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[CANCER RESEARCH 52, 6385-6389. November 15, 1992] Advances in Brief Structural Requirements of Simple Organic Cations for Recognition by Multidrug-resistant Cells1 Marc Dellinger, Berton C. Pressman, Cesar Calderon-Higginson, Niramol Savaraj, Haim Tapiero, Despina Kolonias, and Theodore J. Lampidis2 Museum d'Histoire Naturelle, Departement de Biophysiques, Paris, France ¡M.D.]; Department of Molecular and Cellular Pharmacology, University of ¡Miami, School of Medicine, [B. C. P., C. C-H.J; Veterans Administration Hospital and Division of He matology/Oncology, University of Miami /A'. S.J; ICIG, Hôpital Paul Brousse, Villejuif, France ¡H.TJ; and Department of Medical Oncology, University of Miami, School of Medicine, Miami, Florida 33136 [D. K., T. J. L.J Abstract We previously noted that a wide variety of drugs which are recognized by multidrug-resistant cells (MDR+) are positively charged. However, it remains unclear why and how such a large number of structurally different compounds can be distinguished by MDR+ cells. The majority of the diverse compounds subject to MDR are complex and thereby complicate definitive structure/function characterization of the P-glycoprotein-mediated MDR mechanism. Using a series of simple aromatic (alkypyridiniums) and nonaromatic (alkylguanidiniums) organic cations differing in their lipophilicity by stepwise additions of single alkyl car bons, we demonstrate by growth inhibition studies that a single aromatic moiety and a critical degree of lipophilicity (log P > -1) are required for recognition of these simple organic cations by MDR+ cells. Thus, MDR •¿ cells are not cross-resistant to the nonaromatic guanidiniums but do show cross-resistance to those aromatic pyridiniums with chain lengths >four. Resistance ratios, as determined by comparison of 50% inhibitory doses in MDR—versus MDR+ cells, increase as a function of increasing chain lengths of these latter simple aromatic compounds. Resistance to pyridinium analogues in MDR+ cells is reversible by cotreatment with nontoxic doses of verapamil. Preliminary uptake data with radioactive analogues further implicate the MDR mechanism of lowered drug accumulation in accounting for resistance to the pyridin ium homologues. Utilization of these simple organic cations provides a rational basis for better defining the physical chemical properties of more complex compounds processed by the MDR mechanism and suggests a strategy for designing chemotherapeutic agents with reduced susceptibility to MDR. Introduction MDR3 describes a phenomenon in which a given cell or cell line, when exposed to a single agent, either displays or becomes cross-resistant to a large number of other diverse agents. Spe cific genes have been identified (1, 2) which code for a A/r 180,000 glycoprotein (P-gp) found in the plasma membrane of cells which exhibit MDR (2, 3). P-gp is thought to aid in low ering intracellular drug accumulation and consequently to ren der the cell resistant by functioning either as an active efflux pump (2, 4) or by other mechanisms (5-7). Received 7/29/92: accepted 10/1/92. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accord ance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by NIH Grants CA37109 and GM38920, a Veterans Administration National Grant, and a grant from the Sylvester Comprehensive Cancer Center. 2 To whom requests for reprints should be addressed, at Department of Cell Biology and Anatomy, University of Miami, School of Medicine. Fox Cancer Research Building, 1550 NW 10th Avenue. Miami, FL 33136. 3 The abbreviations used are: MDR, multidrug resistance; P-gp, P-glycoprotein; III. Friend leukemia cells; alkylGu*. alkylguanidiniums; alkylPy*. alkylpyridiniums; octylPy*, octylpyridiniums. The question of why and how such a large number of drugs, which are relatively complex and differ widely from one another in structure, are recognized by MDR+ cells remains unclear. We have previously reported that the cationic charge of xanthene dyes was critical for their differential accumulation in certain normal versus tumor epithelial cell lines (8-10). It was subsequently shown that the normal epithelial cell line (CV-1) that had reduced accumulation of these cationic xanthene dyes expressed high amounts of the MDR-1 gene (11) and also ac cumulated less of the cationic anthracycline, Adriamycin (12). With the xanthene dyes, we found that zwitterionic analogues showed less selectivity between MDR- and MDR+ cells than the positively charged compounds (13). Several independent studies with other types of compounds support the hypothesis that cationic charge is an important feature for MDR recogni tion (14-17). Beck and coworkers (17-19) in a series of papers have suggested that a number of complex compounds which interact (bind and or block) with Pg-p, and thereby modulate MDR, share in common the physical chemical properties of lipophilicity, cationic charge, and at least two planar aromatic domains. These compounds, however, as well as the majority of compounds found to be cross-resistant to MDR+ cells, are complex in structure which makes interpretation of their MDR structure/function activity difficult. Here, by means of a series of simple aromatic and nonaromatic cations, which we have synthesized (Fig. 1), differing in lipophilicity by systematic additions of alkyl carbons, the specific molecular characteristics necessary for recognition by the P-glycoprotein-mediated MDR mechanism have been examined. Materials and Methods Cell Lines. Previously, we reported that the MDR+ cells used here were developed from parental MDR- FLC by exposure to stepwise increases of Adriamycin and had the following typical MDR properties: broad spectrum resistance to cytotoxic drugs (>2000-fold resistance to Adriamycin as compared to MDR- cells); high levels of MDR-1 gene expression; lowered plasma transmembrane potentials; and reduced accumulation of Adriamycin, rhodamine 123, and other lipophilic cat ions compared to their sensitive MDR—counterparts (20). Lower drug accumulation in these MDR+ cells is reversed by the widely used MDR modulator, verapamil. with concomitant reversal of resistance to these agents (21). Compounds. Alkylguanidiniums and alkylpyridiniums were synthe sized as previously described (22). Verapamil was purchased from Sigma (St. Louis, MO). Growth Inhibition Assays. Growth inhibition assays were performed by seeding exponentially growing sensitive (FLC) or resistant (ARN) cells at 1.0 x IO5 cells/ml in Eagle's minimal essential medium sup plemented with 10% fetal bovine serum at 37°Cin 10% CO2air and treating each continuously with the indicated doses of guanidinium 6385 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1992 American Association for Cancer Research. SIMPLE ORGANIC CATIONS AND MDR and pyridinium analogues. At 72 h, cells excluding trypan blue were counted, and survival curves were derived as previously described (23). Partition Coefficient Determinations. Partition coefficients for each of the guanidinium and pyridinium analogues, expressed as log P, were determined spectrophotometrically with the system: octanol-1 (highperformance liquid chromatography grade) versus l M Mg(ClO4)2. The inclusion of the salt provides a lipophilic companion aniónfor the test cation, so that the partition of the cation is a function of its intrinsic lipophilicity. Drug Uptake Assays. Exponentially growing MDR+ and MDRcells at 1.3 x 106/ml were treated, respectively, with either [l4C]guanidinium or l-4-[werA>'/-l4C]octylpyridinium for 60 min at 30°Cin '•A OUA 100 6a.... C2-GUA-Ht%¿A , O tt licity is progressively increased, by the addition of alkyl car bons, the cytotoxic potency of these compounds increases ex ponentially in both MDR- and MDR+ cells. Although these nonaromatic compounds are positively charged, MDR+ cells exhibit relatively little (<3-fold) or no cross-resistance to them. In contrast, we find that MDR+ cells are cross-resistant to a series of aromatic cations, alkylPy+ (Fig. 1) of equivalent lipophilicity to the alkylGu* which differ from them in that U o ' NH~—C —¿ NHR * + Fig. I. Structures of guanidiniums and pyridiniums used in these studies. An alogues differing by stepwise additions of single alkyl carbons were synthesized, ranging in chain length from guanidinium to octylguanidinium. and from methylpyridinium to dodecylpyridinium. C4-PYR0AA« ¿ •¿'£' CS-P'YR-A C3-GUA- •¿*•m *A. •¿ A, . .i.v C4-GUA.\ ' 10050GUANIDINIUMSSflA 100so0 C6-PYR-*8V•I K % ^AA tt C7-PYR*.'*'¡Õ1 100500100150A $ O se o C"""A"f.. their charge is delocalized by a single pyridine ring (Fig. 2). Moreover, in addition to aromaticity, a critical chain length, equivalent to a minimal degree of lipophilicity (log P > —¿1.00; Table 1), is required before MDR+ cells show cross-resistance to these simple aromatic compounds. To determine whether the position of the alkyl chain is also critical for MDR recognition, we synthesized l-methyl-4-octylPy+ and found that MDR+ cells show a similar level of cross-resistance to this compound as to its yV-alkyl counterpart, l-octylPy+. Thus, the chain length, i.e., degree of lipophilicity of the aromatic alkylPy+, but NH •¿ .«&.. . o u lengths of C7, CIO and C12 in MDR+ cells is reversed by cotreatment with nontoxic doses of verapamil, further support the involvement of the MDR efflux mechanism for these com- ' %û ....fe• Results Growth-inhibitory Activity of Alkylguanidiniums and Alkylpyridiniums in MDR+ and MDR- Cells. The inhibition of growth after 72-h exposure to the series of nonaromatic cations, alkylGu+ (Fig. 1), as shown in Fig 2, indicates that as lipophi of MDR is its sensitivity to modulation by the calcium channel antagonist, verapamil (25), and consequently, verapamil has been used to verify P-gp-mediated MDR (4). Thus, the data in Fig. 3, which show that cross-resistance to alkylPy"1"with chain 1 •¿ .¡ft 600100500100so0 5%CO2/95% air. At this time both compounds reach equilibrium in MDR+ and MDR- cells. Intracellular uptake was determined accord ing to the methods previously described (24). not the position of the alkyl groups, determines the recognition of cations by the P-gp-mediated MDR process. Reversal of Cross-Resistance in MDR+ Cells to C7, CIO, and C12 AlkylPy* Analogues by Verapamil. One of the hallmarks C1-PYR 10050010050010050PYRIDINIUMS' * - •¿V4'•s AAAAA so •¿â€¢C10-PYR ,AA. DRUG CONCE NTRATION . (M) Fig. 2. Seventy-two-h growth inhibition assays of guanidiniums (left) and pyridiniums (right), varying in lipophilicity by the stepwise addition of alkyl carbons (GUA to C8-GUA and Cl-PYR to C-IOPYR) in MDR- (•)and MDR+ (A) Friend leukemia cells. Note the lack of difference in growth inhibition between MDR- and MDR+ cells produced by the alkylguanidiniums. In contrast, for the alkylpyridiniums, beginning with ( \ l'\ K. differences in growth inhibition be tween MDR—and MDR+ cells increase as a function of increasing lipophilicity. Each point represents the average cell count of duplicate cultures, and for each analogue experiments were repeated at least twice. pounds. Additional corroboration is supplied by preliminary data indicating that drug retention of l-4-[mefA>'/-14C]octylPy+ is severalfold greater in MDR—than in MDR+ cells. However, accumulation differences between MDR—and MDR+ cells for the nonaromatic cationic [14C]Gu+ are minimal. These results indicate that differences in the level of drug retention correlate qualitatively with differential cytotoxicity. Relationship of Partition Coefficients, Chain Lengths, 50% Inhibitory Doses, and Resistance Ratios. The octanol:H2O partition coefficients (expressed as log P), chain lengths, 50% inhibitory doses, and resistance ratios (MDR+/MDR—) for 6386 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1992 American Association for Cancer Research. SIMPLE ORGANIC CATIONS AND MDR Table 1 Partition coefficients (log P), 50% inhibitory doses in MDR+ and MDR- cells, and resistance ratios (MDR+/MDR-) of each of the listed alkylguanidiniums and alkylpyridiniums with different alkyl chain lengths Note lack of difference between 50% inhibitor) doses in MDR+ and MDRcells (resistance ratio) for the nonaromatic alkylguanidiniums. In contrast, for the aromatic alkylpyridiniums of chain length >4, the resistance ratios increase in accordance with increasing chain length and log P. Fifty % inhibitory doses represent the dose of test compound which yields 50% growth (cell number) after 72-h treatment as compared to the growth of untreated cells. These values are the average of replicate cultures in each experiment, which was repeated at least twice. an aromatic moiety inhibits the ability of these compounds to access or adsorb to the P-gp for subsequent expulsion from the cell. Discussion Previously, Pressman (22) demonstrated that the uptake of the simple nonaromatic orgainic cation, guanidine, with rela tively low lipophilicity, which enters the mitochondrial matrix freely without being trapped within the nonpolar environment of membranes, could serve as an accurate measure of mitochon MDR(M)2.0 MDR+4.0 ratio2.03.01.51.31.31.01.30.81.28.58.671.037.0236.0400.0 CompoundGUAC2-GUAC3-GUAC4-GUAC5-GUAC6-GUAC8-GUACl-PYRC4-PYRC5-PYRC6-PYRC7-PYRC8-PYRC10-PYRC12-PYRLog P-1.00-0.18+0.25+0.58+ drial membrane potential. Furthermore, he showed a relation 10~33.0 x IO-31.0 x ship between an increased inhibitory effect on respiration in 10-"8.0 x I0~45.5 x 10~52.0 x 10~52.6 x isolated mitochondria and increased lipophilicity in the same 10~51.4 x IO'51.1 x series of guanidinium homologues used here (22). He reasoned 10~54.0 x 10~54.0 x 1.00+ that, since electron transport and oxidative phosphorylation 10~61.3 x 10~61.7 x 1.40+2.25-2.10-1.00ND"+0.11+0.60+ 10^63.0 x 10~62.5 x take place within the membranes of mitochondria, the more 10~33.0 x 10^32.5 x lipophilic guanidiniums would accumulate there and thus be 10~58.5 x 10~41.0 x 10~43.0 x 10~43.5 x more potent inhibitors of these processes. 10~57.0 x IO'45.0 x Since we had found that the positive charge for certain more IO'62.3 x 10~48.5 x complex compounds was involved in their recognition by 10~51.3 x 10~65.5 x 1.30ND+2.60lD5o° 10~76.0 x 10-"2.4 x MDR+ cells (12,13,21) and others had shown this for different x 10~5IDso x IO'8Resistance families of equally or more complex compounds (14-19), we " ID50, 50% inhibitory dose; GUA. alkylguanidiniums; PYR. alkylpyridiniums; were prompted to use these simple organic cations to probe the ND, not determined. relationship between lipophilicity and recognition by P-glycoprotein-mediated MDR. In addition, since the guanidiniums each of the compounds tested are presented in Table 1. It is did not contain an aromatic moiety but the pyridiniums did, we apparent that for alkylGu"1" and alkylPy"1", toxicity in both utilized these two related structures to determine the impor MDR- and MDR+ cells increases as a function of their alkyl tance, if any, that a single aromatic ring may have for recogni tion by MDR+ cells. chain length and their correspondingly increasing partition co As noted above, we found that the increased inhibition of efficients. Thus, the more lipophilic the compound, the more growth in intact MDR+ and MDR—cells, which we detect as a cytotoxic it is toward both MDR—and MDR+ cells. However, for the alkylPy+ with chain lengths >4, toxicity for MDR+ cells function of increasing lipophilicities of the guanidinium com lags significantly behind that for MDR—cells. The potencies of pounds tested here, correlates with their potency to inhibit res longer chain alkylPy"1"for MDR— cells continue to increase piration in isolated mitochondria (22). Whether or not inhibi exponentially as a function of their increasing alkyl chain tion of mitochondrial respiration is the primary mechanism by which these compounds inhibit growth in whole cells remains lengths. Thus, a simple correlation exists between lipophilicity and cytotoxicity for the alkylGu"1"in both MDR—and MDR+ to be determined. More importantly, however, we find that the cells. This relationship also applies to MDR- cells for the pyridiniums also inhibit growth as a function of their increasing alkylPy"1",but in MDR+ cells, cytotoxicity due to these aro lipophilicity but only in MDR—cells. In MDR+ cells, we find that the growth-inhibitory potency of pyridinium homologues matic cations increases less sharply when the partition coeffi cients of the alkylPy* rise above a critical value, i.e., log P > with log P > —¿ 1 does not correlate nearly as well with increas —¿ 1.00. Accordingly, the lipophilicity and aromaticity of the ing lipophilicities, since MDR recognizes these aromatic com pounds, which results in lowered drug accumulation. Our data simple cationic compounds examined facilitate either their pre sentation, or binding, to the MDR P-gp. Moreover, the results, with reversal of resistance to the pyridinium analogues to which with the nonaromatic cationic alkylGu"1"suggest that absence of MDR+ cells show cross-resistance, by the standard MDR C7-PYRIDINIUM C10-PYRIDINIUM 100 •¿ Fig. 3. Seventy-two-h growth inhibition assays of pyridiniums with alkyl chain lengths of 7, 10, and 12 incubated with (below) or without (above) cotreatment of 10 »jg/mlof verapamil. Note that differences in sensitivity to these compounds be tween MDR- and MDR+ cells continue to in crease as a function of increasing lipophilicity. These differences are markedly reduced or elimi nated when both cell types are cotreated with ver apamil. Each point represents the average cell count of replicate cultures, and for each analogue experiments were repeated at least twice. C12-PYRIDINIUM 100. zH5OK"OJU)U^oK A A 50 50z0O A$A«UA°100zIIIg ' HA +VP_HDA.4* - ' JL^_ •¿.„••4†•¿ ' 100 150-• 50 50UJO.n'flA' •¿A 10 10" li" If 10" DRUG 10' 1»' l«'5 IO'4 IO'5 IO'2 10 CONCENTRATION 10 7 10 * 10 ! 10 ' If3 (M) 6387 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1992 American Association for Cancer Research. SIMPLE ORGANIC CATIONS AND MDR modulator, verapamil, further support this interpretation. Ad ditionally, our preliminary uptake data with radioactive octylpyridinium and radioactive guanidinium also implicate the MDR mechanism for the pyridiniums to which MDR+ cells are cross-resistant. Thus, in comparison to other studies with substantially more complex organic cations in which it was shown that two planar aromatic moieties were required for modulatory activity in MDR+ cells (18), our results with simple organic cations show that only a single aromatic moiety is necessary for MDR-recognition. Moreover, we define a critical chain length or lipophilicity which must be attained before these compounds can be recognized by the P-gp-mediated MDR mechanism. Although it has been shown that MDR P-gp has homology to a number of eukaryotic and prokaryotic glycoproteins known to function as pumps or transporters of specific proteins and inorganic ions (26-30), the MDR P-gp does not display a typically selective lock-and-key fit, characteristic of enzymesubstrate interactions. The wide variety of compounds which are processed by the MDR P-gp-effluxing system argues rather in favor of a less selective process, such as adsorption. In this regard, Sehested et al. (5) showed that MDR+ cells undergo accelerated endocytosis and proposed a nonspecific adsorptive, lysosomal ion-trapping mechanism to account for expulsion of cationic drugs from MDR-t- cells. However, compounds with hard cationic charges such as rhodamine 123 (and the alkylPy* used here) which are processed by the MDR mechanism are not candidates for ion trapping (31) by acidic lysosomes. Therefore, nonselective ion-trapping alone cannot entirely account for the handling of all MDR drugs. More recently, Warren et al. (6) have demonstrated in an MDR-t- cell line in which P-gp is overexpressed that the content of lysosomal enzymes is reduced and that the relative rates of execretion of these enzymes are significantly greater as compared to those of its MDR—coun terpart. Their data suggest a linkage between accelerated lyso somal enzyme elimination and enhanced drug efflox, since both processes are inhibited by verapamil. Higgins and Gottesman (7) suggested that the MDR P-gp may function as a flippase, which would also invoke an adsorp tive process to explain the wide variety of lipophilic organic compounds effluxed from MDR+ cells. While the invocation of a flippase to explain MDR remains speculative, it is of interest that this mechanism also involves a relatively unselective ad sorptive process. Our data, showing that a critical lipophilicity for the aromatic alkylPy"1"must be reached before MDR+ cells show cross-resistance to them and that cross-resistance in creases as a function of increasing lipophilicity, are more con sistent with an adsorptive process than with a substrate selective pump mechanism. Regardless of the mechanism(s) involved, the data presented here clearly demonstrate that for simple cytotoxic organic cat ions, an aromatic moiety and a critical degree of lipophilicity are required for MDR recognition. The question of whether the same physical chemical properties which we have identified for differential toxicity by MDR+ and MDR-cells will also prevail for MDR modulation toward other chemotherapeutic agents such as Adriamycin, with the pyridinium and guanidinium an alogues used here, remains to be determined. The data presented could serve to provide a rational basis for better understanding the structural requirements of more complex molecules that interact with the MDR mechanism. 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Trevorrow. K. W., Valet, G., Tapiero, H., and Lampidis, T. J. Increased uptake and retention of rhodamine 123 in multidrug resistant Friend leukemie cells induced by the carboxylic ionophores monensin and nigericin. J. Cell Pharmacol., 2: 29-36, 1991. 6389 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1992 American Association for Cancer Research. Structural Requirements of Simple Organic Cations for Recognition by Multidrug-resistant Cells Marc Dellinger, Berton C. Pressman, Cesar Calderon-Higginson, et al. Cancer Res 1992;52:6385-6389. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/52/22/6385 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. 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