Investigation of tumor hypoxia using a two-enzyme system for in vitro generation of oxygen deficiency

Abstract

Background: Oxygen deficiency in tumor tissue is associated with a malign phenotype, characterized by high invasiveness, increased metastatic potential and poor prognosis. Hypoxia chambers are the established standard model for in vitro studies on tumor hypoxia. An enzymatic hypoxia system (GOX/CAT) based on the use of glucose oxidase (GOX) and catalase (CAT) that allows induction of stable hypoxia for in vitro approaches more rapidly and with less operating expense has been introduced recently. Aim of this work is to compare the enzymatic system with the established technique of hypoxia chamber in respect of gene expression, glucose metabolism and radioresistance, prior to its application for in vitro investigation of oxygen deficiency.

Methods: Human head and neck squamous cell carcinoma HNO97 cells were incubated under normoxic and hypoxic conditions using both hypoxia chamber and the enzymatic model. Gene expression was investigated using Agilent microarray chips and real time PCR analysis. 14C-fluoro-deoxy-glucose uptake experiments were performed in order to evaluate cellular metabolism. Cell proliferation after photon irradiation was investigated for evaluation of radioresistance under normoxia and hypoxia using both a hypoxia chamber and the enzymatic system.

Results: The microarray analysis revealed a similar trend in the expression of known HIF-1 target genes between the two hypoxia systems for HNO97 cells. Quantitative RT-PCR demonstrated different kinetic patterns in the expression of carbonic anhydrase IX and lysyl oxidase, which might be due to the faster induction of hypoxia by the enzymatic system. 14C-fluoro-deoxy-glucose uptake assays showed a higher glucose metabolism under hypoxic conditions, especially for the enzymatic system. Proliferation experiments after photon irradiation revealed increased survival rates for the enzymatic model compared to hypoxia chamber and normoxia, indicating enhanced resistance to irradiation. While the GOX/CAT system allows independent investigation of hypoxia and oxidative stress, care must be taken to prevent acidification during longer incubation.

Conclusion: The results of our study indicate that the enzymatic model can find application for in vitro investigation of tumor hypoxia, despite limitations that need to be considered in the experimental design.

Figure 1

Transcriptomics from HNO97 head and neck squamous cell carcinoma cells under normoxia and hypoxia. Gene expression pattern of known and validated HIF-1 target genes [15] before and after 24 h incubation under normoxic and hypoxic conditions (2% O₂) using the enzymatic GOX/CAT system and a hypoxia chamber. The colour scale encodes differential regulation of genes from green (≤- 2-fold downregulated vs. reference normoxia t = 0 RNA) to red (≥+ 2-fold upregulated vs. reference normoxia t = 0 RNA).

Figure 2

Strongest and differentially regulated genes. (A) Strongest regulated genes in HNO97 cells under hypoxia and respective p-values. (B) Differentially regulated genes between the GOX/CAT system and hypoxia chamber in HNO97 cells and respective p-values. The colour scale encodes differential regulation of genes from green (downregulated vs. reference normoxia t = 24 RNA) to red (upregulated vs. reference normoxia t = 24 RNA).

Figure 3

Pathway analysis of hypoxia regulated genes using a hypoxia chamber and the enzymatic GOX/CAT system. The genes PGK1, PGM1, SDS, ENO2, ALDOC, GAPDH, GPI and HKDC1 were upregulated for both hypoxia systems (Thermometer 1: Hypoxia chamber, Thermometer 2: GOX/CAT system). Those genes are involved in glycolytic pathways. p = 4 × E-5.

Figure 4

Quantitative RT-PCR analysis of the expression of hypoxia regulated genes. Expression of carbonic anhydrase IX (CA9) (A) and lysyl oxidase (LOX) (B) in head and neck squamous cell carcinoma cells HNO97 under normoxia and hypoxia (2% O₂) using the enzymatic GOX/CAT system and a hypoxia chamber. mRNA levels were measured by quantitative real time PCR. Columns, average from three independent measurements and show relative expression levels compared with cells at time point t = 0; Bars, SD. * p < 0.05.

Figure 5

Glucose metabolism. Uptake of FDG in HNO97 cells incubated for 24 h under normoxic and hypoxic conditions (2% O₂) using the GOX/CAT system and a hypoxia chamber. Mean values and standard deviation. * p < 0.05.

Figure 6

In vitro cell response to photon irradiation in the 72-h proliferation assay. Cells were incubated for 24 h under normoxia and hypoxia (2% O₂) using the GOX/CAT system and a hypoxia chamber. The ratio vital treated to vital untreated cells was determined. Mean values and standard deviation. * p < 0.05.