Hypoxia-inducible Factor-1 Luciferase Reporter Gene Construction And Anti-cancer Effects Of The Four Traditional Chinese Medicine Injection

BACKGROUND:At present, chemiotherapy is one of the most important methord in cancer therapy. But the adverse reaction made much suffering to the patient. Except the cancer itself we must pay attention to the inhance of patient's immunity and life quality. Now many Chinese compound have that effect. So it is our task to reveal its mechanism in anti-cancer effect.Hypoxia, a decrease in O2 levels, triggers adaptive responses in solid tumors that include induction of angiogenesis and a switch to anaerobic metabolism. Hypoxia inducible factor,(HIF-1α)is the most important transcription factor that related to hypoxia. HIF-1 is a basic helix-loop-helix Per-Arnt-Sim transcription factorcomposed of two subunits, HIF-1ɑand HIF-1β. HIF-1ɑlevels are primarily dependent on the intracellular oxygenconcentration . Under nonhypoxic conditions, HIF-1ɑprotein is rapidly and continuously degraded by ubiquitination and proteosomal degradation. In contrast, HIF-1βalso known as aryl hydrocarbon receptor nucleartranslocator, is constitutively present in normoxic cells. HIF-1 activates transcription of target genes by binding to a HRE that contains the HIF-1 core DNA binding site 5-RCGTG-3.HIF-1 is an attractive molecular target for development of novelcancer therapeutics. To identify small molecule inhibitors of HIF-1transcriptional activation, we have developed a HTS using engineered cell lines that express the luciferase reporter gene in a HIF-1-inducible fashion. C OCL2 is generally used as chemical hypoxia inducible factor. Because Co2+ is a competitive factor of Fe2+ .It decrease the affinity of O2 to haematoglobin.The PathDetect in vivo signal transduction pathway cis-reporting systems include a reporter plasmid and a positive or negative control plasmid.Expression of the Photinus pyralis (firefly)luciferase gene in the reporter plasmid is controlled by a synthetic promoter that contains direct repeats of the HIF-1α. And is useful for identifying drug candidates in a high-throughput format.To screen anti-cancer Chinese drug we have developed a cell-based HTS using engineered cell lines that express the luciferase reporter gene in a COCL2 HIF-1-inducible fashion. To combine the HRE-LUC activity screen results with in vitro validation we have developed Western blot to detect the HIF-1α, and RT-PCR was used to detect the gene transcription level of adenylate kinase-3(AK3), which is one of the target downstream genes of HIF-1. Cell proliferation is detected by MTT.OBJECTIVE: In this study we want to construct HRE-LUC reporter gene, on this basis we want to verify how Chinese medicine injections affects the protein stability and transcriptional activation of HIF-1 induced by cobalt chloride in Hela cell line. RESULTS: We have developed a cell-based HTS to identify small molecule inhibitors of the HIF-1 pathway, which may have antiangiogenic and anticancer activities. The proliferation of Hela cell is apparently repressed by AiDi injection. AIDI injection differentially and dose-relatedly repressed the relative value of luciferase(P<0.05). The results of Western blot showed that AIDI injection dose-relatedly repressed the congregation of HIF-1 protein induced by cobalt chloride. The transcription level of AK3 can be repressed by different dose of AIDI injection.CONCLUTION: We have developed a molecular targeted HTS that coherently identifies small molecule inhibitors of HIF-1 transcriptionalactivity and VEGF expression. Screening of larger chemical libraries may lead to the identification of active compounds that couldfind interesting applications in anticancer treatment. AiDi is related to inhibit HIF-1αaggregation and transcriptional activation,and is a potential anti-cancer drug.