28 Because our and other data26,27 show that EGFR is up-regulated in HCC, we employed a novel TRAIL protein in which scTRAIL was fused to a humanized single-chain variable fragment derived from cetuximab, a chimeric Ab directed against the extracellular domain of EGFR. This fusion protein (αEGFR-scTRAIL) selectively binds to EGFR-positive HCC cells and, through target-antigen binding, mimics membrane-bound TRAIL, which enhances stable TRAIL-R signaling complex formation. With respect to the results of in vitro dose-finding experiments for apoptosis induction and to the pharmacokinetics of TRAIL in vivo, a concentration of 100 ng/mL

of the TRAIL proteins was used in this study.40,41 Higher concentrations did not differ in their ability to induce apoptosis in the presence of BZB. For BZB treatment, we chose a concentration of 500 ng/mL, which, when Vemurafenib datasheet combined with TRAIL, has been shown to be nontoxic in PHHs.24 We CDK inhibitor analyzed EGFR-targeted scTRAIL, compared to non-targeted scTRAIL alone and in combination

with BZB, in Huh7 liver cancer cells that are known to express EGFR. Although nontargeted scTRAIL plus BZB induced significantly higher caspase activity, compared to the respective agents alone, the combination of EGFR-targeted scTRAIL with BZB was most effective. Inhibition of EGFR signaling by itself can induce apoptosis in appropriate cell systems. However, our study shows that apoptosis induction by αEGFR-scTRAIL is entirely ZD1839 dependent on TRAIL signaling, rather than on inhibition of EGFR function, because TRAIL-neutralizing Abs almost completely abolished caspase activation. Furthermore, pretreatment of Huh7 cells with a caspase inhibitor strongly reduced caspase-8 and caspase-3 activation induced by targeted TRAIL, indicating that the observed proapoptotic effects were indeed the result of TRAIL-induced caspase activation. Strikingly, neither scTRAIL

nor EGFR-targeted scTRAIL alone or combined with BZB induced caspase activation in PHHs, indicating that this treatment is nontoxic to healthy hepatocytes. Because the liver is composed of multiple cell types that might modulate TRAIL susceptibility of hepatocytes, we analyzed TRAIL effects in organotypic cultures of fresh liver explants.32 We found a modest increase of caspase activity in HCC liver explants treated with scTRAIL and BZB. In contrast, EGFR-targeted scTRAIL in combination with BZB induced a significant increase of caspase activity in HCC tissues, again indicating its increased antitumor activity. We suggest that the increased antitumor activity of targeted TRAIL is largely conferred by its tumor specificity and increased bioactivity.