A, dose-dependent effects of nilotinib on cell viability. HCC cells were treated with nilotinib at the indicated concentrations for 72 h. Cell viability was measured by MTT assay. … Nilotinib Induces Autophagy in HCC Cells Autophagy is the process MEK162 MEK inhibitor of sequestrating cytoplasmic proteins into lytic compartments and is characterized by the formation of the autophagosome, a double membrane structure that sequesters the target organelle/protein and then fuses with endo/lysosomes where the contents and its major component, LC3, are degraded (18�C19). We therefore investigated whether nilotinib could elicit autophagy in HCC cells by Western blot analysis. As shown in Fig. 2A, significantly increased expression of lipidized LC3 (LC3-II) was observed in nilotinib-treated HCC cell lines in a time-dependent manner.

Next, another distinct marker of autophagy, AVO induction (20), was examined by staining with vital acridine orange. Similarly, the result showed that nilotinib promoted the accumulation of AVOs in the cytoplasm of Hep3B cells (Fig. 2B). Previous studies have proven that 3-methyladenine (3-MA), an inhibitor of phopshatidylinositol 3-kinase, can inhibit autophagy (20). Therefore, we next used 3-MA to investigate nilotinib-induced autophagy in HCC cells. As shown in Fig. 2C (top), nilotinib-induced cleavage of LC3 was attenuated by treatment with 1 mm 3-MA. hydroxychloroquine (HCQ) also inhibits autophagy by raising lysosomal pH leading to inhibition of both the fusion of the autophagosome with the lysosome, and lysosomal protein degradation.

Prevention of autophagy by co-treatment with HCQ further proved that autophagy could be triggered by nilotinib (Fig. 2C, bottom). Furthermore, autophagy-related proteins including ATG3, 5, 7, 12, Beclin-1, and P62 showed constant expression levels in all tested HCC cell lines treated with nilotinib (Fig. 2D). Taken together, the data presented here indicate that nilotinib induces autophagy in HCC cells. FIGURE 2. Nilotinib induces autophagy in HCC. A, top, dose-dependent effects of nilotinib-induced autophagy. HCC cells were treated with nilotinib at the indicated concentrations for 24 h. Bottom, time-dependent effects of nilotinib-induced autophagy. Cells were … Nilotinib-induced Autophagy Relies on the AMPK Signaling Pathway We next validated the target pathway by which nilotinib signals autophagy in HCC cells. Upstream regulation of autophagy occurs via different signaling pathways, including AMP-activated AV-951 protein kinase (AMPK), which is a heterotrimeric complex consisting of a catalytic ��-subunit and regulatory ��- and ��-subunits (21). AMPK is activated by various conditions of stress that are known to induce autophagy.