Unlike the parent compound, 17β-estradiol, the metabolite 2-ME shows anti-growth effects and functions as a potential therapeutic agent in several types of cancer. We have previously shown that 2-ME induces cell death in osteosarcoma cells but not in normal osteoblasts [4
]. In this report, we demonstrate that 2-ME regulates IFN pathway in a number of osteosarcoma cell lines. 2-ME induces IFN gene promoter activity, expression, and IFN-dependent transcriptional activities in low tumorigenic MG63 and high tumorigenic 143B osteosarcoma cells. 2-ME dependent effects on IFN genes and pathways are specific to osteosarcoma cells and are associated with anti-proliferative effects. Whereas 2-ME does not influence IFN and IFN-dependent pathways in normal HOB cells which have been previously shown to be resistant to the anti-growth activities of 2-ME [5
]. Our results also show that the eIF-2α protein and the downstream effectors of IFN pathway are regulated by 2-ME treatment in several osteosarcoma cell lines. In addition, eIF-2α phosphorylation is decreased in osteosarcoma tumor patients compared to normal. Our results suggest that IFN pathway is involved in osteosarcoma growth and regulation.
It has been reported earlier that 2-ME-mediated cell death is accompanied by increases in IFNβ mRNA levels in low metastatic MG63 osteosarcoma cells [5
]. Current investigation shows that 2-ME-induces type I IFN (α and β) mRNA levels in low and high tumorigenic osteosarcoma cells. In MG63 cells, the mRNA levels increase at 16 and 24 h. Whereas, in 2-ME-treated 143B cells, IFNα increases at 16 h and IFNβ mRNA increases at 8 and 24 h. While this biphasic induction needs to be studied further, the late induction may be due to secondary responses and to transcription factors that may be activated during early response. The induction of mRNA appears to be due to the induction of gene promoter activity. 2-ME-mediated induction of IFN promoter suggests that 2-ME might recruit transcription factors (proteins) that could directly bind to regulatory elements on the IFN promoter. Previous studies show that (nuclear factor kappa B) NFκB, (interferon regulatory factor) IRF-1 and P53 proteins directly bind to IFN gene sequences and induce transcription. 2- ME-mediated anti-tumor effects in osteosarcoma cells and in several other systems involve the induction of P53 [6
]. Further work is needed to determine whether P53 or other protein transcription factors are involved in these biological effects of 2-ME and contribute to 2-ME-stimulated responses and IFN promoter activities.
IFN-mediated transcriptional activation and signaling has been well studied by several investigators using various model systems [9
]. IFN actions trigger a cascade of events leading to ISRE and GAS dependent transcriptional events [20
]. While ISRE effect is mainly induced through the type I IFNs, IFNα and IFNβ [22
], the GAS effects are induced by type II IFN family member, IFNγ. [23
]. Our results show that 2-ME treatment increased transcription of ISRE- dependent transcription in MG63, 143B, KHOS and HOS osteosarcoma cells. Similarly, GAS-dependent transcription is increased in MG63, 143 and KHOS osteosarcoma cells but not in HOS cells. This differential response could be due in part to the differences in cell origin and the genetic heterogeneity of osteosarcoma. GAS and ISRE activations are due to specific activation of JAK/STAT family members and formation of dimeric complexes of STAT family members in cytoplasm and translocation into nucleus [9
]. However, detailed investigations are required to identify the nature of hetero- and homo-dimers that might be forming in 2-ME-treated cells and contributing to the activation of ISRE and GAS elements.
2-ME treatment does not affect IFN promoter activity or gene expression and IFN-regulated pathways in normal HOB cells. We have previously demonstrated that 2-ME induces anti-growth activities in several osteosarcoma cell lines but not in normal primary HOB cells [5
]. Current studies suggest that there is an association between 2-ME-induced anti-proliferative effects and the induction of IFN pathways in 2-ME-treated osteosarcoma cells.
IFN was approved nearly three decades ago for clinical applications. IFN-α2 remains a mainstay of treatment for viral infections and certain forms of cancer including osteosarcoma. Studies have shown that the protein products of IFN, the couple of hundred IFN stimulated genes (ISGs), underlie the biological and clinical effects of IFNs [9
]. They provide fundamental cellular defense mechanisms against viral infections and cancer. Studies from other groups have shown that co-treatment of IFN improves the anti-tumor effects of chemotherapeutic agents such as doxorubicin and etoposide [15
]. The European American oncology study group (EURAMOS) is investigating the effect of IFN as a follow up therapy in patients who have undergone conventional chemotherapy[10
]. Furthermore, IFN has been shown to increase the chemotherapy sensitivity of osteosarcoma cell lines [25
]. Although additional work is warranted to delineate these molecular pathways in 2-ME treated osteosarcoma cells, our findings point out that IFN signaling could play important role in the control of osteosarcoma.
IFN-regulated anti-tumor mechanisms are mediated by eukaryotic initiation factor(eIF)-2, and through the down-regulation of protein synthesis [26
]. EIF-2-mediated mechanism has been well documented in IFN-treated cells and in several other cancer models [27
]. EIF-2 forms a ternary complex with GTP and MET-tRNA and delivers the initiation factor tRNA to the ribosomal site of protein synthesis [26
]. The eIF-2 is discharged and released as a complex with GDP. This GDP should be replaced with GTP in order for eIF-2 to form a new ternary complex and trigger a new set of translation initiation. The phosphorylated form of eIF-2 is not able to trigger initiation resulting in the inhibition of protein synthesis [26
]. Our work from previously published reports and from current studies indicate that 2-ME treatment results in the phosphorylation of eIF-2α protein in vitro. The regulation is specific to anti-growth activities of 2-ME since 2-ME does not appear to regulate eIF-2α in HOB cells. Our analyses of tissue specimens from patients show decreased levels of the phosphorylated form of eIF-2α protein in osteosarcoma tissues compared to normal (Table ). The disease specific regulation was observed in 11 out of 15 specimens. The eIF-2α phosphorylation is associated with osteosarcoma independent of organ and histology subtype as summarized in Table . This raises the possibility that a cause for tumor progression could be the decreased eIF-2α phosphorylation which eventually results in decreased inhibition of protein synthesis and apoptosis in tumor cells. Additional data involving a large number of tissues would be necessary to conclude whether the apparent difference in phosphorylation reflects the apparent differences in growth regulation between normal and tumor tissues. These observations further corroborate our findings that exposure to 2-ME leads to increased phosphorylation of eIF-2α resulting in increased apoptotic and anti-tumor effects due to the inhibition of protein synthesis in osteosarcoma cells [18
]. These investigations point out that decreased IFN-signaling could be potentially associated with the disease state in osteosarcoma tissues. Recent studies show that IFN inhibitory activity in osteosarcoma patients could be an important factor that contributes to tumor progression. Inhibition of IFN pathways could lead to increased susceptibility to disease and progression of disease to metastatic stages [31
]. These findings and our current results emphasize that understanding of IFN signaling could help diagnose and provide optimal therapy in osteosarcoma patients.
Analysis of phospo and non-phospo eIF-2α protein levels in osteosarcoma tissues