Published online Apr 12, 2016. doi: 10.5528/wjtm.v5.i1.53
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
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.
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.