Pro-apoptotic Sorafenib signaling in murine hepatocytes depends on malignancy and is associated with PUMA expression in vitro and in vivo.

The multi-kinase inhibitor Sorafenib increases the survival of patients with advanced hepatocellular carcinoma (HCC). Current data suggest that Sorafenib inhibits cellular proliferation and angiogenesis and promotes apoptosis. However, the underlying pro-apoptotic molecular mechanisms are incompletely understood. Here we compared the pro-apoptotic and anti-proliferative properties of Sorafenib in murine hepatoma cells and syngeneic healthy hepatocytes in vitro and in animal models of HCC and liver regeneration in vivo. In vitro, we demonstrate that cell cycle activity and expression of anti-apoptotic Bcl-2 like proteins are similarly downregulated by Sorafenib in Hepa1-6 hepatoma cells and in syngeneic primary hepatocytes. However, Sorafenib-mediated activation of caspase-3 and induction of apoptosis were exclusively found in hepatoma cells, but not in matching primary hepatocytes. We validated these findings in vivo by applying an isograft HCC transplantation model and partial hepatectomy (PH) in C57BL/6 mice. Sorafenib treatment activated caspase-3 and thus apoptosis selectively in small tumor foci that originated from implanted Hepa1-6 cells but not in surrounding healthy hepatocytes. Similarly, Sorafenib did not induce apoptosis after PH. However, Sorafenib treatment transiently inhibited cell cycle progression and resulted in mitotic catastrophe and enhanced non-apoptotic liver injury during regeneration. Importantly, Sorafenib-mediated apoptosis in hepatoma cells was associated with the expression of p53-upregulated-modulator-of-apoptosis (PUMA). In contrast, regenerating livers after PH revealed downregulation of PUMA and were completely protected from Sorafenib-mediated apoptosis. We conclude that Sorafenib induces apoptosis selectively in hepatoma cells but not in healthy hepatocytes and can additionally increase non-apoptotic hepatocyte injury in the regenerating liver.

Synergy between expression of fusogenic membrane proteins, chemotherapy and facultative virotherapy in colorectal cancer.

Using Chou-Talalay median effect analysis, we demonstrated in permanent and short-term cultures of colorectal cancer cells that the expression of measles virus fusogenic membrane glycoproteins (FMGs) in combination with chemotherapy often causes over most of the cytotoxic dose range synergistic cell killing. In this combined treatment, we observed strongly enhanced annexin V binding and caspase-3/7 activity when compared to single-agent treatment. Furthermore, we showed increased expression of heat-shock protein (Hsp)70 and Hsp90alpha, but not of Hsp60. In a subcutaneous HT-29 colorectal xenograft model, we demonstrated that the administration of a replication-defective adenoviral or herpes simplex virus (HSV) amplicon vector (Ad.H/F or HSV.H/F) encoding tumor-restricted FMG in combination with FOLFOX significantly enhanced treatment outcome when compared to treatment with each compound individually. To increase the fraction of tumor cells expressing the FMG, we trans-complemented the Ad.H/F and HSV.H/F vector with the respective oncolytic replication-restricted adenovirus Ad.COXDeltaMK or HSV-1 G47Delta vector. At the end of the observation period (day 100), eight out of 10 animals that received G47Delta, HSV.H/F and FOLFOX were alive and tumor free. Administration of the analogous adenovirus-based regimen resulted in four out of 10 long-term survivors. We demonstrated that the expression of FMG in combination with chemotherapy can significantly enhance treatment outcome, which is further enhanced by combination with trans-complementing oncolytic vectors.