Department of Pediatric Surgery, University Children's Hospital, Hoppe-Seyler-Strasse 3, 72076 Tuebingen, Germany.
Int J Oncol. 2011 Feb;38(2):447-54. doi: 10.3892/ijo.2010.860. Epub 2010 Dec 3.
Multidrug resistance (MDR) is a major reason for poor treatment results in hepatoblastoma (HB). The objective of this study was to establish a drug resistance model for HB to analyse alternative treatment options in vitro. Both HB cell lines HUH6 and HepT1 were xenotransplanted in NMRI mice (nu/nu) and 2 cycles of cisplatin (CDDP) treatment were administered. Thereafter, xenotransplants were excised and viable tumour cells were re-cultured. 3D cultures of HUH6 and HepT1 cells were generated on a low binding culture surface. Cell viability in response to CDDP/DOXO (doxorubicin) and apoptosis was assessed by MTT-assay and caspase 3 activity, respectively. Efflux of doxorubicin was measured by flow cytometry. Cellular levels of ABC-transporters (MDR1, MRP1, cMOAT and BRCP) were determined by real time rt-PCR. Only HepT1 cells isolated from HB xenografts showed resistance to CDDP, but did not survive repeated passages. Culturing HUH6 and HepT1 cells as spheroids was successful and 3D cultures showed an IC50-drift to higher drug concentrations for CDDP and DOXO compared to 2D cultures. Treatment with CDDP and DOXO led to homogeneous apoptosis in spheroids. Increased doxorubicin efflux in HUH6 spheroids was not influenced by the P-glycoprotein inhibitor tariquidar. Expression levels of MDR1, MRP1, cMOAT and BRCP in 3D cultures were similar to those in 2D cultures and were higher in HepT1 than in HUH6 cells. In conclusion, a 3D cell culture model for multidrug resistance was established for hepatoblastoma. The underlying mechanism involves altered accessibility of the cells for drugs rather than up-regulation of ABC-transporters.
多药耐药（MDR）是肝母细胞瘤（HB）治疗效果不佳的主要原因。本研究旨在建立 HB 的耐药模型，以分析体外的替代治疗方案。将 HB 细胞系 HUH6 和 HepT1 分别异种移植到 NMRI 小鼠（nu/nu）中，并进行 2 个周期的顺铂（CDDP）治疗。然后切除异种移植物并重新培养有活力的肿瘤细胞。在低结合培养表面上生成 HUH6 和 HepT1 细胞的 3D 培养物。通过 MTT 测定和 caspase 3 活性分别评估 CDDP/DOXO（阿霉素）和细胞凋亡对细胞活力的影响。通过流式细胞术测量阿霉素的外排。通过实时 rt-PCR 测定 ABC 转运蛋白（MDR1、MRP1、cMOAT 和 BRCP）的细胞水平。只有从 HB 异种移植物中分离的 HepT1 细胞对 CDDP 有耐药性，但不能在反复传代后存活。成功培养 HUH6 和 HepT1 细胞为球体，与 2D 培养相比，3D 培养对 CDDP 和 DOXO 的 IC50 漂移到更高的药物浓度。CDDP 和 DOXO 的治疗导致球体中的细胞凋亡均匀。在 HUH6 球体中，多柔比星外排的增加不受 P-糖蛋白抑制剂 tariquidar 的影响。3D 培养物中 MDR1、MRP1、cMOAT 和 BRCP 的表达水平与 2D 培养物相似，在 HepT1 细胞中高于 HUH6 细胞。总之，建立了肝母细胞瘤多药耐药的 3D 细胞培养模型。潜在的机制涉及细胞对药物的可及性改变，而不是 ABC 转运蛋白的上调。
