Together, these results show inhibitor MEK162 that nilotinib induces autophagy and autophagic cell death by activation of AMPK. Next, we showed that nilotinib-mediated AMPK activation is through PP2A inhibition, rather than being regulated by the canonical LKB1/STAND/Mo-25-complex or CAMKK pathways. It has been reported that PP2A regulates the interaction between the AMPK�� and �� subunits (37), and dephosphorylates AMPK�� in a cell-free system (38). Recently, AMPK inhibition induced by glucose, palmitate, or ethanol was also found to be mediated by PP2A activation (39�C41). One previous finding also revealed that PP2A mediates AMPK inhibition to up-regulate heat shock protein (HSP) 70 expression during stress (26). Exploration of the AMPK-downstream signaling cascades revealed that nilotinib-mediated AMPK activation to induce autophagy is via an mTOR-independent pathway.

The serine/theroine kinase mammalian target of rapamycin (mTOR) is a major negative regulator of autophagy (42), and AMPK serves as one of the main mTOR regulators. Activation of AMPK inhibits mTOR phosphorylation and elicits autophagy. However, recent studies have documented the existence of mTOR-indepenent autophagy (43). For example, a decrease in intracellular inositol or inositol 1,4,5-triphosphate (IP3) levels by lithium is known to induce autophagy in neurobalstoma cells (44). The correlation between autophagy and tumorigenesis has been explored extensively, but whether autophagy acts as a pro-tumorigenic or antitumor player in tumor development and cancer therapy is still unclear.

Recently, it has been suggested that cancer cells have evolved to require autophagy under basal conditions, implying cell-autonomous roles for autophagy in tumor maintenance. For example, autophagy has been demonstrated to be required for continued cell growth in pancreatic cancers (45). Inhibition of autophagy also results in metabolic turbulence, reduced oxidative phosphorylation and decreased ATP production (46). In contrast, accumulating evidence shows that suppression of the proteins involved in autophagy such as Beclin-1 and Atg-5 may cause acceleration of tumorigenesis. Deletion of one copy of an autophagy-related gene (47), or reduced expression level of such genes has been found in certain types of cancer cells (48). Our present data show that nilotinib induces autophagy which contributes to cytotoxicity, rather than drug-resistance in HCC cells.

This finding indicates that autophagy plays a tumor-suppressive role in liver cancer cells treated with nilotinib. Furthermore, as we observed no significant changes in autophagy-related proteins such as Atg-5,-7, or Beclin-1, we speculate that nilotinib induces an unconventional type autophagy. Atg-5/Atg-7-independent alternative autophagy has been discovered in several embryonic tissues revealing that autophagy can occur through at least two different Cilengitide pathways (49).