The Suppression Of Hypoxia-inducible Factor And Vascular Endothelial Growth Factor By SiRNA Using Multicellular Tumor Spheroids, And Its Relationship Research With Radiation

Purpose: The hypoxic microenvironment is closely associated with the radiation resistance of tumor cells. Hypoxia induces several genes such as hypoxia-inducible factor (HIF-1) and vascular endothelial growth factor (VEGF) to promote tumor cell growth and survival. The up-regulated expression levels of HIF-1 and VEGF in tumor cells also correlate with their resistance to radiation, suggesting that these genes are potential therapeutic targets for strategies designed to enhance radiation effects. To further investigate this possibility, we investigated the effects of suppressing these genes upon the radiation sensitivity of cancer cells. Since multicellular spheroids mimic the solid tumors more closely than the monolayer culture, it provide an excellent three-dimensional in vitro model in which hypoxic conditions can be generated to facilitate detailed investigations including the response to various chemical agents and radiation. We conducted these experiments using multicellular spheroids as a three-dimensional in vitro tumor model and RNA interference as the method of gene suppression.Material and methods: SQ⁵ cells were cultured to 95% confluence and an in vitro multicellular tumor spheroid model was developed using agarose-coated 96-well plates and seeded into at a density of 5000 cells per well and cultured for 4 days when the diameter was up to 500um. SQ⁵ human lung carcinoma cells were treated with HIF-1/VEGF siRNA and/or radiation. Forward transfection methods were employed for monolayer cells and reversed transfection for the spheroids. Hypoxic conditions were generated by incubating cells for 6-24 h at 37?in a Gaspak^TM Pouch Anaerobic System or Anaero Pack System and were incubated for 6 h before exposure to radiation. Cells were irradiated using a Cs-137 Gamma Cell irradiator at room temperature under both normoxic and hypoxic conditions. Gene expression was analyzed using quantitative RT-PCR and western blotting. Cell toxicity was qualified by colony formation assay.Results: Compared with monolayer cells, spheroids showed up-regulated expression of HIF-1 and increased radiation resistance. This finding suggests that HIF-1 expression is enhanced during the growth of 3-dimensional cell structures. HIF-1 protein expression was detectable under normoxic conditions. Hypoxic conditions for 6 hours elevated the expression of HIF-1 and VEGF and enhanced the surviving fraction of spheroids after exposure to radiation. HIF-1 mRNA and corresponding protein expression under both normoxic and hypoxic conditions were inhibited completely by HIF siRNA treatment in spheroids. However, when the expression of HIF-1 and VEGF was down-regulated by transfection of targeting siRNA, this did not influence the cytotoxic effects of the radiation under either normoxic or hypoxic conditions.Conclusions: Multicellular spheroids, an appropriate in vitro system for simulating 3-D tumor micro milieu can be used for evaluating and predicting tumor response to therapy and knock down of HIF-1 mRNA and VEGF mRNA by small interference RNA. We have established a method to transfect siRNA into spheroid cells. Our current data indicate that the functions of HIF-1 or VEGF are independent of radiation sensitivity in spheroids under either normoxic or hypoxic conditions. The reasons are following. First of all, we used a spheroid culture system that lacks the microenvironment that would exist around tumors in vivo including a vascular system. We also applied only single doses of radiation and not a fractionated schedule by which it is possible to assess reoxygenation and repopulation in which HIF-1 and VEGF may play important roles. Further studies using tumor animal models and different radiation schedule are therefore warranted.