Acquired resistance to oxaliplatin is not directly associated with increased resistance to DNA damage in SK-N-ASrOXALI4000, a newly established oxaliplatin-resistant sub-line of the neuroblastoma cell line SK-N-AS

Abstract

The formation of acquired drug resistance is a major reason for the failure of anti-cancer therapies after initial response. Here, we introduce a novel model of acquired oxaliplatin resistance, a sub-line of the non-MYCN-amplified neuroblastoma cell line SK-N-AS that was adapted to growth in the presence of 4000 ng/mL oxaliplatin (SK-N-ASrOXALI4000). SK-N-ASrOXALI4000 cells displayed enhanced chromosomal aberrations compared to SK-N-AS, as indicated by 24-chromosome fluorescence in situ hybridisation. Moreover, SK-N-ASrOXALI4000 cells were resistant not only to oxaliplatin but also to the two other commonly used anti-cancer platinum agents cisplatin and carboplatin. SK-N-ASrOXALI4000 cells exhibited a stable resistance phenotype that was not affected by culturing the cells for 10 weeks in the absence of oxaliplatin. Interestingly, SK-N-ASrOXALI4000 cells showed no cross resistance to gemcitabine and increased sensitivity to doxorubicin and UVC radiation, alternative treatments that like platinum drugs target DNA integrity. Notably, UVC-induced DNA damage is thought to be predominantly repaired by nucleotide excision repair and nucleotide excision repair has been described as the main oxaliplatin-induced DNA damage repair system. SK-N-ASrOXALI4000 cells were also more sensitive to lysis by influenza A virus, a candidate for oncolytic therapy, than SK-N-AS cells. In conclusion, we introduce a novel oxaliplatin resistance model. The oxaliplatin resistance mechanisms in SK-N-ASrOXALI4000 cells appear to be complex and not to directly depend on enhanced DNA repair capacity. Models of oxaliplatin resistance are of particular relevance since research on platinum drugs has so far predominantly focused on cisplatin and carboplatin.

Fig 1. Representative fluorescence in situ hybridisation (FISH) images of chromosomes 2 (A, D and G), 12 (B, E and H) and 8 (C, F and I) in SK-N-AS (A-C), SK-N-ASrOALI4000(-) (D-F), and SK-N-ASrOXALI4000 (G-I) neuroblastoma cells.

Scale bar represents 10μm.

Fig 2. Effects of cytotoxic drugs on the viability of SK-N-AS cells, SK-N-AS cells with acquired resistance to oxaliplatin (SK-N-AS^rOXALI⁴⁰⁰⁰), or SK-N-AS^rOXALI⁴⁰⁰⁰ cells that had been cultivated for 10 weeks in the absence of oxaliplatin (SK-N-AS^rOXALI^4000(-)).

Drug concentrations that reduce cell viability by 50% (IC₅₀) or 90% (IC₉₀) were determined by MTT assay after 120h of incubation. * P < 0.05 relative to control; ^# mean ± S.D. (presented when no bar is visible on the chosen scale).

Fig 3. Effects of H1N1 influenza A virus infection on cell viability.

Non-MYCN-amplified SK-N-AS neuroblastoma cells, SK-N-AS cells with acquired resistance to oxaliplatin (SK-N-AS^rOXALI⁴⁰⁰⁰), SK-N-AS^rOXALI⁴⁰⁰⁰ cells that were passaged for 10 passages in absence of oxaliplatin (SK-N-AS^rOXALI^4000(-)), or MYCN-amplified UKF-NB-3 neuroblastoma cells were infected with H1N1 influenza strain A/WSN/33 virus at different multiplicities of infection (MOIs) and cell viability was determined 48h post infection relative to non-treated control. The dotted line indicates the viability of non-infected control cells. * P < 0.05 relative to non-infected control cells.

Fig 4. Phosphorylation status of 49 receptor tyrosine kinases.

Receptor tyrosine kinase phosphorylation was determined by a commercial kit (Proteome Profiler Human Phospho-RTK Array Kit, R&D Systems, Abingdon, UK) with subsequent densitometric analysis using ImageJ software (http://imagej.nih.gov/ij/). A) Receptor tyrosine kinase phosphorylation status expressed as fold change spot density relative to a control membrane area. Images of the membranes are presented in S1 Fig. B) Differential phosphorylation of receptor tyrosine kinases that were found phosphorylated in at least one cell line (as indicated by a fold change spot density relative to a control membrane area >2) in SK-N-AS^rOXALI⁴⁰⁰⁰ or SK-N-AS^rOXALI^4000(-) cells relative to SK-N-AS.

Fig 5. Oxygen consumption by SK-N-AS and SK-N-AS^rOXALI⁴⁰⁰⁰ cells.

Oxygen consumption was determined in intact cells in the absence of treatment (baseline), in response to oligomycin (8 μg/mL), an inhibitor of ATP synthase that causes a leak of protons resulting in inhibition of respiration (leak), and in response to FCCP (10 μM) that uncouples the electron transport chain resulting in maximum oxidative phosphorylation.

Fig 6. Effects of ultraviolet C (UVC) radiation on the viability of SK-N-AS and SK-N-AS^rOXALI⁴⁰⁰⁰ cells.

A) Dose-dependent effects of UVC on SK-N-AS and SK-N-AS^rOXALI⁴⁰⁰⁰ cells as indicated by MTT assay five days post exposure. B) Representative images and quantification of colony formation by SK-N-AS and SK-N-AS^rOXALI⁴⁰⁰⁰ cells, as determined 11 days post exposure to UVC (32 J/m²) relative to non-irradiated control.