Fig 1.
Construction of the recombinant cytostatic/cytotoxic protein XTEN-Killin.
Schematic representation of the DNA/protein sequence of XTEN-Killin (A), the procedure for production (B), and a summary of its intended functions (C).
Fig 2.
Expression, purification, and cleavage of XTEN-Killin.
(A-C) Bacterial lysate expressing XTEN-Killin after heating step (A) and purified XTEN-Killin (B) were analyzed with SDS-PAGE, followed by Coomassie staining. The protein solution was additionally analyzed with HPLC (C) at 210 nm, revealing purity of >90% (peak at 6.3 min). (D) XTEN-Killin coupled with near-infrared dye 6S-IDCC was also analyzed with SDS-PAGE followed by Coomassie staining before (1) and after (2) digestion with MMP-2 enzyme to prove the functionality of the cleavage site. Free XTEN can not be stained with Coomassie, therefore after digestion (2) only CPP killin (approx. 5 kDa, black arrow) and potential complexes formed by the arginine-rich composition of the CPP Killin are visible (approx. 17 kDa). Some additional thin bands at 9 and 13 kDa could represent degradation products after digestion. Markers in A, B and D: 3–260 kDa.
Fig 3.
Uptake of XTEN-Killin-6S-IDCC in cancer cells.
Low density epithelial sarcoma (HT-1080) cells were exposed to 3 μM (A and B) and 5 μM (C) XTEN-Killin-6S-IDCC for 24–96 h. After 24 h, intracellular enrichment of likely cleaved Killin-6S-IDCC in the endosomes of the MMP-2-positive HT-1080 cells was visible (red, A-B). T-cell lymphoma (Jurkat) cells, which are characterized by low MMP-2 expression, were exposed to 5 μM XTEN-Killin-6S-IDCC for 24–96 h. Up to 48 h, Jurkat cells showed only extracellular binding of the XTEN-Killin-6S-IDCC to the membrane (D, red). Intracellular uptake was observed only at 96 h (E, red). 72 h after treatment of HT-1080 cells (C) and 96 h after treatment of Jurkat cells (F), only a few Killin-6S-IDCC-positive cells (red, intranuclear) were found positive for annexin A5, used as assay for apoptosis/cell death (green). Blue color—DAPI stain. Bars: 10 μM.
Fig 4.
Dose-dependent toxicity of XTEN-Killin.
HT-1080 cells were treated with different amounts of XTEN-Killin (Ki) or XTEN-Killin-6S-IDCC (Ki-6S-IDCC) and XTEN-Killin-fluorescein (Ki-fluorescein), as well as XTEN polypeptide, and analyzed by flow cytometry. Relative cell densities of viable and apoptotic annexin A5 positive cells (± SEM) were compared to untreated control cells (100% ±SEM) after 24 h of treatment. At least 3 independent experiments for each group were analyzed using one-way ANOVA with Tukey’s multiple comparison test; *** p<0.001, ** p<0.01. The experiment with XTEN-Killin-fluorescein (Ki-fluorescein) was done without repetitions.
Fig 5.
Treatment effects of XTEN-Killin on normal and cancer cells.
Two cancer cell lines (HT-1080 A-A1, HeLa B-B1) and normal liver BRL3A cells (C-C1) were seeded at a density of 15,000 cm-2, left to adhere for 6–8 h and treated with XTEN-Killin (10 μM). Cell growth and apoptosis level (annexin A5 staining) were measured. Dashed lines represent cell density and amount of apoptotic cells in the cultures at the time of plating (0 h). Statistical difference of cell density means were analyzed using one-way ANOVA with Tukey’s multiple comparison test; * p<0.05, ** p<0.01, *** p<0.001.