Involvement of NF-κB activation in the apoptosis induced by extracellular adenosine in human hepatocellular carcinoma HepG2 cells

Adenosine can exhibit cytotoxic activity in vivo and in vitro, though its mechanisms are still uncertain In this study, we investigated the adenosine-mediated apoptotic signaling pathway and the role of NF-kappa B in human hepatocellular carcinoma HepG2 cells. HepG2 cells were treated with different concentrations of adenosine for 12-48 h, and the effect of adenosine on cell proliferation was evaluated by ivrrr assay. The cytotoxicity of adenosine alone or in combination with an NF-kappa B inhibitor. pyrrolidine dithiocarbamate (PDTC), was also evaluated by MIT assay and the mode of cell death was detected by Hoechst 33342 staining. Cell cycle progress was performed by flow cytometry with PI staining The protein expressions of Bc1-2. p53, NF-kappa B subunit p65. and caspase-3 were assayed by Western blot. Caspase-3 activity was measured by spectrophotomtenc assay. The results showed that adenosine significantly reduced the viability of HepG2 cells in a dose- and time-dependent manner, with IC 50 (24 and 48 h) of 2.52 and I 89 inmol"L-1, respectively. The apoptotie index (percentage of sub-G1 phase) of HepG2 cells in adenosine treatment alone for 12 and 24 +/- 11 or in combination with PDTC were 8 30%. 22.32% and 20 18%. 3089%. respectively All of them were higher than that in the control group (0 81%, p < 001). The characteristic changes of cell apoptosis (chromatin condensation and sub-G1 peak) were observed under fluorescent microscopy and flow cytometry We also found that the apoptotic process triggered by adenosine was involved in G(0)-G(1) cell-cycle arrest, enhanced the activity of caspase-3. upregulated p53 and NF-kappa B p65 expression, and downrceulated Bc1-2 expression Inhibition of NF-kappa B by PDTC decreased NF-kappa B p65 expression, enhanced cell apoptosis ratio, and increased caspasc-3 activity NF-kappa B may play an anti-apoptosis role in adenosine-induced HepG2 cytotoxicity