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

Abstract

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 downregulation. 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 RIPAinsoluble 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.

The role of decellularized cell derived extracellular matrix in the establishment and culture of in vitro breast cancer tumor model

“…Apoptosis and necrosis values of approximately 5%-10% seen in CD-dECM doped organoids were found to be compatible with other 3D culture findings with MCF-7 cells in the literature [37][38][39]. Also, consistently with previous literature findings, it was thought that the significant necrosis and moderately high apoptosis observed in organoids without CD-dECM are the result of both the challenges encountered in mass transfer and diffusion restrictions as a result of the organoid's nature and the high cell-cell contact [40,41]. Finally, the usual and low apoptosis and necrosis values observed in CD-dECM-containing groups were thought to be due to the relatively uniform distribution of nutrients and gases, thanks to the 3D irregular structure evident in microscopy findings.…”

The Role of Biomimetic Hypoxia on Cancer Cell Behaviour in 3D Models: A Systematic Review

“…We identified a total of 42 publications [16,19,22,23,30,41,42,45,49,52,56,59,61,64,68,72,74,80,85,89,[100][101][102][103]105,106,[108][109][110][111][112][113][114][115][116][117][118][119][120][121][122][123] comparing cell response to chemotherapy and other interventions between 2D and 3D in vitro tumour models, using a variety of anticancer agents and taking into account any publications which involved more than one type of anticancer agents, yielding in total 85 separate findings, including 79 outcomes concerning chemotherapy agents, three involving radiotherapy, and a further five exploring natural compound agents such as orange-peel flavones. The definitions for drug response varied throughout the studies in terms of markers used and duration over which drug responses were measured.…”