Involvement of ROS-alpha v beta 3 integrin-FAK/Pyk2 in the inhibitory effect of melatonin on U251 glioma cell migration and invasion under hypoxia

Cheng-Shi Xu, Ze-Fen Wang, Xiao-Dong Huang, Li-Ming Dai, Chang-Jun Cao, Zhi-Qiang Li

Melatonin, a well-known antioxidant, has been shown to possess anti-invasive properties for glioma. However, little is known about the effect of melatonin on glioma cell migration and invasion under hypoxia, which is a crucial microenvironment for tumor progress. In addition, focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2) are closely associated with cell migration and invasion. Therefore, we investigated the possible role of these kinases and its related signaling in the regulation of human U251 glioma cells behavior by melatonin under hypoxia. The abilities of migration and invasion of U251 glioma cells were determined by wound healing and transwell assay in vitro. The intracellular production of reactive oxygen species (ROS) was measured by using the fluorescent probe 6-carboxy-2', 7'-dichorodihydrofluorescein diacetate (DCFH-DA). Immunofluorescence experiments and western blotting analysis were used to detect the expression level of protein. Small interfering RNAs (siRNA) was used to silence specific gene expression. The pharmacologic concentration (1 mM) of melatonin significantly inhibited the migration and invasion of human U251 glioma cells under hypoxia. The inhibitory effect of melatonin was accompanied with the reduced phosphorylation of FAK and Pyk2, and decreased expression of alpha v beta 3 (αvβ3) integrin. Additionally, inhibition of αvβ3 integrin by siRNA reduced the phosphorylation of FAK/Pyk2 and demonstrated the similar anti-tumor effects as melatonin, suggesting the involvement of αvβ3 integrin- FAK/Pyk2 pathway in the anti-migratory and anti-invasive effect of melatonin. It was also found that melatonin treatment decreased the ROS levels in U251 glioma cells cultured under hypoxia. ROS inhibitor apocynin not only inhibited αvβ3 integrin expression and the phosphorylation levels of FAK and Pyk2, but also suppressed the migratory and invasive capacity of U251 glioma cells under hypoxia. These results suggest that melatonin exerts anti-migratory and anti-invasive effects on glioma cells in response to hypoxia via ROS-αvβ3 integrin-FAK/Pyk2 signaling pathways. This provides evidence that melatonin may be a potential therapeutic molecule targeting the hypoxic microenvironment of glioma.