We confirmed NVP-AUY922 datasheet both HBS and HAS at sites HRE1 and HRE3. We also found a typical consensus HRE site in the CypB promoters of the mouse, rat, and monkey. To further confirm the responses of the four putative consensus HRE sites to hypoxic conditions, we designed several luciferase reporter constructs and conducted luciferase assays. The luciferase assay demonstrated that only the pGL3-CypB-350 construct had a significant increase in luciferase

activity under hypoxic conditions, compared with normoxic conditions; pGL3-CypB-350M and pGL3-CypB-150M/350M constructs, containing 5′-AAAG-3′, rather than 5′-CGTG-3′, at the HRE site, exhibited reduced luciferase activity. The empty pGL3 basic plasmid did not alter luciferase activity under either normoxic or hypoxic

conditions (Fig. 2D). To confirm the activity of HIF-1α transactivation on the CypB promoter, we overexpressed HIF-1α via transfection of the pcDNA3-HIF-1α expression vector under normoxic conditions. Results were the same see more as those observed under hypoxic conditions, in terms of luciferase activity (Fig. 2D). We then evaluated the physical interaction between HIF-1α and HRE3 of the CypB promoter via an electrophoretic mobility shift assay (EMSA). As indicated in Fig. 2E, only the WT oligonucleotide incubated with the nuclear extracts from Huh7 cells under hypoxic conditions exhibited a strong, mobility-shifted band, whereas the mutated oligonucleotide and 100-fold excess of cold oligonucleotide yielded noticeably attenuated bands. The nuclear extracts from HepG2 cells also showed similar results (data not shown). To confirm these results, we conducted chromatin immunoprecipitation (ChIP) assays. HIF-1α directly bound to the CypB promoter located at −266 bp (HRE3) (Fig. 2F). These results indicate that CypB is a hypoxia-inducible gene and MCE its expression can be induced transcriptionally by HIF-1α in HCC. HIF-1α activates the transcription of diverse genes related to growth and survival in solid tumor cells.21 We demonstrated that CypB is directly induced by HIF-1α. Therefore, we hypothesized that CypB is involved in HCC cell growth and survival,

particularly in response to cellular stresses induced by hypoxia, cisplatin treatment, and H2O2 treatment. Initially, to examine the direct role of CypB in cell death under these stress conditions, we prepared a stably transfected cell line by transfecting Huh7, PLC/PRF/5, HepG2, and Hep3B cells with Mock and pcDNA-CypB/WT or a CypB knockdown cell line with scrambled siRNA and CypB siRNA. After the hypoxia, cisplatin, and H2O2 treatments, the pcDNA-CypB/WT–transfected cell line evidenced better survival rates than the Mock-transfected cell line, but the CypB siRNA-transfected cell line evidenced markedly reduced cell survival, compared with the scrambled siRNA-transfected cell line (Fig. 3A). Interestingly, the same results were observed in the p53-defective HCC cells, such as Hep3B cells (Fig.