As shown in Fig. 4B, knocking down AR with siRNA in the AR+/y primary cultured HCC cells enhanced cell migration, and addition of AR in L-AR−/y primary cultured HCC cells AZD6738 order reduced cell migration. As knockdown of AR also increased migration, but not cell mobility, in SKAR+ human HCC cells (Fig. 4C; Supporting Fig. 2C), we further examined MMP9 messenger RNA (mRNA)/protein expression in the SKAR+ cells and found that the addition of DHT reduced MMP9 mRNA and protein expression (Fig. 4D). As an early study suggested that prostate epithelial AR could suppress MMP9 expression by way of modulation of NF-κB activity,29 we examined NF-κB expression in the mice HCC.

Indeed, our data showed loss of hepatic AR led to higher expression/activation of NF-κB in 60-week-old livers (Fig. 4E). Mechanistic INK 128 in vitro dissection revealed that AR could suppress the tumor necrosis factor alpha (TNF-α)-induced NF-κBRE-luciferase activity29, 30 and MMP9 promoter-luciferase activity in SKAR+ human HCC cells (Fig. 4F). Together, the results from Fig. 4, Supporting Fig. 2B,C suggest that hepatic AR could also function through the NF-κB-MMP9 pathway to modulate cell migration ability to suppress HCC metastasis. With the contradictory AR functions (tumor initiation versus migration/anoikis) taken into

consideration in HCC therapy, we hypothesized that applying current molecular targeting agents (suppressing cell growth and migration) combined with the addition of AR (suppressing cell migration and anoikis) might benefit the current

PI-1840 therapeutic paradigm. As the above results demonstrated that AR could play negative roles on HCC metastasis, we were encouraged to determine if we could enhance the therapeutic efficacy of HCC survival by targeting hepatic AR. Sorafenib (Bayer), a molecular target agent that has passed phase III clinical trials by way of targeting multiple kinases, yet has higher median inhibitory concentration (IC50) on p38,31 has been applied to treat advanced HCC patients with some benefits and fewer complications.32 We first titrated the sorafenib dose in human SKhep1 HCC cells and found 5 μM of sorafenib had a moderate cytotoxicity effect during 2 days treatment (Fig. 5A). Using this dose, we found sorafenib reduced ERK phosphorylation (pERK) significantly, yet had little influence on p-p38 (Fig. 5B, lane 3 versus 1). However, adding AR with 5 μM of sorafenib resulted in abolished p-p38 (Fig. 5B, lane 4 versus 2). Furthermore, we found that sorafenib treatment alone could enhance cell anoikis and reduce cell migration in the SKhep1 cells (Fig. 5C,D, lane 2 versus 1), and addition of AR alone could also enhance SKhep1 cell anoikis and suppress SKhep1 cell migration (Fig. 5C,D, lane 3 versus 1). As expected, the combination treatment of adding AR plus sorafenib resulted in additive enhancement of cell anoikis and suppression of migration (Fig. 5C,D, lanes 4 versus 3).