Abstract
BackgroundOsteosarcoma is a bone tumor that often affects children and young adults. Although a combination of surgery and chemotherapy has improved the survival rate in the past decades, local recurrence and metastases still develop in 40% of patients. A definite therapy is yet to be determined for osteosarcoma. Anti- tumor compound and a metabolite of estrogen, 2-methoxyestradiol (2-ME) induces cell death in osteosarcoma cells. In this report, we have investigated whether interferon (IFN) pathway is involved in 2-ME-induced anti-tumor effects in osteosarcoma cells.Methods2-ME effects on IFN mRNA levels were determined by Real time PCR analysis. Transient transfections followed by reporter assays were used for investigating 2-ME effects on IFN-pathway. Western blot analyses were used to measure protein and phosphorylation levels of IFN-regulated eukaryotic initiation factor-2 alpha (eIF-2α).Results2-ME regulates IFN and IFN-mediated effects in osteosarcoma cells. 2 -ME induces IFN gene activity and expression in osteosarcoma cells. 2-ME treatment induced IFN-stimulated response element (ISRE) sequence-dependent transcription and gamma-activated sequence (GAS)-dependent transcription in several osteosarcoma cells. Whereas, 2-ME did not affect IFN gene and IFN pathways in normal primary human osteoblasts (HOB). 2-ME treatment increased the phosphorylation of eIF-2α in osteosarcoma cells. Furthermore, analysis of osteosarcoma tissues shows that the levels of phosphorylated form of eIF-2α are decreased in tumor compared to normal controls.Conclusions2-ME treatment triggers the induction and activity of IFN and IFN pathway genes in 2-ME-sensitive osteosarcoma tumor cells but not in 2-ME-resistant normal osteoblasts. In addition, IFN-signaling is inhibited in osteosarcoma patients. Thus, IFN pathways play a role in osteosarcoma and in 2-ME-mediated anti-proliferative effects, and therefore targeted induction of IFN signaling could lead to effective treatment strategies in the control of osteosarcoma.
Highlights
Osteosarcoma is a bone tumor that often affects children and young adults
Normal and osteosarcoma tissues Osteosarcoma tissues and normal muscle tissues were obtained through a protocol submitted and approved by the institutional review board (IRB), an internal review committee at Mayo Clinic represented by several clinical faculty members including pathologists, oncologists, orthopedic surgeons, legal and administrative team members
Our results indicate that the eukaryotic initiation factor-2 alpha (eIF-2a) phosphorylation increases in the presence of 2-ME treatment. 2-ME treatment increases eukaryotic initiation factor (eIF)-2a phosphorylation after 16 h by 15, 4, 2and 2-fold in MG63, 143B, KHOS and HOS cells compared to vehicle (Figure 6A &6B) [18]. 2-ME treatment does not affect eIF-2a and phosphor eIF-2a in human osteoblasts (HOB) cells (Figure 6A)
Summary
Osteosarcoma is the most common malignant primary bone tumor in children and adolescents. It is the 6th leading cancer in children under 15. Three major IFN-regulated pathways involving RNA-dependent protein kinase (PKR)/the eukaryotic initiation factor (eIF)2a system, 2-5A synthetase/RNase L system and Janus Kinase (JAK)/signal transducer and activator of transcription (STAT) system have been identified [9]. Several studies have demonstrated that IFNs block osteosarcoma growth in patients, animal models, and in cultured cells [10,12]. IFNb has been shown to block the growth of osteosarcoma cells in vitro. We have investigated the regulation and association of IFN pathways in 2-ME-mediated effects in osteosarcoma cells
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