RNA-dependent protein kinase is required for interferon-γ-induced autophagy in MG63 osteosarcoma cells.

Osteosarcoma is a bone tumor that mainly affects children and adolescents. Interferons (IFNs) have been shown to exert antitumor effects in osteosarcoma cells, although the molecular mechanisms have not been fully realized. We investigated IFN-γ actions on osteosarcoma cells. Our results show that IFN-γ induces the accumulation of autophagosomes in osteosarcoma cells. IFN-γ treatment leads to the conversion of autophagy marker light chain 3 (LC3)-I to LC3-II in osteosarcoma cells, and this conversion is accompanied by puncta formation. Also, IFN-γ-mediated induction of autophagosome formation and autophagic flux require RNA-dependent protein kinase (PKR) activity. In addition, our findings show that IFN-γ-mediated osteosarcoma cell death is not dependent on PKR. Our study suggests that IFN-γ has differential effects that lead to induction of cell death and autophagy in osteosarcoma cells. Further evaluation of the IFN-γ-mediated molecular mechanism could lead to improved understanding of and targeted treatment strategies for osteosarcoma.

Detection of macromolecular inversion-induced structural changes in osteosarcoma cells by FTIR microspectroscopy.

Fourier transform infrared (FTIR) microspectroscopy provides a biochemical fingerprint of the cells. In this study, chemical changes in 143B osteosarcoma cells were investigated using FTIR analysis of cancer cells after their treatment with polymeric invertible micellar assemblies (IMAs) and curcumin-loaded IMAs and compared with untreated osteosarcoma cells. A comprehensive principal component analysis (PCA) was applied to analyze the FTIR results and confirm noticeable changes in cell surface chemical structures in the fingerprint regions of 1480-900 cm-1. The performed clustering shows visible differences for all investigated groups of cancer cells. It is demonstrated that a combination of FTIR microspectroscopy with PCA can be an efficient approach in determining interactions of osteosarcoma cells and drug-loaded polymer micellar assemblies. Graphical abstract.

Assessment of Renal Osteodystrophy via Computational Analysis of Label-free Raman Detection of Multiple Biomarkers.

Accurate clinical evaluation of renal osteodystrophy (ROD) is currently accomplished using invasive in vivo transiliac bone biopsy, followed by in vitro histomorphometry. In this study, we demonstrate that an alternative method for ROD assessment is through a fast, label-free Raman recording of multiple biomarkers combined with computational analysis for predicting the minimally required number of spectra for sample classification at defined accuracies. Four clinically relevant biomarkers: the mineral-to-matrix ratio, the carbonate-to-matrix ratio, phenylalanine, and calcium contents were experimentally determined and simultaneously considered as input to a linear discriminant analysis (LDA). Additionally, sample evaluation was performed with a linear support vector machine (LSVM) algorithm, with a 300 variable input. The computed probabilities based on a single spectrum were only marginally different (~80% from LDA and ~87% from LSVM), both providing an unacceptable classification power for a correct sample assignment. However, the Type I and Type II assignment errors confirm that a relatively small number of independent spectra (7 spectra for Type I and 5 spectra for Type II) is necessary for a p < 0.05 error probability. This low number of spectra supports the practicality of future in vivo Raman translation for a fast and accurate ROD detection in clinical settings.

STIM1 expression is associated with osteosarcoma cell survival.

OBJECTIVE: To examine the role of store-operated calcium entry (SOCE) and stromal interaction molecule 1 (STIM1) in survival and migration of osteosarcoma cells and investigate what blockade of store-operated Ca2+ contributes to the regulation of osteosarcoma cells. METHODS: First, we examined the expression levels of STIM1 in osteosarcoma cell lines by Western analysis and in tissue specimens by immunohistochemistry. Second, we investigated the effect of SOCE and STIM1 on osteosarcoma cell viability using MTS assays and on cell proliferation using colony formation. Third, we investigated the role of SOCE and STIM1 in cell migration using wound healing assays and Boyden chamber assays. Finally, we studied the effect of SOCE on the nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) activity by luciferase assays. RESULTS: STIM1 was overexpressed in osteosarcoma cell lines and tissue specimens and was associated with poor survival of osteosarcoma patients. Also, inhibition of SOCE and STIM1 decreased the cell viability and migration of osteosarcoma cells. Furthermore, our results showed that blockade of store-operated Ca2+ channels involved down-regulation of NFATc1 in osteosarcoma cells. CONCLUSIONS: STIM1 is essential for osteosarcoma cell functions, and STIM1 and Ca2+ entry pathway could be further explored as molecular targets in the treatment of osteosarcoma.

Inhibition of STAT3 blocks protein synthesis and tumor metastasis in osteosarcoma cells.

BACKGROUND: Osteosarcoma is the most common bone cancer. Despite advances, molecular mechanisms associated with osteosarcoma have not been fully understood. Hence, an effective treatment for osteosarcoma has yet to be developed. Even though signal transducer and activator of transcription3 (STAT3) has been implicated, its role in pathogenesis of osteosarcoma is not fully determined. In this study, we investigated the antitumor effect of napabucasin (NP) (BBI608), an inhibitor of STAT3 on osteosarcoma in vitro and in vivo and studied the underlying molecular mechanism. METHODS: Cell viability, colony formation, apoptosis, tumor growth and metastasis assays were performed to examine the effect of NP on osteosarcoma in vitro and in vivo. Real-time RT-PCR, western analysis, immunofluorescence and reporter assays were used to monitor the expression and activity of proteins and underlying molecular pathways. Protein synthesis, co-immunoprecipitation and CAP binding assays were carried out to understand NP-mediated mechanism of actions in osteosarcoma cells. RESULTS: Our results show that NP treatment decreases cell viability and induces apoptosis in several osteosarcoma cell lines. NP treatment suppresses both expression and phosphorylation of STAT3 in addition to blocking STAT3-mediated transcription and downstream target proteins in osteosarcoma cells. Furthermore, NP inhibits protein synthesis through regulation of the eukaryotic initiation factor 4E (eIF4E) and eIF4E-binding protein 1 (4E-BP1). NP also inhibits the progression of osteosarcoma tumors and metastasis in vivo in an orthotopic tibial model of osteosarcoma. CONCLUSIONS: Taken together, our investigation reveals that NP acts through a novel mechanism and inhibits osteosarcoma growth and metastasis, and could be investigated clinically for treating osteosarcoma patients alone or in combination with other drugs.

Decreased local and systemic levels of sFRP3 protein in osteosarcoma patients.

Osteosarcoma is a malignant bone tumor that occurs mainly in children and adolescents. Because Wnt signaling has been implicated in the pathogenesis of osteosarcoma, we have investigated the circulating and local levels of the Wnt antagonist protein, Secreted Frizzled Related Protein (sFRP) 3, in osteosarcoma patients. Enzyme linked immunosorbent assay (ELISA) analysis of 67 osteosarcoma and age-matched non-diseased control sera showed that sFPR3 protein levels were significantly lower in osteosarcoma than in normal. Analysis of tumor and adjacent normal tissues (9 pairs) from osteosarcoma patients showed a decrease in sFRP3 expression in 5 out of 9 tumor samples compared to normal tissues. Furthermore, immunohistochemical analysis of tissue microarray revealed a significant decrease in sFRP3 levels in tumor compared to normal bone. RNA sequencing analysis in osteosarcoma cells shows suppression of sFRP3 and concomitant expression of multiple Wnt family members mediating canonical or non-canonical Wnt signaling. Taken together, our findings show that the systemic and local levels of sFRP3 protein are downregulated in osteosarcoma and sFRP3 levels could be explored further in the diagnosis and the care of osteosarcoma patients.

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.

Efflux inhibition by IWR-1-endo confers sensitivity to doxorubicin effects in osteosarcoma cells.

Osteosarcoma is the most common bone tumor that affects children and young adults. Despite advances in the use of combination chemotherapy regimens, response to neoadjuvant chemotherapy in osteosarcoma remains a key determinant of patient outcome. Recently, highly potent small molecule inhibitors of canonical Wnt signaling through the poly(ADP-ribose) polymerase (PARP)-family enzymes, tankyrases 1 & 2 (Tnks1/2), have been considered as possible chemotherapy sensitizing agents. The goal of this study was to determine the ability of the highly specific Tnks1/2 inhibitor IWR-1-endo to sensitize chemotherapy-resistant osteosarcoma to doxorubicin. We found that IWR-1-endo significantly inhibited cellular efflux, as measured by cellular retention of Calcein AM and doxorubicin. In a model of doxorubicin resistant osteosarcoma, pre-treatment with IWR-1-endo strongly sensitized to doxorubicin. This sensitization reduced the doxorubicin IC50 in doxorubicin-resistant cells, but not in chemotherapy naïve cells and caused doxorubicin-treated cells to accumulate at the G2/M checkpoint. Further, we found that sensitization with IWR-1-endo produced increased γH2AX foci formation, indicating increased DNA damage by doxorubicin. Taken together, our findings show that IWR-1-endo increases cellular responses to doxorubicin, by blocking efflux transport in a drug-resistant model of osteosarcoma.

Olaratumab for the treatment of advanced soft tissue sarcoma.

INTRODUCTION: Olaratumab, a human monoclonal antibody against platelet derived growth factor receptor alpha (PDGFR- α), is the first drug that in combination with doxorubicin for the treatment of patients with advanced/metastatic soft tissue sarcoma (STS) that has showed an improved overall survival compared to doxorubicin alone. These initial results are exciting and have the potential to change the landscape of treatment for patients with STS. Areas covered: This article reviews the development of olaratumab for oncology use by reviewing articles in PubMed for 'platelet derived growth factor' and 'receptor' and 'soft tissue sarcoma'. We provide an overview of the published studies to date for olaratumab and specifically the use in soft tissue sarcoma. Expert commentary: Olaratumab is a well-tolerated drug that, when combined with doxorubicin, has shown an improved overall survival compared to doxorubicin alone and the phase III confirmatory study is eagerly awaited.

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 ß-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.

Inhibiting DNA-PKCS radiosensitizes human osteosarcoma cells.

Osteosarcoma survival rate has not improved over the past three decades, and the debilitating side effects of the surgical treatment suggest the need for alternative local control approaches. Radiotherapy is largely ineffective in osteosarcoma, indicating a potential role for radiosensitizers. Blocking DNA repair, particularly by inhibiting the catalytic subunit of DNA-dependent protein kinase (DNA-PKCS), is an attractive option for the radiosensitization of osteosarcoma. In this study, the expression of DNA-PKCS in osteosarcoma tissue specimens and cell lines was examined. Moreover, the small molecule DNA-PKCS inhibitor, KU60648, was investigated as a radiosensitizing strategy for osteosarcoma cells in vitro. DNA-PKCS was consistently expressed in the osteosarcoma tissue specimens and cell lines studied. Additionally, KU60648 effectively sensitized two of those osteosarcoma cell lines (143B cells by 1.5-fold and U2OS cells by 2.5-fold). KU60648 co-treatment also altered cell cycle distribution and enhanced DNA damage. Cell accumulation at the G2/M transition point increased by 55% and 45%, while the percentage of cells with >20 γH2AX foci were enhanced by 59% and 107% for 143B and U2OS cells, respectively. These results indicate that the DNA-PKCS inhibitor, KU60648, is a promising radiosensitizing agent for osteosarcoma.

Hypoxia-related microRNA-210 is a diagnostic marker for discriminating osteoblastoma and osteosarcoma.

Osteoblastoma is a benign bone tumor that can often be difficult to distinguish from malignant osteosarcoma. Because misdiagnosis can result in unfavorable clinical outcomes, we have investigated microRNAs as potential diagnostic biomarkers for distinguishing between these two tumor types. Next generation RNA sequencing was used as an expression screen to evaluate >2,000 microRNAs present in tissue derived from rare formalin fixed paraffin embedded (FFPE) archival tumor specimens. MicroRNAs displaying the greatest ability to discriminate between these two tumors were validated on an independent tumor set, using qPCR assays. Initial screening by RNA-seq identified four microRNA biomarker candidates. Expression of three miRNAs (miR-451a, miR-144-3p, miR-486-5p) was higher in osteoblastoma, while the miR-210 was elevated in osteosarcoma. Validation of these microRNAs on an independent data set of 22 tumor specimens by qPCR revealed that miR-210 is the most discriminating marker. This microRNA displays low levels of expression across all of the osteoblastoma specimens and robust expression in the majority of the osteosarcoma specimens. Application of these biomarkers to a clinical test case showed that these microRNA biomarkers permit re-classification of a misdiagnosed FFPE tumor sample from osteoblastoma to osteosarcoma. Our findings establish that the hypoxia-related miR-210 is a discriminatory marker that distinguishes between osteoblastoma and osteosarcoma. This discovery provides a complementary molecular approach to support pathological classification of two diagnostically challenging musculoskeletal tumors. Because miR-210 is linked to the cellular hypoxia response, its detection may be linked to well-established pro-angiogenic and metastatic roles of hypoxia in osteosarcomas and other tumor cell types. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1137-1146, 2017.

2-Methoxyestradiol-Mediated Induction of Frzb Contributes to Cell Death and Autophagy in MG63 Osteosarcoma Cells.

Osteosarcoma is a bone tumor that mainly affects children and adolescents. Although its pathogenesis is still not fully understood, activation of Wnt signaling has been implicated in the development and metastasis of osteosarcoma. In this report, we have investigated the effect of the anti-tumor compound, 2-methoxyestradiol (2-ME) on Wnt antagonist frizzled-related protein b (Frzb), also known as secreted frizzled-related protein (sFRP)3 in human osteosarcoma (MG63) cells. Our results show that 2-ME treatment induces Frzb gene promoter activity, and increases Frzb mRNA and protein levels in osteosarcoma cells. In addition, 2-ME treatment regulates downstream Wnt signaling, increasing the cytoplasmic levels of ß-catenin, and blocking ß-catenin-mediated Wnt activation in osteosarcoma cells. 2-ME-mediated induction of Frzb protein expression is specific to osteosarcoma cells, as it does not affect Frzb expression in normal primary human osteoblasts. Furthermore, 2-ME-induced apoptosis and autophagy are blocked in osteosarcoma cells transfected with Frzb siRNAs. Taken together, these studies demonstrate that Frzb protein plays an important role in 2-ME-mediated anti-tumor mechanisms in osteosarcoma cells. J. Cell. Biochem. 118: 1497-1504, 2017. © 2016 Wiley Periodicals, Inc.

Molecular strategies for modulating Wnt signaling.

The importance of the Wnt signaling cascade in the fields of developmental biology, regenerative medicine, cancer genetics, and neurobiology has fueled a wide search for potent pharmacological agents capable of controlling Wnt signaling. Numerous fields of study have lent assistance to this endeavor, yielding both natural and synthetic compounds that are capable of inducing or inhibiting Wnt at multiple stages within the pathway. Further, there is a large of body research which has investigated endogenous Wnt inducers and inhibitors, namely the secreted Wnts, Dickkof proteins, secreted Frizzled-Related Proteins, and Wnt Inhibitory Factor-1, along with others which may act via indirect means to stimulate or inhibit Wnt (e.g. the Smads, bone morphogenetic proteins, and Hedgehog proteins). This review will summarize the research surrounding currently available small molecules used to target Wnt signaling. These compounds will be classified based upon their ability to stimulate or inhibit Wnt, their derivation (natural or synthetic), and their specific mechanism of action.

Inhibition of Autophagy Increases 2-Methoxyestradiol-Induced Cytotoxicity in SW1353 Chondrosarcoma Cells.

Chondrosarcoma is a cartilage tumor and is the second most common malignant bone cancer. Unlike many tumors, chondrosarcomas are resistant to conventional chemotherapy and radiotherapy. Autophagy is a homeostatic mechanism through which cellular proteins and organelles are subjected to lysosomal degradation and recycling. Autophagy could play a dual role in cancer by facilitating either cell death or cell survival. To determine whether autophagy plays a role in cell death in chondrosarcoma, we have studied the effect of the anti-tumor compound 2-methoxyestradiol (2-ME) in chondrosarcoma cells in culture. Transmission electron microscopy imaging indicates that 2-ME treatment leads to the accumulation of autophagosomes in human chondrosarcoma (SW1353 and Hs819T) cells. Also, 2-ME induces the conversion of microtubule-associated protein LC3-I to LC3-II, a protein marker that is correlated with the formation of autophagosomes. Our results show that siRNAs directed against ATG3 blocks 2-ME-induced autophagosome formation in chondrosarcoma cells. In addition, treatment with Bafilomycin A1 (Baf) and 3-methyladenine (3-MA), the inhibitors of autophagy, further increased the cell death in 2-ME-treated chondrosarcoma cells. Taken together, our studies demonstrate that autophagy causes resistance to cytotoxicity in chondrosarcoma cells, and the efficacy and anti-tumor effects of drugs in chondrosarcoma could be enhanced by modulating autophagy.

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

Osteosarcoma 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.

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.

Tissue engineered constructs: perspectives on clinical translation.

In this article, a "bedside to bench and back" approach for developing tissue engineered medical products (TEMPs) for clinical applications is reviewed. The driving force behind this approach is unmet clinical needs. Preclinical research, both in vitro and in vivo using small and large animal models, will help find solutions to key research questions. In clinical research, ethical issues regarding the use of cells and tissues, their sources, donor consent, as well as clinical trials are important considerations. Regulatory issues, at both institutional and government levels, must be addressed prior to the translation of TEMPs to clinical practice. TEMPs are regulated as drugs, biologics, devices, or combination products by the U.S. Food and Drug Administration (FDA). Depending on the mode of regulation, applications for TEMP introduction must be filed with the FDA to demonstrate safety and effectiveness in premarket clinical studies, followed by 510(k) premarket clearance or premarket approval (for medical devices), biologics license application approval (for biologics), or new drug application approval (for drugs). A case study on nerve cuffs is presented to illustrate the regulatory process. Finally, perspectives on commercialization such as finding a company partner and funding issues, as well as physician culture change, are presented.

Invertible micellar polymer nanoassemblies target bone tumor cells but not normal osteoblast cells.

AIM: To demonstrate the capability of the invertible micellar polymer nanoassemblies (IMAs) to deliver and release curcumin using the recently discovered mechanism of macromolecular inversion to treat bone tumor cells. MATERIALS & METHODS: The effect of IMA-mediated delivery of curcumin on osteosarcoma cell survival was investigated using MTS assays. To assess the effect of IMAs-delivered curcumin on osteosarcoma cell growth, fluorescence-activated cell sorting was performed. The uptake of micellar nanoassemblies was followed using confocal microscopy. RESULTS & DISCUSSION: IMAs-delivered curcumin is effective in blocking osteosarcoma cell growth. It decreases cell viability in human osteosarcoma (MG63, KHOS, and LM7) cells while having no effect on normal human osteoblast cells. It indicates that curcumin-loaded IMAs provide a unique delivery system targeted to osteosarcoma cells.

MicroRNA-34c inversely couples the biological functions of the runt-related transcription factor RUNX2 and the tumor suppressor p53 in osteosarcoma.

Osteosarcoma (OS) is a primary bone tumor that is most prevalent during adolescence. RUNX2, which stimulates differentiation and suppresses proliferation of osteoblasts, is deregulated in OS. Here, we define pathological roles of RUNX2 in the etiology of OS and mechanisms by which RUNX2 expression is stimulated. RUNX2 is often highly expressed in human OS biopsies and cell lines. Small interference RNA-mediated depletion of RUNX2 inhibits growth of U2OS OS cells. RUNX2 levels are inversely linked to loss of p53 (which predisposes to OS) in distinct OS cell lines and osteoblasts. RUNX2 protein levels decrease upon stabilization of p53 with the MDM2 inhibitor Nutlin-3. Elevated RUNX2 protein expression is post-transcriptionally regulated and directly linked to diminished expression of several validated RUNX2 targeting microRNAs in human OS cells compared with mesenchymal progenitor cells. The p53-dependent miR-34c is the most significantly down-regulated RUNX2 targeting microRNAs in OS. Exogenous supplementation of miR-34c markedly decreases RUNX2 protein levels, whereas 3'-UTR reporter assays establish RUNX2 as a direct target of miR-34c in OS cells. Importantly, Nutlin-3-mediated stabilization of p53 increases expression of miR-34c and decreases RUNX2. Thus, a novel p53-miR-34c-RUNX2 network controls cell growth of osseous cells and is compromised in OS.