TRAIL mutant membrane penetrating peptide alike-MuR6-TR enhances the antitumor effects of TRAIL in pancreatic carcinoma both in vitro and in vivo

Abstract

Abstract.To remedy the drug resistance of natural tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and enhance its antitumor effects, we prepared a type of TRAIL mutant membrane penetrating peptide alike (TMPPA)-TRAIL mutant R6 (MuR6-TR) by mutating the N-terminal of the soluble TRAIL gene sequence. The expressed MuR6-TR protein was purified to treat pancreatic carcinoma cell lines BxPC-3 and PANC-1. The inhibitory effects on the proliferation of BxPC-3 and PANC-1 cells was assessed with CCK-8 assay and compared with natural TRAIL. The antitumor effect of MuR6-TR was assessed on implant tumors derived from PANC-1 cells in nude mice and compared with gemcitabine. Finally, the soluble MuR6-TR gene was successfully mutated with 4 amino acids in the N-terminal of TRAIL and had a molecular size of 513 bp. The mutant MuR6-TR was connected to pET32a and verified by enzymatic digestion and sequencing. The recombinant MuR6-TR was transformed and expressed in Escherichia coli. The CCK-8 assay results indicated that MuR6-TR inhibited the growth of BxPC-3 and PANC-1 cells in a dose-dependent manner, with IC 50 values of 4.63 and 7.84 ng/ml, respectively, which were much lower than that of natural TRAIL. MuR6-TR demonstrated a higher inhibitory effect on tumor growth (24.2%) than natural TRAIL (14.4%) and an effect similar to that of gemcitabine at an early period. Thus, the mutant MuR6-TR exhibited a stronger antitumor effect than that of natural TRAIL both in vivo and in vitro and may have potential therapeutic value for pancreatic carcinoma, which requires further validation. IntroductionPancreatic carcinoma is a neoplasm formed by abnormal proliferation of pancreatic cells due to the dysregulation of cellular growth under the effect of multiple tumorigenic factors. Due to its biological complexity and serious threat to patients, it is necessary to develop new therapeutic strategies for pancreatic carcinoma since current approaches have limited efficacy. For example, gemcitabine is associated with serious side effects and resistance is observed in various cases (1). Among tumorigenic factors, a deficiency in cellular apoptosis, the programmed death of cells, plays a critical role in the onset and development of tumors (2). Caspase activation is a key step in apoptosis and can be activated by both intrinsic and extrinsic pathways to induce a catalytic reaction and mediate cellular apoptosis (3). It has been proposed that an increase in the apoptotic threshold through alteration of molecules contributes to the therapeutic resistance of pancreatic carcinoma including apoptosis inducers or antitumor medications (4). Therefore, enhancing the sensitivity of tumor cells to apoptosis inducers is a potential strategy for the development of novel therapeutic strategies.Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), also termed as Apo2 ligand (Apo2L), is a member of the tumor necrosis factor (TNF) superfamily and the immune regulator of congenital and acquired immunity. TRAIL can initiate apoptotic s...

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“…These results also suggest that the cell-penetrating sequence increased the binding affinity between TRAIL and the cell membrane and up-regulated DR5 to activate caspase [ 104 ]. Furthermore, the TRAIL mutant R6 (MuR6-TR) exerted a greater inhibitory effect on tumor growth than did natural TRAIL when used to inhibit activities of pancreatic carcinoma cell lines BxPC-3 and PANC-1, thus indicating that the CPP sequence may have enhanced the ability of TRAIL-Mu3 to penetrate the cell membrane without increasing cell sensitivity to TRAIL [ 140 ].…”