Efficacy of combined axitinib with dacarbazine in a B16F1 melanoma xenograft model

Abstract

In this study, we evaluated the efficacy and intestinal side effects of the selective inhibitor of vascular endothelial growth factor (VEGF) receptors, axitinib and/or dacarbazine (DTIC), in a B16F1 melanoma xenograft model. C57BL/6 mice were subcutaneously inoculated with B16F1 melanoma cells. The study was randomized into four groups receiving either 0.5% carboxyl methylcellulose, DTIC, axitinib or a combination of DTIC and axitinib. When the experimental period was complete, the tumor tissues from each mouse were excised, photographed and weighed. The tumor and intestinal tissues were harvested with 4% paraformaldehyde, and paraffin-embedded sections were prepared for hematoxylin and eosin staining, immunohistochemical staining (with antibody specific to proliferating cell nuclear antibody) and terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling assays. The expression of the VEGF and matrix metalloproteinase 9 genes was analyzed using real-time polymerase chain reaction. No significant benefit to treatment with a combination of axitinib and DTIC, as opposed to axitinib alone, was observed; however, the combined treatment did not enhance the level of enteritis compared with that observed in the axitinb group. In addition, axitinib, as a single agent, demonstrated an improved treatment efficacy compared with DTIC. Therefore, axitinib represents a potential novel, efficient and safe anticancer agent, suggesting a possible use for this schedule in treating melanomas that are less sensitive to DTIC.

Keywords: axitinib; dacarbazine; melanoma.

Figure 1.

Antiproliferative effects of the vehicle, dacarbazine (DTIC), axitinib, and axitinib and DTIC combination treatments in mice inoculated with melanoma xenografts. (A) The tumor volume was monitored and recorded. ^**P<0.01 and ^***P<0.001, compared with the control group. (B) Representative photographs of the tumor sections are shown. (C) Tumors excised at day 14 were weighed. (D) Liver and (E) spleen indices. The experiments were performed three times with identical results. Data are expressed as the mean ± SEM. The number of mice in each group is 10. ^*P<0.05, ^**P<0.01 and ^***P<0.001. NS, not significant.

Figure 2.

Following the administration of axitinib, dacarbazine (DTIC), a combination of axitinib and dacarbazine, or vehicle, tumor tissue sections were obtained from mice that had previously been inoculated with melanoma xenografts. The sections were stained with hematoxylin and eosin (H&E), antibody specific to proliferating cell nuclear antibody (PCNA) and terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling (TUNEL), to detect changes in the levels of necrosis, proliferation and apoptosis, respectively. Original magnification, ×200.

Figure 3.

Axitinib, alone and in combination with dacarbazine (DTIC), affects metastasis-related factors and lifespan in mice inoculated with melanoma xeno-grafts. (A and B) Effect of DTIC, axitinib or a simultaneous combination of DTIC and axitinib on VEGF and MMP9 gene expression by quantitative polymerase chain reaction analysis. Columns, mean; bars, SEM. The experiments were performed twice, with similar results. ^*P<0.05 and ^***P<0.001. (C) Axitinib, alone and in combination with DTIC, had a more prolonged effect than the vehicle or DTIC. C57BL/6 mice were allowed to live until their spontaneous death. The survival rates of mice inoculated with melanoma xenografts in an additional experiment (eight mice per group) were recorded and shown as Kaplan-Meier curves.

Figure 4.

Hisptopathological changes induced by the vehicle, dacarbazine (DTIC), axitinib, or a simultaneous combination of DTIC and axitinib in mice inoculated with melanoma xenografts. The intestine samples were fixed with formalin, embedded in paraffin and sectioned. The hisptopathological sections were stained with hematoxylin and eosin. Original magnification, ×200.