Figure 2
The Src pathway is involved in chondrosarcoma chemoresistance. (A) Immunoblotting showing AKT, pAKT, Fyn, pSrc and loading control α-tubulin (a tub) for untreated chondrosarcoma cell lines and after 24-h 1 μ M dasatinib. MCF-7: breast cancer cell line, shown as positive control. Presence of all kinases in all cell lines phophorylated Src in all cell lines, although levels are very low in L835. After 24-h 1 μ M dasatinib treatment, levels of pSrc (at Y418) are decreased in all cell lines, and of pAKT in OUMS27, L835, L3252, JJ012 and NDCS-1. (B) Immunoblotting showing p53, MDM2 and p21 in untreated chondrosarcoma cell lines (Mock) and after 24-h 1 μ M dasatinib. U2OS (osteosarcoma) cell line is shown as positive control. MDM2 expression is low in all cell lines. TP53 wild-type cell lines are negative for p53 protein expression, with low p21 protein expression, except for CH2879. TP53 mutant cell lines show high TP53 protein expression with low p21 protein expression. No change in protein levels is observed after 24-h 1 μ M dasatinib treatment. (C) Combination of dasatinib (Das) with doxorubicin (DXR) leads to synergistic loss of cell viability at concentrations, which are ineffective on their own in most cell lines. Combination treatment was more effective in TP53 mutant cell lines than in TP53 wild-type cell lines (P=0.002 for cell viability, P=0.043 for combination indices). (D) Apoptosis assay in JJ012 cell line alternating 1 μ M dasatinib (Das) and 1 μ M doxorubicin (DXR) demonstrates the occurrence of apoptosis during combination. Apoptosis is calculated as percentage of AnnexinV-Alexxa633-stained cells per total number of HOECHST-stained cells. (D and E) Dasatinib successfully inhibits migration in chondrosarcoma cell lines in concentrations as low as 200 nM. (E) SW1353 cell line shown as representative over the course of 4 h; (F) bar chart showing migration for all cell lines.
