Hypoxia in tumors is generally associated with chemoresistance and radioresistance. However, the correlation between the heterodimeric hypoxia-inducible factor-1 (HIF-1) and the multidrug resistance transporter P-glycoprotein (P-gp) has not been investigated. Herein, we demonstrate that with increasing size of DU-145 prostate multicellular tumor spheroids the pericellular oxygen pressure and the generation of reactive oxygen species decreased, whereas the alpha-subunit of HIF-1 (HIF-1alpha) and P-gp were up-regulated. Furthermore, P-gp was up-regulated under experimental physiological hypoxia and chemical hypoxia induced by either cobalt chloride or desferrioxamine. The pro-oxidants H2O2 and buthionine sulfoximine down-regulated HIF-1alpha and P-gp, whereas up-regulation was achieved with the radical scavengers dehydroascorbate, N-acetylcysteine, and vitamin E. The correlation of HIF-1alpha and P-gp expression was validated by the use of hepatoma tumor spheroids that were either wild type (Hepa1) or mutant (Hepa1C4) for aryl hydrocarbon receptor nuclear translocator (ARNT), i.e., HIF-1beta. Chemical hypoxia robustly increased HIF-1alpha as well as P-gp expression in Hepa1 tumor spheroids, whereas no changes were observed in Hepa1C4 spheroids. Hence, our data demonstrate that expression of P-gp in multicellular tumor spheroids is under the control of HIF-1.

Electrochemotherapy (ECT) is a promising new approach to fighting cancer and has successfully been applied to regressing tumours in several animal models and clinical trials with squamous cell carcinomas, basal cell carcinomas, metastatic melanomas and liver tumours (Sersa et al, 1995;Glass et al, 1996;Heller et al, 1996;Mir et al, 1997;Ramirez et al, 1998, Belehradek et al, 1993. During ECT treatment, antineoplastic drugs with low cell membrane permeability, e.g. bleomycin (Poddevin et al, 1991), are delivered into cells by short and intense electric field pulses. These field pulses with a field strength of approximately 130 000 Vm -1 are applied for microseconds and have been discussed to transiently increase the permeability of cell membranes by electroporation Nishi et al, 1997). Owing to the high field strength of the applied electrical fields, pulses have to be administered locally at the solid tumour site of avoid serious side-effects of the electrical field on the patient. This implicates that the enhanced delivery of the anticancer agent is restricted to the area that has been electrically treated. Unlocalized tumours, however, remain untreated, making desirable an ECT approach that uses fairly lower field strengths which can be more generally applied to tissues or whole organs.In the present study, we used multicellular prostate tumour spheroids of the DU-145 cell line to investigate whether treatment with low electrical field strengths increases the uptake and toxicity of the cell membrane-permeant anthracycline doxorubicin. Tumour spheroids were treated for 90 s with a single electrical field pulse of a field strength that has previously been shown to stimulate tumour cell proliferation Wartenberg et al, 1997). An experimental approach for ECT based on stimulation of cell proliferation should be promising since chemotherapeutics are known to be far more effective in rapid cycling as compared to slow-cycling tumour cells.  Culture technique of multicellular tumour spheroidsThe human prostate cancer cell line DU-145 was kindly provided by Dr J Carlsson, Uppsala, Sweden. Cells were cultured in Ham's F10 medium (Gibco, Live Technologies, Helgerman Court, MD, USA) as described previously (Sauer et al, 1997) Spheroids were grown from single cells (passages 2-15) seeded in 250 ml siliconated spinner flasks (Tecnomara, Fernwald, Germany) at 1 × 10 5 cells ml -1. The spinner flask medium (175 ml) was stirred (20 rpm) using a stirrer system (Tecnomara) and partly changed every day. For the experiments, tumour spheroids of a size class of 150 ± 30 µm were used. Enzymatic dissociation of multicellular tumour spheroidsDissociation of multicellular spheroids was performed as described previously . In brief, spheroids were washed in phosphate-buffered saline (PBS) and placed in a Summary Electrochemotherapy (ECT) is a new approach to the treatment of tumours. In the present study, multicellular prostate tumour spheroids were treated with non-lethal direct current (DC) electrical fields, and upt...