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Recombinant TAT-gelonin fusion toxin: synthesis and characterization of heparin/protamine-regulated cell transduction.

ABSTRACT: Protein toxins, such as gelonin, are highly desirable anti-cancer drug candidates due to their unparalleled potency and repetitive reaction mechanism in inhibiting protein translation. However, for its potential application in cancer therapy, there remains the cell membrane barrier that allows permeation of only small molecules, which must be overcome. To address this challenge, we conjugated gelonin with a protein transduction domain (PTD), the TAT peptide, via genetic recombination. The chimeric TAT-gelonin fusion protein (TAT-Gel) retained equipotent N-glycosidase activity yet displayed greater cell uptake than unmodified recombinant gelonin (rGel), thereby yielding a significantly augmented cytotoxic activity. Remarkably, TAT-Gel displayed up to 177-fold lower IC?? (avg. 54.3 nM) than rGel (avg. IC?? : 3640 nM) in tested cell lines. This enhanced cytotoxicity, however, also raised potential toxicity concerns due to the non-selectivity of PTD in its mediated cell transduction. To solve this problem, we investigated the plausibility of regulating the cell transduction of TAT-Gel via a reversible masking using heparin and protamine. Here, we demonstrated, both in vitro and in vivo, that the cell transduction of TAT-Gel can be completely curbed with heparin and yet this heparin block can be efficiently reversed by the addition of protamine. This reversible tight regulation of the cell transduction of TAT-Gel by heparin and protamine sheds light of possible application of TAT-Gel in achieving a highly effective yet safe drug therapy for the treatment of tumors.

SUBMITTER: Shin MC

PROVIDER: S-EPMC4198515 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Recombinant TAT-gelonin fusion toxin: synthesis and characterization of heparin/protamine-regulated cell transduction.

Shin Meong Cheol MC Zhao Jingwen J Zhang Jian J Huang Yongzhuo Y He Huining H Wang Mei M Min Kyoung Ah KA Yang Victor C VC

Journal of biomedical materials research. Part A 20140423 1

Protein toxins, such as gelonin, are highly desirable anti-cancer drug candidates due to their unparalleled potency and repetitive reaction mechanism in inhibiting protein translation. However, for its potential application in cancer therapy, there remains the cell membrane barrier that allows permeation of only small molecules, which must be overcome. To address this challenge, we conjugated gelonin with a protein transduction domain (PTD), the TAT peptide, via genetic recombination. The chimer ...[more]

PMID: 24733757

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Project description:Essentials Heparin-protamine balance (HPB) modulates bleeding after neonatal cardiopulmonary bypass (CPB). HPB was examined in 44 neonates undergoing CPB. Post-operative bleeding occurred in 36% and heparin rebound in 73%. Thrombin-initiated fibrin clot kinetic assay and partial thromboplastin time best assessed HPB.SummaryBackground Neonates undergoing cardiopulmonary bypass (CPB) are at risk of excessive bleeding. Blood is anticoagulated with heparin during CPB. Heparin activity is reversed with protamine at the end of CPB. Paradoxically, protamine also inhibits blood coagulation when it is dosed in excess of heparin. Objectives To evaluate heparin-protamine balance in neonates undergoing CPB by using research and clinical assays, and to determine its association with postoperative bleeding. Patients/Methods Neonates undergoing CPB in the first 30 days of life were studied. Blood samples were obtained during and after surgery. Heparin-protamine balance was assessed with calibrated automated thrombography, thrombin-initiated fibrin clot kinetic assay (TFCK), activated partial thromboplastin time (APTT), anti-FXa activity, and thromboelastometry. Excessive postoperative bleeding was determined by measurement of chest tube output or the development of cardiac tamponade. Results and Conclusions Of 44 neonates enrolled, 16 (36%) had excessive postoperative bleeding. The TFCK value was increased. By heparin in neonatal blood samples, but was only minimally altered by excess protamine. Therefore, it reliably measured heparin in samples containing a wide range of heparin and protamine concentrations. The APTT most closely correlated with TFCK results, whereas anti-FXa and thromboelastometry assays were less correlative. The TFCK and APTT assay also consistently detected postoperative heparin rebound, providing an important continued role for these long-established coagulation tests in the management of postoperative bleeding in neonates requiring cardiac surgical repair. None of the coagulation tests predicted the neonates who experienced postoperative bleeding, reflecting the multifactorial causes of bleeding in this population.

Dataset Information

Recombinant TAT-gelonin fusion toxin: synthesis and characterization of heparin/protamine-regulated cell transduction.

Publications

Recombinant TAT-gelonin fusion toxin: synthesis and characterization of heparin/protamine-regulated cell transduction.

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