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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.

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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.

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