Characterisation of the Morphological, Functional and Molecular Changes in Sunitinib-Resistant Renal Cell Carcinoma Cells

Abstract

Sunitinib resistance is a major clinical problem hampering the treatment of renal cell carcinoma (RCC). Studies on the comprehensive characterisation of morphological, functional and molecular changes in sunitinib-resistant RCC cells are lacking. The aim of the current study was to develop sunitinib resistance in four human RCC cell lines (786-0, Caki-1, Caki-2 and SN12K1), and to characterise the changed cell biology with sunitinib resistance. RCC cells were made resistant by continuous, chronic exposure to 10 μM of sunitinib over a period of 12 months. Cell proliferation, morphology, transmigration, and gene expression for interleukin-6 (IL-6), interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), Bcl-2 and Bax were studied. There was no significant difference in growth rate or transmigration between the parental and resistant cells. Sunitinib-resistant cells were significantly hypertrophic compared with parental cells as evidenced by increases in the surface areas of the whole cells and the nuclei. IL-6 was significantly increased in all resistant cells. IL-8 was increased in sunitinib-resistant Caki-2 and SN12K1 cells and decreased in 786-0 without any significant changes in Caki-1. VEGF was increased in resistant Caki-2 and SN12K1 cells but not in 786-0 and Caki-1. The Bcl2/Bax ratio was increased in Caki-1, Caki-2 and SN12K1 cells but decreased in 786-0 cells. The increased IL-6 may contribute to sunitinib resistance either via VEGF-mediated angiogenesis or through shifting of the Bcl2/Bax balance in favour of anti-apoptosis.

Keywords: angiogenesis; anti-apoptosis; interleukin-6; renal cell carcinoma; sunitinib resistance.

Figure 1.

Sunitinib-resistant cells are hypertrophic. RCC cell lines that are resistant to 10 μM of sunitinib showed significant increases in the surface areas of the whole cells and the nuclei. Results are presented as percentage of the control group. **P < 0.01 and ***P < 0.001.

Figure 2.

Morphological heterogeneity and nuclear atypia in sunitinib-resistant cells. Representative H&E images showing increased cell and nuclear size in resistant cells. The 786-0 appeared spindle-shaped whereas the SN12K1 showed multi-lobular nucleus or nuclear blebs.

Figure 3.

Phase contrast microscopy of live cells showing hypertrophy of sunitinib-resistant cells. Representative phase contrast microscopy further confirms the hypertrophic nature of sunitinib-resistant cells.

Figure 4.

Growth rate showed no significant difference between control and sunitinib-resistant cells. In general, resistant cells showed a slightly decreased growth rate but this was not statistically significant. Results are expressed as percentage of control.

Figure 5.

Lack of difference in transmigration between control and sunitinib-resistant cells. After 24 h of transmigration, in response to 10% FBS as a chemoattractant, no statistically significant difference was observed between the parental and sunitinib-resistant cells. Results are expressed as percentage of control.

Figure 6.

IL-6 is increased in all resistant cells. IL-6 was the common molecule that was increased in all sunitinib-resistant cells. Regarding IL-8, heterogeneity in expression was observed. ***P < 0.001.

Figure 7.

Differential expression of proangiogenic and apoptosis-regulatory genes in resistant cells. There was an increased expression of the proangiogenic gene VEGF in Caki-2 and SN12K1 cells and an increased Bcl2/Bax ratio in Caki-1, Caki-2 and SN12K1 cells. *P < 0.05, **P < 0.01 and ***P < 0.001.