CHK/MATK overexpression inhibits osteosarcoma cell migration induced by sublethal concentration of doxorubicin
To determine the effect of overexpressing CHK/MATK in osteosarcoma, we transduced WT cells with GFP-MATK lentiviral particles and created a stable cell line. WT+MATK stable cell line was validated with qPCR and western blot (Figure 5a). After validation, we conducted wound closure assays on U2OS WT and WT+MATK stable cells with and without 0.4 µM doxorubicin treatment for 48 hours. The untreated wound closure assay did not show a significant difference in cell migration between U2OS WT and WT+MATK stable cell lines as these CHK/MATK cells were able to migrate (Figure 5b). Interestingly, however, when WT+MATK cells were treated with 0.4 µM doxorubicin, we observed a significant reduction, rather than enhancement, in cell migration, with only 30% wound closure observed (Figure 5c). Our results indicate that the overexpression of CHK/MATK in U2OS cells efficiently hinders cell migration induced by sublethal concentrations of doxorubicin.
To investigate the role of CHK/MATK in inhibiting the phosphorylation of Src at Tyr-416, we treated WT+MATK stable cells with 0.4 µM doxorubicin and examined pSrc Tyr-416 levels. Our results indicated that doxorubicin treatment caused minimal phosphorylation at Tyr-416, which was not significantly different from the untreated WT+MATK cells (Figure 5d). We also examined the phosphorylation level of Tyr-527 to determine the inhibitory mechanism of CHK/MATK in osteosarcoma. Likewise, western blot analysis showed non-significant phosphorylation at Tyr-527 with or without doxorubicin in the presence of CHK/MATK overexpression (Figure 5d). These results suggest that the inhibitory effect of CHK/MATK on Src phosphorylation on Tyr-416 is not due to an increase in phosphorylation at Tyr-527. Overall, our results demonstrate that MMP-2 knockout resulted in substantial re-expression of CHK/MATK in U2OS cells. This re-expression plays a crucial role in inhibiting cell migration induced by sublethal concentrations of doxorubicin by regulating Src phosphorylation/activation.