Glutathione-s-transferase modified electrodes for detecting anticancer drugs

Highlights

• Glutathione-s-transferase (GST) as a general probe for anticancer drugs.

• Both optical and electrochemical detection demonstrated.

• Many anticancer drugs are tested as substrate and target for GST.

Abstract

With the fast growth of cancer research, new analytical methods are needed to measure anticancer drugs. This is usually accomplished by using sophisticated analytical instruments. Biosensors are attractive candidates for measuring anticancer drugs, but currently few biosensors can achieve this goal. In particular, it is challenging to have a general method to monitor various types of anticancer drugs with different structures. In this work, a biosensor was developed to detect anticancer drugs by modifying carbon paste electrodes with glutathione-s-transferase (GST) enzymes. GST is widely studied in the metabolism of xenobiotics and is a major contributing factor in resistance to anticancer drugs. The measurement of anticancer drugs is based on competition between 1-chloro-2,4-dinitrobenzene (CDNB) and the drugs for the GST enzyme in the electrochemical potential at 0.1 V vs. Ag/AgCl by square wave voltammetry (SWV) or using a colorimetric method. The sensor shows a detection limit of 8.8 μM cisplatin and exhibits relatively long life time in daily measurements.

Introduction

In the past few decades, a number of important anticancer drugs have been approved for clinical use, including cisplatin, carboplatin, oxaliplatin, gemcitabine, pirarubicin and doxorubicin. The efficacy of these drugs however depends on the intrinsic and acquired resistance of patients during treatment, which is a major problem in cancer therapy (Koberle et al., 2010). Drug resistance is partly attributed to reactions catalyzed by glutathione-s-transferases (GSTs). In humans, GSTs are expressed in large amounts, counting for ~4% of the total soluble proteins in the liver, which is the main detoxification organ (Smith et al., 2006). GST is a detoxification enzyme, catalyzing glutathione (GSH) conjugation reactions. GSH is a tri-peptide that maintains a stable redox environment of cells and therefore protects cells from xenobiotic substances.

A GST has two substrate binding domains: one domain is highly selective for GSH and the other binds anticancer drugs by providing a hydrophobic environment (Stewart 2007). Since most anti-cancer drugs are quite hydrophobic, they act as good substrates for GST. At the same time, GST activates the thiol group on GSH for nucleophilic attacking drug substrates, yielding GSH adducts (see Fig. 1A for reaction with cisplatin). This type of reaction decreases the concentration of free anti-cancer drugs inside cells and thus decreases toxicity. Therefore, an increased concentration of GSH or GST might be related to drug resistance in cancer therapy (Nussbaumer et al., 2011).

Monitoring the concentration of anticancer drugs is required to optimize therapy and avoid over or under dosage. It is also critical to have convenient analytical methods to monitor the effect of GST. In the past few decades, a variety of methods for detecting anticancer drugs have been reported. These mainly include high-performance liquid chromatography coupled with various kinds of detectors (Desai et al., 2004, Jacquet et al., 1992, Khuhawar and Arain, 2005, Martincic et al., 2012, Yaroshenko et al., 2013), such as fluorescence (Shire and Loppnow 2012), UV–vis absorption (Menon et al., 2012) and electrochemistry (Dospivova et al., 2012, Teradal et al., 2012, Ting et al., 2009). These methods are very accurate, but are quite expensive to carry out. Biosensors may be another option for detection and provide complementary information. In recent years, many new biomolecules including enzymes, antibodies and aptamers have been developed to serve as probes (Chen et al., 2012, Li et al., 2010, Liu et al., 2009, Wang et al., 2013, Willner and Zayats, 2007). Given the structural diversity of anticancer drugs (Fig. 1B), it is quite challenging to come up with a different probe for each drug molecule. Since GST is a general detoxification enzyme, we hypothesize that it might detect all these drugs. The activity of GST can be measured using chromogenic substrates such as 1-chloro-2,4-dinitrobenzene (CDNB), which turns a yellow color upon reaction with GSH (Fig. 2A), where GST serves as a catalyst (Zhao et al., 1993). Since anticancer drugs compete for GSH and GST, they may act as inhibitors for the CDNB reaction. This could provide an optical means for monitoring the drugs. While optical assays might work in clean buffers, clinical samples are likely to contain blood serum or cell extract, which strongly scatter light and interfere with optical detection (Petrlova et al., 2006). To this end, electrochemistry-based methods are more useful since they can work well in complex sample matrix. To the best of our knowledge, electrochemistry detection of anticancer drugs has only been reported using surfactants (Teradal et al., 2012), DNA (Dospivova et al., 2012), multi-walled carbon nanotubes (Wang et al., 2012) and nanoparticles (Ting et al., 2009, Zhou et al., 2007). In those methods, drug recognition was based on simple adsorption or DNA intercalation, leading to compromised selectivity. On the other hand, no previous studies explored GST-based biosensors for anticancer drug detection. We reason that using GST as a probe might be more relevant to the native cellular environment. In this work, we modify carbon paste electrodes with GST to measure cisplatin and others drugs.