Implication of necrosis-linked p53 aggregation in acquired apoptotic resistance to 5-FU in MCF-7 multicellular tumour spheroids

Abstract

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 μM) 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 -700 μm 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.