[10] To gain insight into variable status between autophagy and apoptosis, we compared the apoptotic effect of GANT61 with other chemotherapeutic agents which have been reported to have cytotoxic effects in HCC cells at indicated concentrations (listed in Supporting Table 1). As shown in Fig. 7A, inhibition of autophagy by 3-MA and CQ partially reversed the cytotoxic effect Nutlin-3a solubility dmso induced by GANT61,

sorafenib (an FDA-approved multikinase inhibitor for treatment of HCC in patients) and other chemotherapeutic/chemopreventive agents in Huh7 cells. However, in HepG2 and Hep3B cells, 3-MA and CQ exhibited variable effects depending on specific chemotherapeutic/chemopreventive agents. Thus, autophagy may contribute to cell survival or death depending on specific context and different cell types. We observed that 3-MA and CQ prevented cytotoxicity induced by GANT61 and sorafenib consistently in all three cell lines. The latter finding is consistent with the data of flow cytometry using Annexin-V/propidium iodide staining showing that inhibition JNK pathway inhibitors of autophagy by 3-MA and

CQ prevented GANT61 and sorafenib-induced apoptosis in Huh7 cells (Fig. 7B). These results are noteworthy, given the role of the Gli inhibitor GANT61 for induction of autophagy and apoptosis in HCC cells as documented in the current study and the fact that sorafenib is the only therapeutic agent currently available for systemic therapy of HCC in patients. To assess the antitumor potential of GANT61 and the role of autophagy in vivo, we employed a tumor xenograft model in which Huh7 cells were inoculated into SCID mice and the animals were

treated intraperitoneally with vehicle control, GANT61, or GANT61 in combination with 3-MA click here (intraperitoneal injection, started 1 week after inoculation, performed every other day for 4 weeks). As shown in Fig. 8A,B, GANT61 treatment significantly inhibited Huh7 tumor growth and the effect was attenuated by the autophagy inhibitor 3-MA. Induction of autophagy in GANT61-treated tumor was confirmed by immunofluorescent staining and immunoblotting for LC3II (Fig. 8C,D). GANT61 treatment increased the cleavage of caspase-3 and caspase-8 in tumor tissues and the effect was partially reversed by cotreatment with 3-MA. These results suggest that GANT61-induced autophagy contribute to HCC cell apoptosis and cytotoxicity in vivo and that the activity of autophagy is a key factor that determines the efficacy of Hh-targeted therapy. This study provides novel evidence that the Hh signaling pathway is a key regulator of autophagy in HCC cells. Although Hh signaling activation in HCC cells may exert an effect on other cell types in the liver (such as hepatic stellate cells, liver progenitor cells, and tumor-initiating stem-like cells),[24] our data provide the first evidence for an autocrine action of Hh signaling in HCC cells.