Abstract
Osteosarcoma (OS) is the most common type of bone tumor, and has limited therapy options. 15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) has striking anti-tumor effects in various tumors. Here, we investigated molecular mechanisms that mediate anti-tumor effects of 15d-PGJ2 in different OS cell lines. Human U2-OS and Saos-2 cells were treated with 15d-PGJ2 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 H2DCFDA. 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-PGJ2-treated OS cells, followed by immunohistochemistry with hematoxylin/eosin and Ki-67. 15d-PGJ2 substantially decreased cell viability, colony formation and wound closure capability of OS cells. Non-malignant human osteoblast was less affected by 15d-PGJ2. 15d-PGJ2 induced rapid intracellular ROS production and time-dependent activation of MAPKs (pERK1/2, pJNK and pp38). Tempol efficiently inhibited 15d-PGJ2-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-PGJ2, 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-PGJ2 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-PGJ2 as a promising natural compound to interfere with OS tumor growth.
Highlights
Osteosarcoma (OS), a tumor of mesenchymal origin, represents the most common primary malignancy of bones and the most frequent malignant bone tumor [1,2]
We demonstrated that 15d-PGJ2 has pronounced cytotoxic effects in U2-OS and Saos-2 OS cell lines
Quantification revealed that significantly smaller tumors formed from 15d-PGJ2 pre-treated U2-OS (57%, p = 0.0004), Saos-2 (38%, p = 0.035), and MG-63 (35%, p = 0.0015) cells compared to vehicle-pretreated cells (Figure 5B)
Summary
Osteosarcoma (OS), a tumor of mesenchymal origin, represents the most common primary malignancy of bones and the most frequent malignant bone tumor [1,2]. The final but stable degradation product of PGD2 , 15-deoxy-∆12,14 -PGJ2 (15d-PGJ2 ), has been shown to regulate a variety of cellular events ranging from cell growth to apoptosis [18]. These characteristics of 15d-PGJ2 are based on its electrophilic character as the α,β-unsaturated ketone moiety may form covalent protein adducts via Michael addition with cellular nucleophiles, preferentially thiol groups. We demonstrated that 15d-PGJ2 has pronounced cytotoxic effects in U2-OS and Saos-2 OS cell lines These effects were mainly based on ROS-mediated apoptosis and were more pronounced in OS cell lines as compared to a non-malignant osteoblast cell line. We showed substantial inhibitory effects of 15d-PGJ2 on OS-tumor growth by employing the avian ex ovo chorioallantoic membrane (CAM) assay, providing an important step towards preclinical in vivo models that meet 3R requirements and allow high throughput development of new treatment modalities
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
