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2.  Regulation of interferon pathway in 2-methoxyestradiol-treated osteosarcoma cells 
BMC Cancer  2012;12:93.
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.
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).
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.
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.
PMCID: PMC3414746  PMID: 22429849
2-Methoxyestradiol; osteosarcoma; Interferon; ISRE; GAS
3.  Double-Stranded RNA-Dependent Protein Kinase Is Involved in 2-Methoxyestradiol–Mediated Cell Death of Osteosarcoma Cells 
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.
Osteosarcoma is the most common primary bone tumor and most frequently develops during adolescence. 2-Methoxyestradiol (2-ME), a metabolite of 17β-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 17β-estradiol, 4-hydroxyestradiol, and 16α-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)-2α. 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-2α. 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.
PMCID: PMC1955766  PMID: 17014383
estrogen metabolite; MG63 cells; interferon; protein kinase; double-stranded RNA-dependent protein kinase
4.  Potential therapeutic application of gold nanoparticles in B-chronic lymphocytic leukemia (BCLL): enhancing apoptosis 
B-Chronic Lymphocytic Leukemia (CLL) is an incurable disease predominantly characterized by apoptosis resistance. We have previously described a VEGF signaling pathway that generates apoptosis resistance in CLL B cells. We found induction of significantly more apoptosis in CLL B cells by co-culture with an anti-VEGF antibody. To increase the efficacy of these agents in CLL therapy we have focused on the use of gold nanoparticles (GNP). Gold nanoparticles were chosen based on their biocompatibility, very high surface area, ease of characterization and surface functionalization. We attached VEGF antibody (AbVF) to the gold nanoparticles and determined their ability to kill CLL B cells. Gold nanoparticles and their nanoconjugates were characterized using UV-Visible spectroscopy (UV-Vis), transmission electron microscopy (TEM), thermogravimetric analysis (TGA) and X-ray photoelectron spectroscopy (XPS). All the patient samples studied (N = 7) responded to the gold-AbVF treatment with a dose dependent apoptosis of CLL B cells. The induction of apoptosis with gold-AbVF was significantly higher than the CLL cells exposed to only AbVF or GNP. The gold-AbVF treated cells showed significant down regulation of anti-apoptotic proteins and exhibited PARP cleavage. Gold-AbVF treated and GNP treated cells showed internalization of the nanoparticles in early and late endosomes and in multivesicular bodies. Non-coated gold nanoparticles alone were able to induce some levels of apoptosis in CLL B cells. This paper opens up new opportunities in the treatment of CLL-B using gold nanoparticles and integrates nanoscience with therapy in CLL. In future, potential opportunities exist to harness the optoelectronic properties of gold nanoparticles in the treatment of CLL.
PMCID: PMC1876244  PMID: 17488514

Results 1-4 (4)