Bax and

Bcl-2 proteins play a central regulatory role in apoptotic cell death. Therefore, the expression levels of Bax and selleck products Bcl-2 following NX treatment were measured by western blot analyses. As shown in Fig. 7A, NX treatment (2.5–10.0 μg/ml) resulted a dose-dependent increase in the expression level of Bax and decrease in the expression level of Bcl-2. To further confirm whether modulation of Bax/Bcl-2 ratio is correlated with the release of cytochrome c in cytosol, the levels of cytochrome c in the cytosolic fraction were measured. We found the levels of cytochrome c were significantly elevated in a dose-dependent manner following NX treatment as shown in Fig. 7A. It is well documented that the apoptotic process is executed by

cysteinyl aspartate-specific proteases known as caspases, which PCI-32765 solubility dmso demolish the cell in an orderly fashion by cleaving a large number of cellular protein substrates [21]. Therefore, activation of caspases 3 and 9 was assessed after NX treatment by western blot analyses. Results indicated that NX treatment resulted in increased levels of cleaved-caspases 3 and 9 in a dose-dependent manner, while there was no change in expression level of caspase 8 ( Fig. 7A). Altered expression of cell cycle regulatory protein such as CDKs and cyclins has been implicated in tumorigenesis [22] and [23]. As our results demonstrated inhibition of cell proliferation upon NX treatment, we further examined it’s effect

on the expression of cell regulatory proteins. As shown in Fig. 7B, NX exposure caused a decrease in cyclinE, cyclinD1, CDK2 and CKD4 levels in liver cancer cells. During cell cycle analysis we found that NX treatment caused G1 phase cell cycle arrest. We also found from immunoblot analysis that NX treatment caused significant induction of p21WAF, a key regulator of G1-S phase transition, in a dose-dependent manner (Fig. 7B). Kip1/p27 is another important CDK inhibitor that regulates Cdk-cyclin activity at G1-S transition [24]. Protein levels of Kip1/p27 were also strongly upregulated after NX exposure. In addition, we found that NX treatment to liver cancer cells caused a dose-dependent increase expression of p53 (Fig. 7B). Further, we investigated the level medroxyprogesterone of activated (phosphorylated) and total ERK1/2, JNK and p38 kinases in NX-treated HepG2 cells and found phosphorylation of ERK1/2, JNK, and p38 kinase levels were downregulated by NX without any change in their total protein levels (Fig. 7C) The present study we have shown that NX inhibited 2-AAF-mediated liver tumor promotion in DEN-initiated rats, which was correlated with a decrease in proliferation index together with inhibition of COX-2, iNOS and PCNA expression. Besides its anti-tumor promoting activity, we also observed that NX causes apoptotic cell death to human liver cancer cells. Cancer development is a sequential event which often involves chronic inflammation and hyperplasia.