Hypoxia enhances 5HRE and AFPp-regulated NTR/CB1954 suicide gene system in killling hepatocellular carcinoma cell line HepG2

Objective: To evaluite the inhibitory effect of Nitro-reductase/CB1954 (NTR/CB1954) suicide gene system, which contains 5 copies of hypoxia-responsive element (5 HRE) and promoter of alpha-fetoprotein gene (AFPp), gainst human hepatocellular carcinoma cell line HepG2 under hypoxia condition in vitro. Methods: 5HRE- and AFPp-regulated nitroreductase (NTR) gene eukaryotic expression vector was transfected into AFP-positive human hepatocellular carcinoma cell line HepG2 and AFP-negative human gastric carcinoma cell line MKN45 by Lipofectamine 2000. Stably transfected cell lines were selected by G418. RT-PCR and Western blotting were employed to examine the expression of NTR mRNA and NTR protein, respectively. Pro-drug CB1954 was added into stably-transfected cell lines; inhibitory activity of its derivative product (4-hydroxylamine) was observed by MTT. Results: Monoclonal HepG2 cells stably expressing NTR were successfully obtained. Expression of NTR mRNA and NTR protein in monoclonal HepG2 cells under hypoxia condition was significantly higher than those under normoxia condition as confirmed by RT-PCR and Western blotting, respectively. Monoclonal MKN45 cells did not express NTR protein under either hypoxia condition or normoxia condition. NTR effectively activated CB1954 under hypoxia condition, resulting in dose-dependent inhibition on the proliferation of HepG2 cells as shown by MIT assay (P <0.05). But CB1954 did not inhibit proliferation of MKN45 cells and wild type HepG2 cells. Conclusion: 5HRE- and AFPp-regulated NTR/CB1954 suicide gene system can specifically kill human hepatocellular carcinoma cell line HepG2 under hypoxia condition.