Archives

  • 2018-07
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • 2021-05
  • 2021-06
  • 2021-07
  • 2021-08
  • 2021-09
  • 2021-10
  • 2021-11
  • 2021-12
  • 2022-01
  • 2022-02
  • 2022-03
  • 2022-04
  • 2022-05
  • 2022-06
  • 2022-07
  • 2022-08
  • 2022-09
  • 2022-10
  • 2022-11
  • 2022-12
  • 2023-01
  • 2023-02
  • 2023-03
  • 2023-04
  • 2023-05
  • 2023-06
  • 2023-07
  • 2023-08
  • 2023-09
  • 2023-10
  • 2023-11
  • 2023-12
  • 2024-01
  • 2024-02
  • 2024-03
  • Two major mechanisms including apoptosis and oxidative stres

    2019-07-02

    Two major mechanisms including apoptosis and oxidative stress have been suggested for MTX-induced cytotoxicity (Hafez et al., 2015; Neuman et al., 1999; El-Sheikh et al., 2015). It has been reported that MTX promotes cell death via apoptosis in both cancerous and non-transformed cells (Mazur et al., 2009). However, the most common reported mechanism for MTX induced toxicity is the oxidative stress (Ali et al., 2014; Şener et al., 2006a; Uraz et al., 2008). MTX decreases the NADPH content of cells, which in turn sensitizes hepatocytes to the oxidative stress. On the other hand, prolonged use of MTX leads to the accumulation of MTX polyglutamate derivatives in hepatocytes that result in the generation of reactive oxygen species (ROS). The elevated ROS levels and decreased antioxidant defense of cells results in the oxidative stress. This in turn stimulates the development of hepatotoxicity (Yin et al., 2009; Armagan et al., 2015). Although, it has been assumed that antioxidant compounds are able to decrease the corresponding toxicity (Şener et al., 2006b; Vardi et al., 2010; Vardi et al., 2013; De et al., 2015; Abdel-Daim et al., 2017), there is an urgent need to investigate novel therapies preventing the accumulation of MTX in non-target cells. Carboxypeptidase-G2 (CPG2) an enzyme from Pseudomonas sp. (strain RS-16) has been used for the treatment of elevated plasma concentrations of MTX in patients with renal delayed MTX clearance (Tuffaha and Al Omar, 2012). CPG2 is a zinc-dependent dimeric protein with no mammalian analogue (Rowsell et al., 1997). CPG2 rapidly hydrolyzes extracellular MTX to its non-toxic metabolites, called 2, 4-diamino-N10-methypteroic Puromycin and glutamic acid (Mitrovic et al., 2016). To cleave MTX into its non-toxic derivatives inside the cellular compartment, it is reasonable to guide the CPG2 enzyme into the cells. However, because of its large molecular size, CPG2 is unable to pass through the cell membrane by passive diffusion (Trifilio et al., 2013). Until now, various carriers such as cell-penetrating peptides (CPPs) have been used to deliver therapeutic cargos into the cells (Bechara and Sagan, 2013; Gautam et al., 2016; Fonseca et al., 2009; Farkhani et al., 2014; Wang et al., 2014; Zhang et al., 2015). Trans-activator transduction domain (TAT) as an extensively studied CPP has 11 amino acids (YGRKKRRQRRR), which has been derived from the HIV- TAT protein. Various biological molecules fused to the TAT peptide have been trans-located through the plasma membrane rapidly and efficiently (Vives et al., 1997; Gupta et al., 2005; Wadia and Dowdy, 2005; Gump and Dowdy, 2007; Rapoport et al., 2011). Proteins ranging from 10 to 120 KDa have been delivered into almost all the cells and tissues with TAT as a CPP (Zhang et al., 2016).
    Materials and methods
    Results
    Discussion MTX is widely used in the treatment of cancer and autoimmune diseases. However, side effects such as hepatotoxicity have limited its applications (Abo-Haded et al., 2017). Although the CPG2 enzyme effectively detoxifies MTX, it is unable to enter the cells. Therefore, we have constructed a TAT–CPG2 fusion protein to evaluate its inhibitory effect against MTX inside the cellular compartment. To determine the transduction efficiency of TAT-CPG2, Western blot analysis and fluorescence staining have been used. Our results showed that both native and denatured TAT-CPG2 were successfully transduced into the HepG2 cells in a concentration and time-dependent manner. Fluorescence staining results showed that TAT-CPG2 protein transduced into approximately 100% of the cells. Also, 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 ones. Similar observations have been reported in other studies. Jin et al. has reported that denatured TAT-CAT and 9ARG-CAT enzymes have been efficiently transduced into the HeLa and PC12 cells, respectively (Jin et al., 2001). Kim et al. showed that in contrast to the native TAT-SOD, the denatured enzyme has been successfully delivered into the cells (Kim et al., 2006). They have demonstrated that the unfolded form of protein has superior properties for the efficient transduction of TAT-SOD into the HeLa cells. In another study, Nagahara et al. showed that the transduction of denatured TAT-p27 protein into HepG2 cells induces cell migration at low concentrations; while correctly folded TAT-p27 could not induce any cell migration (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 the attributed mechanisms to the higher transduction efficiency of denatured proteins (Becker-Hapak et al., 2001).