Basic Study
Copyright ©The Author(s) 2016. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Transl Med. Apr 12, 2016; 5(1): 53-58
Published online Apr 12, 2016. doi: 10.5528/wjtm.v5.i1.53
Prednisolone inhibits SaOS2 osteosarcoma cell proliferation by activating inducible nitric oxide synthase
Alessandra Cazzaniga, Jeanette A M Maier, Sara Castiglioni
Alessandra Cazzaniga, Jeanette A M Maier, Sara Castiglioni, Dipartimento di Scienze Biomediche e Cliniche Luigi Sacco, Università di Milano, 20157 Milano, Italy
Author contributions: Cazzaniga A, Maier JAM and Castiglioni S designed the research; Cazzaniga A and Castiglioni S performed the research and analyzed the data; Maier JAM wrote the paper.
Institutional review board statement: The study was reviewed and approved by the Institutional Review Board of the Department of Biomedical and Clinical Sciences - University of Milan.
Conflict-of-interest statement: We declare no conflict of interest in the study design, its interpertation and presentation of its scientific content.
Data sharing statement: No additional data are available.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Dr. Jeanette AM Maier, Dipartimento di Scienze Biomediche e Cliniche Luigi Sacco, Università di Milano, via GB Grassi 74, 20157 Milano, Italy. jeanette.maier@unimi.it
Telephone: +39-2-50319648 Fax: +39-2-50319659
Received: October 5, 2015
Peer-review started: October 5, 2015
First decision: November 24, 2015
Revised: December 14, 2015
Accepted: January 29, 2016
Article in press: January 31, 2016
Published online: April 12, 2016
Processing time: 188 Days and 20.2 Hours
Abstract

AIM: To investigate the effect of prednisolone, a synthetic glucocorticoid used in inflammatory diseases, on the growth of cultured osteosarcoma cells.

METHODS: Two osteosarcoma cell lines with different degree of differentiation were used. SaOS2 show a rather mature phenotype, while U2OS are negative for almost all osteoblastic markers. The cells were exposed to different concentrations of prednisolone (1-9 μmol/L) with or without antioxidants or the inhibitor of inducible nitric oxide synthase (iNOS) l-N6-(iminoethyl)-lysine-HCl (L-NIL). Cell growth was assessed by counting viable cells. The production of nitric oxide (NO) was measured in the conditioned media by the Griess method. The production of reactive oxygen species was quantified using 2’-7’-dichlorofluorescein diacetate. Western blot with specific antibodies against NOSs was performed on cell extracts.

RESULTS: Prednisolone inhibited SaOS2 cell growth in a dose dependent manner. No significant effects were observed in U2OS. The inhibition of SaOS2 growth is not due to oxidative stress, because antioxidants do not rescue cell proliferation. Since high concentrations of NO inhibit bone formation, we also measured NO and found it induced in SaOS2, but not in U2OS, exposed to prednisolone, because of the upregulation of iNOS as detected by western blot. Therefore, we treated SaOS2 with prednisolone in the presence or in the absence of L-NIL. L-NIL prevented NO release induced by prednisolone at all the concentrations apart from 9 μmol/L. At the same concentrations, we found that L-NIL rescued SaOS2 growth after exposure to prednisolone. In U2OS cells, prednisolone did not induce NO production nor affected cell growth. All together, these data indicate that a link exists between increased amounts of NO and growth inhibition in response to prednisolone in SaOS2.

CONCLUSION: Prednisolone inhibited SaOS2 proliferation by increasing the release of NO through the upregulation of iNOS, while no effect was exerted on U2OS.

Keywords: Osteosarcoma cells; Prednisolone; Nitric oxide; Inducible nitric oxide synthase; Endothelial nitric oxide synthase; Reactive oxygen species

Core tip: Since prednisolone, a widely used synthetic glucocorticoid, inhibits osteoblast proliferation, we evaluated its effects on osteosarcoma cells. In particular, we used two osteoblastic osteosarcoma cell lines with different degree of differentiation, i.e., SaOS2, which have a rather mature phenotype, and U2OS, which are less differentiated. We found that prednisolone inhibited SaOS2 proliferation by increasing the release of nitric oxide (NO) through the upregulation of inducible NO synthase (iNOS). Indeed, pharmacological inhibition with the iNOS inhibitor l-N6-(iminoethyl)-lysine-HCl restored the normal proliferation rate of the SaOS2. On the contrary, prednisolone did not modulate NO production nor cell growth in U2OS.