Establishation Of A Cellular Model To Screen For New Antitumor Drugs Targeting HIF-1

Hypoxia is a hallmark of solid tumors, due to the rapid growth of cancer cells and the abnormal angiogenesis within them. Accumulating evidence have shown that hypoxic cells are more resistant to killing by ionizing radiation and chemotherapy, are more invasive and metastatic, and are resistant to apoptosis.The cause of this phenomenon is the upregulation of a series of genes and proteins mediated by hypoxia. The Hypoxia Inducible Factor 1(HIF-1) plays a very important role through this progress.HIF-1 is a heterodimer of HIF-1αand HIF-1β. Unlike HIF-1β, which is constitutively expressed, HIF-1αis a oxegen regulated subunit. In normoxia, HIF-1αis hydroxylated in a oxegen-dependent way once it is translated, which triggers the association of HIF-1αand pVHL E3 ligase complex,leading to HIF-1αdegradation via ubiquitin-proteasome pathway(half time <5 min). In hypoxia however, HIF-1αis stabilized and translocate to nucleus, where it interacte with HIF-1βto form HIF-1 which then binds to the Hypoxia Responsible Element (HRE) of the target genes, activating the transcriptions. Within tumors HIF-1 regulates a series of genes transcription that participate in angiogenesis, glucose metabolism, cell proliferation, and cell migration/invasion. The activation of HIF-1 involves lots of signal pathways and there are various post translation modifications to the HIF-1αsubunit such as hydroxylation, acetylation, phosphorylation, ubiquitination, SUMOlation, etal. As a result, the HIF-1 inhibitors discovered so far are mostly non-specific. Looking for the specific ones has been the focus recently. This paper aims to express the protein-protein binding domain (bHLH-PAS domain) of HIF-1αand HIF-1βin vitro, and synthesize the canonical HRE sequence(5'-GTGCTACGTGCTGCCCTG -3'), and further establish a model that can be utilized to screen for specific HIF-1 inhibitors.1. The construction of the recombinant expression vector of HIF-1α/βbHLH-PAS domains.Firstly, using the RT-PCR method, we amplified cDNA of the bHLH-PAS domains of HIF-1αand HIF-1βfrom the human breast cancer cell MDA 468 cDNA. Secondly, after digestion and ligation we ligated the amplified cDNAs into the pFastBac HTb vector respectively. We then selected the correct vector(s) through digesting and sequencing. Nextly, the DH10αwas transfected with the selected recombinant vectors, and we finally got the positive clones after blue/white color selection. The positive Bacmids were extracted for later experiments.2. The expression of HIF-1α/βbHLH-PAS domains.In this part we expressed the recombinant priteins using the Bac-to-Bac Baculoviruse Expression System of Invitrogen Company. After that we purified the RPs utilizing the Ni-NTA column. At last the RPs were identified via SDS-PAGE and Western blot. The result is that we got the bHLH-PAS domains of HIF-1αand HIF-1βsubunit successfully, nominated as HIF-1α–RP and HIF-1β-RP.3. Evaluation of the activity of HIF-1α–RP and HIF-1β-RP and establishation of the cellular model ELISA.Firstly, we synthesized the wild type HRE sequence (5'-GTGCTACGTGCTG-CCCTG-3') and mutant HRE sequence (5'-GTGCTA- AAAGCTGCCCTG -3') and labeled the wtHRE with biotin at 3'. Then we tested the specific interaction between the RPs and the synthesized HRE using EMSA assy. According to the principle of Enzyme-Linked Immunosorbent Assay( ELISA) and utilizing Echinomycin as the positive control, we established a ELISA assay which could be used as a promising model to screen for specific HIF-1 inhibitor. The result showed that the RPs worked well and the model was founded.