An arginine-rich cell penetrating peptide contained anti-gelatinase scFv-LDM fusion protein shows potent antitumor efficacy in pancreatic cancer

Abstract

Pancreatic cancer (PC) is one of the most dangerous cancers with less than 5% survival rate in 5 years. This study was to evaluate the antitumor activities of dFv-LDP-AE and dFv-R-LDP-AE, two energized fusion protein targeting gelatinases, on pancreatic cancer. The fusion protein dFv-LDP-AE consists of two tandem anti-gelatianses scFv and an enediyne antibiotic lidamycin (LDM) for receptor binding and cell killing. To improve the penetration capability, the fusion protein dFv-LDP-AE was integrated with an arginine-rich cell penetrating peptide (Arg)9 and then generated the fusion protein dFv-R-LDP-AE. The current study demonstrated that dFv-LDP and dFv-R-LDP had high affinity with the antigen gelatinases and PC cells, the integration of (Arg)9 could increase the penetration rate of fusion protein in SW-1990 and PANC-1 cells. After enediyne-energized with chromophore of lidamycin, the energized fusion protein dFv-LDP-AE and dFv-R-LDP-AE showed potent cytotoxicity to PC cells and could induced the robust cell apoptosis and necrosis in vitro. Western blot showed that dFv-R-LDP-AE could increase PARP cleavage, and inhibited the expression of VEGF, Cyclin D1, Cox-2 and Bcl-2 in SW-1990 and PANC-1 cells. In vivo, at a tolerated dosage, dFv-LDP, dFv-LDP-AE and dFv-R-LDP-AE inhibited tumor growth by 20.42%, 56.31% (P < 0.01, compared to that of control) and 74.2% (P < 0.05, compared to that of dFv-LDP-AE) in pancreatic cancer SW-1990 xenografted mice, respectively. Moreover, the results of in vivo optical imaging showed that fusion protein dFv-R-LDP displayed prominent accumulation in the tumor in SW-1990 xenografted mice and Capan-2 orthotopic transplanted mice. These results showed that dFv-R-LDP-AE possessed potent antitumor efficacy on PC, which indicating it could be a promising candidate for targeting therapy of PC.

Membrane Internalization Mechanisms and Design Strategies of Arginine-Rich Cell-Penetrating Peptides

“…Deshpande et al [ 113 ] proved that the attachment of an arginine-rich CPP octaarginine (R 8 ) and transferrin (Tf) to the surface of DOX-loaded liposomes improved the targeting of A2780 ovarian carcinoma cells and controlling tumor growth in an A2780 ovarian xenograft model. Besides ovarian carcinoma [ 114 ], the arginine-rich CPPs also showed their application potential in the therapy of other cancers, such as colorectal cancer [ 115 , 116 , 117 ], lung cancer [ 36 , 118 , 119 , 120 , 121 , 122 , 123 ], oral cancer [ 75 , 124 , 125 ], breast cancer [ 122 , 126 , 127 , 128 , 129 ], prostate cancer [ 130 , 131 , 132 , 133 ], pancreatic cancer [ 134 , 135 ], renal carcinoma [ 121 , 136 ], etc.…”

Inhibitor of Differentiation-2 Protein Ameliorates DSS-Induced Ulcerative Colitis by Inhibiting NF-κB Activation in Neutrophils

“…Then, IPTG (isopropyl-β-D-thiogalactoside) was added to the culture and incubated for another 3 hours to induce high expression of recombinant human ID2 protein. The preparation of recombinant protein was described in our previous study ( 30 ). The bacteria were collected by centrifugation and broken by ultrasound.…”

DataSheet_1.doc

“…Then, IPTG (isopropylb-D-thiogalactoside) was added to the culture and incubated for another 3 hours to induce high expression of recombinant human ID2 protein. The preparation of recombinant protein was described in our previous study (30). The bacteria were collected by centrifugation and broken by ultrasound.…”

“…Peptide mass fingerprinting and MS/MS queries were performed using the MASCOT engine (Matrix Science, London, UK; version 2.4) (32,33). The method for labelling hID2 with FITC was previously described (30). In brief, the recombinant human ID2 protein and FITC were shaken at 4°C in carbonate buffer solution [100 mmol/L NaHCO 3 , 10 mmol/L Na 2 CO 3 (pH 9.0)] for 12 hours, and then the FITC-labelled hID2 protein was isolated in a Millipore hyperfiltration tube with a cut-off of 5,000 Daltons.…”