Modulator on Tumor Cells by Influencing Both P-Glycoprotein BIBW 22, a Dipyridamole Analogue, Acts as a Bifunctional

BIBW 22, a phenylpteridine analogue of dipyridamole (DPM), en hanced » ini risiine cytotoxicity approximately 10 times more than DPM in a multidrug-resistant (MDR) KB V20C cell line. Using rhodamine 123 accumulation in KB V20C cells as an indicator of MDR phenotype, BIBW 22 was shown to be about 100 times more potent than DPM in inhibiting the MDR-associated efflux of rhodamine 123. Photolabeling of P-glycoprotein in KB V20C plasma membranes with 0.2 UM['H]azidopine was strongly inhibited by 1 UMBIBW 22, indicating that this compound re verses the MDR phenotype by interfering with MDR-associated P-glycoprotein. In addition, BIBW 22 at 1 MMcould also enhance the cytotoxicity of 5-fluorouracil in KB cells about 20-fold. Its potency in inhibiting nucleoside transport is 7-fold more potent than that of DPM. These results suggest that BIBW 22 is a potent bifunctional modulator which influences both P-glycoprotein and nucleoside transport in tumor cells. Potential use of this compound as a modulator of combination che motherapy involving antimetabolites and drugs affected by MDR should be explored. Introduction DPM' has been used therapeutically in humans for many years as a coronary vasodilator and platelet aggregation inhibitor (1). The major biochemical effects of DPM are inhibition of nucleoside mem brane transport and cyclic AMP phosphodiesterase inhibition (1, 2). DPM has recently been reported to augment the activity of such anticancer agents as 5-fluorouracil, methotrexate, and cisplatin (3-5) and to sensitize MDR cells to MDR-associated drugs (6, 7). Since 5-FUra is often used in combination with MDR-associated drugs, the inclusion of a bifunctional modulator, such as DPM, is postulated to improve the therapeutic potential of drug combinations by reversing the MDR phenotype as well as inhibiting nucleoside transport; the latter mechanism is known to decrease the cytotoxicity of such antimetabolites as 5-FUra and methotrexate (8, 9). The com bined use of 5-FUra and DPM for cancer treatment has been studied before, but no therapeutic advantage has been reported (10). Whether the addition of another modulator of 5-FUra such as leucovorin may improve the therapeutic index remains to be determined (10). One of the possible reasons may be due to insufficient DPM concentrations; therefore, more potent agents with pharmacological properties similar to those of DPM are needed. Recently a number of DPM analogues were evaluated with respect to their ability both to reverse MDR phenotype and to inhibit nucle oside transport. BIBW 22 (Fig. 1) was identified as the most effective DPM analogue in blocking P-glycoprotein function and inhibiting Received 1/19/93: accepted 3/19/93. The costs of publication of this article were defrayed in pan by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This study is supported by Grant CA 44358 from the National Cancer Institute. 2 To whom requests for reprints should be addressed. ' The abbreviations used are: DPM, dipyridamole; VCR, vincristine; MDR, multidrug resistance: 5-FUra, 5-fluorouracil. nucleoside transport. It could also enhance the cytotoxicity of 5-FUra and sensitize MDR tumor cells to VCR. Materials and Methods Materials. Pancreatin (Sigma Chemical Co., St. Louis, MO), VCR (Sig ma), 5-FUra (Sigma), verapamil (Sigma), rhodamine 123 (Eastman Kodak Co., Rochester, NY), [l4C]thymidine (56 mCi/mmol; ICN Radiochemicals, Irvine, CA), and [3H]azidopine (56 Ci/mmol; Amersham, Arlington Heights, IL) were obtained from the sources indicated. Both DPM and BIBW 22 were synthe sized by Dr. Karl Thomae GmbH, Biberach/Riss, Germany. Tumor Cell Lines. The KB cells from the American Type Tissue Culture Collection (Rockville, MD) were maintained in RPMI 1640 supplemented with 5% fetal bovine serum and 0.1 mg/ml kanamycin and incubated under humid ified air with 5% CO2 at 37°C. KB V20C cells, a subclone of the VCR-resistant cell line KB V20, were developed from the parental KB cells by stepwise selection for resistance with increasing concentrations of VCR. They were maintained under the same condition as outlined above with media supplemented with 20 nM VCR. These cells have been shown to overexpress P-glycoprotein by Western blot (data not shown). In Vitro Drug Sensitivity. The KB V20C cells were maintained in drugfree media 3 days before determination of the concentration capable of inhib iting 50% of growth. Cells (IO4) were seeded in 2 ml of culture media/well in 24-well plates (Corning Glass Works, Coming, NY). The cells were incubated with different concentrations of anticancer drugs in the absence or presence of either BIBW 22 or DPM at 5% CO2 and 37°Cfor 72 h. Subsequently, the méthylà ̈ne blue cell staining method described by Finlay el al. (11) was used to measure the cytotoxic effect. All experiments were carried out in triplicate. Nucleoside Transport. The transport of [MC]thymidine (56 mCi/mmol) at 37°Cwas used as an indicator of nucleoside transport. KB cells (4 x IO5) were seeded in a 35-mm plastic dish (Becton Dickinson, Lincoln Park, NJ) and incubated for 48 h at 37°C.The cells were preincubated for 15 min at 37°C with either BIBW 22 or DPM at appropriate concentrations, and then exposed to l UM[l4C]thymidine for 30 s. Each dish was placed on ice and washed 5 times with ice-cold phosphate-buffered saline containing 20 UMDPM. Cells were harvested with 1 ml of 1% Sarkosyl and mixed thoroughly with 8 ml of scintillation solution (National Diagnostics, Manville, NJ); their radioactivity was then assayed (6). Rhodamine 123 Retention Assay. KB or KB V20C cells (2 x IO5) were seeded in 1 ml of culture media/well in 24-well plates and incubated overnight at 37°Cand then for 15 min at 37°Cwith serial concentrations of BIBW 22, DPM, or verapamil in fresh media. Subsequently, 5 ug of rhodamine 123 were added, and the cells were further incubated at 37°Cfor 30 min. The cells were then rinsed once with media and incubated again in fresh media without rhodamine 123, but with BIBW 22, DPM, or verapamil at the indicated concentrations at 37°Cfor 20 min. Cells were then removed from the wells after pancreatiti treatment and collected by centrifugation (500 x g). The cells were resuspended in fresh media and analyzed by flow cytometry with a FACS IV (Becton Dickinson, Mountain View, CA) at 488 nm and 360 mW. Green fluorescence of rhodamine 123 was measured using 530/30 nm and 580 band pass filters. A minimum of IO4 cells/sample were analyzed and fluorescence was plotted on a 5-decade logarithmic scale. The median fluorescence was used as a quantitative measure of intracellular fluorochrome accumulation and used as a parameter for P-glycoprotein inhibition. The logarithmically meas ured fluorescence intensity was recalculated to its linear value (12). 1974 American Association for Cancer Research Copyright © 1993 on February 20, 2013 cancerres.aacrjournals.org Downloaded from BIBW 22 AS B1FUNCTIONAL MODULATOR OF TUMOR CELLS Fig. 1. Chemical structure of BIBW 22. Preparation and l'h<>to:iffinity Labeling of Plasma Membranes. Prepa ration of the plasma membranes from KB or KB V20C cells and subsequent photolabeling with [3H]azidopine in the presence or absence of BIBW 22, DPM, or verapamil were performed as described previously (13). Briefly, the cells were washed and disrupted with a ground glass homogenizer (Fisher Scientific, Pittsburgh, PA). The homogenate was then centrifuged at 1000 x g for 10 min. The supernatant was overlaid on a 35% sucrose solution and centrifuged for 60 min at 18,000 X g. The membrane fraction at the interface was then centrifuged for another 60 min at 100,000 X g. Pellets were resuspended and stored at -70°C until their use. The protein concentration was determined by the method of Bradford (14), using the Bio-Rad protein assay kit (Bio-Rad, Richmond, CA). Fifty ug of the plasma membrane protein were photolabeled in 40 mm Tris-HCl buffer (pH 7.4), containing 4% dimethyl sulfoxide and 200 nM [3H]azidopine in a final volume of 50 ul in the absence or presence of BIBW 22, DPM, and verapamil, respectively. Photolabeled membrane proteins were then analyzed using 7.5% sodium dodecyl sulfatepolyacrylamide gel electrophoresis; a total of 25 ug of protein was loaded onto each line. The gel was fixed, treated with the fluorographic reagent Amplify (Amersham). then dried, and finally exposed for 7-10 days at -70°C using Kodak XAR-5 film (Eastman Kodak).