Cetuximab potentiates oxaliplatin cytotoxic effect through a defect in NER and DNA replication initiation

Abstract

Preclinical studies have demonstrated that the chemotherapeutic action of oxaliplatin, a third generation platinum derivative, is improved when combined with cetuximab, a monoclonal antibody inhibitor of epidermal growth factor receptors. To explore the mechanism of this synergistic benefit, we used HCT-8 and HCT-116, two human colon cancer cell lines, respectively, responsive and non-responsive to the oxaliplatin/cetuximab combination. We examined the effect of drug exposure on glutathione-S-transferase-mediated oxaliplatin detoxification, DNA-platinum adducts formation, cell cycle distribution, apoptosis, and the expression of multiple targets involved in DNA replication, recombination, and repair. The major changes we found in HCT-8 were a stimulation of oxaliplatin-DNA adduct formation associated with reduced expression of the key enzyme (excision repair cross complementation group1: ERCC1) in the key repair process of oxaliplatin-DNA platinum adduct, the nucleotide excision repair (NER), both at the mRNA and protein levels. We also observed a reduced expression of factors involved in DNA replication initiation, which correlates with an enrichment of cells in the G1 phase of the cell cycle as well as an acceleration of apoptosis. None of these changes occurred in the non-responsive HCT-116 cell that we used as a negative control. These findings support the fact that cetuximab potentiates the oxaliplatin-mediated cytotoxic effect as the result of inhibition of NER and also DNA replication initiation.

Figure 1

Effect of oxaliplatin combined with cetuximab on platinum–DNA adduct formation and repair. DNA was extracted and platinum content was measured by atomic absorption spectrophotometry. Quantitation of platinum–DNA adducts was performed after (A) exposure to either 5 μM oxaliplatin alone or combined with 20 μg ml⁻¹ cetuximab for 24 h (n=3); (B) exposure of the HCT-8 and HCT-116 cell lines for only 1 h (T0) to 40 μM oxaliplatin alone (L-OHP, ) or combined with cetuximab (C225, ) and followed after medium change by either vehicle (□); or 20 μg ml⁻¹ cetuximab () exposure for 6 and 24 h (n=6). Dilution effect of proliferation on the DNA adduct content was corrected by [³H]-thymidine incorporation. Values are mean±s.e.m. of at least three independent experiments. ^***P<0.005; ^*P<0.05. Student's t-test compared to oxaliplatin alone.

Figure 2

Effect of cetuximab, oxaliplatin, and the combination of both on ERCC1 expression at the mRNA and protein levels in the HCT-8 cell line. (A) Relative quantitation of ERCC1 mRNA was performed with GAPDH as the housekeeping gene and untreated cells as control reference. HCT-8 cells were exposed to 20 μg ml⁻¹ cetuximab (C225, □), 5 μM oxaliplatin (L-OHP, ), or a combination of both (▪) for 6 and 24 h. Results are expressed as the mean±s.e.m. of three independent experiments. (B) Western blot analysis of ERCC1 protein expression after 24 h exposure to 5 μM oxaliplatin, 20 μg ml⁻¹ cetuximab, or a combination of both drugs and representative of three independent experiments. ^*P<0.05 Student's t-test compared to the effect of oxaliplatin alone.

Figure 3

Effect of cetuximab, oxaliplatin, or combination exposure on cell cycle repartition of HCT-8 and HCT-116 cell lines. The cell cycle distribution of HCT-8 and HCT-116 cells was determined after (A) 24 h exposure to drug-free medium (), 20 μg ml⁻¹ cetuximab (C225, ), 5 μM oxaliplatin alone (L-OHP, ), or a combination of both (). (A representative experiment of three independent assessments is shown), and (B) kinetic evolution until 72 h of the percent of cells in G0/G1, S, and G2/M phases after exposure to either vehicle (control), 20 μg ml⁻¹ cetuximab daily (C225), 5 μM oxaliplatin for 24 h followed by drug-free medium (L-OHP), or the combined treatment of oxaliplatin plus cetuximab for 24 h followed by 20 μg ml⁻¹ cetuximab daily (L-OHP+C225). Results are expressed as the mean±s.e.m. of three independent experiments.

Figure 4

Proapoptotic effect of the oxaliplatin/cetuximab combination. (A) HCT-8 and HCT-116 cells were exposed to 5 μM oxaliplatin (L-OHP) or 5 μM oxaliplatin plus 20 μg ml⁻¹ cetuximab (L-OHP+C225) for 24 h. Cells were then stained with DAPI to visualise the nuclear morphology and to detect any pattern of chromatin condensation and nuclear DNA fragmentation that characterises apoptotic cells (a representative experiment of three independent assessments is shown). (B) Quantitation of phosphorylated AKT variations in HCT-8 and HCT-116 cells after 24 h exposure to 20 μg ml⁻¹ cetuximab (□), 5 μM oxaliplatin (), or a combination of both (▪) compared to untreated cells. Results are expressed as the mean±s.e.m. of three independent experiments. ^*P<0.05. Student's t-test compared to the effect of oxaliplatin alone.

Figure 5

Multigene analysis of cetuximab, oxaliplatin, or a combination of both on expression of multiple genes involved in DNA replication, repair, and recombination. (A) Reverse transcription quantitative PCR analysis was performed on various genes involved in DNA repair, replication, and recombination in HCT-8 and HCT-116 cells exposed to either 20 μg ml⁻¹ cetuximab (C225), 5 μM oxaliplatin (L-OHP), or a combination of both (Combo) for 24 h. Gene expression variation factors <2 () and increased effect of oxaliplatin by more than 30% (▪) when combined with cetuximab are represented. (B) Focus on relative expression of genes involved in the initiation of DNA replication after 24 h exposure to 20 μg ml⁻¹ cetuximab (□), 5 μM oxaliplatin (), or a combination of both (▪). To normalise gene expression between treated and control samples, control genes of GAPDH and HPRT were used. Relative quantitation of mRNA expression of each gene was performed in triplicate.