Vandetanib restores head and neck squamous cell carcinoma cells' sensitivity to cisplatin and radiation in vivo and in vitro

Abstract

Purpose: We investigated whether vandetanib, an inhibitor of the tyrosine kinase activities of vascular endothelial growth factor receptor-2 (VEGFR-2), epidermal growth factor receptor (EGFR), and rearranged during transfection (RET), could augment the antitumor activity of radiation with or without cisplatin in preclinical in vitro and in vivo models of human head and neck squamous cell carcinoma (HNSCC).

Experimental design: OSC-19 and HN5 HNSCC cells that were cisplatin and radioresistant were treated with vandetanib, cisplatin, and radiation alone or in combination in vitro and in vivo using an orthotopic nude mouse model. Treatment effects were assessed using clonogenic survival assay, tumor volume, bioluminescence imaging, tumor growth delay, survival, microvessel density, tumor and endothelial cell apoptosis, and EGFR and Akt phosphorylation data.

Results: Vandetanib plus cisplatin radiosensitized HNSCC cells in vitro and in vivo. The combination treatment with vandetanib, cisplatin, and radiation was superior to the rest of treatments (including the double combinations) in antitumoral effects, prolonging survival, decreasing cervical lymph node metastases in vivo. It also increased both tumor and tumor-associated endothelial cell apoptosis and decreased microvessel density in vivo. An analysis of tumor growth delay data revealed that vandetanib plus cisplatin enhanced radioresponse in vivo. All vandetanib-containing treatments inhibited EGFR and Akt phosphorylation in vitro and in vivo.

Conclusion: The addition of vandetanib to combination therapy with cisplatin and radiation was able to effectively overcome cisplatin and radioresistance in in vitro and in vivo models of HNSCC. Further study of this regimen in clinical trials may be warranted.

Figure 1

Effects of vandetanib and/or cisplatin on radiosensitivity of head and neck squamous cell carcinoma cells. OSC-19 and HN5 cells in culture were exposed to cisplatin (4 µM and 9 µM, respectively) for 1 h, vandetanib (2 µM and 2.5 µM, respectively) for 6 h and then irradiated at 2 Gy, 4 Gy, or 6 Gy. After treatments, clonogenic survival assays were performed. Points indicate the means of triplicate experiments; bars, standard errors.

Figure 2

In vivo effects of treatment with radiation, vandetanib, cisplatin, and their combinations on tumor growth, bioluminescence imaging, and survival time in mice. A, The in vivo effects of treatments on tumor growth in OSC-19-luc mice. B, The in vivo effects of treatments on tumor growth in HN5 mice. OSC-19-luc and HN5 human HNSCC cells were injected into the tongues of nude mice. After tumor nodules had developed, mice were treated. Tumors were measured with microcalipers twice a week. Points indicate means; bars, standard errors. C, The effects of treatment on OSC-19-luc orthotopic tumor followed by bioluminescence imaging. Points, mean; bars, SE. Photon counts were calculated from the imaging data using the IVIS Living Image software. D, The in vivo effects of treatments on survival time in OSC-19 mice. E, The in vivo effects of treatments on survival time in HN5 mice. Animals were euthanized when they had lost more than 20% of their initial body weight or at 50 d after cell inoculation. Survival was analyzed by the Kaplan-Meier method and compared with log-rank tests. F, Representative bioluminescence images corresponding to OSC-19-luc tumors from each treatment group, 17 days after cell inoculation.

Figure 3

Immunohistochemical analyses of OSC-19 xenograft tumors in nude mice. A, Tumors were harvested after 7 d of treatment, and representative sections obtained from OSC-19 tumors were immunostained for expression of CD31 (endothelial cell marker) and TUNEL (tumor cell apoptosis) (magnification × 200). Double staining for CD31 (red)/TUNEL (green) was also performed to reveal apoptosis in tumor-associated endothelial cells (magnification × 400). Results of quantitative analysis for B, CD31 staining (microvessel density); C, TUNEL staining; and D, endothelial cells apoptosis. Columns indicate means; bars, standard errors; *, P < 0.05; **, P < 0.01 as compared with controls; ***, P < 0.001 as compared with controls.

Figure 4

Vandetanib alone or in combination with cisplatin and/or radiation inhibited epidermal growth factor receptor and Akt phosphorylation in vivo and in vitro (OSC-19). A, Tumors were harvested after 7 d of treatment, and snap-frozen in liquid nitrogen. They were then homogenized in lysis buffer before being subjected to Western immunoblotting. B, Cells were treated with 2 µM of vandetanib for 6 h, 4 µM of cisplatin for 1 h, and irradiation (3 Gy). Whole-cell lysates were obtained and subjected to Western immunoblotting to resolve proteins. Antibodies to total (unphosphorylated) receptors and β-actin were used as protein loading controls.