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Unlabelled: We studied the involvement of interferon-regulated, PKR on 2-ME-mediated actions in human osteosarcoma cells. Our results show that PKR is activated by 2-ME treatment and is necessary for 2-ME-mediated induction of osteosarcoma cell death., Introduction: Osteosarcoma is the most common primary bone tumor and most frequently develops during adolescence. 2-Methoxyestradiol (2-ME), a metabolite of 17beta-estradiol, induces interferon gene expression and apoptosis in human osteosarcoma cells. In this report, we studied the role of interferon-regulated double-stranded (ds)RNA-dependent protein kinase (PKR) protein on 2-ME-mediated cell death in human osteosarcoma cells., Materials and Methods: Western blot analyses were used to measure PKR protein and phosphorylation levels. Cell survival and apoptosis assays were measured using trypan blue exclusion and Hoechst dye methods, respectively. A transient transfection protocol was used to express the dominant negative PKR mutants., Results and Conclusions: PKR was increased in 2-ME-treated MG63 cells, whereas 17beta-estradiol, 4-hydroxyestradiol, and 16alpha-hydroxyestradiol, which do not induce cell death, had no effect on PKR protein levels. Also, 2-ME treatment induced PKR kinase activity as indicated by increased autophosphorylation and phosphorylation of the endogenous substrate, eukaryotic initiation factor (eIF)-2alpha. dsRNA poly (I).poly (C), an activator of PKR protein, increased cell death when osteosarcoma cells were treated with a submaximal concentration of 2-ME. In contrast, a serine-threonine kinase inhibitor SB203580 and a specific PKR inhibitor 2-aminopurine (2-AP) blocked the 2-ME-induced cell death in MG63 cells. A dominant negative PKR mutant protein conferred resistance to 2-ME-induced cell death to MG63 osteosarcoma and 2-ME-mediated PKR regulation did not require interferon gene expression. PKR protein is activated in cell free extracts by 2-ME treatment, resulting in autophosphorylation and in the phosphorylation of the substrate eIF-2alpha. We conclude from these results that PKR is regulated by 2-ME independently of interferon and is essential for 2-ME-mediated cell death in MG63 osteosarcoma cells.

Osteosarcoma is the most common primary malignant bone tumor in children and young adults. Surgical resection and adjunctive chemotherapy are the only widely available options of treatment for this disease. Anti-tumor compound 2-Methoxyestradiol (2-ME) triggers cell death through the induction of apoptosis in osteosarcoma cells, but not in normal osteoblasts. In this report, we have investigated whether autophagy plays a role in 2-ME actions on osteosarcoma cells. Transmission electron microscopy imaging shows that 2-ME treatment leads to the accumulation of autophagosomes in human osteosarcoma cells. 2-ME induces the conversion of the microtubule-associated protein LC3-I to LC3-II, a biochemical marker of autophagy that is correlated with the formation of autophagosomes. Conversion to LC3-II is accompanied by protein degradation in 2-ME-treated cells. 2-ME does not induce autophagosome formation in normal primary human osteoblasts. In addition, 2-ME-dependent autophagosome formation in osteosarcoma cells requires ATG7 expression. Furthermore, 2-ME does not induce accumulation of autophagosomes in osteosarcoma cells that express dominant negative mutant RNA-dependent protein kinase (PKR) and are resistant to anti-proliferative and anti-tumor effects of 2-ME. Taken together, our study shows that 2-ME treatment induces PKR-dependent autophagy in osteosarcoma cells, and that autophagy could play an important role in 2-ME-mediated anti-tumor actions and in the control of osteosarcoma. [ABSTRACT FROM AUTHOR]

Osteosarcoma is a bone tumor that mainly affects children and adolescents. Interferons (IFNs) have been shown to exert antitumor effects in osteosarcoma cells, although the molecular mechanisms have not been fully realized. We investigated IFN-γ actions on osteosarcoma cells. Our results show that IFN-γ induces the accumulation of autophagosomes in osteosarcoma cells. IFN-γ treatment leads to the conversion of autophagy marker light chain 3 (LC3)-I to LC3-II in osteosarcoma cells, and this conversion is accompanied by puncta formation. Also, IFN-γ–mediated induction of autophagosome formation and autophagic flux require RNA-dependent protein kinase (PKR) activity. In addition, our findings show that IFN-γ–mediated osteosarcoma cell death is not dependent on PKR. Our study suggests that IFN-γ has differential effects that lead to induction of cell death and autophagy in osteosarcoma cells. Further evaluation of the IFN-γ–mediated molecular mechanism could lead to improved understanding of and targeted treatment strategies for osteosarcoma. [ABSTRACT FROM AUTHOR]