Overexpression of the GLI1 gene has frequently been found in various cancer types, particularly in brain tumors, in which aberrant GLI1 induction promotes cancer cell growth. Therefore, identifying the molecular players controlling GLI1 expression is of clinical importance. Previously, we reported that AMPK directly phosphorylated and destabilized GLI1, resulting in the suppression of the Hedgehog signaling pathway. The current study not only demonstrates that AMPK inhibits GLI1 nuclear localization, but further reveals that β-TrCP plays an essential role in AMPK-induced GLI1 degradation. We found that activation of AMPK promotes the interaction between β-TrCP and GLI1, and induces β-TrCP-mediated GLI1-ubiquitination and degradation. Inhibiting AMPK activity results in the dissociation of the β-TrCP and GLI1 interaction, and diminishes β-TrCP-mediated-GLI1 ubiquitination and degradation. On GLI1, substitution of AMPK phosphorylation sites to aspartic acid (GLI13E) results in stronger binding affinity of GLI1 with β-TrCP, accompanied by enhanced GLI1 ubiquitination and later degradation. In contrast, the GLI1 alanine mutant (GLI13A) shows weaker binding with β-TrCP, which is accompanied by reduced β-TrCP-mediated ubiquitination and degradation. Together, these results demonstrate that AMPK regulates GLI1 interaction with β-TrCP by phosphorylating GLI1 and thus both post-translational modifications by AMPK and β-TrCP ultimately impact GLI1 degradation.

Pardaxin (H-GFFALIPKIISSPLFKTLLSAVGSALSSSGGQE-OH), a 33-amino-acid polypeptide, is an antimicrobial peptide (AMP) isolated from the marine fish species Pardachirus marmoratus. Pardaxin shows antibacterial and antitumor activities. However, pardaxin-induced inhibition of oral cancer and the mechanism of tumor reduction in buccal pouch carcinogenesis after pardaxin painting remain undetermined. Additionally, the toxic effects of pardaxin on normal tissue remain unclear. The present study investigated the anticancer activity of pardaxin in oral squamous cell carcinoma (OSCC) cells in the hamster buccal pouch model with or without 7,12-dimethylbenz[a]anthracene (DMBA) pretreatment. This is the first study to confirm the effects of pardaxin on normal tissue and its nontoxic effects in vivo. Cell viability assays and colony formation tests in OSCC cell lines (SCC-4) demonstrated that pardaxin reduced cell viability in a dose-dependent manner. Immunofluorescence staining of cleaved caspase-3 in SCC-4 cells revealed that expression of activated caspase-3 in SCC-4 cells significantly increased after 24-h treatment with pardaxin. Additionally, a cell cycle analysis indicated that pardaxin treatment resulted in the cell cycle arrest of SCC-4 cells in the G2/M phase, thereby limiting cell proliferation. Furthermore, pardaxin treatment substantially alleviated carcinogenesis in the DMBA-induced hamster buccal pouch model by lowering prostaglandin E2 levels. These results suggest that pardaxin is a potential marine drug for adjuvant chemotherapy for human OSCC and oral cancer.