Progress in pathophysiology and related drug development of hypoxia-inducible factor-1 / 药学实践杂志

Hypoxia-inducible factor-1 (HIF-1) is a critical nuclear transcriptional factor mediating cell adaptive response to hypoxia in mammalian and human .It is the key mediator which modulates oxygen homeostasis exclusively .In the one hand , HIF-1 can protect and promote kinds of physiological processes , such as embryo normal development , cartilage and bone formation .In the other hand, it is also involved in lots of human deceases which is caused by ischemia and hypoxia , such as tumor, diabetes and its complica-tions.The molecular mechanisms of HIF-1 involved in these diseases have become a research hotspot and such studies will provide the new therapeutic means for these diseases , recent new drug researches have been focused on HIF-1 related signal pathway inhibitors , HIF-1 activity inhibitors, HIF-1 targeted therapy, etc.

Long-term and stable correction of uremic anemia by intramuscular injection of plasmids containing hypoxia-regulated system of erythropoietin expression

Relative deficiency in production of glycoprotein hormone erythropoietin (Epo) is a major cause of renal anemia. This study planned to investigate whether the hypoxia-regulated system of Epo expression, constructed by fusing Epo gene to the chimeric phosphoglycerate kinase (PGK) hypoxia response elements (HRE) in combination with cytomegalovirus immediate-early (CMV IE) basal gene promoter and delivered by plasmid intramuscular injection, might provide a long-term physiologically regulated Epo secretion expression to correct the anemia in adenine-induced uremic rats. Plasmid vectors (pHRE-Epo) were synthesized by fusing human Epo cDNA to the HRE/CMV promoter. Hypoxia-inducible activity of this promoter was evaluated first in vitro and then in vivo in healthy and uremic rats (n = 30 per group). The vectors (pCMV-Epo) in which Epo expression was directed by a constitutive CMV gene promoter served as control. ANOVA and Student's t-test were used to analyze between-group differences. A high-level expression of Epo was induced by hypoxia in vitro and in vivo. Though both pHRE-Epo and pCMV-Epo corrected anemia, the hematocrit of the pCMV-Epo-treated rats exceeded the normal (P < 0.05), but that of the pHRE-Epo-treated rats didn't. Hypoxia-regulated system of Epo gene expression constructed by fusing Epo to the HRE/CMV promoter and delivered by plasmid intramuscular injection may provide a long-term and stable Epo expression and secretion in vivo to correct the anemia in adenine-induced uremic rats.

Construction and identification of a double-regulated replicative adenovirus AdTPHre-hEndo / 中华肝胆外科杂志

Objective To develop a double-regulated replicative adenovirus carrying the Human endostatin gene(hEndo). Methods The plasmid pTPHre-hEndo was constructed by gene engineering technique, carrying human endostatin gene, in which El A gene and E1B gene were driven by human telomerase reverse transcriptase (hTERT) promoter and hypoxia response element (HRE) promoter,respectively. The pTPHre-hEndo was co-transfected with pBHGE3 in 293 cells to generate recombinant adenovirus AdTPHre-hEndo. Virus titer was measured by the TCID50 method. Virus replication assay was performed to evaluate the selective replication ability of AdTPHre-hEndo. The transgene expression of endostatin was detected by ELISA assay. Results A novel gene-viral therapeutic system AdTPHre-hEndo was constructed by gene engineering technique and its titer was 3. 25 X 1010 pfu/ml.Proliferative test revealed that AdTPHre-hEndo could proliferate selectively in telomerase positive tumors. Furthermore, in comparison with non-replicative adenovirus Ad-hEndo, the transgene expression of endostatin mediated with AdTPHre-hEndo was significantly increased (P < 0. 01).Conclusion The novel gene-viral therapeutic system AdTPHre-hEndo has the capacity to replicate in pancreatic cancer cells and expresses the endostatin efficiently, and may provide a new strategy for pancreatic cancer gene therapy.

Therapeutic effect of triple regulated adenovirus vector expressing mda7/IL24 on liver cancer in vitro / 肿瘤

Objective: To construct an E1 A-deleted 24-bp triple regulated replicative adenovirus vector SG600/interleukin24 (IL24), which was driven by both hTERT promoter and HRE promoter. The level of IL24 in liver cancer cells was determined and the replication capacity of SG600/ IL24 and its killing effects on liver cancer cells were observed. Methods: SG600-IL24 vector was constructed using DNA cloning and recombination techniques. The IL24 gene expression in liver cancer cell lines SMMC-7721 and BEL-7404 and normal cell line BJ was detected by ELISA assay. The replications of SG600/IL-24 in different cell lines were determined by evaluating TCID50 (50% tissue culture infectious dose) at 49 and 96 h. In vitro cell-killing effects of SG600/IL24 on the three liver cancer cell lines were analyzed by MTT assay and CPE (cytopathic effect) staining method at different MOI values. Results: IL24 was over-expressed in both SMMC-7721 and BEL-7404 cells but was weakly expressed in BJ cells. At 48 and 96 h post infection the replication of SG600/IL-24 were 794 and 7940 folds in SMMC-7721 cells; 622 and 7 810 folds in BEL-7404 cells; 20 and 200 folds in BJ cells. MTT assay showed that the MOI values of SG600/IL24 for killing 50% and 90% cells were 0.3 and 5 for SMMC-7721 cells; 3 and 20 for BEL-7404 cells; 50 and 150 for BJ cells. CPE staining demonstrated that SG600/IL24 had significant killing effects on both liver cancer cells SMMC-7721 and BEL-7404 but had no significant influence on BJ cells. The cell-killing capability of SG600/IL24 was superior than that of replicative adenovirus ZD55/IL24 and non replicative adenovirus Ad-IL24. Conclusion: After SMMC-7721 and BEL-7404 liver cancer cells are infected with SG600/1124 at high efficiency, the virus replication is active and the expression of IL24 increases greatly. SG600/IL24 has specific cell-killing effects on the two liver cancer cell lines but has no significant influence on normal cells. This study provides a basis for further investigating the effect of SG600/IL24 on liver cancer in vivo.

Response of hypoxia-response element in human liver cancer cells to different microenvironments / 肿瘤

Objective: Bel-7402 cells were stably transfected with a vector constructed with multiple copies of the hypoxia response- element (HRE) sequence of the human vascular endothelial growth factor (VEGF) gene and with the enhanced green fluorescent protein (EGFP) to establish a human hepatoma cell line Bel-7402/5HRE-EGFP. This paper aimed to study the responses of HRE in human liver cancer cells to different microenvironments by observing the changes in hypoxia-inducible factor 1 (HIF-1α) and vascular endothelial growth factor (VEGF) expression in Bel-7402/5HRE-EGFP cell lines under hypoxic conditions. Methods: The expression vector was constructed with 5 copies of HRE sequence and a minimal cytomegalovirus (CMV) as promoter and green fluorescent protein (GFP) as a reporter gene. The effect of different microenvironments such as hypoxia, H2 O2 or acidic pH on the activity of HRE in Bel-7402 cells and the changes in the expression levels of HIF-1α and VEGF under hypoxic condition were determined by using flow cytometry and immunohistochemical staining method. The association of the staining intensity and the distribution of pimonidazole, a hypoxic probe, with the expression and the distribution of HIF-1α, VEGF, and GFP were analyzed. The influence of hypoxia in tumor tissues of nude mice on the activity of HRE and expression of related genes were observed. Results: The HRE in liver cancer cells was very sensitive to hypoxia, which induces up-regulation of HIF-1α and VEGF expressions in tumor cells or in tumor tissues. Both distribution regions of HIF-1α and VEGF were almost the same. Conclusion: Hypoxia plays a pivotal role in controlling the expression of angiogenesis-related factors in human liver cancer cells.