Double-Stranded RNA-Dependent Protein Kinase Is Involved in 2-Methoxyestradiol—Mediated Cell Death of Osteosarcoma Cells

Abstract

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-MEmediated 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 17␤-estradiol, induces interferon gene expression and apoptosis in human osteosarcoma cells. In this report, we studied the role of interferon-regulated doublestranded (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 17␤-estradiol, 4-hydroxyestradiol, and 16␣-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)-2␣. 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-2␣. We conclude from these results that PKR is regulated by 2-ME independently of interferon and is essential for 2-MEmediated cell death in MG63 osteosarcoma cells.

eIF2α Kinase PKR Modulates the Hypoxic Response by Stat3-Dependent Transcriptional Suppression of HIF-1α

“…In regard to hypoxia, 2-methoxyestradiol has been shown to act as an inhibitor of tumor growth and vascularization through its ability to downregulate Stat3 and HIF-1α (44,45). Interestingly, 2-methoxyestradiol is a potent inducer of apoptosis in tumor cells by activating PKR (46). We observed that 2-methoxyestradiol treatment of hypoxic MEFs resulted in the downregulation of HIF-1α in a manner that was dependent on PKR ( Supplementary Fig.…”

RNA-Dependent Protein Kinase Is Essential for 2-Methoxyestradiol-Induced Autophagy in Osteosarcoma Cells

“…We have previously demonstrated that PKR plays a role in 2-ME-mediated cell cycle arrest and apoptosis in osteosarcoma cells [24], [25]. Osteosarcoma cells expressing transdominant mutant PKR is resistant to anti-cellular and anti-tumor effects of 2-ME [24]. In this report, we show that 2-ME-mediated autophagosome formation and the conversion of LC3-I to LC3-II are inhibited in cells that express dominant negative mutant PKR protein.…”

“…2-ME treatment induces PKR expression and PKR activity in osteosarcoma cells [24]. To determine whether PKR is required for 2-ME-mediated autophagic flux, we have investigated the effect of 2-ME on autophagosome formation and LC3-I to LC3-II conversion, in osteosarcoma cells that have been stably-transfected with trans- dominant mutant PKR cDNAs and are defective for PKR activity and resistant to 2-ME-mediated anti-proliferative effects.…”

“…Also, PKR has been shown to induce apoptosis in cancer cells [38], [39]. We have previously demonstrated that PKR plays a role in 2-ME-mediated cell cycle arrest and apoptosis in osteosarcoma cells [24], [25]. Osteosarcoma cells expressing transdominant mutant PKR is resistant to anti-cellular and anti-tumor effects of 2-ME [24].…”

“…2-Methoxyestradiol (2-ME) is a naturally occurring metabolite of 17β-estradiol and is an inhibitor of tumor cell proliferation in various types of cancer [15]–[21]. 2-ME has been shown to block cell proliferation and induce cell cycle arrest and apoptosis in osteosarcoma cells [22]–[24]. 2-ME-mediated anti-tumor actions require RNA-dependent protein kinase (PKR) in osteosarcoma cells.…”