IL FARMACO 59 (2004) 679–684 www.elsevier.com/locate/farmac
Acetylsalicylic acid as a potential regulator of prolidase-convertible pro-drugs in control and neoplastic cells Krzysztof Chrzanowski a, Anna Bielawska a, Krzysztof Bielawski a, Jerzy Pałka a,*, Sławomir Wołczyn´ski b b
a Department of Medicinal Chemistry, Medical Academy of Białystok, Kilinskiego 1, 15 230 Białystok, Poland Department of Gynaecological Endocrinology, Medical Academy of Białystok, Skłodowskiej 24 A, 15 276 Białystok, Poland
Received 14 February 2004; accepted 25 April 2004 Available online 15 June 2004
Abstract Proline analogue of melphalan (Mel-pro) is one of the pro-drugs activated by prolidase, cytoplasmic imidodipeptidase highly expressed in some neoplastic tissues. In order to limit the action of prolidase on the pro-drug in normal cells, prolidase inhibitor, acetylsalicylic acid (ASA), was tested in fibroblasts (showing average prolidase activity for normal cells) and in MDA-MB 231 breast cancer cells (showing elevated activity of the enzyme). The effect of Mel-pro in the presence and absence of ASA on prolidase activity (colorimetric assay), DNA biosynthesis (3H-thymidine incorporation assay), cytotoxicity (tetrazoline assay) and ability to penetrate cell membrane (thin layer chromatography) in both type of cells was measured. It has been found that 5 mM ASA significantly decreased conversion of Mel-pro to Mel in cultured fibroblasts as well as it decreased cytotoxicity and the effect of this drug on DNA synthesis. In contrast, 5 mM ASA had relatively lower effect on the conversion of Mel-pro into Mel in MDA-MB 231 cells as well it had little effect on Mel-pro-induced inhibition of DNA synthesis and cell death. It suggests that ASA may serve as an inhibitor of prolidase-convertible pro-drugs in normal cells. © 2004 Elsevier SAS. All rights reserved. Keywords: Pro-drug; Proline analogue of melphalan; Prolidase; Acetylsalicylic acid; Fibroblast; Breast cancer cells
1. Introduction
prolidase [7] as well for prolidase from normal [8] and neoplastic cells [9,10].
Melphalan (Mel) belongs to the class of antitumor agents with an alkylating and cross-linking action on guanine and possibly other bases of deoxyribonucleic acid that result in arresting cell division [1]. It has the wide spectrum of antineoplastic activity, however its action is accompanied by a wide variety of untoward side effects [1–3]. In order to minimize its side effects, efforts were undertaken to construct pro-drug (the drug was conjugated with proline through N-methyl-carbonyl connector forming imino-bond) [4–6]. Our previous results have shown that proline analogue of melphalan (Mel-pro), N-[[[[(S)-carboxy]pyrrolidin1yl]carbonyl]methyl]-4-[bis(2-chloroethyl)amino]-2phenylalanine dilithium salt (Fig. 1), conjugated through imidobond may serve as a substrate for purified pig kidney
* Corresponding author. E-mail address:
[email protected] (J. Pałka). © 2004 Elsevier SAS. All rights reserved. doi:10.1016/j.farmac.2004.04.008
Fig. 1. The chemical structure of Mel-pro. An arrow indicates imido-bond susceptible to the action of prolidase.
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Prolidase [E.C.3.4.13.9] is a cytosolic exopeptidase that cleaves imidodipeptides with C-terminal proline [11,12]. The biological function of the enzyme involves the metabolism of proline-containing protein degradation products and the recycling of proline from imidodipeptides for prolinecontaining protein resynthesis, mainly collagen [13,14]. The presence of prolidase in cytoplasm allows to suspect that it may be targeted as a Mel-pro-converting enzyme. This strategy should be of benefit particularly in case of anti-neoplastic pro-drug, since at least some tumour tissues evoke increased prolidase activity compared to normal tissues [15,16]. In such a case the release of the drug from the pro-drug would be more efficient in neoplastic tissues than in normal tissues. In order to limit the action of prolidase on the pro-drug in normal cells, prolidase inhibitor, acetylsalicylic acid (ASA) [17], was tested in fibroblasts showing medium prolidase activity for normal human cells and in MDA-MB 231 breast cancer cells showing elevated activity of the enzyme [10]. The specific objective of present studies was to examine the effect of ASA on susceptibility of Mel-pro to the action of prolidase, the ability of Mel-pro to penetrate cell membrane, cytotoxicity and DNA biosynthesis in fibroblasts and MDA-MB 231 cells. 2. Experimental 2.1. Materials ASA was the product of Fluka Chemie AG (Germany). Glycyl-proline (Gly-pro), trypsin, bovine serum albumin (BSA), pig kidney prolidase, 3-(4,5-dimethylthiazole-2-yl)2,5-diphenyltetrazolium bromide (MTT), Dulbecco’s minimal essential medium (DMEM) and foetal bovine serum (FBS) used in cell culture were products of Gibco (USA). Glutamine, penicillin and streptomycin were obtained from Quality Biological Inc. (USA). [3H]-thymidine (6.7 Ci/mmol) was the product of NEN (USA). 2.2. Fibroblast cultures Normal human skin fibroblasts were maintained in DMEM supplemented with 10% FBS, 2 mmol/l glutamine, 50 U/ml penicillin and 50 µg/ml streptomycin at 37 °C in a 5% CO2 in an incubator. The cells were used between 12th and 14th passages. The fibroblasts were subcultivated by trypsinization. Subconfluent cells from Costar Flasks were detached with 0.05% trypsin, 0.02% ethylenodiaminetetraacetic acid (EDTA) in the calcium-free phosphate-buffered saline (PBS). For the prolidase assay, cells were cultured in six-well plates (Costar). For these experiments cells were counted in hemocytometers and cultured at 1 × 105 cells per well in 2 ml of growth medium. Cells reached confluency at day 6 and in most cases such cells were used for the experiments. 2.3. MDA-MB 231 cell culture Breast cancer MDA-MB 231 cells were maintained in DMEM supplemented with 10% fetal FBS, 50 U/ml penicil-
lin, 50 µg/ml streptomycin at 37 °C. Cells were cultured in Costar flasks and subconfluent cells were detached with 0.05% trypsin and 0.02% EDTA in calcium-free PBS, counted in hemocytometers and plated at 5 × 105 cells per well of six-well plates (Nunc) in 2 ml of growth medium (DMEM without phenol red with 10% CPSR1). Cells reached about 80% of confluence at day 3 and in most cases such cells were used for the assays. 2.4. Determination of prolidase activity The activity of prolidase was determined according to the method of Myara et al. [18], which is based on the measurement of proline by Chinard’s reagent [19]. Briefly, the monolayer was washed three times with 0.15 mol/l NaCl. Cells were collected by scraping and suspended in 0.15 mol/l NaCl, centrifuged at low speed (200 g) and the supernatant was discarded. The cell pellet (from six-wells) was suspended in 0.3 ml of 0.05 mol/l Tris–HCl, pH 7.8, and sonicated three times for 10 s at 0 °C. The samples were then centrifuged (18,000 g, 30 min) at 4 °C. Supernatant was used for protein determination and then prolidase activity assay. The activation of prolidase requires preincubation with manganese, therefore 0.1 ml of the supernatant was incubated with 0.1 ml of 0.05 mol/l Tris–HCl, pH 7.8, containing 2 mmol/l MnCl2 for 2 h at 37 °C. After the preincubation, the prolidase reaction was initiated by adding 0.1 ml of the preincubated mixture to 0.1 ml of 94 mmol/l Gly-pro to a final concentration of 47 mmol/l. After the additional incubation for 1 h at 37 °C, the reaction was terminated with 1 ml of 0.45 mol/l trichloroacetic acid. In the parallel tubes reaction was terminated at time “zero” (without incubation). The released proline was determined by adding 0.5 ml of the trichloroacetic acid supernatant to 2 ml of a 1:1 mixture of glacial acetic acid/Chinard’s reagent (25 g of ninhydrin dissolved at 70 °C in 600 ml of glacial acetic acid and 400 ml of 6 mol/l orthophosphoric acid) and incubated for 10 min at 90 °C. The amount of proline released was determined colorimetrically by reading an absorbance at 515 nm and calculated by using the proline standards. Protein concentration was measured by the method of Lowry et al. [20]. Enzyme activity was calculated as nanomoles of released proline per minute per milligram of supernatant protein. 2.5. Cell viability assay The assay was performed according to the method of Carmichael et al. [21] using 3-(4,5-di-methylthiazole-2-yl)2,5-diphenyltetrazolium bromide (MTT). The cells were cultured for 24 h with various concentrations of drugs studies in six-well plates, washed three times with PBS and then incubated for 4 h in 1 ml of MTT solution (0.5 mg/ml of PBS) at 37 °C. The medium was removed and 1 ml of 0.1 mol/l HCl in absolute isopropanol was added to the attached cells. Absorbance of converted dye in living cells was measured at a wavelength of 570 nm. Cell viability in the presence of drugs was calculated as a percent of control cells.
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2.6. Mitogenic assay To examine the effect of the studied drugs on cell proliferation, the fibroblasts were plated in 24-well tissue culture dishes at 1 × 105 cells/well and MDA-MB 231 at 1 × 106 cells/well in 1 ml of growth medium. After 48 h the cells were incubated with varying concentrations of proline analogue of Mel with or without 5 mM ASA and 0.5 µCi of [3H]thymidine for 24 h at 37 °C. Cells were rinsed three times with PBS, solubilized with 1 ml of 0.1 mol/l sodium hydroxide containing 1% SDS, scintillation fluid (9 ml) was added and radioactivity incorporation into DNA was measured in scintillation counter. 2.7. Drug accumulation in the cells Growth medium was removed from the plates and the monolayer was washed three times with 1 ml of medium. For accumulation studies, 50 µl of the drug (40 mmol/l) was added in dimethyl sulphoxide (1%, final concentration) to 1 ml of fresh medium with or without 5 mM ASA and the cells were incubated for 4 h. After that, the medium and cells were separated by centrifugation (200 g, 5 min). The medium was evaporated to dryness in a vacuum and the residue was dissolved in 0.5 ml of methanol. The cells were washed three times with fresh medium, suspended in 0.5 ml of methanol, sonicated and centrifuged at 16,000 g for 10 min. The respective samples were submitted to thin layer chromatography on DC-Alufolien Kieselgel 60 F254 (0.2 mm) in methanol. The chromatograms were analysed at UV (254 nm).
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in MDA-MB 231 cells (Fig. 3B) it inhibited the enzyme activity by about 25% only. As can be seen from Fig. 4 Mel-pro shows similar susceptibility to the action of prolidase from fibroblasts (Fig. 4A, lane 3) and MDA-MB 231 cells (Fig. 4B, lane 3), compared to standard prolidase substrate—glycyl-L-proline (Gly-pro) and about fivefold higher susceptibility, compared to another its substrate—glycyl-L-hydroxyproline (Gly-hyp). In the presence of 5 mM ASA, prolidase from fibroblasts (Fig. 4A, lane 4) loses ability to convert Mel-pro by about fivefold while prolidase from MDA-MB 231 cells (Fig. 4B, lane 4) by about twofold, only. We have compared the transport of Mel-pro (in the absence or presence of 5 mM ASA into fibroblast (Fig. 5A) and MDA-MB 231 cells (Fig. 5B). The cells were cultured for 4 h in the presence of 2 mM Mel-pro with or without 5 mM ASA
Fig. 2. Prolidase activity in human skin fibroblasts and oestrogenindependent breast cancer MDA-MB 231 cells.
2.8. Statistical analysis In all experiments, the mean values for six independent experiments ± standard deviation (S.D.) were calculated, unless otherwise indicated.
3. Results Preparation of the Mel-pro, N-[[[[(S)-carboxy]pyrrolidin1yl]carbonyl]methyl]-4-[bis(2-chloroethyl)amino]-2phenylalanine dilithium salt was satisfactorily achieved by standard chemical transformations according to the method described previously [7]. The study on the effect of ASA on prolidase-dependent conversion of Mel-pro into Mel was performed in human skin fibroblasts and breast cancer MDA-MB 231 cells, based on the differences in their prolidase activity. In contrast to fibroblasts, MDA-MB 231 cells express almost threefold higher prolidase activity (Fig. 2). Experiment presented on Fig. 3 shows the effect of ASA on prolidase activity in human skin fibroblasts and breast cancer MDA-MB 231 cells cultured for 24 h. It has been found that ASA used at 5 mM concentration inhibited prolidase activity in fibroblasts by about 55% (Fig. 3A); however,
Fig. 3. Prolidase activity in fibroblasts (A) and breast cancer MDA-MB 231 cells (B) incubated for 24 h in the presence of different concentrations of ASA. Mean values ± S.D. from three independent experiments done in duplicates are presented.
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Fig. 4. Susceptibility of Mel-Pro, Gly-pro, Gly-hyp and Mel-pro in the presence of 5 mM ASA to the action of prolidase from fibroblasts (A) and MDA-MB 231 cell (B) homogenate. The susceptibility of Gly-pro to the action of prolidase was considered as 100%. Mean values ± S.D. from six assays are presented.
and after that time the presence of the drug in the medium and the cells was analysed by thin layer chromatography (Fig. 5). The result shows that 5 mM ASA do not affect ability of Mel-pro to penetrate the cell membrane in fibroblasts (Fig. 5A, lane 4) and MDA-MB 231 cells (Fig. 5B, lane 4). However, it was found that ASA completely inhibited conversion of Mel-pro in fibroblasts (Fig. 5, lane 6) and limited this process in MDA-MB 231 cells (Fig. 5, lane 6). ASA abolishes the ability of Mel-pro to inhibit DNA synthesis in fibroblasts (Fig. 6A) and significantly reduces this ability in MDA-MB 231 cells (Fig. 6B). In fibroblasts IC50 of Mel-pro for DNA synthesis (measured by thymidine incorporation assay) was found at about 25 µM, while in the presence of 5 mM ASA, Mel-pro used at this concentration does not produce significant inhibition of DNA synthesis. In breast cancer MDA-MB 231 cells IC50 of Mel-pro for DNA synthesis was found at about 12 µM, while in the presence of 5 mM ASA the pro-drug used at this concentration evoked decreased the potency to inhibit DNA synthesis by about 20%. Mel-pro in the absence of ASA evokes higher cytotoxicity than in the presence of 5 mM ASA during 24 h of incubation in fibroblasts (Table 1) and breast cancer MDA-MB 231 cells (Table 2). In fibroblast 5 mM ASA significantly reduced cytotoxicity of Mel-pro. At 25 µM Mel-pro produced reduction in cell viability by about 40% and in the presence of 5 mM ASA by about 10% only, compared to controls (Table 1). In contrast, in MDA-MB 231 cells 5 mM ASA
Fig. 5. Thin layer chromatography. Transport of Mel-pro into fibroblasts (A) and breast cancer MDA-MB 231 cells (B) during 4-h incubation with 2 mM concentrations of the drug in the absence or presence of 5 mM ASA: (1) growth medium, (2) methanol extract of control cells, (3) growth medium from cells treated with Mel-pro, (4) methanol extract of cells treated with Mel-pro, (5) growth medium from cells treated with Mel-pro + 5 mM ASA, (6) methanol extract of cells treated with Mel-pro + 5 mM ASA, (7) Mel-pro standard, (8) N-methyl-carbonyl-melphalan and (9) ASA.
slightly reduced cytotoxicity of Mel-pro. At 25 µM concentration Mel-pro produced about 55% while in the presence of 5 mM ASA about 42% reduction in cell viability, compared to controls (Table 2). 4. Discussion The N-acylproline linkage is unique in peptides in that it involves a tertiary amide. Most proteases cannot cleave that bond except specific, cytosolic imidodipeptidase, prolidase [7]. Cytosolic location of this imidodipeptidase suggests that it may serve as a pro-drug-converting enzyme. It may have importance particularly in case of anti-neoplastic pro-drugs, since at least in some tumour tissues the enzyme activity is several fold higher than in normal tissues [15,16]. In fact, conjugation of melphalan with proline through imido-bond resulted in formation of a good substrate for prolidase. Moreover, Mel-pro was found to be more effectively transported into the normal [8] and some neoplastic [9] cells than the free drug. Although the nature of the transport is unknown at present, it is accepted that the uptake of alkylating agents occurs by a passive transport mechanism [22,23]. It suggests that Mel-pro may serve as a prolidase-convertible pro-drug.
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MDA-MB 231 breast cancer cells (showing high activity of the enzyme). Several aspects of biological actions of the Mel-pro in the absence and presence of ASA were studied. At first we found that ASA strongly inhibited prolidase activity in fibroblasts and slightly in MDA-MB 231 cells. The presence of ASA do not affect the ability of Mel-pro to penetrate cell membrane in fibroblasts and MDA-MB 231 cells, but completely inhibited the conversion of proline analogue of melphalan in fibroblasts and limited this process in MDA-MB 231 cells. Furthermore, ASA abolished the ability of Mel-pro to inhibit DNA synthesis and significantly reduced the cytotoxicity of Mel-pro in fibroblasts. However, in breast cancer MDA-MB 231 cells ASA only slightly affected Mel-pro-dependent cytotoxicity and inhibition of DNA synthesis. The data postulate that ASA may drastically reduce Melpro-induced cytotoxicity in normal cells without significant effect on the process in neoplastic cells that evoke elevated prolidase activity. It suggests that ASA may serve as an inhibitor of prolidase-convertible pro-drugs in normal cells.
Fig. 6. DNA synthesis in fibroblasts (A) and breast cancer MDA-MB 231 cells (B) cultured for 24 h with different concentrations of Mel-pro in the absence or presence of 5 mM ASA. Mean values ± S.D. from three independent experiments done in duplicates are presented. Table 1 Viability of fibroblasts treated for 24 h with different concentrations of Mel-pro in the absence and presence of 5 mM ASA Concentration of Mel-pro (µmol/l) 0 5 10 15 20 25
Viability of cells a (% of control) Mel-pro Mel-pro + 5 mM ASA 100 100 97 ± 2 99 ± 2 83 ± 2 96 ± 2 66 ± 2 94 ± 2 60 ± 3 92 ± 3 59 ± 2 91 ± 2
a
Mean values ± S.D. from three independent experiments done in duplicates are presented. Table 2 Viability of MDA-MB 231 cells treated for 24 h with different concentrations of Mel-pro in the absence and presence of 5 mM ASA Concentration of Mel-pro (µmol/l) 0 5 10 15 20 25
Viability of cells a (% of control) Mel-pro Mel-pro + 5 mM ASA 100 100 60 ± 2 95 ± 2 55 ± 2 86 ± 2 50 ± 2 74 ± 2 47 ± 1 66 ± 3 45 ± 1 58 ± 3
a Mean values ± S.D. from three independent experiments done in duplicates are presented.
In order to limit the action of prolidase on the pro-drug in normal cells, prolidase inhibitor, ASA, was tested in normal fibroblasts (showing medium prolidase activity) and in
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