New phenothiazine-type multidrug resistance modifiers: anti-MDR activity versus membrane perturbing potency

BBRC Biochemical and Biophysical Research Communications 304 (2003) 260–265 www.elsevier.com/locate/ybbrc

New phenothiazine-type multidrug resistance modifiers: anti-MDR activity versus membrane perturbing potencyq Andrzej B. Hendrich,a,* Olga Wesołowska,a Noboru Motohashi,b Joseph Moln
ar,c and Krystyna Michalaka b

a Department of Biophysics, Wrocław Medical University, ul. Chałubi
nskiego 10, Wrocław 50-368, Poland Department of Medicinal Chemistry, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose-shi, Tokyo 204-8588, Japan c Institute of Microbiology, Albert Szent-Gy€ orgyi Medical University, Dom ter 10, Szeged H-6720, Hungary

Received 26 March 2003

Abstract The phenothiazine multidrug resistance (MDR) modulators are chemically diversified but share the common feature to be hydrophobic cationic molecules. Molecular mechanisms of their action may involve interactions with either P-glycoprotein or membrane lipid matrix. In the present work we study the anti-MDR and biophysical membrane effects of new phenothiazine derivatives differing in the type of group substituting phenothiazine ring at position 2 (H–, Cl–, CF3 –) and in the side chain group (NHCO2 CH3 or NHSO2 CH3 ). Within each phenothiazine subset we found that anti-MDR activity (determined by P-glycoprotein inhibition assessed by flow cytometry) correlates with the theoretically calculated hydrophobicity value (log P ) and experimental parameters (determined by calorimetry and fluorescence spectroscopy) of lipid bilayers. It is concluded that the biological and biophysical activity of phenothiazine derivatives depends more on the type of ring substitution than on the nature of the side chain group. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Phenothiazine derivatives; Multidrug resistance reversal; Drug–lipid interactions; Lipid bilayer fluidity; P-glycoprotein

The phenomenon of multidrug resistance (MDR) of cancer cells is characterized in vitro by cross-resistance to many different anti-cancer agents, which do not share either a common chemical structure, pharmacological target or metabolization pathway [1]. The best characterized mechanism contributing to MDR is a decrease in the permeability of the plasma membrane for anti-cancer agents, along with the overexpression of a drug q Abbreviations: MDR, multidrug resistance; P-gp, P-glycoprotein; PhDs, phenothiazine derivatives; PhMC, phenothiazine methoxycarbonylamide; PhMS, phenothiazine methanesulfonylamide; PS, phosphatidylserine; DPPC, dipalmitoylphosphatidylcholine; DMPE, dimyristoylphosphatidylethanolamine; DMPG, dimyristoylphosphatidylglycerol; DPH, 1,6-diphenyl-1,3,5-hexatriene; NPN, N-phenyl-1naphthylamine; DiOC2 ð3Þ, 3,30 -diethyloxacarbocyanine iodide; DMSO, dimethyl sulfoxide; FAR, fluorescence activity ratio; log P , logarithm of molar partition coefficient; Tm , main phase transition temperature. * Corresponding author. Fax: +48-71-784-0088. E-mail address: hendrich@biofiz.am.wroc.pl (A.B. Hendrich).

transporting plasma membrane protein, P-glycoprotein (P-gp) [2,3]. According to the ‘‘vacuum cleaner’’ hypothesis, P-gp substrate drug molecules are attached to transport protein and transported out of the cell during their passage through the cell membrane. The rate of drug diffusion across a membrane depends on the fluidity of the lipid bilayer: the greater the disorder in the membrane, the greater its permeability. Most of the substances that modulate multidrug resistance share one common feature—they are cationic amphiphiles. Thus, modulators are able to infiltrate into the lipid bilayer and influence its properties. Also P-gp, an integral protein embedded in the membrane, may be sensitive to changes in the biophysical features of membranes. It has been shown that substrate recognition, P-gp conformational stability, and its ATPase activity are dependent on the biophysical properties of the membrane lipid phase (reviewed in [1]). Multidrug resistance modulators, similar to P-gp substrates, constitute an extremely diverse group of

0006-291X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0006-291X(03)00580-1

A.B. Hendrich et al. / Biochemical and Biophysical Research Communications 304 (2003) 260–265

compounds. The only common features shared by MDR modulators of different chemical structures are their relative hydrophobicity and positive charge at physiological pH [4,5]. The presence of hydrogen bond donor/ acceptor groups and their spatial arrangement in the modulatorÕs molecule have also been recognized to correlate with anti-MDR potency [6–8]. It is not surprising, therefore, that interactions between membranes and amphiphilic modulators were found to be important in the anti-MDR activity of various groups of modifiers. The influence on the physical state of the lipid matrix [9], membrane permeability [10], and passive transbilayer drug kinetics [11] has been found to correlate with a modulatorÕs potency. Phenothiazines, apart from their clinical use as tranquillizers, are able to restore the sensitivity of multidrug resistant cells to chemotherapy [12–14]. The mechanism of the anti-MDR action of phenothiazines is not yet fully understood. The extremely wide scope of the effects of phenothiazines on membranes (e.g., influence on lipid phase transitions [15–19], erythrocyte shape changes [20,21], and hemolysis [22]) suggests, however, that their MDR reversing action could possibly be exerted, not by directly inhibiting P-gp, but by indirectly perturbing the lipid matrix in which P-glycoprotein is embedded. The ability of phenothiazines to alter the properties of model and natural membranes has been extensively investigated, but these studies were mostly performed using commercial therapeutic compounds: chlorpromazine (CPZ) [23] and trifluoperazine (TFP) [24]. Only a few articles have dealt with the problem of the relationship between the structure and the activity of phenothiazine derivatives [25–28]. It has been found that both the type of substituent at position 2 of the phenothiazine ring and the length of the alkyl bridge connecting the ring system with the side group play some role in the activity of phenothiazine. The aim of this paper was to study the effects on membranes and the anti-MDR effects of phenothiazine derivatives differing both with regard to the type of side chain groups and to the character of the substituent at position 2 of the phenothiazine ring. We also investigated the correlation between biophysical effects on membranes and the MDR reversal potency of these two kinds of phenothiazines. We have shown experimentally that the extent of P-glycoprotein inhibition in cancer cells measured by flow cytometry and the membrane perturbing potency determined by fluorescence spectroscopy and microcalorimetry are similar. A positive relation between the experimental results and theoretical predictions for PhDs lipophilicity was also found. Considering the structural features of the compounds studied, we concluded that the type of ring substituent is more important than the type of side chain group in defining the biological activities of PhD investigated.

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Materials and methods 1,2-Dimyristoyl-sn-glycero-3-phosphatidylglycerol (DMPG) was purchased from Avanti Polar Lipids (Alabaster, AL, USA). Bovine brain L -a-phosphatidylserine (PS), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), and 1,2-dimyristoyl-sn-glycero-3-phosphatidylethanolamine (DMPE) were purchased from Sigma (St. Louis, MO, USA). The lipids were used as delivered, without further purification. Fluorescent probes: 1,6-diphenyl-1,3,5-hexatriene (DPH), N-phenyl-1naphthylamine (NPN), and verapamil hydrochloride were obtained from Sigma (St. Louis, MO, USA). 3,30 -Diethyloxacarbocyanine iodide (DiOC2 ð3Þ) was purchased from Molecular Probes (Eugene, OR, USA). All the other chemicals used in the experiments were of analytical grade. Phenothiazine derivatives (PhDs): methoxycarbonylamides (PhMCs) and methanesulfonylamides (PhMSs) were synthesized as described in [29,30]. The chemical structures and abbreviations of individual PhMCs and PhMSs are shown in Fig. 1. Within each subgroup there are three compounds differing with respect to the type of substituent at position 2 (H–, Cl–, CF3 –) of the phenothiazine ring. Calculation of the partition coefficient. Octanol/water partition coefficients of phenothiazine derivatives were calculated according to the method of Ghose et al. [31] using Titan 1.0.8 software (Wavefunction, Irvine, USA & Schrodinger, Portland, USA). Calorimetric measurements. Stock solutions of PhDs (3.5 mM) were prepared in chloroform:methanol (1:1; v:v). For each calorimetric sample 2 mg of the appropriate lipid was dissolved in the PhD stock solution. The amount of PhD stock solution was chosen in order to obtain a drug:lipid molar ratio of 0.06 in the sample. The samples were dried under a stream of nitrogen and the residual solvent was removed under vacuum for at least 3 h. Samples were hydrated with 20 ll of a 20 mM Tris–HCl buffer (150 mM NaCl, 0.5 mM EDTA, pH 7.4). The hydrated mixtures were heated to a temperature of approximately 10 °C higher than the main phase transition temperature of a given lipid and vortexed until homogeneous dispersion was obtained. Calorimetric measurements were performed using a Rigaku calorimeter, which was partially rebuilt in our laboratory. The scanning rate was 1.25 °C/min. At least two separate samples of each PhD were made, each sample being scanned at least three times, immediately after preparation. Fluorescence spectroscopy. Bovine brain phosphatidylserine suspensions were sonicated (UP 200s sonicator, Dr. Hilscher GmbH, Berlin, Germany) in a 1/15 M Michaelis phosphate buffer (pH 7.4) to obtain small unilamellar liposomes. Fluorescent probe stock solutions (1 mM) were prepared in DMSO (NPN) and tetrahydrofuran (DPH). PhD stock solutions (5 mM) were prepared in DMSO. Liposomes were incubated with a fluorescent probe in darkness for 30 min or 15 min (for DPH and NPN, respectively) at room temperature. PhDs were then added (at a concentration varying from 5 to 100 lM) and incubation was continued under the same conditions for the next 20 (DPH) or 10 min (NPN). In all the experiments, the final phospholipid

Fig. 1. The chemical structure and abbreviations of phenothiazine derivatives studied: methoxycarbonylamides (PhMCs) and methanesulfonylamides (PhMSs).

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concentration was 200 lM and the concentration of the fluorescent probe was 5 lM. Fluorescence measurements were performed by a LS 50B spectrofluorimeter (Perkin–Elmer, Beaconsfield, UK) using emission and excitation slits of width 5 nm. The DPH excitation and emission wavelengths were 380 and 450 nm, respectively. NPN fluorescence was excited at 350 nm and emission spectra were recorded over a range of 360–580 nm. As PhDs were found to display intrinsic fluorescence within this wavelength range, the emission spectra of PhDs in PS liposomes were recorded in the absence of NPN and removed from the spectra obtained in the presence of NPN. Data were collected and processed using FLDM Perkin–Elmer software. Flow cytometry. 3,30 -Diethyloxacarbocyanine iodide (DiOC2 ð3Þ) was used as a P-gp fluorescent substrate in flow cytometric experiments. Its specificity as a P-glycoprotein substrate was demonstrated recently [32,33]. Stock solutions of DiOC2 (3) (75 ng/ml) and PhDs (1 mg/ml) were prepared in DMSO. A L5178Y mouse T lymphoma parent cell line was transfected with the pHa MDR1/A retrovirus as previously described in [34]. Cell lines expressing MDR1 were selected by culturing the infected cells with 60 ng/ml colchicine to maintain the expression of the MDR phenotype. The L5178 MDR cell line and the L5178Y parent cell line were grown in McCoyÕs 5A medium with a 10% heat inactivated horse serum, L -glutamine, and antibiotics. The cell concentration was regulated to 2  106 =ml and the cells were resuspended in McCoyÕs 5A medium without any serum. Then, the cells were distributed into 0.5 ml aliquots in Eppendorf centrifuge tubes and the compounds investigated were added (2 ll). After 10 min of incubation at room temperature, 10 ll of the fluorescent indicator solution was added to the samples. The final DiOC2 (3) concentration was 1:63  1012 M. The cells were incubated for a further 20 min at 37 °C, washed twice, and resuspended in 0.5 ml phosphate-buffered saline (PBS) for analysis. The fluorescence of the cell population was measured by flow cytometry using a Beckton–Dickinson FACScan instrument equipped with an argon laser. The fluorescence excitation and emission wavelengths were 488 and 520 nm, respectively. Verapamil was used as a positive control. The influence of DMSO on the cells was also monitored. The fluorescence intensity was calculated for parental and MDR cell lines as a percentage of the control mean for untreated cells. The fluorescence activity ratio (FAR) was calculated from the following equation [35] on the basis of the fluorescence values (FL) measured FAR ¼

ðFLmdr ðFLparent

treated Þ=ðFLmdr control Þ treated Þ=ðFLparent control Þ

methoxycarbonylamides are slightly more hydrophobic than methanesulfonylamides. Within each subset the hydrophobicity of compounds with respect to the substituted derivatives increases in the following order: H– < Cl– < CF3 –. According to the calculations of log P , all the PhDs studied are hydrophobic and in water/lipid systems should intercalate into lipid bilayers. This property of the molecules studied is confirmed experimentally by calorimetric and fluorescence spectroscopic measurements. Calorimetry Calorimetric measurements revealed that all the phenothiazine derivatives studied alter the character of the main phase transition of lipids when mixed with DPPC, DMPE, or DMPG. Pretransition disappears in lipids that show this phenomenon (i.e., DPPC and DMPG) when the PhDs studied were added. The main transition peaks are broadened (data not shown) and their maxima shifted toward lower temperatures with respect to the transition temperatures of pure lipids. The influence of PhDs on the main transition parameters: temperature (Tm ) and enthalpy (DH ) of the lipids studied is presented in Figs. 2A and B, respectively. Looking at the decrease of transition temperatures induced

:

Results and discussion Theoretical partition coefficients The theoretical octanol/water log P values of the phenothiazine derivatives studied are presented in Table 1. According to these calculations, phenothiazine Table 1 Theoretical octanol/water log P values of the phenothiazine derivatives studied Phenothiazine derivative

log P

HPhMC ClPhMC FPhMC HPhMS ClPhMS FPhMS

2.959 3.517 3.880 2.209 2.767 3.123

Fig. 2. The influence of PhMCs and PhMSs on the thermotropic properties ((A) transition temperature and (B) enthalpy) of the main phase transition of DPPC, DMPE, and DMPG. The phenothiazine derivative/lipid molar ratio was 0.06 in all mixtures studied.

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by phenothiazine methoxycarbonylamides, we found that the most pronounced changes are caused by CF3 -substituted compounds. Smaller Tm changes are observed for compounds with a chlorine substitute at position 2 of the phenothiazine ring, while H-substituted phenothiazines have a lesser effect on the transition temperature. In the case of methanesulfonylamides, all PhDs influence Tm in a similar way, while Cl- and CF3 substituted compounds cause a dramatic drop in Tm only in DMPE model membranes. Analysis of the effects exerted by phenothiazine derivatives on the transition enthalpy of all the lipids studied does not clearly indicate which are the most effective derivatives. Individually, the greatest decrease of DH is induced by FPhMC in DPPC bilayers. All other PhDs, except for FPhMC, alter the transition enthalpy of charged DMPG to a greater extent than other lipids. In the case of DMPE, the influence of the compounds studied on DH is small or even negligible. The broadening of transition peaks described above, together with a decrease in transition temperature accompanied by small (or almost non-existent) changes of enthalpy, resembles our previous results obtained in a study of trifluoperazine/lipid mixtures [24] and by other authors on the subject of chlorpromazine/lipid interactions [23,27]. Thus, we may simply conclude that, like other phenothiazine derivatives, PhMCs and PhMSs intercalate into lipid bilayers and presumably are located close to the polar/apolar interface. Fluorescence spectroscopy The addition of all the phenothiazine derivatives studied to phosphatidylserine liposomes causes a strong quenching of NPN fluorescence. H- and Cl-substituted compounds from both groups quench NPN fluorescence to a similar extent. The ratios of the maximal intensities of NPN fluorescence spectra measured in the absence (F0 ) and in the presence of 100 lM of the modifier (F) are as follows: F0 =F ¼ 13:75 and 15.02 for ClPhMC and HPhMC; F0 =F ¼ 8:54 and 6.40 for ClPhMS and HPhMS, respectively. However, FPhMS (F0 =F ¼ 68:73) is twice as strong a quencher than FPhMC (F0 =F ¼ 24:74). Within each group of PhDs the most lipophilic trifluoromethyl-substituted derivative is the strongest quencher, the Cl-substituted compound acts more weakly and the H–PhD is the weakest membrane perturbant. In the case of PhMSs, the effect exerted by trifluoromethylsubstituted phenothiazine is almost twice as strong as the effect of the H-substituted derivative. Additionally, the maximum NPN fluorescence emission is slightly redshifted in the presence of PhDs when compared with the fluorescence maximum in pure PS liposomes (kmax ¼ 411:2 nm). As before, the strongest effect (ca. 5 nm) is observed for CF3 -substituted compounds.

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The fluorescence polarization of DPH in PS liposomes is increased in the presence of all the PhDs studied. Both PhMCs and PhMSs influence DPH polarization in the same way and to a similar extent. The increase in phenothiazine derivative-induced DPH polarization is concentration dependent up to a PhD concentration of 50 lM. At greater concentrations the degree of DPH polarization remains constant and is ca. 50–60% higher than DPH polarization measured in PS liposomes without PhD. The role of the substituent at position 2 of the phenothiazine ring is less pronounced in the case of the change in DPH polarization than for NPN fluorescence quenching. For both groups of phenothiazine derivatives CF3 -substituted compounds are the most active membrane perturbing agents, which can be observed especially at lower concentrations of PhDs (i.e., before ‘‘saturation’’ point). At a concentration of 25 lM of the CF3 -substituted phenothiazine derivatives, the degree of DPH polarization reaches about 130– 140% of the control values, while for other PhDs the degrees of polarization are below 120%. At high concentrations of PhDs, the DPH polarization values obtained for H-, Cl-, and CF3 -substituted compounds converge. The localization of phenothiazine molecules in membranes proposed on the basis of calorimetric experiments is also supported by fluorescence spectroscopy. DPH molecules are located in membranes within the hydrophobic region of lipid hydrocarbon chains [36]. The phenothiazine derivative-induced increase in DPH fluorescence polarization suggests that the mobility of fluorescent probe molecules is reduced, i.e., PhDs partially immobilize the hydrocarbon chains of lipids. On the other hand, NPN molecules are located close to the polar/apolar interface of the lipid bilayer [37] and changes in their spectral properties reflect any alterations in this region of the membrane. The NPN fluorescence quenching observed, together with a slight red-shift of its maximum emission, may result from the increase in the polarity of the NPN environment. This effect might arise either from the displacement of the fluorescent probe from its previous location or from the loosening of the bilayer structure and subsequent increase of water intercalation into membranes caused by phenothiazine derivatives. Flow cytometry Flow cytometric experiments clearly show that the multidrug resistant lymphoma cell line L5178 MDR in mice accumulates less fluorescent dye DiOC2 (3) (mean fluorescence value 11) than the parental cell line L5178Y (mean fluorescence value 68). Treatment of multidrug resistant cells with both PhMCs and PhMSs results in partial MDR reversal that manifests itself in increased DiOC2 ð3Þ accumulation by treated cells in comparison

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Fig. 3. MDR reversal induced by PhDs in multidrug resistant lymphoma cells of mice, expressed as fluorescence activity ratio (FAR). The phenothiazine derivative concentration was 4 lg/ml.

to the control (untreated MDR cells). The fluorescence activity ratio (FAR) values obtained for PhMCs and PhMSs at a concentration of 4 lg/ml are shown in Fig. 3. The results presented above show that all the phenothiazine derivatives studied are effective MDR modulators in cancer cells overexpressing P-glycoprotein. PhMSs are slightly more active than PhMCs. In all cases anti-MDR activity increases in the following order: H– < Cl– < CF3 – substituted derivatives. This is in agreement with the increasing lipophilicity of a compound. These results show that in the presence of PhMCs and PhMSs resistant cells accumulate more DiOC2 ð3Þ than untreated resistant cells, i.e., P-gp transport activity is partially blocked by all the phenothiazine derivatives studied.

usually they are considered to be specific P-gp inhibitors. Direct binding of trifluoperazine to P-gp has recently been demonstrated by fluorescence quenching of both an external MIANS probe [38] and intrinsic P-gp tryptophan fluorescence [39]. The results presented here suggest that the indirect inhibition of P-glycoprotein activity by affecting the lipid matrix of the protein surrounding the membrane should also be taken into account in the case of PhMCs and PhMSs. The hydrophobicity of an individual PhD is certainly not the only determinant of its activity. Studying PhMCs and PhMSs, we observe a similar potency in both groups for affecting model membranes and MDR reversal in the lymphoma cells of mice, in spite of PhMCs being more hydrophobic than PhMSs. The lipophilicity of the compound seems to determine its activity only with respect to the group of phenothiazine derivatives it belongs to. Other factors, such as the spatial arrangement of polar and hydrophobic groups in a PhD molecule, should be considered when trying to relate structural features to biological activity. In general, the results obtained are in agreement with those of other studies on the relationship between structure and activity carried out for phenothiazines [25,26,28].

Acknowledgments Our research was supported by the State Committee for Scientific Research (KBN): Grant No. 6 P05A 01221. Olga Wesołowska was additionally supported by KBN Grant No. 3 P04A 05322. Olga Wesołowska is grateful to the Foundation for Polish Science for the scholarship.

Factors governing the level of MDR reversal activity of phenothiazine derivatives References Looking at the effects of individual PhDs, we have found that CF3 -substituted compounds are the most effective in perturbing the bilayer structure as recorded by the changes in NPN fluorescence, DPH polarization, and enthalpy of the main phase transition of lipids. CF3 substituted PhDs are also the most effective P-glycoprotein blockers. The only exception from this pattern is the PhD-induced decrease in the main phase transition temperatures of phospholipids. This transition parameter of all the phospholipids studied is most strongly affected by chlorine-substituted derivatives. The results of the present study indicate that the hydrophobicity of a given compound is correlated with both its effects on membranes and anti-MDR activity for both PhMCs and PhMSs. It was expected that the most lipophilic compounds would be the best membrane perturbants. The dependence of the potency of MDR reversal of PhDs on their hydrophobicity was, however, less expected. The exact molecular mechanism of the anti-MDR action of phenothiazines is unknown, but

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