15d-PGJ2 Promotes ROS-Dependent Activation of MAPK-Induced Early Apoptosis in Osteosarcoma Cell In Vitro and in an Ex Ovo CAM Assay

Osteosarcoma (OS) is the most common type of bone tumor, and has limited therapy options. 15-Deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) has striking anti-tumor effects in various tumors. Here, we investigated molecular mechanisms that mediate anti-tumor effects of 15d-PGJ(2) in different OS cell lines. Human U2-OS and Saos-2 cells were treated with 15d-PGJ(2) and cell survival was measured by MTT assay. Cell proliferation and motility were investigated by scratch assay, the tumorigenic capacity by colony forming assay. Intracellular ROS was estimated by H(2)DCFDA. Activation of MAPKs and cytoprotective proteins was detected by immunoblotting. Apoptosis was detected by immunoblotting and Annexin V/PI staining. The ex ovo CAM model was used to study growth capability of grafted 15d-PGJ(2)-treated OS cells, followed by immunohistochemistry with hematoxylin/eosin and Ki-67. 15d-PGJ(2) substantially decreased cell viability, colony formation and wound closure capability of OS cells. Non-malignant human osteoblast was less affected by 15d-PGJ(2). 15d-PGJ(2) induced rapid intracellular ROS production and time-dependent activation of MAPKs (pERK1/2, pJNK and pp38). Tempol efficiently inhibited 15d-PGJ(2)-induced ERK1/2 activation, while N-acetylcystein and pyrrolidine dithiocarbamate were less effective. Early but weak activation of cytoprotective proteins was overrun by induction of apoptosis. A structural analogue, 9,10-dihydro-15d-PGJ(2), did not show toxic effects in OS cells. In the CAM model, we grafted OS tumors with U2-OS, Saos-2 and MG-63 cells. 15d-PGJ(2) treatment resulted in significant growth inhibition, diminished tumor tissue density, and reduced tumor cell proliferation for all cell lines. Our in vitro and CAM data suggest 15d-PGJ(2) as a promising natural compound to interfere with OS tumor growth.

Automated summary

The study revealed that 15-Deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) exhibits significant anti-tumor effects on osteosarcoma cells by promoting apoptosis, increasing intracellular ROS levels, and activating the MAPK pathway, offering a promising natural compound to interfere with osteosarcoma tumor growth.