Methotrexate al., 2017; Asci et al., 2017). According

Methotrexate (MTX) is an antagonistof folate that inhibits the activity of dihydrofolatereductase and blocks the synthesis of DNA which in turn leadsto 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 agentin the treatment of many types of cancers (Huang et al., 2011a) and several autoimmune disorders, includingpsoriasis and  rheumatoid arthritis (Abdel-Raheem and Khedr, 2014;Favalli et al., 2014; Kivity et al., 2014; Hafez et al., 2015).

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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 asliver 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 frequentlyobserved form of renal toxicity is acute renal failure following high-dose MTX (HD-MTX)therapy (Vardi et al.

, 2013). HD-MTX is wildlyused in various cancers such as osteosarcoma, leukemia, central nervous system(CNS) lymphoma, and leptomeningeal cancer (Schwartz et al., 2007).  Oxidative stress has been claimed asthe possible mechanism for MTX hepatotoxicity (?ener et al., 2006a; Uraz et al., 2008). Ithas been reported that MTX decreases the nicotinamide adenine dinucleotidephosphate (NADPH) content of the cells, which in turn sensitizeshepatocytes to oxidative stress. Also, long-term use of MTX leadsto the accumulation of polyglutamate forms of MTX in hepatocytes that result inthe generation of reactive oxygen species (ROS).

Theincreased generation of ROS, together with the decreased antioxidant defense ofthe cells leads to an oxidative stress, stimulating the development ofhepatotoxicity (Yin et al., 2009; Armagan et al.,2015). (Hagag et al., 2016) Although, it has been hypothesized thatantioxidant 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 preventingthe 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 fromfolic acid and its analogues (Jeyaharan etal., 2016).Recombinant form of carboxypeptidase G2 received approval from FDAfor 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 twosubunits of 41 KDa has no mammalian analogue (Rowsell et al.

, 1997). CPG2 rapidly hydrolyses extracellular MTX to its non-toxicmetabolites 2, 4-diamino-N10-methypteroicacid (DAMPA) and glutamic acid that results in reduced plasma MTX concentration(Mitrovic et al.,2016). DAMPA and glutamic acid aremetabolized by liver, creating a non-nephrotoxic pathwayfor the elimination of MTX. (Patterson and Lee, 2010). CPG2 is notable to discriminate between MTX and tetrahydrofolateM3  (the circulating form offolate in the blood stream).

To preventaccumulation of MTX in the cellular compartment intracellular delivery of CPG2 resultsin the conversion of MTX to its non-toxic metabolites in the cytoplasm. Proteins are polar and largemolecules, which cannot passively pass through the cell membrane. Therefore, some other methods for intracellular delivery ofproteins are required (D’Astolfo et al.

, 2015; Yang and Hinner, 2015). To date, various carriers have been used to delivertherapeutic cargos into the cells, among which cell-penetratingpeptides (CPPs) have attracted lots of attention due to their high efficiency (Fonseca et al., 2009; Farkhani etal., 2014; Wang et al., 2014).

CPPs are short peptides, generally with 5–30 amino acids. These peptides containa high percentage of basic residues having the ability to cross the cellmembrane with very limited or without any significant toxicity (Milletti, 2012; Shi et al., 2014). Since discovery, CPPs have been used widely for intracellular deliveryof various therapeutic molecules such as peptides, proteins, andoligonucleotides in vitro and in vivo (Wadia and Dowdy, 2005; Bechara andSagan, 2013; Zhang et al.

, 2015;Gautam et al., 2016). Transactivatortransduction domain (TAT) an extensively studied CPP, is an 11-amino acidpeptide (YGRKKRRQRRR) derived from the HIV- TAT protein. TAT is rich in arginineand lysine (Green and Loewenstein, 1988; Herceand Garcia, 2007; Rizzuti et al.,2015). Variousbiological molecules fused to the TAT peptide have been translocated throughthe 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). Proteinsranging from 10 to 120 kDa have been delivered into almost all the cells and tissuessuccessfully (Zhang et al., 2016). The TAT peptide has been extensively used for delivery of proteinsin 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), CNSdisorders (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 CPG2into the cell could convert MTX to its non-toxic metabolites. In this study, we designed, expressed and purified a TAT-CPG2fusion protein for direct transduction into the cells.

Furthermore, weevaluated possible inhibitory effects of TAT-CPG2 fusion protein against MTX-inducedtoxicity in HepG2 cells. To our knowledge this is the first report on the intracellulardelivery of the CPG2 protein and evaluation of the protective effects of TAT-CPG2on MTX-induced hepatotoxicity. M1Glucarpidase(voraxaze®) or carboxypeptidase G2 (CPG2)is an enzyme M1 from M2CPG2  is a zinc-dependent dimeric protein M3??? ??? ?????????


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