Figure 4.

T84.66-Hep/protamine-mediated modulation of cytotoxicity by TAT-Gel. (A) T84.66-Hep blocking of TAT-Gel cell transduction. When LS174T cells were incubated with TAT-Gel/T84.66-Hep complex, prepared by mixing TAT-Gel with increasing TAT-to-Hep molar ratios (from 5:1 to 1:3) of T84.66-Hep, a significantly reduced cytotoxicity was observed, compared with that of TAT-Gel. (B) Protamine-induced release of TAT-Gel. Addition of protamine to TAT-Gel/T84.66-Hep-treated cells, with increasing Hep-to-Pro molar ratios (from 10:1 to 1:2), cytotoxicity effects were significantly augmented. At above 5:1 molar ratio, the anti-cancer effect (IC₅₀) was similar to that induced by TAT-Gel alone. (C) Time dependency of the protamine addition time on effects of protamine-triggered release. Cells were incubated with TAT-Gel/T84.66-Hep for 6 h, and, after wash, at intended time points (0, 2, 6, 24 and 48 h), protamine (Hep : Pro molar ratio of 1:2) was added to the wells, and the cells were incubated with protamine further up to total 72 h. ***P < 0.001 and n.s.: not significant by 1-way ANOVA (Tukey's multiple comparison test as the post hoc test). (TAT-Gel: recombinant TAT-gelonin fusion chimera, T84.66-Hep: T84.66-heparin chemical conjugate, Hep: heparin, Pro: protamine)