Enrofloxacin enhances the effects of chemotherapy in canine osteosarcoma cells with mutant and wild-type p53

Abstract

Adjuvant chemotherapy improves survival time in dogs receiving adequate local control for appendicular osteosarcoma, but most dogs ultimately succumb to metastatic disease. The fluoroquinolone antibiotic enrofloxacin has been shown to inhibit survival and proliferation of canine osteosarcoma cells in vitro. Others have reported that fluoroquinolones may modulate cellular responses to DNA damaging agents and that these effects may be differentially mediated by p53 activity. We therefore determined p53 status and activity in three canine osteosarcoma cell lines and examined the effects of enrofloxacin when used alone or in combination with doxorubicin or carboplatin chemotherapy. Moresco and Abrams canine osteosarcoma cell lines contained mutations in p53, while no mutations were identified in the D17 cells or in a normal canine osteoblast cell line. The addition of enrofloxacin to either doxorubicin or carboplatin resulted in further reductions in osteosarcoma cell viability; this effect was apparent regardless of p53 mutational status or downstream activity.

Keywords: chemotherapy; comparative oncology; oncology; small animal; tumour biology.

Figure 1

p53 protein sequence alignment of canine OSA cell lines. (A) p53 protein sequence of canine OSA cell lines Abrams, D17, and Moresco aligned to published wild-type p53 protein sequences corresponding to NCBI accession numbers AB537893.1 and NP_001003210.1. (B) Table showing exon and nucleotide positions of p53 missense mutations observed in Abrams and Moresco along with resulting nucleotide and amino acid changes.^a nucleotides are numbered from the ATG translation initiation codon, A = +1.^b wild-type sequence corresponds to NCBI accession number AB537893.1

Figure 2

p53 pathway activation in canine OSA cell lines. MDM2, p53, and p21 expression in canine OSA cell lines and CaOBs following 24 h of exposure to increasing concentrations of Dox. For canine OSA cell lines, two blots of p53 are shown from two independent assays in which longer incubation and exposure times were used in the second run. For CaOBs, weak p53 induction could only be observed in one blot with a very long exposure time.

Figure 3

Concentration dependent decrease in proliferation of OSA cell lines following Dox and Carbo treatment. Absorbance data from a representative MTT assay is shown as a percent of untreated (UT) control cells. Error bars reflect SD and statistical analysis was performed using two-way ANOVA with Tukey’s multiple comparisons test. P-values reflect significance as compared to preceding drug concentration for each cell line and drug. *P < 0.05, ** P < 0.01, ***P < 0.001, ****P < 0.0001

Figure 4

p53 pathway and H2Ax protein expression in canine OSA cell lines following combination Enro and chemo treatment. Abrams, D17, Moresco, and CaOBs were treated with Enro (40 μg/mL), Dox (100nM), or Carbo (100 μM) alone and in combination. Protein was extracted at 6 and 24 h after initiation of treatment and analysed by western blot. D + E = Dox and Enro combination treatment, C + E = Carbo and Enro combination treatment.

Figure 5

Decreased cell proliferation in canine OSA and control normal CaOB cells following combination Enro and chemo treatment. Abrams, D17, Moresco and normal CaOBs were treated with increasing concentrations of Enro, Dox, or Carbo alone and in combination for 72 h. Cell proliferation was measured with an MTT assay and absorbance data is shown as a percentage of untreated (UT) control cells. Error bars reflect SD and statistical analysis was performed using two-way ANOVA with Tukey’s multiple comparisons test. P-values are displayed for combination treatment groups and reflect the lower significance value when compared to both concentration matched single treatment groups (Enro and corresponding Dox or Carbo). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Figure 6

Comparison of high concentration Enro and Dox or Carbo combination treatment effects on cell proliferation in canine OSA cell lines and CaOBs. MTT assay data from the highest concentrations of drugs used (same as represented in Figure 5) were compared between cell lines. Measurements, error bars, and statistical analysis were done similarly as in Figure 5. Concentrations of drugs were: Enro (40 μg/mL), Dox (30 nM) and Carbo (100 μM). *P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001