A decrease in the CAT activity in MTX-treatedcells 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 increasein the CAT activity in TAT-CPG2pretreated cells has been observed. Therefore, transduced TAT-CPG2 prevents theaccumulation of MTX inside the cells and maintains the balance between oxidantsand antioxidants hence protects cells against the oxidative stress induced byMTX.Based on the in vitro results, and reported mechanismsfor MTX cell toxicity, also considering HepG2 as a proliferating cell line, one might conclude that in the cell death induced by MTX in HepG2 cells both mechanismsof cell cycle suppression caused by the inhibition of dihydrofolatereductase andoxidative stress caused by the accumulation of MTX are involved.Therefore, transduced TAT-CPG2 convertsMTX into its non-toxic metabolites and prevents the accumulation of MTX in the cell and thus itstoxic effect.Delivery ofCPG2 into the cells by protein transduction is potentially valuable for a strategy known as enzyme/prodrug therapy.
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CPG2 is able to hydrolyzespecifically the amido, carbonyl or ureido bonds betweenL-glutamic acid and the carboxyl-, phenol or aniline-substituted aromaticrings, respectively (1,2). Several prodrugs such as4-(2-chloroethyl)(2-mesyloxyethyl)amino-benzoyl-L-glutamic acid (CMDA) andZD2767P have been synthesized to release potent DNA-alkylatingmustard drugs. These prodrugs are utilized in CPG2-medited strategies, such as antibody- or gene-directed enzyme prodrug therapy (ADEPTor GDEPT) (Jamin et al.,2011; Capucha et al., 2012; Karjoo et al., 2016).
Because of disadvantagesof cancer gene therapy such as safety problems, it hasbeen proposed that direct delivery of proteins into the cell is an alternative to gene therapy, especially gene therapy of those type of cancers that do not require long-term sustained and regulatedexpression of the transgene (Ford et al., 2001). Therefore, we will propose thatTAT-CPG2 fusion protein can be used as an alternative to the GDEPT approach. ConclusionWe have shown the construction, expression andpurification of CPG2 fused to HIV-1 TAT peptide (TAT–CPG2) in this study. Wehave demonstrated for the first time that TAT-CPG2 in both native and denaturedforms can be efficiently transduced into the HepG2 cells. Also, we have providedevidences for the enzyme activity of transduced TAT-CPG2 fusion protein.Furthermore, we have shown that TAT-CPG2 fusion protein strongly protects HepG2 cells against MTX-induced cell death.We assume that transduced CPG2 converts MTX to non-toxic metabolites andprevents the accumulation of MTX in cells and therefore prevents the cell proliferation suppression and oxidative stress caused byMTX.
However, further studiesare required to elucidate the involved cellular mechanisms. Our success in the protein transduction of TAT-CPG2 may provide a new strategy forprotecting against cell toxicity resulting from MTX in various organs andalso may provide an opportunity for the development of therapeutic methods forthe treatment of cancer by enzyme/prodrug strategy.