Association between tissue hypoxia and elevated non-protein sulphydryl concentrations in human cervical carcinoma xenografts

A double staining technique was developed for the simultaneous measurement of tissue hypoxia and the concentration of non-protein sulphydryls (NPSH), based on the fluorinated nitroimidazole EF5 and the fluorescent histochemical NPSH stain 1-(4-chloromercuriphenoylazo)-naphthol-2 (mercury orange). Cryostat sections of tumour tissue were examined by fluorescence image analysis, using a computer-controlled microscope stage to generate large tiled field images of the cut tumour surface. This method was applied to the human cervical squamous cell carcinoma lines ME180 and SiHa, grown as xenografts in severe combined immunodeficient (SCID) mice, in order to determine if there is a systematic relationship between tissue hypoxia and NPSH levels. Hypoxic regions of the tumours, defined by EF5 labelling, were found to show greater NPSH concentrations relative to better oxygenated regions. This is probably due to increases in glutathione, since the ME180 and SiHa xenografts contained low levels of cysteine and metallothionein; the other major cellular thiols that can bind to mercury orange. Because the effects of glutathione on radiation and chemotherapy resistance are likely to be greater under hypoxic conditions, these results have potentially important implications for the study of resistance mechanisms in solid tumours. © 1999 Cancer Research Campaig

HIF-Independent Regulation of Thioredoxin Reductase 1 Contributes to the High Levels of Reactive Oxygen Species Induced by Hypoxia

Cellular adaptation to hypoxic conditions mainly involves transcriptional changes in which hypoxia inducible factors (HIFs) play a critical role. Under hypoxic conditions, HIF protein is stabilized due to inhibition of the activity of prolyl hydroxylases (EGLNs). Because the reaction carried out by these enzymes uses oxygen as a co-substrate it is generally accepted that the hypoxic inhibition of EGLNs is due to the reduction in oxygen levels. However, several studies have reported that hypoxic generation of mitochondrial reactive oxygen species (ROS) is required for HIF stabilization. Here, we show that hypoxia downregulates thioredoxin reductase 1 (TR1) mRNA and protein levels. This hypoxic TR1 regulation is HIF independent, as HIF stabilization by EGLNs inhibitors does not affect TR1 expression and HIF deficiency does not block TR1 hypoxic-regulation, and it has an effect on TR1 function, as hypoxic conditions also reduce TR1 activity. We found that, when cultured under hypoxic conditions, TR1 deficient cells showed a larger accumulation of ROS compared to control cells, whereas TR1 over-expression was able to block the hypoxic generation of ROS. Furthermore, the changes in ROS levels observed in TR1 deficient or TR1 over-expressing cells did not affect HIF stabilization or function. These results indicate that hypoxic TR1 down-regulation is important in maintaining high levels of ROS under hypoxic conditions and that HIF stabilization and activity do not require hypoxic generation of ROS

Transient Alteration of Cellular Redox Buffering before Irradiation Triggers Apoptosis in Head and Neck Carcinoma Stem and Non-Stem Cells

Background: Head and neck squamous cell carcinoma (HNSCC) is an aggressive and recurrent malignancy owing to intrinsic radioresistance and lack of induction of apoptosis. The major focus of this work was to design a transient glutathione depleting strategy during the course of irradiation of HNSCC in order to overcome their radioresistance associated with redox adaptation. Methodology/Principal Findings: Treatment of SQ20B cells with dimethylfumarate (DMF), a GSH-depleting agent, and L-Buthionine sulfoximine (BSO), an inhibitor of GSH biosynthesis 4 h before a 10 Gy irradiation led to the lowering of the endogenous GSH content to less than 10 % of that in control cells and to the triggering of radiation-induced apoptotic cell death. The sequence of biochemical events after GSH depletion and irradiation included ASK-1 followed by JNK activation which resulted in the triggering of the intrinsic apoptotic pathway through Bax translocation to mitochondria. Conclusions: This transient GSH depletion also triggered radiation-induced cell death in SQ20B stem cells, a key event to overcome locoregional recurrence of HNSCC. Finally, our in vivo data highlight the relevance for further clinical trials o

Effect of mild hyperglycemia +/- meta-iodo-benzylguanidine on the radiation response of R3230 Ac tumors.

The effects of glucose or meta-iodo-benzylguanidine (MIBG) on oxygen utilization (QO2) of several tumor cell lines were studied using a Clark-type electrode chamber. For in vivo studies, rats bearing R3230 Ac rat mammary adenocarcinomas were utilized. To evaluate changes in tumor oxygenation induced by glucose or MIBG, intratumoral pO2 and skeletal muscle pO2 were measured using Eppendorf Histography. To find the effect of mild hyperglycemia (i.p., 1 g/kg) +/- MIBG (i.p., 20 mg/kg) on the radiation response, a growth delay assay was used. Glucose alone produced a approximately 20% inhibition of QO2 in several tumor cells we tested except Q7 tumor cells. MIBG inhibited QO2 in R3230 Ac tumors. The median tumor pO2 for glucose + MIBG was increased from 5.3 mm Hg to 13.8 mm Hg. We hypothesized that combined treatment with glucose + MIBG significantly enhanced radiation-induced tumoricidal effects on R3230 Ac tumors, mainly due to reduction in QO2 and increase in tumor pO2