Application of tumor model multicellular spheroids for combination therapy of siRNA targeting HIF-VEGF and radication

目的：缺氧诱导因子（HIP）和血管内皮生长因子（VEGF）在乏氧肿瘤细胞中的表达升高，表现为放射抵抗。因此，我们假设抑制HIF和VEGF功能可能会增强放射的细胞毒性作用。为了阐明这一点，我们用小干扰RNA（siRNA）抑制这些基因的功能，并以多细胞球体作为三维肿瘤模型。材料和方法：人肺癌细胞SQS单层和球体，用HIF/VEGF siRNA和/或放射线处理。采用定量RT-PCR和Western blotting分析基因表达。结果：与单层细胞相比，在球状体中HIF和VEGF的表达水平稳定上调。HIF或VEGF siRNA处理显示单层细胞和球体中这些mRNA表达的抑制。低氧条件可明显诱导单层细胞和球状体的辐射抗性。HIP siRNA增强了缺氧条件下单层细胞的辐射效应，而VEGF siRNA则无此作用。结论：肿瘤球体可用于评价siRNA下调基因表达对肿瘤细胞的辐射效应。HIF siRNA对缺氧条件下的单层细胞有辐射增强作用，诱导细胞产生辐射抗性。

Purpose: Expression of hypoxia-inducible factor (HIP) and vascular endothelial growth factor (VEGF) elevated in hypoxic tumor cells that show resistant to radiation. Therefore, we hypothesized inhibiting HIF and VEGF function may enhance the cytotoxic effects of radiation. To clarify this point, we use small interference RNA (siRNA) to inhibit those gene function and multicellular spheroid as a 3D tumor model.Materials and methods : Monolayer and spheroids of human lung carcinoma cell SQS, were treated with HIF/VEGF siRNA and/or radiation. Quantitative RT-PCR and western blotting were done to analyze gene expression. Cell toxicity was qualified by colony formation assay.Results : In spheroid, the expression levels of both HIF and VEGF were stably up-regulated compared with monolayer cells. Treatment with HIF or VEGF siRNA displayed inhibition of those mRNA expressions in both monolayer cells and spheroid. Hypoxic condition apparently induced radiation resistance of monolayer cells and spheroid. HIP siRNA but not VEGF siRNA enhanced radiation effect on monolayer cell under hypoxic condition. Either of HIF or VEGF siRNA did not enhance radiation effect on spheroid either normoxic or hypoxic condition.Conclusion : Spheroids can be applied for evaluating tumor response to down-regulation gene expression by siRNA. HIF siRNA showed radiation enhancement effect on monolayer cells under hypoxic condition in which radiation resistance of cells were induced.