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.