Publications by authors named "Bibiane Steinecker-Frohnwieser"

10 Publications

  • Page 1 of 1

Effects of a combined therapy of bortezomib and ionizing radiation on chondrosarcoma three-dimensional spheroid cultures.

Oncol Lett 2021 Jun 30;21(6):428. Epub 2021 Mar 30.

Department of Radiation Oncology, Medical University of Vienna, A-1090 Vienna, Austria.

Chondrosarcomas represent a heterogeneous group of primary bone cancers that are characterized by hyaline cartilaginous neoplastic tissue and are predominantly resistant to radiation and chemotherapy. However, adjuvant radiotherapy is often recommended in inoperable cases or after incomplete resections. To improve the efficiency of treatment, the present study tested a combination therapy with ionizing radiation (IR) and the proteasome inhibitor bortezomib. Using a three-dimensional (3D) spheroid model, 0-20 Gy of IR was applied to chondrosarcoma cells and healthy human chondrocytes. Following combined treatment with IR and bortezomib, the cell cycle distribution, apoptotic induction, the survivin pathway, autophagy and DNA damage were evaluated. Both cell types exhibited a slight decrease in viability following increasing doses of IR; the chondrosarcoma cells demonstrated a significant dose-dependent increase in the expression levels of the DNA damage marker histone H2AX phosphorylation at serine 139 (γH2AX). The combination treatment with bortezomib significantly decreased the cell viability after 48 h compared with that in irradiated cells. High-dose IR induced a G/M phase arrest, which was accompanied by a decrease in the number of cells at the G and S phase. Co-treatment with bortezomib changed the distribution of the cell cycle phases. The mRNA expression levels of the proapoptotic genes Bcl-2-associated X protein (Bax) and Bak were significantly increased by bortezomib treatment and combination therapy with IR. In addition, the combination therapy resulted in a synergistic decrease of the expression levels of survivin and its corresponding downstream pathway molecules, including heat shock protein 90, X-linked inhibitor of apoptosis protein, smad 2 and smad 3. Comparative analyses of γH2AX at 1 and 24 h post-IR revealed efficient DNA repair in human chondrosarcoma cells. Therefore, additional bortezomib treatment may only temporarily improve the radiation sensitivity of chondrosarcoma cells. However, the inhibition of the survivin pathway by the combined treatment with IR and bortezomib, observed in the present study, revealed a novel aspect in the tumor biology of chondrosarcoma 3D spheroid cultures and may represent a potential target for therapy.
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http://dx.doi.org/10.3892/ol.2021.12689DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8045153PMC
June 2021

The Effect of Body Mass Index and Metformin on Matrix Gene Expression in Arthritic Primary Human Chondrocytes.

Cartilage 2020 Oct 7:1947603520962558. Epub 2020 Oct 7.

Department for Rehabilitation, Ludwig Boltzmann Institute for Arthritis and Rehabilitation, Gröbming, Austria.

Objective: Obesity is a known risk factor for knee osteoarthritis (OA). Diabetes has been associated with progression of OA and metformin is the first-line treatment in type 2 diabetes. The effect of the body mass index (BMI) and metformin on the expression of certain matrix genes in human chondrocytes is unclear. The purpose of this study was to investigate the effect of BMI and metformin on the expression of matrix genes in primary human chondrocytes.

Design: Adult female patients undergoing knee arthroplasty for end-stage OA were enrolled. Primary chondrocytes were cultivated and stimulated with metformin. Matrix gene expression was analyzed using polymerase chain reaction. Clinical data were used in multivariable regression models to assess the influence of BMI and metformin stimulation on gene expression.

Results: A total of 14 patients were analyzed. BMI was a predictor of increased expression in ADAMTS5 (β = -0.11, = 0.03). Metformin slightly reduced expression in ADAMTS5 (β = 0.34, = 0.04), HIF-1a (β = 0.39, = 0.04), IL4 (β = 0.30, = 0.02), MMP1 (β = 0.47, < 0.01), and SOX9 (β = 0.37, = 0.03). The hip-knee-ankle angle and proton pump inhibitors (PPIs) intake were associated with reduced SOX9 expression (β = 0.23, < 0.01; β = 2.39, < 0.01). Higher C-reactive protein (CRP) levels were associated with increased MMP1 expression (β = -0.16, = 0.02).

Conclusion: We found that BMI exerts a destructive effect via induction of ADAMTS5. Metformin reduced the expression of catabolic genes ADAMTS5 and MMP1 and might play a role in disease prevention. Limb malalignment and PPI intake was associated with a reduced expression of SOX9, and higher CRP levels correlated with increased MMP1 expression, indicating a destructive process.
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http://dx.doi.org/10.1177/1947603520962558DOI Listing
October 2020

Mechanical exposure and diacerein treatment modulates integrin-FAK-MAPKs mechanotransduction in human osteoarthritis chondrocytes.

Cell Signal 2019 04 21;56:23-30. Epub 2018 Dec 21.

Ludwig Boltzmann Department for Rehabilitation, Ludwig Boltzmann Cluster for Arthritis and Rehabilitation, Saalfelden, Austria; Department of Biophysics, Medical University Graz, Graz, Austria.

Background: Progression of osteoarthritis (OA) is characterized by an excessive production of matrix degrading enzymes and insufficient matrix repair. Despite of active research in this area, it is still unclear how the combination of mechanical exposure and drug therapy works. This study was done to explore the impact of the disease modifying OA drug (DMOAD) diacerein and moderate tensile strain on the anabolic metabolism and the integrin-FAK-MAPKs signal transduction cascade of OA and non-OA chondrocytes.

Methods: Cyclic tensile strain was applied in terms of three different intensities by the Flexcell tension system. Influence on catabolic parameters such as MMPs, ADAMTS, and IL-6 were assessed by qPCR. Changes in phosphorylation of FAK, STAT3 as well as MAP kinases were verified by western blot analysis. Intracellular calcium was measured fluorimetrically using fura-2.

Results: Tensile strain at moderate intensity (SM/SA profile) proved to be most efficient in terms of reducing production of matrix degrading enzyme and IL-6 expression. Treatment with diacerein by itself and diacerein in combination with SM/SA stimulation reduced phosphorylation of FAK and STAT3, which is more pronounced in OA cells. Pretreatment with diacerein for 7 days resulted in an increase in the sensitivity to Yoda1, the agonist for the mechanically activated ion channel Piezo1. However, in OA chondrocytes a significant reduction in Piezo1 expression was observed following treatment with diacerein.

Conclusion: Our results demonstrated for the first time that diacerein intensively intervenes in the regulation of FAK and STAT3 and influences components considered relevant for the progression of OA, even in the presence of mechanical stimulation.
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http://dx.doi.org/10.1016/j.cellsig.2018.12.010DOI Listing
April 2019

The therapeutic nuclear magnetic resonance changes the balance in intracellular calcium and reduces the interleukin-1β induced increase of NF-κB activity in chondrocytes.

Clin Exp Rheumatol 2018 Mar-Apr;36(2):294-301. Epub 2017 Nov 28.

Department of Special Anaesthesia and Pain Therapy, Medical University of Vienna, Austria.

Objectives: Osteoarthritis as the main chronic joint disease is characterised by the destruction of articular cartilage. Developing new, more effective and in particular non-invasive methods to achieve pain reduction of OA patients are of exceptional interest. Clinical observations demonstrated positive effects of therapeutically applied low nuclear magnetic resonance (NMRT) for the treatment of painful disorders of the musculoskeletal system. In this study the cellular mechanism of action of NMRT was examined on chondrocytes.

Methods: Cal-78 human chondrosarcoma cells were kept under inflammatory conditions by application of IL-1β. NMRT treated cells were tested for changes in histamine induced Ca2+ release by fura-2 calcium imaging. The effects of IL-1β and of NMRT treatment were further tested by determining intracellular ATP concentrations and the activity of MAP-kinases and NF-κB.

Results: NMRT influenced the intracellular calcium signalling by elevating the basal [Ca2+]i. The peak calcium concentration evoked by 10 μM histamine was increased by IL-1β and this increase was reversed under NMRT treatment. Screening of different kinase-activities revealed an apparent increase in activity of MAPK/ERK and MAPK/JNK in NMRT stimulated cells, p38 was downregulated. The IL-1β-induced decline in intracellular ATP and the elevated NF-κB activity was reversed under NMRT stimulation.

Conclusions: Under inflammatory conditions, NMRT influenced cellular functions by modulating cellular calcium influx and/or calcium release. Further, NMRT induced changes in MAPK activities such as down-regulation of NF-κB and increasing intracellular ATP might help to stabilise chondrocytes and delay cartilage damage due to OA.
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June 2018

Pharmacological treatment with diacerein combined with mechanical stimulation affects the expression of growth factors in human chondrocytes.

Biochem Biophys Rep 2017 Sep 1;11:154-160. Epub 2017 Jul 1.

Department of Orthopaedic Surgery, Medical University of Graz, Graz, Austria.

Background: Osteoarthritis (OA) as the main chronic joint disease arises from a disturbed balance between anabolic and catabolic processes leading to destructions of articular cartilage of the joints. While mechanical stress can be disastrous for the metabolism of chondrocytes, mechanical stimulation at the physiological level is known to improve cell function. The disease modifying OA drug (DMOAD) diacerein functions as a slowly-acting drug in OA by exhibiting anti-inflammatory, anti-catabolic, and pro-anabolic properties on cartilage. Combining these two treatment options revealed positive effects on OA-chondrocytes.

Methods: Cells were grown on flexible silicone membranes and mechanically stimulated by cyclic tensile loading. After seven days in the presence or absence of diacerein, inflammation markers and growth factors were analyzed using quantitative real-time PCR and enzyme linked immune assays. The influence of conditioned medium was tested on cell proliferation and cell migration.

Results: Tensile strain and diacerein treatment reduced interleukin-6 (IL-6) expression, whereas cyclooxygenase-2 (COX2) expression was increased only by mechanical stimulation. The basic fibroblast growth factor (bFGF) was down regulated by the combined treatment modalities, whereas prostaglandin E2 (PGE2) synthesis was reduced only under OA conditions. The expression of platelet-derived growth factor (PDGF) and vascular endothelial growth factor A (VEGF-A) was down-regulated by both.

Conclusions: From our study we conclude that moderate mechanical stimulation appears beneficial for the fate of the cell and improves the pharmacological effect of diacerein based on cross-talks between different initiated pathways.

General Significance: Combining two different treatment options broadens the perspective to treat OA and improves chondrocytes metabolism.
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http://dx.doi.org/10.1016/j.bbrep.2017.06.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5614688PMC
September 2017

The Proteasome Inhibitor Bortezomib Affects Chondrosarcoma Cells via the Mitochondria-Caspase Dependent Pathway and Enhances Death Receptor Expression and Autophagy.

PLoS One 2016 15;11(12):e0168193. Epub 2016 Dec 15.

Division of Biomedical Research, Medical University of Graz, Graz, Austria.

High grade chondrosarcoma is characterized by its lack of response to conventional cytotoxic chemotherapy, the tendency to develop lung metastases, and low survival rates. Research within the field prioritizes the development and expansion of new treatment options for dealing with unresectable or metastatic diseases. Numerous clinical trials using the proteasome inhibitor bortezomib have shown specific efficacy as an active antitumor agent for treating a variety of solid tumors. However, as of yet the effect of bortezomib on chondrosarcoma has not been investigated. In our study, bortezomib decreased cell viability and proliferation in two different chondrosarcoma cell lines in a time- and dose dependent manner. FACS analysis, mRNA- and protein expression studies illustrated that induction of apoptosis developed through the intrinsic mitochondria-caspase dependent pathway. Furthermore, bortezomib treatment significantly increased expression of the death receptors TRAILR-1 and TRAILR-2 in chondrosarcoma cells. An increased expression of the autophagy markers Atg5/12, Beclin, and LC3BI-II supports the interpretation that bortezomib functions as a trigger for autophagy. Our results demonstrated for the first time that bortezomib reduced viability and proliferation of chondrosarcoma cells, induced apoptosis via the mitochondria-caspase dependent pathway and enhanced death receptor expression and autophagy.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0168193PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5158315PMC
July 2017

Diacerein retards cell growth of chondrosarcoma cells at the G2/M cell cycle checkpoint via cyclin B1/CDK1 and CDK2 downregulation.

BMC Cancer 2015 Nov 10;15:891. Epub 2015 Nov 10.

Ludwig Boltzmann Institute for Rehabilitation of Internal Diseases, Ludwig Boltzmann Cluster for Rheumatology, Balneology and Rehabilitation, Saalfelden, Austria.

Background: Chondrosarcoma is characterized for its lack of response to conventional cytotoxic chemotherapy, propensity for developing lung metastases, and low rates of survival. Research within the field of development and expansion of new treatment options for unresectable or metastatic diseases is of particular priority. Diacerein, a symptomatic slow acting drug in osteoarthritis (SYSADOA), implicates a therapeutic benefit for the treatment of chondrosarcoma by an antitumor activity.

Methods: After treatment with diacerein the growth behaviour of the cells was analyzed with the xCELLigence system and MTS assay. Cell cycle was examined using flow cytometric analysis, RT-PCR, and western blot analysis of specific checkpoint regulators. The status for phosophorylation of mitogen-activated protein kinases (MAPKs) was analyzed with a proteome profiler assay. In addition, the possible impact of diacerein on apoptosis was investigated using cleaved caspase 3 and Annexin V/PI flow cytometric analysis.

Results: Diacerein decreased the cell viability and the cell proliferation in two different chondrosarcoma cell lines in a dose dependent manner. Flow cytometric analysis showed a classical G2/M arrest. mRNA and protein analysis revealed that diacerein induced a down-regulation of the cyclin B1-CDK1 complex and a reduction in CDK2 expression. Furthermore, diacerein treatment increased the phosphorylation of p38α and p38β MAPKs, and Akt1, Akt2, and Akt 3 in SW-1353, whereas in Cal-78 the opposite effect has been demonstrated. These observations accordingly to our cell cycle flow cytometric analysis and protein expression data may explain the G2/M phase arrest. In addition, no apoptotic induction after diacerein treatment, neither in the Cal-78 nor in the SW-1353 cell line was observed.

Conclusions: Our results demonstrate for the first time that the SYSADOA diacerein decreased the viability of human chondrosarcoma cells and induces G2/M cell cycle arrest by CDK1/cyclin B1 down-regulation.
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http://dx.doi.org/10.1186/s12885-015-1915-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4641423PMC
November 2015

Extended ultrastructural characterization of chordoma cells: the link to new therapeutic options.

PLoS One 2014 5;9(12):e114251. Epub 2014 Dec 5.

Center for Medical Research, Medical University of Graz, Graz, Austria.

Chordomas are rare bone tumors, developed from the notochord and largely resistant to chemotherapy. A special feature of this tumor is the heterogeneity of its cells. By combining high pressure freezing (HPF) with electron tomography we were able to illustrate the connections within the cells, the cell-cell interface, and the mitochondria-associated endoplasmic reticulum membrane complex that appears to play a special role among the characteristics of chordoma. These lipid raft-like regions are responsible for lipid syntheses and for calcium signaling. Compared to other tumor cells, chordoma cells show a close connection of rough endoplasmic reticulum and mitochondria, which may influence the sphingolipid metabolism and calcium release. We quantified levels of ceramide and glycosylceramide species by the methyl tert-butyl ether extraction method and we assessed the intracellular calcium concentration with the ratiometric fluorescent dye Fura-2AM. Measurements of the changes in the intracellular calcium concentration revealed an increase in calcium due to the application of acetylcholine. With regard to lipid synthesis, glucosylceramide levels in the chordoma cell line were significantly higher than those in normal healthy cells. The accumulation of glycosylceramide in drug resistant cancer cells has been confirmed in many types of cancer and may also account for drug resistance in chordoma. This study aimed to provide a deep morphological description of chordoma cells, it demonstrated that HPF analysis is useful in elucidating detailed structural information. Furthermore we demonstrate how an accumulation of glycosylceramide in chordoma provides links to drug resistance and opens up the field for new research options.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0114251PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4257693PMC
July 2015

Sesquiterpene lactones downregulate G2/M cell cycle regulator proteins and affect the invasive potential of human soft tissue sarcoma cells.

PLoS One 2013 14;8(6):e66300. Epub 2013 Jun 14.

Department of Orthopedic Surgery, Medical University of Graz, Graz, Austria.

Soft tissue sarcomas (STS) represent a rare group of malignant tumors that frequently exhibit chemotherapeutic resistance and increased metastatic potential. Many studies have demonstrated the great potential of plant-derived agents in the treatment of various malignant entities. The present study investigates the effects of the sesquiterpene lactones costunolide and dehydrocostus lactone on cell cycle, MMP expression, and invasive potential of three human STS cell lines of various origins. Both compounds reduced cell proliferation in a time- and dose-dependent manner. Dehydrocostus lactone significantly inhibited cell proliferation, arrested the cells at the G2/M interface and caused a decrease in the expression of the cyclin-dependent kinase CDK2 and the cyclin-dependent kinase inhibitor p27(Kip1). In addition, accumulation of cells at the G2/M phase transition interface resulted in a significant decrease in cdc2 (CDK1) together with cyclin B1. Costunolide had no effect on the cell cycle. Based on the fact that STS tend to form daughter cell nests and metastasize, the expression levels of matrix metalloproteinases (MMPs), which play a crucial role in extracellular matrix degradation and metastasis, were investigated by Luminex® technology and real-time RT-PCR. In the presence of costunolide, MMP-2 and -9 levels were significantly increased in SW-982 and TE-671 cells. Dehydrocostus lactone treatment significantly reduced MMP-2 and -9 expression in TE-671 cells, but increased MMP-9 level in SW-982 cells. In addition, the invasion potential was significantly reduced after treatment with both sesquiterpene lactones as investigated by the HTS FluoroBlock™ insert system.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0066300PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3682952PMC
January 2014

The sensitivity of G protein-activated K+ channels toward halothane is essentially determined by the C terminus.

J Biol Chem 2004 Aug 2;279(33):34240-9. Epub 2004 Jun 2.

Medical University of Vienna, Department of Anesthesia and Intensive Care Medicine (B), Währinger Gürtel 18-20, A-1090 Vienna, Austria.

G protein-activated K(+) channels (GIRKs or Kir3.x) are targets for the volatile anesthetic, halothane. When coexpressed with the m(2) acetylcholine (ACh) receptor in Xenopus oocytes, agonist-activated GIRK1(F137S)- and GIRK2-mediated currents are inhibited by halothane, whereas in the absence of ACh, high concentrations of halothane induce GIRK1(F137S)-mediated currents. To elucidate the molecular mechanism of halothane action on GIRK currents of different subunit compositions, we constructed deletion mutants of GIRK1(F137S) (GIRK1(Delta363*)) and GIRK2 (GIRK2(Delta356)) lacking the C-terminal ends, as well as chimeric GIRK channels. Mutated GIRK channels showed normal currents when activated by ACh but exhibited different pharmacological properties toward halothane. GIRK2(Delta356) showed no sensitivity against the inhibitory action of halothane but was activated by halothane in the absence of an agonist. GIRK1(Delta363*) was activated by halothane more efficiently. Currents mediated by chimeric channels were inhibited by anesthetic concentrations that were at least 30-fold lower than those necessary to decrease GIRK2 wild type currents. Glutathione S-transferase pulldown experiments did not show displacement of bound Gbetagamma by halothane, indicating that halothane does not interfere with Gbetagamma binding. Single channel experiments revealed an influence of halothane on the gating of the channels: The agonist-induced currents of GIRK1 and GIRK2, carried mainly by brief openings, were inhibited, whereas higher concentrations of the anesthetic promoted long openings of GIRK1 channels. Because the C terminus is crucial for these effects, an interaction of halothane with the channel seems to be involved in the mechanism of current modulation.
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http://dx.doi.org/10.1074/jbc.M403448200DOI Listing
August 2004