivity to bafilomycin at low concentrations (Fig. 4), indicating that bafilomycin can effectively inhibit V-ATPases which are overexpressed in cisplatin-resistant cells to induce intracellular acidification. Bafilomycin is a highly specific inhibitor of V-ATPases, but inhibition of proton translocation is reversible,32 suggesting why bafilomycin cannot kill the cells completely. Bafilomycin inhibits drug efflux in MDR-type cells.35 KB/CP4 cells express 1 member of the MRP family, which functions as a drug efflux pump.36 However, we did not observe inhibition of LTC4 transport by bafilomycin in KB/CP4 membrane vesicles (data not shown). It remains to be determined if alkalinization of the acidic compartment by inhibitors will release cisplatin or its metabolites from acidic organelles. Interaction of the 16 kDa subunit of V-ATPase with 1 integrin has been reported,37 but 1 integrin–mediated signaling prevented lung-cancer cells from chemotherapy-induced apoptosis.38 These results suggest that V-ATPases have pleiotropic functions that modulate signal transduction for cell growth control, angiogenesis, metastasis and drug resistance. Thus, V-ATPases might provide valuable information for understanding proton pumps in tumor cells and suggest a promising molecular target for cancer chemotherapy.
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