Having demonstrated the superior activity of EGFR-targeted scTRAIL, we next compared the apoptosis-inducing effects of the scTRAIL proteins in intact, unfixed tissue explants from HCC and healthy livers by measuring caspase activation in liver tissue extracts. Combined treatment of HCC tissues with scTRAIL and BZB resulted in a moderate, but not significant increase in caspase-3 activation (3.64- ± 0.92-fold of untreated control; n = 8), compared to the single treatment with both agents alone (1.86- ± 0.64- and 2.92- ± 0.72-fold, respectively; Fig. 5A). In contrast, treatment of HCC tissues (n = 11) with EGFR-targeted scTRAIL and BZB significantly (P < 0.05) increased caspase-3 activation

(10.57- ± 2.80-fold), ��-catenin signaling compared to BZB or EGFR-targeted scTRAIL alone (3.53- ± 0.72- and 3.46- ± 0.87-fold; Fig. 5B). Similar to our observation in HCC cells, we found a significant (P < 0.05) increase of caspase-3 activity in HCC tissues treated with EGFR-targeted scTRAIL and BZB, compared to treatment with nontargeted scTRAIL and BZB. In contrast, no significant differences in caspase-3 activation were found between

targeted and nontargeted scTRAIL treatment without BZB (Fig. 5C). Importantly, neither Pexidartinib supplier scTRAIL nor EGFR-targeted scTRAIL alone or in combination with BZB significantly increased caspase-3 activation in intact healthy liver tissues (n = 7; Fig. 5A, B). To further support these results, we performed IHC analyses for caspase-3 activation and caspase-mediated CK-18 cleavage in HCC (n = 5) and healthy liver tissues (n = 5) after TRAIL and BZB treatment. Almost no caspase-3 activation was found in healthy liver tissues treated with scTRAIL or EGFR-targeted scTRAIL in the presence of BZB (Fig. 6A). In contrast, HCC liver tissues treated with

EGFR-targeted scTRAIL and BZB revealed a higher number of active Methocarbamol caspase-3-positive hepatocytes, compared to scTRAIL and BZB (Fig. 6A). In line with this, HCC tissues incubated with targeted scTRAIL and BZB also showed higher levels of caspase-cleaved CK-18, compared to HCC tissues treated with nontargeted scTRAIL and BZB, whereas no CK-18 fragments were found in healthy liver tissues treated with the respective agents (Fig. 6B). To quantify the IHC results, cells positive for caspase-3 activation or CK-18 fragments were counted at ×400 magnification in four microscopic fields of the HCC liver explants (n = 3; Fig. 6C, D). Compared to untreated HCC tissues, treatment with BZB alone resulted in no significant increase of caspase-3 activation and CK-18 cleavage, and also scTRAIL combined with BZB induced neither a significant increase of caspase-3 activation (6.33% ± 0.51%; Fig. 6C) nor of CK-18 fragments (5.35% ± 0.48%; Fig. 6D), compared to treatment with scTRAIL alone. EGFR-targeted scTRAIL significantly (P < 0.01) induced caspase-3 activation (4.04% ± 0.03%), but not CK-18 cleavage (4.79% ± 0.43%) in HCC tissues, compared to untreated control (data not shown).