Fn14•TRAIL is more potent than soluble forms of Trail and Fn14 alone or in combination.

<p>(<b>A</b>) SK-HEP-1 [A, left panel], HepG2 [A, middle panel] and Huh7 [A, right panel] HCC cell lines, as well as NKNT3 [B, left panel] and FHB [B, right panel] hepatocyte cell lines, were incubated with 0, 3, 30 or 300 ng/ml of Fn14•TRAIL, TRAIL, Fn14-Fc or combination of the later two for 48 hours. Viable cells were stained with trypan blue and counted. The results represent the mean +/- SD of three independent experiments (* <i>p</i> ≤ 0.05). (<b>B</b>) HepG2 HCC cells were incubated with 30ng/ml Fn14•TRAIL for 24 hours, in the presence or absence of anti Fn14 or anti TRAIL blocking antibodies. Treated cells were stained by Annexin V-FITC and Propidium Iodide, and counted by flow cytometer (2x10⁴ cells per sample). The results represent the mean +/- SD of two independent experiments (* p ≤ 0.05).</p

Fn14•TRAIL induces death of HCC cell lines in a dose dependent manner.

<p>SK-HEP-1 (<b>A</b>) HCC cells were incubated with indicated concentrations of Fn14•TRAIL for 72 [left panel], 48 and 24 hours [right panel]. (<b>B</b>,<b>C</b>) HepG2 [B, left panel] and Huh7 [B, right panel] HCC cell lines, as well as NKNT3 [C, left panel] and FHB [C, right panel] hepatocyte cell lines, were incubated with indicated concentrations of Fn14•TRAIL for 48 and 24 hours. Viable cells were stained with trypan blue and counted. Results represent the mean +/- SE of 3 independent experiments (* <i>p</i> < 0.05 ** <i>p</i> < 0.01 vs no Fn14•TRAIL). </p

HCC and hepatocyte cell lines express TRAIL, TRAIL receptors, Fn14 and TWEAK.

<p>(<b>A</b>) The mRNA expression level of TRAIL, TRAIL receptors (DR4, DR5, DCR-1, DCR-2, OPG), Fn14 and TWEAK was determined by quantitive real-time PCR analysis. A representative experiment of three independent experiments is shown. Data are shown as average of triplicates (SD < 0.3), normalized against two endogenous control human genes, TBP and Actin-B, as calculated by Dataassist v2.0 software. (<b>B</b>,<b>C</b>) Protein expression of TRAIL, TRAIL receptors (DR4, DR5, DcR1, DcR2), Fn14 and TWEAK was determined by flow cytometeric analysis. (<b>D</b>) Fn14•TRAIL binds to HCC cells – HepG2 cells were incubated with Fn14•TRAIL, soluble TRAIL, Fn14 or the combination of the latter for 30 min at 4°c, immune-stained with PE-conjugated anti-Fn14, and analyzed by flow-cytometry. The results represent the mean +/- SD of triplicates (* <i>p</i> ≤ 0.05). </p

No hepatotoxicty is observed in Fn14•TRAIL treated mice.

<p>HepG2 Nude mice were treated daily with subcutaneous injections of 200μg of Fn14•TRAIL (n=4) or vehicle (n=4) for 8 days. One hour after last injection blood counts (<b>A</b>) liver enzymes and urea (<b>B</b>) were measured. Livers were harvested and H&E stained sections were tested (<b>C</b>). Data are presented as mean +/- S.E. </p

Molecular analysis of the Fn14•TRAIL.

<p>(A) The amino-acid sequence of the Fn14-TRAIL protein. The amino-acid sequence of the extra-cellular domain of human Fn14 (amino-acids 1-52 of the mature protein, marked in bold letters) are directly linked to the extra-cellular domain of human TRAIL (amino-acids 53-217 of the mature protein, non-bold letters). The underlined sequence represents the signal-peptide of the human Urokinase protein, utilized to secrete Fn14-TRAIL out of the cell and removed from the mature protein. (B) Fn14-TRAIL separated at denaturizing conditions on SDS-PAGE, Coomassie gel staining. (C) Western blot analysis with anti-TRAIL and anti-Fn14 primary antibodies. </p