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    • Assessment of the activity of native and

    2020-01-21

    Assessment of the activity of native and denatured TAT-CPG2 after penetration suggests that TAT-CPG2 has been successfully delivered into the cells, and the transduced enzyme is functional inside the cells. The enzyme activity of both native and denatured TAT-fusion proteins in the cultured HepG2 Gambogic Acid sale was increased in a concentration and time-dependent manner. Presence of the enzyme activity with denatured TAT-CPG2 indicated that transduced denatured proteins were correctly refolded inside the cells into their native form and their biological activities have been restored. HSP90 like chaperons are probably responsible for refolding of denatured proteins in the cells (Becker-Hapak et al., 2001; Kim et al., 2005). Interestingly, we have observed that the enzyme activity in the cells that were transduced with denatured TAT-CPG2 fusion protein was significantly higher than the activity in the cells transduced with the native protein except at shorter incubation times (15 and 30 min). The lower enzyme activity observed at the first 30 min might be due to the time required for the complete refolding of transduced protein by the intracellular chaperons. Transduction of denatured proteins into the cells has two advantages. Denatured proteins would be transduced into the cells with higher efficiencies than the native ones. This allows the use of lower protein doses. On the other hand, expression of recombinant proteins in prokaryotes such as E. coli results in the formation of inclusion bodies, which require complex refolding process to restore the activity. Hence, using denatured form of proteins in transduction technology would lead to a significant saving in time and cost (Singh and Panda, 2005). To investigate the inhibitory effect of TAT-CPG2 against MTX induced cell death we have used cell viability test and flow cytometry analyses. An increase in the 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 that of the untreated control cells. A significant decrease in the cell viability of transduced cells 48 h after incubation compared to 24 h has been 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 h. According to our observations transduction of the fusion protein at the selected concentrations did 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 (Schepelmann et al., 2005). In agreement with other reports (Yin et al., 2009; Chang et al., 2013), our flow cytometry results showed that MTX mediates cell death by inducing apoptosis in HepG2 cells. We have confirmed that TAT-CPG2 in both native and denatured forms can strongly inhibit the apoptosis effects of MTX. 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; cell cycle 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.