Sorafenib and Doxorubicin Show Synergistic Effects in Human and Canine Osteosarcoma Cell Lines

Abstract

Osteosarcoma (OSA) is the most common bone tumor in both humans and dogs and has a nearly ten-fold higher incidence in dogs than humans. Despite advances in the treatment of other cancers, the overall survival rates for OSA have stagnated for the past four decades. Therefore, there is a great need to identify novel and effective treatments. We screened a series of tyrosine kinase inhibitors and selected sorafenib, a multi-kinase inhibitor, for further evaluation alone and in combination with cisplatin, carboplatin, and doxorubicin on canine and human OSA cell lines. Our data point to synergistic effects when sorafenib is combined with doxorubicin, but not when combined with cisplatin or carboplatin, in both human and canine OSA. Based on current findings, clinical trials using a combination of doxorubicin and sorafenib in proof-of-concept studies in dogs are warranted. These studies can be carried out relatively quickly in dogs where case load is high and, in turn, provide useful data for the initiation of clinical trials in humans.

Keywords: combination therapy; doxorubicin; osteosarcoma; sorafenib.

Figure 1

Cell viability was measured by CellTiter-Glo assay on all seven OSA cell lines, including four canine OSA (D17, Abrams, Gracie, and BZ) and three human OSA (SAOS2, U2OS, and MG63) cell lines. All cell lines were treated with drugs for 72 h. (A) Ten TKIs demonstrate different levels of inhibition on OSA cell viability. (B) OSA cell lines were treated with sorafenib for 72 h. (C) OSA cell lines were treated with doxorubicin.

Figure 2

Photomicrographs taken with Nikon camera at 40× magnification, comparing wound healing in three OSA cell lines with and without sorafenib for up to 48 h. (D17: 3 μM, Abrams: 4 μM, SAOS2: 3 μM). **: p < 0.01, *: p < 0.05, and ns (not significant) for sorafenib treatment compared to control as determined by two-way ANOVA with Tukey’s multiple comparison test.

Figure 3

Sorafenib decreases expression of p-STAT3 and p-ERK in protein analysis. Canine osteosarcoma Abrams cells were treated with either DMSO (control) or various concentrations of sorafenib (0.1, 1, 10, 20 μM) for 24 h then subjected to Western blot analysis. β-actin and β-tubulin were used as loading controls. X: JQ1 0.1 μM, Y: bortezomib 25 nM.

Figure 4

Three different ratios of sorafenib and doxorubicin were examined with combination index (CI) assay on SAOS2 and D17 cells. (A) Normalized isobologram. We included three different ratios of sorafenib and doxorubicin (20:1, 50:1, and 100:1). (B) The combination of sorafenib and cisplatin was examined on D17 and SAOS2 cells at a ratio of 4:1. (C) The combination of sorafenib and carboplatin was examined on D17 and SAOS2 cells at a ratio of 1:5.

Figure 5

The combination of sorafenib and doxorubicin caused inhibition of cell cycle progression, resulting in G2/M arrest in D17 cells. Cell cycle distribution of D17 OSA cells treated with either (A) DMSO (control), (B) sorafenib 5 µM, (C) doxorubicin 100 nM, or (D) both sorafenib 5 µM plus doxorubicin 100 nM for 24 h. Representative flow histograms demonstrate changes in the cell cycle progression on canine OSA D17 cell line. The combination of sorafenib and doxorubicin resulted in a cell arrest at the G2/M phase. Representative cell cycle distribution graphs show G2/M cell arrest in (E) D17, (F) Abrams and human OSA, and (G) SAOS2 cell lines. ***: p < 0.001 for G2/M arrest compared to the combined treatment (5 µM sorafenib and 100 nM doxorubicin) as determined by one-way ANOVA with Dunnett’s multiple comparison test.