Methotrexate (MTX) is an antagonist
of folate that inhibits the activity of dihydrofolate
reductase and blocks the synthesis of DNA which in turn leads
to cell cycle arrest in the G1 and/or S phases (Mazur et al., 2009; Nihal et al.,
2014).  MTX is extensively used as an anticancer agent
in the treatment of many types of cancers (Huang et al., 2011a) and several autoimmune disorders, including
psoriasis and  rheumatoid arthritis (Abdel-Raheem and Khedr, 2014;
Favalli et al., 2014; Kivity et al., 2014; Hafez et al., 2015).
Since the effect of MTX is not selective for the cancer  cells, prolonged usage of MTX causes  various organ toxicity, including liver, kidney,
testis, lung, bone marrow and brain (Abo-Haded et al., 2017; Asci et al.,
2017). According to previous reports, hepatotoxicity is 
one of the most frequently reported side effects of MTX (Ali et al.,
2014).  Low to high doses of MTX  may lead to disorders such as
liver cirrhosis or fibrosis (Mhatre and Marar, 2016). (Mukherjee et al., 2013).
Nephrotoxicity is another major side effect of MTX because most of the drug is excreted in urine. The most frequently
observed form of renal toxicity is acute renal failure following high-dose MTX (HD-MTX)
therapy (Vardi et al., 2013). HD-MTX is wildly
used in various cancers such as osteosarcoma, leukemia, central nervous system
(CNS) lymphoma, and leptomeningeal cancer (Schwartz et al., 2007).  Oxidative stress has been claimed as
the possible mechanism for MTX hepatotoxicity (?ener et al., 2006a; Uraz et al., 2008). It
has been reported that MTX decreases the nicotinamide adenine dinucleotide
phosphate (NADPH) content of the cells, which in turn sensitizes
hepatocytes to oxidative stress. Also, long-term use of MTX leads
to the accumulation of polyglutamate forms of MTX in hepatocytes that result in
the generation of reactive oxygen species (ROS). The
increased generation of ROS, together with the decreased antioxidant defense of
the cells leads to an oxidative stress, stimulating the development of
hepatotoxicity (Yin et al., 2009; Armagan et al.,
2015). (Hagag et al., 2016) Although, it has been hypothesized that
antioxidant compounds could decrease the corresponding toxicity (?ener 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.

Glucarpidase (voraxaze®) is a carboxypeptidase G2 (CPG2) enzymeM1  from Pseudomonas sp. strain RS-16  that cleaves the glutamic acid moiety from
folic acid and its analogues (Jeyaharan et
al., 2016).

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Recombinant form of carboxypeptidase G2 received approval from FDA
for the treatment of elevated plasma MTX levels in  patients with renal delayed  MTX clearance (Goda et al.,
2009). (Goda et al., 2009). CPG2 a zincM2 -dependent dimeric protein with two
subunits of 41 KDa has no mammalian analogue (Rowsell et al., 1997). CPG2 rapidly hydrolyses extracellular MTX to its non-toxic
metabolites 2, 4-diamino-N10-methypteroic
acid (DAMPA) and glutamic acid that results in reduced plasma MTX concentration
(Mitrovic et al.,
2016). DAMPA and glutamic acid are
metabolized by liver, creating a non-nephrotoxic pathway
for the elimination of MTX. (Patterson and Lee, 2010). CPG2 is not
able to discriminate between MTX and tetrahydrofolateM3  (the circulating form of
folate in the blood stream). To prevent
accumulation of MTX in the cellular compartment intracellular delivery of CPG2 results
in the conversion of MTX to its non-toxic metabolites in the cytoplasm.

Proteins are polar and large
molecules, which cannot passively pass through the cell membrane. Therefore, some other methods for intracellular delivery of
proteins are required (D’Astolfo et al., 2015; Yang and Hinner, 2015). To date, various carriers have been used to deliver
therapeutic cargos into the cells, among which cell-penetrating
peptides (CPPs) have attracted lots of attention due to their high efficiency (Fonseca et al., 2009; Farkhani et
al., 2014; Wang et al., 2014).
CPPs are short peptides, generally with 5–30 amino acids. These peptides contain
a high percentage of basic residues having the ability to cross the cell
membrane with very limited or without any significant toxicity (Milletti, 2012; Shi et al., 2014). Since discovery, CPPs have been used widely for intracellular delivery
of various therapeutic molecules such as peptides, proteins, and
oligonucleotides in vitro and in vivo (Wadia and Dowdy, 2005; Bechara and
Sagan, 2013; Zhang et al., 2015;
Gautam et al., 2016). Transactivator
transduction domain (TAT) an extensively studied CPP, is an 11-amino acid
peptide (YGRKKRRQRRR) derived from the HIV- TAT protein. TAT is rich in arginine
and lysine (Green and Loewenstein, 1988; Herce
and Garcia, 2007; Rizzuti et al.,
2015). Various
biological molecules fused to the TAT peptide have been translocated 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
successfully (Zhang et al., 2016). The TAT peptide has been extensively used for delivery of proteins
in cancer research therapy (Sethuraman and Bae, 2007; Huang et al., 2011b; Orzechowska et al., 2014; Fu et al., 2015; Lu et al.,
2016; Zhang et al., 2016),
inflammation (Kwon et al., 2011; Kim et al.,
2015a; Kim et al., 2015c), ischemia
(Kim et al., 2009; Kim et al.,
2010), CNS
disorders (Kim et al., 2009; Zhu et al.,
2014; Kim et al., 2015b),
and enzyme replacement (Yoon et al., 2002; Lee et al.,
2005; Rapoport et al., 2011).

Based on our hypothesis, delivery of CPG2
into the cell could convert MTX to its non-toxic metabolites. 
In this study, we designed, expressed and purified a TAT-CPG2
fusion protein for direct transduction into the cells. Furthermore, we
evaluated possible inhibitory effects of TAT-CPG2 fusion protein against MTX-induced
toxicity in HepG2 cells. To our knowledge this is the first report on the intracellular
delivery of the CPG2 protein and evaluation of the protective effects of TAT-CPG2
on MTX-induced hepatotoxicity.