In the present study, we observed that both COX-2 and iNOS protein expression were elevated in DEN/2-AAF-treated rat liver (Fig. 2 and Fig. 3) respectively. Interestingly, dietary exposure of NX (300 and 600 ppm) resulted in substantial decrease in COX-2 and iNOS expression in DEN/2-AAF-treated rat liver (Fig. 2 and Fig. 3) respectively. These results suggest that NX suppresses DEN/2-AAF-induced inflammation by down regulating COX-2 and iNOS expression see more in the rat liver. PCNA is an auxiliary protein of DNA polymerase-delta and higher level of its expression is correlated with cell proliferation, suggesting PCNA is an excellent marker of cellular proliferation [20].

In our study, the PCNA antigen was not expressed in liver sections of control rats (Fig. 4A). However, liver sections from DEN/2-AAF-treated MEK inhibitor rats were positive for the PCNA staining, indicative of active cell proliferation in liver tissue (Fig. 4B). We observed lower PCNA expression (Fig. 4C–D) in the treatment

groups of NX with DEN/2-AAF suggesting NX has an anti-proliferative effect on DEN/2-AAF-induced liver tumorigenesis in rats. An apoptotic response of NX in the liver tissue of DEN/2-AAF-induced rats was investigated using TUNEL staining. Representative photographs for TUNEL-positive cells in DEN/2-AAF-treated alone or NX with DEN/2-AAF-treated animals are shown in Fig. 5. There was an increase in the number of TUNEL positive cells in the livers of NX +DEN/2-AAF treated rats (Fig. 5C–D) compared to DEN/2-AAF-treated rats (Fig. 5B). However, the apoptotic induction by NX was more pronounced in the group where 600 ppm of NX was given along with DEN/2-AAF

(Fig. 5D). The inhibitory effect of NX (0.5–20.0 μg/ml) on the growth of liver cancer cells was assessed by MTT assay and is shown in Fig. 6A. Treatment with NX (0.5–20.0 μg/ml) for 24 h decreased the cell viability by 12–66%; while, at 48 h, the decrease in cell viability was IMP dehydrogenase even more pronounced (16–88%). Based on these findings, we selected NX doses of 2.5, 5.0 and 10.0 μg/ml and 48 h time point for further studies. In view of above mentioned growth inhibitory effect, we were interested in determining whether NX also induces apoptosis in liver cancer cells. It was observed that treatment of liver cancer cells for 48 h with 2.5–10.0 μg/ml NX increases the number of apoptotic cells from 3.7 to 16.0%. The total percent of apoptotic cells was directly related to NX concentration increasing from 3.7% (control) to 16.0% (10 μg/ml), indicating that NX-induced apoptosis of liver cancer cell is dose-dependent (Fig. 6C). As the induction of apoptosis might also be mediated through the regulation of the cell cycle, we also examined the effect of NX treatment on cell cycle perturbations compared with the vehicle alone treatment. As shown in Fig. 6B, exposure of NX (2.5–10.