The effect of some antineoplastic agents on glutathione S -transferase...

711

Introduction

Chemotherapy is a method applied to the treatment of cancer targeting to kill the tumor cells by the agents called as antineoplastics. Many of those agents inhibit the growth and proliferations of malignant cells by cyto-toxic effects and lead to their death. However, the drug reduced the sensitivity of tumor cells, in other words, the development of resistance to the drug is an important factor limiting the therapeutic efficacy of an antineoplas-tic agent. However, the development of tumor resistance, such as some types of cancer can be spontaneous (natu-ral, or primary resistance), may also develop after che-motherapy (acquired or secondary resistance)1,2.

The development of resistance to antineoplastic agents were associated with increasing reduced glutathi-one (GSH) and glutathione S-transferase (GST) levels in cells and changes in permeability to the drug. Although the GSH concentration is 0.03–3 mM in plasma, it has reached up to 10 mM in the tumor cells. This increase in concentration has attracted the attention of researchers

to the enzymes of glutathione reductase (GR) which pro-duces GSH as a product and GST that consume GSH as a substrate3,4.

Glutathione S-transferases (GSTs, EC 2.5.1.18) are a family of multifunctional enzymes that involve in the detoxification processes through several different mechanisms. These enzymes can catalyze the conjuga-tion of endogenous and exogenous electrophilic xeno-biotics to reduced glutathione (GSH), and remove toxic compounds from circulation through covalent and non-covalent binding5,6.

Mammalian cytosolic GSTs have been divided into alfa (α), pi (π), mü (µ), sigma (σ), teta (θ), zeta (ζ), and omega (ω) classes on the basis of primary structure, substrate specificity and immunochemical properties, and further new classes of GSTs are still being discovered6–10.

It is indicated that GSTP1-1, the most ubiquitous and prevalent of the GST isozymes is secreted heavily in many different types of human tumors such as lung, breast, colon, kidney, ovary, esophagus, and stomach7,11–14. By

ReseaRch aRTIcLe

The effect of some antineoplastic agents on glutathione S-transferase from human erythrocytes

Mustafa Erat1 and Halis Şakiroğlu2

1Atatürk University, Erzurum Vocational College, Chemistry and Chemical Processing Technologies, Erzurum-Turkey and 2Atatürk University, Science Faculty, Department of Chemistry, Erzurum-Turkey

abstractGlutathione S-transferase was purified from human erythrocytes and effects of some antineoplastic agents were investigated on the enzyme activity. The purification procedure was composed of Glutathione-Agarose affinity chromatography after preparation of erythrocytes hemolysate. Using this procedure, the enzyme, having the specific activity of 16.00 EU/mg proteins, was purified 1143-fold with a yield of 80%. The purified enzyme showed a single band on the SDS-PAGE. The effects of paclitaxel, cyclophosphamide, and gemcitabine, are antineoplastic agents, were examined on the in vitro enzyme activity of glutathione S-transferase and were determined to be inhibitors for the enzyme. IC50 values were 0.23 mM for paclitaxel, 5.57 mm for cyclophosphamide, and 6.35 mM for gemcitabine. These constants were 0.182 ± 0.028 mM and 0.162 ± 0.062 mM for paclitaxel, 6.97 ± 0.49 mM and 10.50 ± 5.43 mM for cyclophosphamide, and 6.71 mM and 7.93 mM for gemcitabine, with GSH and CDNB substrates, respectively. Inhibition types of all inhibitors were noncompetitive.

Keywords: Glutathione S-transferase, inhibition, paclitaxel, cyclophosphamide, gemcitabine

Address for Correspondence: Mustafa Erat, Atatürk University, Erzurum Vocational College, Chemistry and Chemical Processing Technologies, 25240 Erzurum-Turkey. Phone: +90 442 2311966, Fax: +90 442 2360948. E-mail: [email protected]

(Received 06 February 2012; revised 12 March 2012; accepted 12 March 2012)

Journal of Enzyme Inhibition and Medicinal Chemistry, 2013; 28(4): 711–716© 2013 Informa UK, Ltd.ISSN 1475-6366 print/ISSN 1475-6374 onlineDOI: 10.3109/14756366.2012.677837

Journal of Enzyme Inhibition and Medicinal Chemistry

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© 2013 Informa UK, Ltd.

10.3109/14756366.2012.677837

GENZ

677837

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712 M. Erat and H. Şakiroğlu


accelerating metabolism of many drugs (adriamycin, chlorambucil, melphalan and other nitrogen mustards) metabolized in this system and used in chemotherapy treatment of increased GSH/GST levels, it is shown that the point targeted with drug has not been reached, in other words, it has caused the development of resistance to the drug15,16. For this reason, in regulating the efficiency of conventional electrophilic cancer drugs in chemotherapy, It came to mind that the use of GST inhibitors may be use-ful. Fort this purpose, various compounds were developed and were tested both experimentally and in clinic17–19.

The purpose of this study is to determine GST inhibi-tors of antineoplastic drugs used available, especially determining those which are GSTP inhibitors, and to find out the inhibition types of the agents determined to be inhibitors. If the results obtained from this in vitro study are supported by in vivo studies, of all antineoplas-tic agents used in the chemotherapy of cancer patients, inhibitors of the enzyme may be predicted to be preferred to the others.

Materials and methods

MaterialGlutathione-Agarose, Sephadex G-100, GSH, 1-chloro-2,4-dinitrobenzene (CDNB), other chemicals used as inhibitors, protein assay reagents, and chemicals for electrophoresis were obtained from Sigma Chem. Co. All other chemicals used were analytical grade and obtained from either Sigma-Aldrich or Merck.

Purification of the enzymeFresh human blood was collected into centrifuge tube with 0.1 M Na-sitrate, 0.16 M glucose, 0.016 M Na-phosphate, 2.59 mM adenine for anticoagulation. It was centrifuged at 2500g for 15 min and the plasma and leukocyte coat were removed. The erythrocytes were washed three times with 0.16 M KCl solution including 1 mM EDTA, the sample was centrifuged at 2500g each time and supernatant was removed. The washed erythrocytes were hemolysed with 5 volumes of ice-cold distilled water containing 2.7 mM EDTA and 0.7 mM β-mercaptoethanol and centrifuged at 4°C, 20,000g for 30 min to remove residual intact cells and membranes20. Purification of the enzyme was per-formed on Glutathione-Agarose affinity gel. For this aim, the gel lyophilized powder was incubated in deionized water a night and packed in a column. After precipitation of the gel, it was equilibrated 10 mM K-phosphate buffer including 0.15 M NaCl, pH 7.4, (equilibration buffer) by means of a peristaltic pump. The flow rate of the column was adjusted as 15 mL/h. The prepared hemolysate was loaded onto the column and washed with equilibration buffer until the final absorbance difference became 0.05 at 280 nm. The enzyme was eluted successively with a gradient of 0 to 10 mM GSH in 50 mM Tris-HCl, pH 9.5, buffer. Active fractions were collected and dialyzed with the equilibration buffer. All of the experiments were per-formed at 4°C21,22.

Activity assayGST activity was determined as described by Habig et al.23 The reaction medium contained 0.1 M potassium phos-phate buffer pH 6.5, 1.0 mM GSH, 1.0 mM CDNB, and 1% absolute ethanol in a total volume of 1.0 mL. The reac-tion was monitored by increase in A

340 with a Beckman

Spectrophotometer (DU 730). All reactions were initi-ated by the addition of the enzyme solution. One unit of activity is defined as the formation of 1.0 μmol product min−1 (extinction coefficient at 340 is 9.6 mM−1 cm−1 for GSDNB).

Protein determinationQuantitative protein determination was measured spec-trophotometrically at 595 nm according to the method of Bradford, with bovine serum albumin as a standard24.

Sodium dodecil sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)To check the purity of the enzyme, SDS-PAGE was per-formed by Laemmli’s procedure25. E. coli β-galactosidase (116 kDa), rabbit phosporylase B (97.4 kDa), bovine albumin (66 kDa), chicken ovalbumin (45 kDa), and bovine carbonic anhydrase (29 kDa) were used as stan-dards (Sigma: MW-SDS–200). The acrylamide concentra-tion of the stacking and the separating gels were 3% and 10%, respectively, and 1% SDS was also added to the gel solution. The gel was stabilized in a solution containing 50% propanol + 10% TCA + 40% distilled water for 30 min. Staining was made for about 2 h in a solution of 0.1% Coomassie Brillant Blue R-250 + 50% methanol + 10% acetic acid + 39.9% distilled water. The gel was washed with several changes of the same solvent without dye until protein bands were cleared.

In vitro inhibition studiesIn order to determine the effects of the antineoplastics on human erythrocytes GST, enzyme activities were assayed for paclitaxel (0.09–1.06 mM), cyclophosphamide (1.8–14.4 mM), and gemcitabin (3.17–25.36 mM) at the cuvette concentrations. An experiment in the absence of inhibitor was used as control for each antineoplastic agent. Control cuvette activity was taken as 100%. For each antineoplastic agent having inhibitory effect, an [Inhibitor]-Relative Activity graph was drawn and inhibi-tor concentrations causing 50% inhibition (IC

50) were

calculated from these graphs.For determining K

i constants (dissociation constant of

enzyme-inhibitor complex), 3 fixed inhibitor concentra-tions (0.053 mM, 0.105 mM, and 0.211 mM for paclitaxel, 3.6 mM, 7.2 mM, and 10.8 mM for cyclophosphamide, and 4.2 mM, 8.5 mM, and 12.7 mM for gemcitabin) were tested. In these experiments, both GSH and CDNB were used as substrates with their 5 different concentrations (1, 0.5, 0.25, 0.125, and 0.0625 mM of GSH and 0.8, 0.4, 0.2, 0.1, and 0.05 mM of CDNB). Three assays were performed for each data point. Analysis of data obtained was made by t-test and they were given as X ± SD. The Lineweaver-

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The effect of some antineoplastics on human GST 713


Burk graphs were obtained for each inhibitor by using 1/V and 1/[S] values26. K

i constants and inhibition types

were estimated from these graphs.

Results

Glutathione S-transferase enzyme, after preparing the haemolysate from human erithrocytes, was purified 1143-fold with a 80% recovery of the total activity hav-ing a specific activity of 16 EU/mg proteins by using glutathione-agarose affinity column. Affinity is the most specific method in chromatographic methods for mak-ing purification. As shown in Table 1, purification of GST was performed on glutathione-agarose gel in single-step. The purity of the enzyme was confirmed by SDS-PAGE (Figure 1). As highlighted in our previous report, the enzyme was thought to be isozyme GSTP owing to the substrate/inhibitor interaction properties. Inhibitory effect of three selected antineoplastic agents (paclitaxel, cyclophosphamide, and gemcitabin) was detected on the purified enzyme. [I]-% Activity graphs were drawn by measuring the enzyme activities in different inhibi-tor concentrations. IC

50 values was calculated as 0.23

mm for paclitacsel, 5.57 mM for cyclophosphamide, and 6.35 mM for gemcitabin with the help of these equations from graphics (Tables 2 and 3). To explain the effect of inhibition mechanisms of drug having inhibitory effects, their inhibition types and K

i constants were determined.

For this purpose, the three reasonable fixed inhibitor concentrations were detected and activities were mea-sured in each fixed inhibitor concentration by decreas-ing substrate concentrations. Lineveawer-Burk graphs were drawn using the data obtained. K

i constants were

calculated using the equations of this graphs. When GSH was used as the variable substrate, the mean K

i

constants of 0182 ± 0028 mM for paclitacsel, 6.97 ± 0.49 mM for cyclophosphamide, and the 6.71 ± 0.31 mM for gemcitabin were found. When the variable substrate was

the CDNB, the mean Ki constants were 0162 ± 0068 mM

for paclitacsel, 10.50 ± 5.49 mM for cyclophosphamide, and 7.93 ± 0.56 mM for gemcitabin. The data obtained from the studies performed with two substrates showed that all of the three antineoplastics were noncompetitive inhibitors.

Discussion

GSH and its related enzymes as an effective detoxifica-tion system that play a pivotal role in the protection of

Figure 1. SDS-PAGE of pure human erythrocytes GST. Lane 1, Molecular mass standarts; lane 2, The enzyme purified from human erythrocytes by Glutathione-Agarose affinity column.

Table 1. Purification of glutathione S-transferase from human erythrocytes.

Purification stepTotal

volume (mL)Activity

(EU/mL)Total

activity (EU)Protein

(µg/mL)Total

protein (µg)Specific activity

(EU/ µg) Yield (%)Purification

foldHemolysate 60 0.08 4.80 5.88 352.80 0.014 100 1Affinity chromatography

8 0.48 3.84 0.03 0.24 16.000 80 1143

Table 2. IC50

, Ki values and inhibition types for paclitaxel, cyclophosphamide, and gemcitabine toward human erythrocyte GST in fixed

CDNB.

Inhibitors IC50

values (mM) Ki constants (mM) Avarage K

i constants (mM) Inhibition Type

Paclitaxel 0.23 0.185 0.182 ± 0.028 Non-competitive0.1530.208

Cyclophosphamide 5.57 7.23 6.97 ± 0.49 Non-competitive7.286.41

Gemcitabine 6.35 6.40 6.71 ± 0.31 Non-competitive6.727.02

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cells against damage are induced by free radicals and are hypothesized to influence the response to adjuvant can-cer therapies, including irradiation and chemotherapy4,27. Of the GSH-related enzymes, GSTs are important cellular drug metabolizing enzymes, catalyzing conjugation of some electrophiles to the tripeptide glutathione with the formation of compounds that are generally less reactive, but effective in resistance to anticancer agents15.

The GSH/GST detoxification system appears to be important for the development of a drug-resistant phe-notype of tumor cells to water-soluble CENUs, platinum compounds and a number of CENU-different alkylating compounds, including melphalan, cyclophosphamide, chlorambucil, nitrogen mustard and doxorubicin15,28 and, therefore, might be a pivotal factor in the known clinical nonresponsiveness of a subset of brain tumors to these compounds29.

The over-expression of GST in tumors should in prin-ciple be linked to enhanced detoxification of akylating agents, and therefore be responsible for the development of resistance30,31. In fact, many acquired akylating agents and cisplatin resistant tumor cells have a high GSH con-centration and/or an increase in the activity of enzymes such as GST4. In particular the isoform GSTP has been associated with early stages of human carcinogenesis and there is an expanding body of evidence that overex-pression of this isoenzyme plays an important role in the malignant cellular transformation of various tissues32.

GST inhibitors are emerging as promising thera-peautic agents for managing the development of resis-tance amongst anticancer agents. For this aim, various compound groups have been determined targeting this system as GST inhibitors and investigated its effects experimentally and clinically17,18. These groups are combined with ethacrynic acid and its derivatives, glu-tathione analogues, plant polyphenolic compounds, bifunctional inhibitors, antimalarial drugs, tocopherols, haloenol lactones, 7-nitro-2,1,3-benzoksadiazole deriva-tives, and prodrugs activated by GST such as, TLK 286, purine analogues, and nitric oxide donors1.

The importance of identified GST inhibitor stems from the possible use of it with antineoplastic agents to eliminate the resistance caused by GST activity. Because of the fact that GSTs play a pivotal role in the detoxification system, inhibition of their all isozymes

is not desirable. Isoenzyme selective GST inhibitors may therefore be used to improve drug response and decrease resistance33.

Ethacrynic acids the first compound studied as an inhibitor of the GST. This compound and its deriva-tives were not used as antineoplastics because of hav-ing a diuretic effect and not being isosim specificity34. Glutathione analogues are another most studied group. The most important of these compounds is γ-glutamyl-S-benzylcysteinyl-phenylglycyl diethylester (TLK 199). It was reported that the compound transforms to its active form after entering into the cell hydrolyzed by intrac-ellular esterases and its new form (TLK 117) inhibits GSTP1-135 and that it increased the efficiency of alkylat-ing agent in many cell lines secreting this enzyme more. Glutathione conjugates, despite being good GST inhibi-tors, cannot be used in vivo, because they are eliminated rapidly. Some flavonoids, such as galangin, kaempferol, eriodictyol and quercetin have been reported to inhibit the activity of GSTP1-116. Of all flavonoids, 25 µM concen-tration of galanginin was observed to inhibit the activity of in almost all cellular GSTP1-136.

Lyon and colleagues37, benefited from quaternary structure of the proteins for the design of more isozyme selective inhibitors of GST. They synthesized sym-metrical bifunctional compounds by changing the binding groups that can interact with each active site of GST monomer. In addition, it has been shown that GSTP was inhibited by antimalarial substances, such as pyrimethamine, artemisinin, quinine, quinidine, and tetracycline, and IC

50 values for these compounds

were 1, 2, 4, 1 and 13 mM, respectively1. GSTP activity has been identified to be inhibited noncompetitively by some tocopherol compounds and to have low IC

50 val-

ues. Researches show that R,R,R-α-tocopherol may be precursor for the development of new GST inhibitors38. A group of compounds having haloenol lactone struc-ture were indicated as mechanism-based inhibitors of the GSTs. It was reported that this group of compounds are compounds like substrates transforming into reac-tive electrophills inactivating the enzyme by binding covalently to essential aminoacids through the normal catalytic mechanism of the enzyme39. New inhibitor compounds, able to pass through the cell membrane due to their lipophilic properties and effectively connected

Table 3. IC50

, Ki values and inhibition types for paclitaxel, cyclophosphamide, and gemcitabine toward human erythrocyte GST in fixed

GSH.

Inhibitors IC50

values (mM) Ki constants (mM) Avarage K

i constants (mM) Inhibition type

Paclitaxel 0.23 0.238 0.162 ± 0.068 Non-competitive0.1430.106

Cyclophosphamide 5.57 16.24 10.50 ± 5.43 Non-competitive9.835.43

Gemcitabine 6.35 7.32 7.93 ± 0.56 Non-competitive8.048.42

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The effect of some antineoplastics on human GST 715


with GSTs, were designed. These compounds are not similar to GSH. For this purpose, a group of compounds, having the sutructure of 7-nitro-2,1,3-benzoxadiazole derivatives have been synthesized40. Of the synthesized compounds, 6-(7-Nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol has shown to be the most effective inhibitor for GSTP1-1 with an IC

50 value of 0.8 μM.

An antineoplastic agent, which is also a GSTP inhibitor, may have the advantage of removing the drug resistance without using an additional GSTP inhibitor. In this work, the effects of some antineoplastics on GSTP were investi-gated. The enzyme was purified from human erythrocytes by the procedure mentioned in our previous report41. Of all the tested antineoplastic agents, paclitacsel, gemcit-abine, and cyclophosphamide were determined to be noncompetitive inhibitors of the enzyme.

Paclitaxel is from taxenes that are widely used in clini-cal application in the treatment of cancer chemotherapy especially ovarian, cervical, endometrial, breast, gastric, and non-small-cell lung cancers. Paclitaxel interfere with spindle microtubuledynamics causing cell cycle arrest and apoptosis. However, it is likely that some patients do not benefit from the treatment due to the chemoresistance mechanisms42. Gemcitabine is a deoxy-cytidine analog and is metabolized in tumor cells by deoxycytidine kinase (dCK) to yield active gemcitabine diphosphate (dFdCDP) and gemcitabine triphosphate (dFdCTP). These gemcitabine nucleosides inhibit DNA synthesis in tumor cells43. In recent years, gemcitabine and its combination with other anticancer drugs have become popular regimens for the treatment of cholang-iocarcinoma44. Cyclophosphamide is an akylating agent. Akylating agents currently used in cancer chemotherapy are highly reactive small molecules that bind covalently to electron-rich nucleophilic moieties. This binding to cellular DNA is believed to explain these drugs’ cytotoxic effects27.

If the influence of these drugs studied in vitro in this report are supported by in vivo studies, these drugs can be used preferably in cancer chemotherapy rather than those which are not GST inhibitors.

acknowledgments

This research was supported by Atatürk University Scientific Research Projects Institution (BAP-2008/259).

Declaration of interest

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The effect of some antineoplastic agents on glutathione S -transferase...

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