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Hypoxia enhances 5HRE and AFPpregulated NTR/CB1954 suicide gene system in killing hepatocellular carcinoma cell line HepG2
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Abstract:
Objective:To evaluate the inhibitory effect of Nitroreductase/CB1954 (NTR/CB1954) suicide gene system，which contains 5 copies of hypoxiaresponsive element (5HRE) and promoter of alphafetoprotein gene (AFPp), against human hepatocellular carcinoma cell line HepG2 under hypoxia condition in vitro. Methods: 5HRE and AFPpregulated nitroreductase (NTR) gene eukaryotic expression vector was transfected into AFPpositive human hepatocellular carcinoma cell line HepG2 and AFPnegative human gastric carcinoma cell line MKN45 by Lipofectamine 2000. Stably transfected cell lines were selected by G418. RTPCR and Western blotting were employed to examine the expression of NTR mRNA and NTR protein, respectively. Prodrug CB1954 was added into stablytransfected cell lines; inhibitory activity of its derivative product (4hydroxylamine) 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 RTPCR 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 dosedependent inhibition on the proliferation of HepG2 cells as shown by MTT assay (P<0.05). But CB1954 did not inhibit proliferation of MKN45 cells and wild type HepG2 cells. Conclusion: 5HRE and AFPpregulated NTR/CB1954 suicide gene system can specifically kill human hepatocellular carcinoma cell line HepG2 under hypoxia condition.
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Project Supported:
Supported by the National Natural Science Foundation of China （No.30672070, No.30400430）
Clc Number:
