Department of Molecular Biology, College of Natural Sciences, Pusan National University, Pusan 609-735, Korea.
Oncol Rep. 2010 Jul;24(1):73-9.
Three-dimensional (3D) multicellular tumour spheroids (MTS) have been used as an in vitro model of solid tumours for drug resistance studies because they mimic the growth characteristics of in vivo tumours more closely than in vitro two-dimensional (2D) culture of cancer cell lines. As observed in solid tumours, MTS exhibits a proliferation gradient with outer regions consisting of proliferating cells that surround inner quiescent cells. The innermost cells in core regions undergo cell death mostly by necrosis to form necrotic core due to insufficient supply of oxygen and nutrient such as glucose with increasing size of spheroids. Tumour necrosis is thought to indicate a poor prognosis and to contribute to acquisition of chemoresistance in solid tumours; however, the mechanism underlying necrosis-mediated chemoresistance remains unclear. In this study, we examined the chemoresistance to 5-Fluorouracil (5-FU) using MCF-7 breast cancer MTS. 5-FU (400 microM) induced apoptosis in MCF-7 cell monolayer as determined by HO/PI staining, PARP cleavage, p53 induction, Bax induction, and Bcl-2 down-regulation. When MCF-7 breast tumour spheroids were cultured on agarose for 8 days, they reached approximately 700 microm in diameter, with a necrotic core. We found that 5-FU-induced apoptosis is markedly reduced in spheroids that were cultured for 9 days and had necrotic core, compared with MCF-7 monolayer cells and spheroids that were cultured for 6 days and had no necrotic core, indicating that the formation of necrotic core may be linked to acquisition of chemoresistance to 5-FU. We also found that a specific set of cellular proteins including p53 was aggregated into a RIPA-insoluble form during MTS culture. Furthermore, most of p53 induced by 5-FU was aggregated in MTS with necrotic core. Our results suggest that necrosis-linked p53 aggregation may contribute to acquired apoptotic resistance to 5-FU in MTS model system.
三维（3D）多细胞肿瘤球体（MTS）已被用作药物耐药性研究的体外模型，因为它们比体外二维（2D）培养的癌细胞系更能模拟体内肿瘤的生长特征。与实体瘤一样，MTS 表现出增殖梯度，外区由增殖细胞组成，内区由静止细胞组成。随着球体的增大，核心区域的最内层细胞由于氧气和葡萄糖等营养物质供应不足而主要通过坏死发生细胞死亡，形成坏死核心。肿瘤坏死被认为预示着预后不良，并有助于实体瘤获得化疗耐药性；然而，坏死介导的化疗耐药性的机制尚不清楚。在这项研究中，我们使用 MCF-7 乳腺癌 MTS 研究了对 5-氟尿嘧啶（5-FU）的化疗耐药性。HO/PI 染色、PARP 切割、p53 诱导、Bax 诱导和 Bcl-2 下调表明，5-FU（400 μM）诱导 MCF-7 细胞单层中的细胞凋亡。当 MCF-7 乳腺癌球体在琼脂糖上培养 8 天时，它们的直径约为 700 μm，形成坏死核心。我们发现，与 MCF-7 单层细胞和培养 6 天且无坏死核心的球体相比，培养 9 天且有坏死核心的球体中 5-FU 诱导的细胞凋亡明显减少，这表明坏死核心的形成可能与对 5-FU 的化疗耐药性有关。我们还发现，一组特定的细胞蛋白，包括 p53，在 MTS 培养过程中聚集形成 RIPA 不溶性形式。此外，5-FU 诱导的 p53 大部分在有坏死核心的 MTS 中聚集。我们的结果表明，与坏死相关的 p53 聚集可能有助于在 MTS 模型系统中获得对 5-FU 的凋亡抵抗。
