MTX delayed MTX clearance (Goda et al., 2009;

MTX as an antagonist of folate remains one of
the most commonly used medications in the treatment of cancer and autoimmune
diseases. Due to its lack of specificity toward malignant cells the efficacy of this drug is limited (Ali et al., 2014). Although it has been suggested that long term usage of MTX results in the
accumulation of polyglutamate forms of MTX in hepatocytes that reduces folate levels and ultimately
leads to hepatotoxicity, another mechanism suggests that oxidative stress plays a major role in MTX
induced hepatotoxicity (Uraz et al., 2008). The major approach to combat
these problems is the usage of antioxidants to reduce MTX side effects (Vardi et al., 2010; Vardi et al., 2013; De et al., 2015).
However, it is favorable to investigate for the
treatment approaches that prevent toxic accumulation of MTX in non-target cells.

CPG2 as a recombinant enzyme rapidly
hydrolyses extracellular MTX to its non-toxic metabolites thus is used as a novel
drug for reducing elevated plasma MTX concentrations in patients with renal delayed
MTX clearance (Goda et al., 2009; Mitrovic et al., 2016). Therefore, intracellular delivery of CPG2 can convert MTX to its
non-toxic metabolites and prevents the accumulation of toxic levels of MTX. Glucarpidase as a dimeric protein with two 41 KDa subunits is not
able to enter the cells.  Therefore,
for intracellular delivery of CPG2, we have evaluated the possibility of
protein transduction using the TAT peptide as an effective CPP. To produce TAT-fusion protein, cpg2
gene was fused to the HIV-1 TAT peptide encoding gene. The production of purified
recombinant TAT- CPG2 fusion protein was
confirmed by Western blot analysis. To
determine the transduction efficiency of the TAT-CPG2, Western blot analysis
and fluorescence staining have been used. Results showed that both native and denatured TAT-CPG2 were successfully transduced into the HepG2 cells in a concentration and
time-dependent manner. Stability of proteins is very important in the host for
therapeutic applications. Significant intracellular
stability of TAT-CPG2 protein continued for up to 36 h after initial
transduction into the cells. Fluorescence staining results indicated that TAT-CPG2
protein transduced into approximately 100% of the cells.   A direct comparison of transduction
efficiency of native and denatured TAT-CPG2 protein by Western blot
analysis and fluorescence
microscopy showed that denatured TAT-CPG2 transduced into the cells more efficiently than the native TAT-CPG2. Similar observations
have been reported in other studies. Jin et al. has reported that denatured Tat-CAT
and 9Arg-CAT efficiently transduced into HeLa and PC12 cells (Jin et al., 2001). Kim et al. showed that in contrast to the native TAT-SOD, denatured
TAT-SOD has been successfully delivered into the cells (Kim et al., 2006). They have demonstrated
that unfolding of protein is needed for efficient transduction of TAT-SOD into HeLa cells. Nagahara et al.
showed that transduction of denatured TAT-p27 protein into HepG2
cells induced cell migration at low concentrations; while
correctly folded TAT-p27 protein could not induce cell migration at even higher
concentrations. This might be due to the low efficiency of correctly folded TAT-p27 to achieve the required intracellular threshold level to elicit cell
migration, not the complete disability to
enter the cell (Nagahara et al., 1998). Lower structural barriers
against denatured proteins and higher values of the Gibbs free energy in
comparison to the correctly folded proteins are probable attributed mechanisms
to the higher transduction efficiency of denatured proteins (Becker-Hapak et al., 2001).

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Functionality of transduced proteins in the cells is essential for their action. We have investigated the activity of both native and denatured TAT-CPG2 after penetration into the cells. All data suggested that TAT-CPG2 has been successfully delivered into the HepG2 cells, and the transduced enzyme was functional in the cells. The enzyme activity of both native and denatured TAT-fusion proteins in the cultured HepG2 cells was increased in a concentration and time-dependent manner. Intracellular stability and enzymatic activity analyses of transduced TAT-CPG2 into the HepG2 cells have shown that significant levels of transduced protein and enzyme activity remained in HepG2 cells up to 36 h. These results indicate that denatured TAT-CPG2 protein transduced into the HepG2 cells whereinM1  denatured TAT-CPG2 protein was correctly refolded to its native form and restored its biological activity. HSP90 like chaperons are probably responsible for the refolding of denatured proteins in the cells (Becker-Hapak et al., 2001; Kim et al., 2005). Our results showed that the enzyme activity in cells transduced with denatured TAT-CPG2 fusion protein except at shorter incubation times (15, 30 min) was significantly higher than the activity in cells transduced with the native protein. The differences between results of Western blot and fluorescence microscopy and enzyme activity at 15 and 30 min may be due to the time required for complete refolding of transduced protein by intracellular chaperons. It has been demonstrated that approximately one hour is required to obtain the highest transduction into the cells  (Kim et al., 2006). Formation of misfolded proteins as inclusion bodies after recombinant expression in E. coli needs a complicated procedure for refolding. Since denatured proteins have higher transduction efficiencies in comparison to the native forms one should note the importance of transduction denatured protein (Singh and Panda, 2005). Hence, using denatured proteins for transduction is commercially valuable.

It has been reported that MTX promotes cell death
through apoptosis in many cells (Mazur et al., 2009). Therefore, we have used cell
viability test and flow cytometry analysis to investigate the possibility of TAT-CPG2 fusion protein against
MTX-induced toxicity. In agreement with other reports (Yin et al., 2009; Chang et al., 2013), our data showed that MTX treatment decreased
viability of HepG2 cells due to the induction of cell death by apoptosis in a
concentration and  time-dependent manner. The viability of cells treated with MTX was
significantly increased when pretreated with the TAT-CPG2 fusion protein. The increasing viability was observed in cells
pretreated with both native and denatured TAT-CPG2 protein. The viability of
transduced cells 24 h after incubation was approximately equal to the viability
of untreated control group. A significant decrease in the cell viability of
transduced cells 48 h after incubation compared to 24 h was observed. We assume
that this decrease in the cell viability at 48 h is probably due to the decrease in the levels
of fusion protein
as a result of degradation within 48 hours, which is in agreement with the results of stability analysis. Our results show that transduction of fusion protein at the selected
concentration does not have any toxic effect on the HepG2 cell viability. This
has been shown in another report evaluating potentials of CPG2 in gene-directed
enzyme prodrug therapy (GDEPT), where CPG2 expression did not cause any toxicity
in HepG2 cells (Schepelmann et al., 2005). We have further examined the inhibitory
effects of TAT-CPG2 fusion protein against MTX-induced cell death using flow cytometry.
In
line with the results of
cell viability analysis, flow cytometry results confirmed that TAT-CPG2 in both
native and denatured form can strongly inhibit the apoptosis effects of MTX on
HepG2 cells. Therefore, our results indicate that transduction of TAT-CPG2
fusion protein efficiently protects HepG2 cells against cell death caused by MTX.

It has been widely reported that increased oxidative stress is one
of the major mechanisms involved in MTX toxicity (Yiang et al., 2014). Therefore, we decided to investigate the effect of some stress markers to check whether protection effects of TAT-CPG2 against MTX-induced
cell death is correlated with the inhibition of oxidative
stress. We have observed that
MTX boosted intracellular ROS generation in cultured HepG2 cells. Previous reports have revealed
that oxidative damage caused by ROS generation is the major factor of MTX
tissue injury (Yiang et al., 2014; Hafez et al., 2015). However, in our study cells
which were pretreated with TAT-CPG2
had a significant decrease in the production of ROS.

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). However, in our study, pretreatment of HepG2 cells with
TAT-CPG2 ameliorated
GSH content. A decrease in the CAT activity in MTX-treated cells compared to that of the untreated
cells has been observed. MTX ratchets down the
activity of the CAT as an antioxidant enzyme (Çetin et al.,
2008; Chang et al., 2013). In our experiment a significant increase in the CAT
activity in 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 hence protects cells against the oxidative stress induced by MTX.

Based on the in vitro results, and reported mechanisms
for MTX cell toxicity, also considering HepG2 as a proliferating cell line, one might conclude that in the cell death induced by MTX in HepG2 cells both
mechanisms of cell cycle suppression caused by the inhibition of dihydrofolate
reductase and oxidative stress caused by the accumulation of MTX
are involved. Therefore, transduced TAT-CPG2 converts
MTX into its non-toxic metabolites and prevents the accumulation of MTX in the cell and thus its
toxic effect.

Delivery of
CPG2 into the cells by protein transduction
is potentially valuable for a strategy known as enzyme/prodrug therapy. CPG2 is able to
hydrolyze specifically the amido, carbonyl or ureido
bonds between L-glutamic acid and the carboxyl-, phenol or aniline-substituted
aromatic rings, respectively (1,2). Several prodrugs such as
4-(2-chloroethyl)(2-mesyloxyethyl)amino-benzoyl-L-glutamic acid (CMDA) and
ZD2767P have been synthesized to release potent DNA-alkylating
mustard drugs. These prodrugs are utilized in CPG2-medited strategies, such as antibody- or gene-directed enzyme prodrug therapy (ADEPT
or GDEPT) (Jamin et al., 2011) (Capucha et al., 2012) (Karjoo et al., 2016). Because of disadvantages
of cancer gene therapy such as safety problems, it has
been proposed that direct delivery of protein into the cell is an alternative to gene therapy, especially gene
therapy of those type of cancers that do not require long-term sustained and regulated expression of the transgene (Ford et al.,
2001). Therefore,
we will propose that TAT-CPG2 fusion protein can be used as an alternative to the GDEPT approach.

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