The estrogen metabolite 2-methoxyestradiol regulates eukaryotic initiation factor 4E (eIF4E) and inhibits protein synthesis in MG63 osteosarcoma cells

AbstractOsteosarcoma is a primary bone tumor that affects children and young adults. The estrogen metabolite 2-methoxyestradiol (2-ME) induces cell death in osteosarcoma cells. To determine whether 2-ME actions involve the control of protein synthesis, we studied the effect of 2-ME on eukaryotic initiation factor 4E (eIF4E) and eIF4E-binding protein 1 (4E-BP1) in MG63 osteosarcoma cells. Our results show that 2-ME treatment increases the association of eIF4E with 4E-BP1 in osteosarcoma cells. Also, 2-ME decreases the binding of eIF4E protein to 7-methyl-guanosine cap structure, indicating that 2-ME treatment results in the inhibition of translational initiation. These findings are further supported by the inhibition of protein synthesis in 2-ME-treated osteosarcoma cells. Taken together, our studies show that 2-ME-mediated antitumor effects in osteosarcoma cells involve the regulation of protein synthesis, and translational machinery could serve as a target in the treatment of osteosarcoma

FH535 Suppresses Osteosarcoma Growth In Vitro and Inhibits Wnt Signaling through Tankyrases

Osteosarcoma (OS) is an aggressive primary bone tumor which exhibits aberrantly activated Wnt signaling. The canonical Wnt signaling cascade has been shown to drive cancer progression and metastasis through the activation of β-catenin. Hence, small molecule inhibitors of Wnt targets are being explored as primary or adjuvant chemotherapy. In this study, we have investigated the ability of FH535, an antagonist of Wnt signaling, to inhibit the growth of OS cells. We found that FH535 was cytotoxic in all OS cell lines which were tested (143b, U2OS, SaOS-2, HOS, K7M2) but well tolerated by normal human osteoblast cells. Additionally, we have developed an in vitro model of doxorubicin-resistant OS and found that these cells were highly responsive to FH535 treatment. Our analysis provided evidence that FH535 strongly inhibited markers of canonical Wnt signaling. In addition, our findings demonstrate a reduction in PAR-modification of Axin2 indicating inhibition of the tankyrase 1/2 enzymes. Moreover, we observed inhibition of auto-modification of PARP1 in the presence of FH535, indicating inhibition of PARP1 enzymatic activity. These data provide evidence that FH535 acts through the tankyrase 1/2 enzymes to suppress Wnt signaling and could be explored as a potent chemotherapeutic agent for the control of OS

Regulation of interferon pathway in 2-methoxyestradiol-treated osteosarcoma cells

BACKGROUND: Osteosarcoma 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. METHODS: 2-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). RESULTS: 2-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. CONCLUSIONS: 2-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

Curcumin and Osteosarcoma: Can Invertible Polymeric Micelles Help?

Systematic review of experimental and clinical data on the use of curcumin in the treatment of osteosarcoma is presented. The current status of curcumin’s therapeutic potential against bone cancer is analyzed in regard to using polymeric micelles (including recently developed invertible, responsive, micelles) as a platform for curcumin delivery to treat osteosarcoma. The potential of micellar assemblies from responsive macromolecules in a controlled delivery of curcumin to osteosarcoma cells, and the release using a new inversion mechanism is revealed

RNA-dependent protein kinase is essential for 2-methoxyestradiol-induced autophagy in 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

Raman Spectroscopic and Microscopic Analysis for Monitoring Renal Osteodystrophy Signatures

Defining the pathogenesis of renal osteodystrophy (ROD) and its treatment efficacy are difficult, since many factors potentially affect bone quality. In this study, confocal Raman microscopy and parallel statistical analysis were used to identify differences in bone composition between healthy and ROD bone tissues through direct visualization of three main compositional parametric ratios, namely, calcium content, mineral-to-matrix, and carbonate-to-matrix. Besides the substantially lower values found in ROD specimens for these representative ratios, an obvious accumulation of phenylalanine is Raman spectroscopically observed for the first time in ROD samples and reported here. Thus, elevated phenylalanine could also be considered as an indicator of the disease. Since the image results are based on tens of thousands of spectra per sample, not only are the average ratios statistically significantly different for normal and ROD bone, but the method is clearly powerful in distinguishing between the two types of samples. Furthermore, the statistical outcomes demonstrate that only a relatively small number of spectra need to be recorded in order to classify the samples. This work thus opens the possibility of future development of in vivo Raman sensors for assessment of bone structure, remodeling, and mineralization, where different biomarkers are simultaneously detected with unprecedented accuracy

Effect of proteasome inhibitor on protein degradation in 2-ME-treated MG63

<p><b>cells. </b>¹⁴C-valine-labeled MG63 cells were treated with vehicle and 2-ME (10 µM) in the presence and absence of MG132 (2.5 µM) for 24 hrs and the protein degradation was analyzed as described in Methods. *P<0.05 compared to Veh.</p