1C) in 50-week-old primary WT tumor cells treated with anisomycin, we tested AR and p38 effects on cell anoikis. As shown in Fig. 2F, we found that
anisomycin reduces anoikis in the 50-week-old WT mice scramble (sc)-treated hepatic cells (57% ± 8% to 39% ± 4%; P = 0.04). However, when comparing cells in the AR siRNA-treated groups we found that anisomycin has a more dramatic impact on reducing cell anoikis (36% ± 4% to 18% ± 0.4%; P = 0.01) (Fig. 2F). The AR-related anisomycin suppression on cell anoikis reached statistical significance (P = 0.03). Our data consistently show that anisomycin reduces anoikis and AR enhances anoikis; furthermore, 3-deazaneplanocin A solubility dmso anisomycin treatment of sc/siAR-infected WT primary cells (Fig. 2F) showed the anoikis between sc versus siAR RNA P = 0.003, which is consistent with our hypothesis. When comparing anisomycin PD0325901 in vitro effect on scAR (lane 1 versus 2; P = 0.04) and siAR (lane 3 versus 4; P = 0.01) cells, the anisomycin was differentially impacted in sc versus siAR cells. Together, the results from Fig. 2E,F suggest that the hepatic AR enhanced cell anoikis, at least in part, by modulating p38 phosphorylation.
To further confirm that AR could enhance cell anoikis in the HCC cells, we repeated those experiments using mouse HCC cells with human HCC cells using previously established SKAR+ cells7 (SKhep1 cell with AR stable expression) and HepG2-AR cells.25 We demonstrated that addition of AR in the human HCC SKAR+ and HepG2-AR cells resulted in increased cell anoikis (Fig. 3A,D). We also demonstrated that addition of AR led to suppression of p-p38 (Fig. 3B,E), and addition of the p38 agonist anisomycin reduced
cell anoikis, whereas expression of AR reversed that effect (Fig. 3C,F). A previous report indicated FasL expression medchemexpress was associated with cell anoikis,26 which was also observed in our system (Supporting Fig. 2A). Furthermore, anisomycin could reduce, although addition of AR could enhance, FasL expression while the cells were detached (Supporting Fig. 2A). Together, the results from Figs. 2 and 3 strongly suggested that AR might increase cell anoikis by way of suppression of p-p38. As early studies suggested that cells with anoikis resistance ability is positively correlated with increased tumor metastasis,23, 27 it is possible that higher AR expression could negatively modulate p38-mediated cell anoikis resistance in HCC progression, which might be one reason why hepatic AR could switch from promotion of HCC initiation to suppression of HCC metastasis at the metastatic stage. In addition to cell anoikis, cell invasiveness (from one foci to multiple foci within the liver) is another important step contributing to the liver tumor metastasis.27 We noticed that the expression of MMP9, an important liver cancer migration marker,13, 28 was higher in the HCC tumors of L-AR−/y mice as compared with those in AR+/y mice (Fig. 4A).