Abstract
The presence of hypoxic regions within solid tumors is caused by an imbalance between cell proliferation and angiogenesis. Such regions may facilitate the onset of recurrence after radiation therapy and chemotherapy, as hypoxic cells show resistance to these treatments. We found that tempol, a nitroxide, strongly induces the accumulation of hypoxia-inducible factor (HIF)-1α, particularly under conditions of hypoxia. We, therefore, evaluated whether tempol enhances the gene expression via HIF-1α, potentially leading to various applications for cancer gene therapy targeting hypoxic cells. Consequently, following treatment with tempol under hypoxia, the luciferase (Luc) activity in the cells transfected with the plasmid containing the luc gene with the oxygen-dependent degradation domain and a promoter composed of hypoxia-responsive elements increased up to approximately 10-fold compared to that observed in cells treated identically with the exception of tempol. The plasmid constructed by replacing the luc gene with the fcy::fur fusion gene as a suicide gene, strongly induced the accumulation of the Fcy::Fur fusion protein, only when incubated in the presence of the hypoxic mimic CoCl2 and tempol. The transfected cells were successfully killed with the addition of 5-fluorocytosine to the cell culture according to the fcy::fur fusion gene expression. As similar but lesser enhancement of the Luc activity was also observed in solid tumor tissues in nude mice, this strategy may be applied for hypoxic cancer eradication.
Highlights
Solid tumors are highly heterogeneous in terms of oxygenation and contain hypoxic cell regions as a result of an imbalance between cell proliferation and angiogenesis
It forms a heterodimeric transcriptional complex with the hypoxia‐inducible factor (HIF)-1β subunit and directly binds to the hypoxia-responsive element (HRE, 5'-A/GCGTG-3') to express a number of its target genes involved in angiogenesis, metabolic adaptation, tolerance of acidosis, cell survival and metastasis, such as vascular endothelial growth factor (VEGF), SLC2A1, which encodes the glucose transporter GLUT1, carbonic anhydrase (CA9), insulin-like growth factor-2 (IGF2) and transforming growth factor-α (TGF-α), respectively [1,8,9,10,11]
In order to determine the rates of enhancement of the HIF-1α activity induced by tempol under hypoxia, the p4HRE‐Luc‐oxygen‐dependent degradation domain (ODD) construct was co-transfected with pGL-4.74, an internal control vector containing the Renilla luc gene (Promega, Madison, WI, USA) at a ratio of 50:1 (p4HRE-Luc‐ODD vs. pGL-4.70) using the Effectene transfection reagent (Qiagen, Valencia, CA, USA), followed by incubation for 8 h
Summary
Solid tumors are highly heterogeneous in terms of oxygenation and contain hypoxic cell regions as a result of an imbalance between cell proliferation and angiogenesis. Nitro-imidazole compounds, such as misonidazole, pimonidazole and nimorazole, have been synthesized as hypoxic cell radiosensitizers causing hypoxic tumor cells to be more sensitive to radiation therapy, and these agents have subsequently been tested in clinical trials These drugs exhibit significant radiosensitizing effects in vitro, they show low levels of radiosensitization in vivo and introduce harmful side effects, including peripheral neuropathy [16]. Tirapazamine (3-amino-1,2,4-benzotriazine 1,4-dioxide, TPZ), which produces damage to hypoxic cells by ROS produced following one-electron reduction by cytochrome P(450) reductase‐enriched microsomes, is currently undergoing evaluation in phase III clinical trials In addition to those mentioned above, new hypoxia-targeted treatments combined with gene therapy have been developed. The goal of this study was to evaluate the enhancement of the cell killing effect in vitro applying the plasmids that can regulate the suicide gene expression under a combination of tempol and hypoxic conditions and to assess the possibility of its application to gene therapy using tumor‐bearing mice boosted with tempol
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