We have observed that MTX boosted intracellular ROS

We have observed that MTX boosted intracellular ROS generation in cultured HepG2 cells. Previous reports have revealed that oxidative damage caused by the ROS generation is the major cause of MTX tissue injury (Yiang et al., 2014; Hafez et al., 2015). In the cells which were pretreated with TAT-CPG2, the ROS production has been decreased significantly. In addition, a significant decrease in the GSH content in MTX-treated cells compared to that of the untreated cells was observed. These results are in agreement with previous studies reporting the depletion of intracellular GSH content by MTX (Chang et al., 2013; Ewees et al., 2015). It has been demonstrated that GSH plays an important role in the cellular antioxidant defense, and its reduction may cause oxidative injury in hepatocytes (Mukherjee et al., 2013). Pretreatment of HepG2 cells with TAT-CPG2 ameliorated the GSH content. In MTX-treated cells the CAT enzyme activity has been decreased compared to that of the untreated cells. MTX ratchets down the activity of CAT as an antioxidant enzyme (Çetin et al., 2008; Chang et al., 2013). In the current study a significant increase in the CAT activity of TAT-CPG2 pretreated cells has been observed. Therefore, transduced TAT-CPG2 prevents the accumulation of MTX inside the cells and maintains the balance between oxidants and antioxidants. Considering HepG2 as a proliferating cell line and based on the reported mechanisms for MTX cytotoxicity, one might conclude that MTX induces cell death in HepG2 cells by two mechanisms; sodium salt suppression (caused by the inhibition of dihydrofolate reductase) and oxidative stress (caused by the accumulation of MTX). Therefore, transduced TAT-CPG2 converts MTX into its non-toxic metabolites and prevents the accumulation of MTX in the cell and its cytotoxic effect.
Conclusion In this study, we have shown the construction, expression and purification of CPG2 fused to the HIV-1 TAT peptide (TAT–CPG2). We have demonstrated for the first time that TAT-CPG2 in both native and denatured forms could be efficiently transduced into the HepG2 cells. Also, we have provided evidences for the enzyme activity of transduced TAT-CPG2 fusion protein. We assume that transduced CPG2 converts MTX to the non-toxic metabolites, which prevents the cell proliferation suppression and the oxidative stress caused by MTX. However, further investigations especially in vivo studies are required to elucidate the involved cellular mechanisms in depth. Our success in the protein transduction of TAT-CPG2 may provide a new strategy for protecting against cell toxicity resulting from MTX in various organs. Therefore, we propose that the TAT-CPG2 fusion protein could be used as an alternative to the gene-directed enzyme prodrug therapy.
Introduction Carboxypeptidase G2 (CPG2) is a 42 kDa, zinc-dependent metalloenzyme from Pseudomonas that cleaves the glutamic acid moiety from folic acid and its analogues. CPG2 is currently being exploited in a rescue therapy following high-doses of the highly cytotoxic drug methotrexate (MTX), commonly used in the treatment of cancer and autoimmune diseases, to metabolise the drug into two non-toxic metabolites: 2,4-diamino-N [10]-methylpteroic acid (DAMPA) and glutamate (Fig. 1A) [1], [2]. CPG2 has also played a key role in the development of antibody directed enzyme pro-drug therapy (ADEPT, see Fig. 1B and caption for full description) [3], an anti-cancer therapy aimed at limiting the action of cytotoxic drugs to tumour sites, thus amplifying their selectivity and effectiveness and diminishing the lethal effects on normal tissue [4]. It is envisaged that CPG2 will be used to generate active drugs (i.e benzoic acid mustard drugs) from a variety of glutamated pro-drugs (i.e glutamated benzoic acid mustard pro-drugs). CPG2 is ideal for use in ADEPT because it has no mammalian homologue, thus no endogenous enzymes would act on a pro-drug specific for CPG2, and being a bacterial enzyme has the advantage of enhanced kinetics with substrate turnover [5].