MMP-2 gene mediates doxorubicin-induced phosphorylation of Src at Tyr-416
Mohammad et al. (2021) showed that increases in Src phosphorylation at Tyr-416, representing active Src, occurs when cancer cells are treated with sublethal concentrations of doxorubicin (23). The increase in phosphorylated Src at Tyr-416 serves as an indicator of Src activation, which plays an important role in cytoskeletal reorganization and cell migration (12,27). We sought to investigate Src activation induced by sublethal concentrations of doxorubicin in both WT and MMP-2-KO cells. As a result, the WT and MMP-2 KO cells were treated with 0.4 µM doxorubicin for 24 hours and pSrc at Tyr-416 was measured (Figure 3a). Western blots show Src phosphorylation at Tyr-416 was indeed significantly increased when WT cells were treated with a sublethal concentration of doxorubicin. On the other hand, increased Src phosphorylation at Tyr-416 was disrupted in MMP-2 KO cells when treated with the same doxorubicin concentration (Figure 3b).
Previous research also revealed that increases in Src phosphorylation at Tyr-527 are observed in cases when Src is inhibited by specific upstream kinases (28). To determine whether Src at Tyr-527 was phosphorylated in the WT or MMP-2 KO cells, the levels in both untreated and 0.4 µM doxorubicin treated cells were examined (Figure 3a, b). Western blots and corresponding quantifications show non-significant change in phosphorylation of Src at Tyr-527 among the WT and MMP-2 KO, either untreated or treated with doxorubicin (Figure 3a, b). Therefore, the results suggest the disruption of Src phosphorylation at Tyr-416 in MMP-2 KO cells is not as a result of increased phosphorylation at Tyr-527 residue. However, our results indicate that MMP-2 gene is an upstream mediator of a signaling pathway that regulates doxorubicin-induced activation and phosphorylation of Src at Tyr-416 in osteosarcoma cells.