2B). The sensitizing effect was further evidenced by an increase of cell apoptosis in response to treatment with sorafenib and lapatinib (Fig. 2C). We thus conclude that targeting Pirfenidone price NPM sensitizes HCC cells to oncogenic kinase inhibitors, such as sorafenib and lapatinib. NPM exerts its death evasion

activity via a mechanism independent of p53 function. NPM was up-regulated in Huh7, Hep3B, and Mahlavu cells following treatment with UV, cisplatin, and doxorubicin (Fig. 3A). Meanwhile, we also observed the induction of BAX expression, a key effector initiating mitochondria-mediated cell death, in all three cell lines (Fig. 3A). Simultaneous induction of NPM (antiapoptosis) and BAX (proapoptosis) represents hormetic mechanisms regulating cell survival versus death in response to stress.7, 26 To understand how NPM helps HCC cells evade death, we first inspected subcellular distribution of NPM and BAX in response to cell stress. Before UV irradiation, NPM was mainly located in the nucleoli and partially in the nucleoplasm (Fig. 3B, left), whereas BAX was primarily located in nucleoplasm and some in the cytoplasm (Fig. 3C, upper left).

Following UV irradiation, NPM was translocated from nucleoli to nucleoplasm, and a set of NPM was further translocated to the cytoplasm (Fig. 3B, right). On the other hand, BAX was translocated to the cytosol and accumulated in the mitochondria (Fig. 3C), particularly in cells undergoing apoptosis (Fig. 3C, right). Notably, as being transfected with siRNA to down-regulate the expression of NPM, cells with relatively Doxorubicin order low NPM expression usually presented with aggregation of BAX in the mitochondria and undergoing apoptosis (Fig. 4A), whereas cells with relatively high NPM level presented with less degree of mitochondrial accumulation of BAX and more resistance Bay 11-7085 to apoptosis induction (Fig. 4A). Interestingly, colocalization of NPM and BAX in the cytoplasm was

noted in some cells presenting with cytoplasmic NPM (Fig. 4B). These findings suggest that the antiapoptosis activity of NPM is involved in blockade of mitochondrial translocation of BAX. The observation that colocalization of a set of NPM with BAX in cytoplasm in HCC cells with relative resistance to death stimuli intrigued us to examine the role of NPM in mitochondrial translocation of BAX in HCC cells. As shown in Fig. 5A, the NPM level in the cytosol of Mahlavu cells was increased after UV irradiation, while NPM was not detected in the mitochondria either before or after UV irradiation. On the other hand, the amount of BAX was increased in both the cytosol and the mitochondria after UV irradiation. Silencing of NPM expression decreased the cytosolic BAX, but increased the mitochondrial BAX, suggestive of blockade of BAX mitochondrial translocation by NPM in response to UV treatment. Prohibitin and glyceraldehyde 3-phosphate dehydrogenase were used as the markers for mitochondrial and cytosolic components, respectively.