Thaliblastine, a Plant Alkaloid, Circumvents Multidrug Resistance by Direct Binding to P-Glycoprotein1

The effect of thaliblastine (TBL, NSC-68075), a plant alkaloid, in over coming multidrug resistance was investigated in doxorubicin (ADR)-resistant murine leukemic P388/R-S4 cells. In the soft agar clonogenic assay, u nontoxic concentration of TBL (2 UMIreduced the 50% inhibitory dose of ADR ( l-h exposure) from 10.8 to 1.4 UMwith a dose modification factor of 7.7. Continuous treatment of P388/R-84 cells with ADR and TBL for 24 h further lowered the 50% inhibitory dose from 3.5 to 0.07 UM, the resistance level being decreased from 233-fold in the absence of TBL to 4.7-fold in the presence of TBL as compared to the parental P388 cells. Although ADR or TBL individually had no detectable effects on cell cycle traverse, the combination of the two drugs caused a significant <... block. Flow cytometric analysis showed that TBL enhanced ADR retention in P388/R-84 cells in a doseand time-dependent manner. TBL partially blocked the photolabeling of P-glycoprotein with [JH]azidopine, and this blocking effect was further enhanced in combination with ADR. Our results indicate that TBL can reverse multidrug resistance by direct in teraction with P-glycoprotein, thereby increasing cellular ADR retention. INTRODUCTION MDR4 is characterized by decreased intracellular drug retention as a result of the increased efflux mediated by the overexpression of the M, 170.000 P-gp (1,2). P-gp acts as a membrane-bound, ATP-dependent, drug-efflux pump and is believed to transport a number of functionally and structurally unrelated drugs (3-5), resulting in a wide spectrum of cross-resistance. The specific binding of Vinca alkaloids to P-gp was first demonstrated by using a radiolabeled photoactive analogue of vinblastine (6, 7). Since then a number of compounds including verapamil, a typical calcium channel blocker (8), progester one (9), a quinoline derivate (MS-073) (10). and cyclosporin A (II) have been shown to inhibit this photoaffinity labeling and reverse MDR in several cell lines. Thus, it has been proposed that the pho tolabeling method may be used for rapid screening of new compounds for MDR reversal (7. 11). Thalicarpine (TBL) is a dimeric aporphine benzylisoquinoline al kaloid originally isolated from several species of the genus Thalictrum (family Ranunculaceae) (12). TBL has antitumor activity against sev eral experimental tumors both in vitro and in vivo (13, 14). Recently. TBL was found to have a higher cytotoxic effect in a cisplatinresistant rat ovarian tumor cell line than in the parental drug-sensitive cell line (15). This effect could be further increased in combination with hyperthermia.s Because of its lower toxicity (16) and structural similarity (Fig. 1) with some photouffinity analogues of P-gp-inhibitory chemicals ( 11), we have investigated the ability of TBL to reverse MDR in ADR-resistant murine leukemic P388/R-84 cells. Received 11/16/92: accepted 3/24/93. The cosls of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked adveniseinem in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by NIH Grant CA-29360. 2 Present address: Dana-Farber Cancer Institute. JF-525, 44 Binney Street. Boston. MA 02115. 1To whom requests for reprints should he addressed, at Division of Experimental Therapeutics. Department of Radiation Oncology. University of Miami School of Medi cine, P. O. Box 016960 (R-711. Miami. FL 331(11. 4 The abbreviations used are: MDR. multidrug resistance: P-gp. P-glycoprolein: TBL, thaliblastine; ADR. adriamycin; ID^n. 50% inhibitory dose; MI. modulator index. s G. Chen el al., unpublished data. MATERIALS AND METHODS Chemicals and Cell Culture. Doxorubicin hydrochloride (ADR, NSC123127) was purchased from Adria Laboratories, Inc., Columbus, OH. A stock solution of ADR ( I mg/ml), prepared in distilled water and kept at -20°C, was diluted in RPMI medium just before use. Thaliblastine hydrochloride (thalicarpine. TBL; Pharmachin. Bulgaria) was obtained from Dr. D. K. Todorov, National Oncological Center. Sofia. Bulgaria, and dissolved in medium before use. Tritium-labeled, photoactive 2.6-dimelhyl-[4-(2'-trifluoromethyl)phenyl]l.4-dihydropyridine-3,5-dicarboxylic acid, ethyl, (/V-4'-azido|3',5'-'H] benzoylaminoethyljdiester (['HJazidopine) (stock solution, 1 HIM)was purchased from Amersham Corporation (Arlington Heights, IL). Mouse leukemic P388 and the doxorubicin-resistam P388/R-84 cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 units/ml of penicillin. 100 ug/ml of streptomycin, and IO UM 2-mercaptoethanol. Cytotoxicity Assay. Soft agar clonogenic and growth inhibition assays were carried out for determination of longand short-term cytotoxicity of ADR and TBL. respectively. Following l-h exposure to ADR in the presence or absence of 2 UMTBL, cells were washed twice with fresh medium and resuspended in RPMI medium containing 15% fetal bovine serum and soft agar at a final concentration of 0.3%. Cells were seeded at a density of 40,000 per well (2 ml) in 6-well plates (Costar, Cambridge, MA) in triplicate. Colonies larger than 50 urn were counted on Day 5: plating efficiency in the control group of both lines was between 45 and 55% (17). The effect of 2 UMTBL on ADR (0.01 to 10 UM)cytotoxicity was determined by the trypan blue dye exclusion method (18), after incubating 1 x IO6 cells with the drug(s) in 1 ml of medium for 24 h at 37°C. DNA Histogram Analysis. Following drug exposure for 24 h, cells were centrifuged and resuspended in propidium iodide-hypotonic citrate solution for l h before flow cytometric analysis for cell cycle distribution (19). Cellular ADR Content. Log-phase cells ( I x W/ml) were exposed to 3.6 UMADR in the absence or presence of TBL (2 or 8 UM)for either 1 or 4 h at 37°C. Cellular ADR fluorescence was monitored after excitation with a 488-nm argon laser line in a FACScan (Becton and Dickinson. San Jose, CA) (17. 20). Fluorescence emission (above 530 nm) from at least 10.000 cells was collected, amplified, and scaled to generate a single-parameter histogram. Photolabeling of Plasma Membrane. Plasma membranes from P388/R-84 cells were prepared according to the procedures of Gerlach el at. (21) and Yusa and Tsuruo (22) with minor modifications. Cells were washed 3 times with ice-cold phosphate-buffered saline [0.15 MNaCl:20 mM sodium phosphate (pH 7.4)] at 200 x g and 4°Cfor IO min and incubated at about 5 x IO7 cells/ml in the hypotonie lysis buffer [10 ITIMKClil.S m.MMgCK:2 imi phenylmethylsulfonyl fluoride: 10 IHMTris (pH 7.4)] on ice for IO min. The swollen cells were disrupted with a T-line homogenizer (Talboys Engineering Corporation, Emerson. NJ). The homogenate was subjected to centrifugation at 40(X) x g for 10 min to remove the nuclei (pellet). The supernatant was centrifuged for 90 min at 4°Cand 50,000 x g. The pellet was stored at -70°C until use. The protein content was determined by the Bradford assay (23). Photolabeling of plasma membranes was performed according to the method described by SafaeÃ-al. (24). The membrane vesicles (50 ug of protein) were photolabeled in 40 ITIMpotassium phosphate buffer (pH 7.0) containing 10 UMCaCl2, 4% dimethyl sulfoxide, and 0.2 UM|3H]azidopine (10 pCi) in a final volume of 50 ul. This mixture preincubated for l h at room temperature in the dark (in the absence or presence of 3.6 H.MADR and/or 2 or 8 |JMTBL) was irradiated for 20 min using a UV cross-linker (UV Stratalinker 1800. Stratagene, La Jolla. CA). The photolabeled membrane protein preparations were separated on a 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Covalent incorporation of ('HJazidopine was detected by fluorography using En'hance (Du Pont. MA).