Publications by authors named "Birgit Lohberger"

52 Publications

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3892/ol.2021.12689DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8045153PMC
June 2021

Surface Modifications of Titanium Aluminium Vanadium Improve Biocompatibility and Osteogenic Differentiation Potential.

Materials (Basel) 2021 Mar 23;14(6). Epub 2021 Mar 23.

Department of Orthopedics and Trauma, Medical University Graz, 8036 Graz, Austria.

Osteogenic cells are strongly influenced in their behaviour by the surface properties of orthopaedic implant materials. Mesenchymal stem and progenitor cells (MSPCs) migrate to the bone-implant interface, adhere to the material surface, proliferate and subsequently differentiate into osteoblasts, which are responsible for the formation of the bone matrix. Five surface topographies on titanium aluminium vanadium (TiAlV) were engineered to investigate biocompatibility and adhesion potential of human osteoblasts and the changes in osteogenic differentiation of MSPCs. Elemental analysis of TiAlV discs coated with titanium nitride (TiN), silver (Ag), roughened surface, and pure titanium (cpTi) surface was analysed using energy-dispersive X-ray spectroscopy and scanning electron microscopy. In vitro cell viability, cytotoxicity, adhesion behaviour, and osteogenic differentiation potential were measured via CellTiter-Glo, CytoTox, ELISA, Luminex technology, and RT-PCR respectively. The Ag coating reduced the growth of osteoblasts, whereas the viability of MSPCs increased significantly. The roughened and the cpTi surface improved the viability of all cell types. The additive coatings of the TiAlV alloy improved the adhesion of osteoblasts and MSPCs. With regard to the osteogenic differentiation potential, an enhanced effect has been demonstrated, especially in the case of roughened and cpTi coatings.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ma14061574DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8005140PMC
March 2021

Synthesis and Pharmacological In Vitro Investigations of Novel Shikonin Derivatives with a Special Focus on Cyclopropane Bearing Derivatives.

Int J Mol Sci 2021 Mar 9;22(5). Epub 2021 Mar 9.

Department of Pharmacognosy, Institute of Pharmaceutical Sciences, University of Graz, Beethovenstr. 8, 8010 Graz, Austria.

Melanoma is the deadliest form of skin cancer and accounts for about three quarters of all skin cancer deaths. Especially at an advanced stage, its treatment is challenging, and survival rates are very low. In previous studies, we showed that the constituents of the roots of as well as a synthetic derivative of the most active constituent showed promising results in metastatic melanoma cell lines. In the current study, we address the question whether we can generate further derivatives with optimized activity by synthesis. Therefore, we prepared 31, mainly novel shikonin derivatives and screened them in different melanoma cell lines (WM9, WM164, and MUG-Mel2 cells) using the XTT viability assay. We identified ()-1-(1,4-dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl 2-cyclopropyl-2-oxoacetate as a novel derivative with even higher activity. Furthermore, pharmacological investigations including the ApoToxGlo Triplex assay, LDH assay, and cell cycle measurements revealed that this compound induced apoptosis and reduced cells in the G1 phase accompanied by an increase of cells in the G2/M phase. Moreover, it showed hardly any effects on the cell membrane integrity. However, it also exhibited cytotoxicity against non-tumorigenic cells. Nevertheless, in summary, we could show that shikonin derivatives might be promising drug leads in the treatment of melanoma.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms22052774DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7967198PMC
March 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1177/1947603520962558DOI Listing
October 2020

Cobalt Chromium Molybdenum Surface Modifications Alter the Osteogenic Differentiation Potential of Human Mesenchymal Stem Cells.

Materials (Basel) 2020 Sep 25;13(19). Epub 2020 Sep 25.

Department of Orthopedics and Trauma, Medical University Graz, 8036 Graz, Austria.

Surface roughness on orthopedic implant materials has been shown to be highly influential on the behavior of osteogenic cells. Mesenchymal stem and progenitor cells (MSPCs) migrate to the interface, adhere, proliferate, and differentiate into osteoblasts, which subsequently form bone matrix. Modifications of the implant surfaces should accelerate this process and improve biocompatibility. In this study, five surface topographies on cobalt chromium molybdenum (CoCrMo) were engineered to examine the influence on MSPCs. Scanning electron microscopy revealed significant differences in the morphology of untreated CoCrMo discs in comparison with CoCrMo with a titanium nitride (TiN) coating, polished and porous coated CoCrMo surfaces, and CoCrMo with a pure titanium (cpTi) coating. Elemental analysis was performed using energy-dispersive X-ray spectroscopy (EDX). Human primary MSPCs were expanded from tissue samples of spongiosa bone and characterized according to the criteria of the International Society for Cellular Therapy. The characteristic phenotype of MSPC was confirmed by flow cytometry and multilineage differentiation. Alcaline phosphatase and osteopontin expression increased significantly in all groups about 5-fold and 10-fold, respectively, in comparison to the undifferentiated controls. The porous coated surface showed a reduced expression of osteogenic markers. Due to the osteogenic differentiation, the expression of integrin α5β1, which is particularly important for cell-material contact, increased 4-7-fold. In the dynamic process of bone biology, MSPCs cultured and differentiated on cpTi, showed significant upregulation of IL6 and leptin.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ma13194292DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7579014PMC
September 2020

Enhanced Osteogenic Differentiation of Human Primary Mesenchymal Stem and Progenitor Cultures on Graphene Oxide/Poly(methyl methacrylate) Composite Scaffolds.

Materials (Basel) 2020 Jul 5;13(13). Epub 2020 Jul 5.

School of Medicine, National University of Ireland, H91 CF50 Galway, Ireland.

Due to its versatility, small size, large surface area, and ability to interact with biological cells and tissues, graphene oxide (GO) is an excellent filler for various polymeric composites and is frequently used to expand their functionality. Even though the major advantage of the incorporation of GO is the enhancement of mechanical properties of the composite material, GO is also known to improve bioactivity during biomineralization and promote osteoblast adhesion. In this study, we described the fabrication of a composite bone cement made of GO and poly(methyl methacrylate) (PMMA), and we investigated its potential to enhance osteogenic differentiation of human primary mesenchymal stem and progenitor cells. Through the analysis of three differentiation markers, namely alkaline phosphatase, secreted protein acidic and rich in cysteine, and bone morphogenetic protein-2 in the presence and in the absence of an osteogenic differentiation medium, we were able to indicate a composite produced manually with a thick GO paper as the most effective among all investigated samples. This effect was related to its developed surface, possessing a significant number of voids and pores. In this way, GO/PMMA composites were shown as promising materials for the applications in bone tissue engineering.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ma13132991DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7372355PMC
July 2020

The role of stretch, tachycardia and sodium-calcium exchanger in induction of early cardiac remodelling.

J Cell Mol Med 2020 08 22;24(15):8732-8743. Epub 2020 Jun 22.

Department of Cardiology, Medical University of Graz, Graz, Austria.

Stretch and tachycardia are common triggers for cardiac remodelling in various conditions, but a comparative characterization of their role in the excitation-transcription coupling (ETC) and early regulation of gene expression and structural changes is lacking. Here, we show that stretch and tachycardia directly induced hypertrophy of neonatal rat cardiac myocytes and also of non-myocytes. Both triggers induced similar patterns of hypertrophy but had largely distinct gene expression profiles. ACTA1 served as good hypertrophy marker upon stretch, while RCAN1 was found increased in response to tachycardia in a rate-dependent fashion. Mechanistically, several calcium-handling proteins, including the sodium-calcium exchanger (NCX), contributed to ETC. Phosphorylation of the calcium/calmodulin-dependent protein kinase II (CaMKII) was elevated and occurred downstream of NCX activation upon tachycardia, but not stretch. Microarray profiling revealed that stretch and tachycardia regulated around 33% and 20% genes in a NCX-dependent manner, respectively. In conclusion, our data show that hypertrophy induction by stretch and tachycardia is associated with different gene expression profiles with a significant contribution of the NCX.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/jcmm.15504DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7412684PMC
August 2020

Periplocin mediates TRAIL-induced apoptosis and cell cycle arrest in human myxofibrosarcoma cells via the ERK/p38/JNK pathway.

Phytomedicine 2020 Jun 5;76:153262. Epub 2020 Jun 5.

Division of Biomedical Research, Medical University Graz, Roseggerweg 48, 8036Graz, Austria; Institute of Pharmaceutical Sciences, Department of Pharmacognosy, University of Graz, Universitaetsplatz 4/1, 8010Graz, Austria.

Background: Periploca sepium is traditionally used in Chinese medicine to treat particularly rheumatic disorders and as a tonic. Periplocin was found as the most cytotoxic compound of its root bark and induced death receptor mediated apoptosis in liposarcoma cells. Sarcomas are a rare type of cancer with only a few treatment options. The five-year survival rate of advanced tumors is low.

Purpose: In this study, we investigated the effects of periplocin in two myxofibrosarcoma (MFS)cell lines, MUG-Myx2a and MUG-Myx2b, which are subclones of the same tumor and reflect the tumor´s heterogeneity, and in T60 primary myxofibrosarcoma cells.

Methods: The xCELLigence system and the CellTiter 96® AQ assay were used for studying cell viability. FACS and Western blot experiments were used to investigate the effects of periplocin on apoptosis induction, cell cycle distribution, and the expression of cleaved PARP, caspase 3, p53, phospho-histone γH2AX, ERK/phospho ERK, p38/phospho p38, and, finally, JNK/phospho JNK. Additionally, the expression of the apoptotic markers Bim, NOXA, Bak, Bcl-2, Bcl-xl, and the death receptors IGFR, FADD, TRADD, TNFR1A, TRAIL-R1, and TRAIL-R2 were evaluated using reversed real-time PCR.

Results: Periplocin decreased dose-dependently the viability of all MFS cell lines and was more effective than the standard chemotherapeutic doxorubicin. It arrested the cells in the G2/M phase and led to caspase activation. Moreover, periplocin increased the mRNA expression of NOXA, Bak, Bcl-2, and death receptors such as TRAIL-R1 and TRAIL-R2 and the protein expression of ERK/phospho ERK, p38/phospho p38, and JNK/phospho JNK. In all cases, differences in the effects in the different subclones were observed.

Conclusion: Periplocin showed promising effects in MFS cells. The higher effectiveness compared to doxorubicin is an important aspect for further research with regard as a treatment option. The different effects of periplocin in the two subclones showed the great importance of intratumoral heterogeneity in MFS therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.phymed.2020.153262DOI Listing
June 2020

CoCrMo surface modifications affect biocompatibility, adhesion, and inflammation in human osteoblasts.

Sci Rep 2020 02 3;10(1):1682. Epub 2020 Feb 3.

Department of Orthopedics and Trauma, Medical University Graz, Graz, Austria.

In this study, different surface modifications were performed on a Cobalt-Chrome-Molybdenum (CoCrMo) alloy and the effects on cell viability and cytotoxicity as well as the adhesion potential of human osteoblasts (hFOB) and their inflammation reaction were investigated in vitro. CoCrMo discs were coated with TiN, with polished and porous coated surfaces, or with pure titanum (cpTi) surfaces and examined by Scanning Electron Microscopy to evaluate surface modifications. In vitro cell viability, adhesion behaviour, and expression of inflammation markers of hFOB human osteoblasts were measured via CellTiter-Glo, CytoTox, ELISA, and RT-PCR respectively. All results were compared to CoCrMo without surface modifications. The biocompatibility data showed high compatibility for the TiN hard coatings. Likewise, the porous surface coating increased cell viability significantly, compared to an untreated CoCrMo alloy. None of the investigated materials influenced cytotoxicity. Different surface modifications did not influence expression of fibronectin, although TiN, porous surface coatings and polished surfaces showed highly significant reductions in integrin subunit expression. In addition to the regulation of adhesion potential these three surfaces stimulated an anti-inflammatory response by osteocytes. Improved biocompatibility and adhesion properties may contribute to better osteointegration of prosthetics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-020-58742-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6997456PMC
February 2020

Human melanoma brain metastases cell line MUG-Mel1, isolated clones and their detailed characterization.

Sci Rep 2019 03 11;9(1):4096. Epub 2019 Mar 11.

Department for Biomedical Research, Medical University of Graz, Graz, Austria.

Melanoma is a leading cause of high mortality that frequently spreads to the brain and is associated with deterioration in quality and quantity of life. Treatment opportunities have been restricted until now and new therapy options are urgently required. Our focus was to reveal the potential heterogeneity of melanoma brain metastasis. We succeeded to establish a brain melanoma metastasis cell line, namely MUG-Mel1 and two resulting clones D5 and C8 by morphological variety, differences in lipidome, growth behavior, surface, and stem cell markers. Mutation analysis by next-generation sequencing, copy number profiling, and cytogenetics demonstrated the different genetic profile of MUG-Mel1 and clones. Tumorigenicity was unsuccessfully tested in various mouse systems and finally established in a zebra fish model. As innovative treatment option, with high potential to pass the blood-brain barrier a peptide isolated from lactoferricin was studied in potential toxicity. Brain metastases are a major clinical challenge, therefore the development of relevant in vitro and in vivo models derived from brain melanoma metastases provides valuable information about tumor biology and offers great potential to screen for new innovative therapies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-019-40570-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6411871PMC
March 2019

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cellsig.2018.12.010DOI Listing
April 2019

Periplocin, the most anti-proliferative constituent of Periploca sepium, specifically kills liposarcoma cells by death receptor mediated apoptosis.

Phytomedicine 2018 Dec 11;51:162-170. Epub 2018 Oct 11.

Institute of Pharmaceutical Sciences, Department of Pharmacognosy, University of Graz, Universitaetsplatz 4/1, 8010 Graz, Austria.

Background: During a screening of Chinese plants traditionally used for the treatment of cancer and related diseases, extracts of the root bark of Periploca sepium Bunge showed strong cytotoxic activity.

Purpose: Isolate and identify cytotoxic compounds from P. sepium and investigate the effects and mechanism of action on different cancer cell lines.

Methods: Extracts obtained with solvents of different polarities of the root bark of P. sepium were tested for their anti-proliferative effects. The most active extract was subjected to activity-guided fractionation using different chromatographic methods. The most active compound was further investigated on sarcoma cell lines regarding its effects concerning apoptosis, DNA damage and death receptor expression.

Results: We isolated the cardiac glycosides periplocin, glucosyl divostroside, periplogenin, periplocymarin and periplocoside M with periplocin exhibiting the lowest IC value against leukemia and liposarcoma cells. Liposarcomas are rare tumors within the heterogeneous group of soft tissue sarcomas and respond poorly to conventional treatments. Periplocin led to growth inhibition and apoptosis induction by changing the expression of death receptors and inducing DNA double strand breaks in SW-872 cells.

Conclusion: Periplocin displays a promising mechanism of action in sarcoma cells because altering the death receptor expression is an interesting target in sarcoma treatment especially to overcome TRAIL resistance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.phymed.2018.10.008DOI Listing
December 2018

Comparative Gene Expression Analysis in WM164 Melanoma Cells Revealed That --Dimethylacrylshikonin Leads to ROS Generation, Loss of Mitochondrial Membrane Potential, and Autophagy Induction.

Molecules 2018 Oct 30;23(11). Epub 2018 Oct 30.

Institute of Pharmaceutical Sciences, Department of Pharmacognosy, University of Graz, Universitaetsplatz 4/1, 8010 Graz, Austria.

Skin cancer is currently diagnosed as one in every three cancers. Melanoma, the most aggressive form of skin cancer, is responsible for 79% of skin cancer deaths and the incidence is rising faster than in any other solid tumor type. Previously, we have demonstrated that dimethylacrylshikonin (DMAS), isolated from the roots of (Boraginaceae), exhibited the lowest IC values against different tumor types out of several isolated shikonin derivatives. DMAS was especially cytotoxic towards melanoma cells and led to apoptosis and cell cycle arrest. In this study, we performed a comprehensive gene expression study to investigate the mechanism of action in more detail. Gene expression signature was compared to vehicle-treated WM164 control cells after 24 h of DMAS treatment; where 1192 distinct mRNAs could be identified as expressed in all replicates and 89 were at least 2-fold differentially expressed. DMAS favored catabolic processes and led in particular to p62 increase which is involved in cell growth, survival, and autophagy. More in-depth experiments revealed that DMAS led to autophagy, ROS generation, and loss of mitochondrial membrane potential in different melanoma cells. It has been reported that the induction of an autophagic cell death represents a highly effective approach in melanoma therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/molecules23112823DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6278572PMC
October 2018

Synthesis of Novel Shikonin Derivatives and Pharmacological Effects of Cyclopropylacetylshikonin on Melanoma Cells.

Molecules 2018 10 30;23(11). Epub 2018 Oct 30.

Institute of Pharmaceutical Sciences, Department of Pharmacognosy, University of Graz, Universitaetsplatz 4, 8010 Graz, Austria.

Despite much research in the last centuries, treatment of malignant melanoma is still challenging because of its mostly unnoticeable metastatic spreading and aggressive growth rate. Therefore, the discovery of novel drug leads is an important goal. In a previous study, we have isolated several shikonin derivatives from the roots of Bureau & Franchet (Boraginaceae) which evolved as promising anticancer candidates. ,-Dimethylacrylshikonin () was the most cytotoxic derivative and exhibited strong tumor growth inhibitory activity, in particular, towards melanoma cells. In this study, we synthesized eighteen novel shikonin derivatives in order to obtain compounds which exhibit a higher cytotoxicity than . We investigated their cytotoxic potential against various melanoma cell lines and juvenile skin fibroblasts. The most active compound was ()-1-(1,4-dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl cyclopropylacetate (cyclopropylacetylshikonin) (). It revealed significant stronger tumor growth inhibitory activity towards two melanoma cell lines derived from metastatic lesions (WM164 and MUG-Mel2). Further investigations have shown that induced apoptosis caspase-dependently, increased the protein levels of cleaved PARP, and led to double-stranded DNA breaks as shown by phosphorylation of H2AX. Cell membrane damage and cell cycle arrest were not observed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/molecules23112820DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6278577PMC
October 2018

Influence of silibinin and β-β-dimethylacrylshikonin on chordoma cells.

Phytomedicine 2018 Oct 12;49:32-40. Epub 2018 Jun 12.

Department of Pharmacognosy, Institute of Pharmaceutical Sciences, University of Graz, Graz, Austria.

Background: Chordoma, slow growing bone tumours originating from remnants of the notochord, leave affected patients with a median survival of six years. The high recurrence rate of chordoma, together with limited treatment options and bad overall prognosis, make the development of new treatment options urgently necessary.

Purpose: In this study, the potential of two natural products, silibinin and β-β-dimethylacrylshikonin (DMAS), was tested on clival (MUG-CC1 and UM-Chor1) as well as sacral (MUG-Chor1 and U-CH2) chordoma cell lines. The treatment was administered both as single- and combined therapy.

Methods: For investigation of cell viability, the Cell Titer 96 Aqueous Non-Radioactive Cell Proliferation Assay Kit was used. Apoptosis induction was studied by flow cytometry, (Annexin V/SYTOX Green, caspase-3) and RT-qPCR. Pathway analyses were performed by western blot.

Results: Both drugs were found to reduce cell viability alone as well as in combination in a dose dependent manner, with DMAS being more efficient than silibinin. The mode of cell death was mainly apoptosis in DMAS treated samples, while the combination therapy led to apoptosis as well as late-apoptosis/necrosis. Silibinin therapy alone, although reducing cell viability, did not lead to significant apoptotic effects in the performed assays. Focussing on the molecular mechanism of DMAS induced apoptosis, it was found that major genes of the mitochondrial apoptosis pathway, like NOXA and PUMA were overexpressed. Additionally, western blot experiments showed a decrease of ERK/pERK, STAT3/pSTAT3 (Tyr705) and AKT/pAKT expression/activation levels under DMAS treatment.

Conclusion: DMAS is a promising new candidate for chordoma therapy, while silibinin or a combination of both is less favourable.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.phymed.2018.06.005DOI Listing
October 2018

Characterization of the endolysosomal system in human chordoma cell lines: is there a role of lysosomes in chemoresistance of this rare bone tumor?

Histochem Cell Biol 2018 Jul 3;150(1):83-92. Epub 2018 May 3.

Division of Biomedical Research, Medical University of Graz, Roseggerweg 48, 8010, Graz, Austria.

Chordoma is a rare tumor of the bone derived from remnants of the notochord with pronounced chemoresistance. A common feature of the notochord and chordoma cells is distinct vacuolization. Recently, the notochord vacuole was described as a lysosome-related organelle. Since lysosomes are considered as mediators of drug resistance in cancer, we were interested whether they may also play a role in chemoresistance of chordoma. We characterized the lysosomal compartment in chordoma cell lines by cytochemistry, electron microscopy (ELMI) and mutational analysis of genes essential for the physiology of lysosomes. Furthermore, we tested for the first time the cytotoxicity of chloroquine, which targets lysosomes, on chordoma. Cytochemical stainings clearly demonstrated a huge mass of lysosomes in chordoma cell lines with perinuclear accumulation. Also vacuoles in chordoma cells were positive for the lysosomal marker LAMP1 but showed no acidic pH. Genetic analysis detected no apparent mutation associated with known lysosomal pathologies suggesting that vacuolization and the huge lysosomal mass of chordoma cell lines is rather a relict of the notochord than a result of transformation. ELMI investigation of chordoma cells confirmed the presence of large vacuoles, lysosomes and autophagosomes with heterogeneous ultrastructure embedded in glycogen. Interestingly, chordoma cells seem to mobilize cellular glycogen stores via autophagy. Our first preclinical data suggested no therapeutically benefit of chloroquine for chordoma. Even though, chordoma cells are crammed with lysosomes which are according to their discoverer de Duve "cellular suicide bags". Destabilizing these "suicide bags" might be a promising strategy for the treatment of chordoma.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00418-018-1673-xDOI Listing
July 2018

Surface modification and characterization of GO/polymer thin coatings as excellent bio-active platforms for tissue regeneration.

Mater Sci Eng C Mater Biol Appl 2018 Mar 23;84:130-139. Epub 2017 Nov 23.

Department of Orthopedic Surgery, Medical University Graz, Auenbruggerplatz 5, Graz, Austria.

Osteo-integration and tissue regeneration are vital for the longevity, durability, and unremitting functionality of medical implants/scaffolds implanted in vivo. It's essential for biomaterials used for in vivo implantation to induce the cellular secretion of growth factors, necessary for the desired tissue generation, since the administration of artificial growth factors, in vivo, is largely prohibited. Plasma functionalized (N and O) and stabilized Graphene Oxide (GO) thin layers in a hybrid with amorphous carbon (aC) induced the expression of vascular endothelial growth factor (VEGF) and osteoprotegerin (OPG) growth factors in fibroblasts (hGF) and, more remarkably, in osteoblasts (hFOB) cells confirming the suitability for tissue regeneration and osteo-integration applications. We also observed a negative trend between hGF fibroblasts, but not hFOB osteoblasts, cellular viability and GO presence in the hybrid films that might indicate the phenomenon of oxidative stress. We traced that back to the presence of higher concentrations of carboxyl and the carbonyl groups on the surface of the GO rich coatings. The above described properties provided by GO coatings might be desirable for bio-selectivity applications and for the reduction of the undesired fibrosis process that is associated with medical implants in vivo environment. Moreover, novel plasma functionalized GO/polymer hybrid thin coating hybrid compositions are promising candidates for tissue engineering and bioengineering applications as excellent antimicrobial and anticancer platforms.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.msec.2017.11.030DOI Listing
March 2018

Histone deacetylase inhibitors vorinostat and panobinostat induce G1 cell cycle arrest and apoptosis in multidrug resistant sarcoma cell lines.

Oncotarget 2017 Sep 24;8(44):77254-77267. Epub 2017 Aug 24.

Department of Orthopedics and Trauma, Medical University of Graz, 8036 Graz, Austria.

Synovial sarcoma and high grade chondrosarcoma are characterized by their 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 histone deacetylases inhibitors (HDACi) have shown specific efficacy as an active antitumor agent for treating a variety of solid tumors. However, as of yet the effect of different HDACi on synovial- and chondrosarcoma cells has not been investigated. In this study, vorinostat (SAHA), panobinostat (LBH-589), and belinostat (PXD101) decreased cell viability of synovial sarcoma (SW-982) and chondrosarcoma (SW-1353) cells in a time- and dose dependent manner and arrested SW-982 cells in the G1/S phase. Western blot analysis determined the responsible cell cycle regulator proteins. In addition, we found apoptotic induction by caspase 3/7 activity, caspase 3 cleavage, and PARP cleavage. In SW-1353 cells only SAHA showed comparable effects. Noteworthy, all HDACi tested had synergistic effects with the topoisomerase II inhibitor doxorubicin in SW-1353 chondrosarcoma cells making the cells more sensitive to the chemotherapeutic drug. Our results show for the first time that SAHA and LBH-589 reduced viability of sarcoma cells and arrested them at the G1/S checkpoint, while also inducing apoptosis and enhancing chemotherapeutic sensitivity, especially in chondrosarcoma cells. These data demonstrate the exciting potential of HDACi for use in sarcoma treatment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.18632/oncotarget.20460DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5652778PMC
September 2017

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbrep.2017.06.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5614688PMC
September 2017

Attachment and growth of human osteoblasts on different biomaterial surfaces.

Int J Comput Dent 2017;20(3):229-243

Objectives: To prove the biocompatibility of biomaterials applied in biomedical devices, in vitro testing is crucial to render a material fit for medical application. The material of choice for dental implants is commercially pure titanium (cp-Ti), while other materials such as zirconia and polyetheretherketone (PEEK) are considered highly promising due to their functional and esthetic properties. The aim of this study was to determine whether PEEK with defined mean surface roughness and composition could achieve results equal to titanium or zirconia.

Materials And Methods: Disks measuring 14 mm in diameter and 1 mm in thickness made from cp-Ti, yttria-stabilized zirconia (Y-TZP), and filled PEEK with a smooth surface finish were used for cell culture experiments. Human fetal osteoblasts (hFOB) were cultured in vitro on each material to observe changes after 1, 3, and 7 days regarding cell viability and lactate dehydrogenase (LDH) release. Additionally, mRNA expression of proliferative factors PCNA and Ki67 and cellular adhesion (vinculin mRNA expression and immunofluorescence staining) were analyzed after 3 days in the culture.

Results: In hFOB cultures, adhesion and viability were decreased on PEEK platelets, while LDH release remained stable. No significant difference was observed in cp-Ti and Y-TZP when compared to the control.

Conclusions: The performance of cp-Ti and Y-TZP was equal to the control in all tests. It seems that highly polished PEEK in this particular composition cannot be recommended for osseointegrated implant applications due to decreased osteoblast attachment. Further investigations are recommended, especially in surface structures optimized for osseointegration.
View Article and Find Full Text PDF

Download full-text PDF

Source
November 2017

Expanded molecular profiling of myxofibrosarcoma reveals potentially actionable targets.

Mod Pathol 2017 12 4;30(12):1698-1709. Epub 2017 Aug 4.

Institute of Pathology, Medical University of Graz, Graz, Austria.

Myxofibrosarcomas are morphologically heterogeneous soft tissue sarcomas lacking a specific immunohistochemical expression profile and recurrent genetic changes. The study was designed to gain further insights into the molecular landscape of myxofibrosarcomas by targeted re-sequencing of known cancer driver hotspot mutations and the analysis of genomewide somatic copy number alterations. A well-defined group of myxofibrosarcomas, including myxofibrosarcomas G1 (n=6), myxofibrosarcomas G3 (n=7), myxofibrosarcomas with morphologically heterogeneous and independently selectable G1 and G3 areas within a tumor (n=8), and myxofibrosarcomas G3 with subsequent tumor recurrence (n=1) or metastatic disease (n=3) were evaluated. Mutational analysis demonstrated mutations in TP53, PTEN, FGFR3, CDKN2A, and RB1. TP53 mutations were seen in 11 (44%) of patients and detected in myxofibrosarcomas G1, G3, with heterogeneous morphology and G3 with subsequent metastases in 1 patient (16%), 3 patients (42%), 2 patients (62.5%), and 3 patients (75%), respectively. Additional mutations were detected in 2 patients, intratumoral mutational heterogeneity in 1 patient. We observed a variety of copy number alterations typical for myxofibrosarcomas, with higher numbers in G3 compared with G1 myxofibrosarcomas. Cluster analysis revealed distinctive features especially in metastatic and recurrent disease. Focal alterations affected CDKN2A, CCND1, CCNE1, EGFR, EPHA3, EPHB1, FGFR1, JUN, NF1, RB1, RET, TP53, and additional novel amplifications in CCNE1, KIT, EGFR, RET, BRAF, NTRK2 were seen in G3 compared with the G1 tumor areas. The total number of focal events in G1 versus G3 tumors differed significantly (P=0.0014). TRIO and RICTOR co-amplification was seen in 8 (44%) G3 and 1 (10%) G1 myxofibrosarcomas and RICTOR amplification alone in 4 (40%) G1 myxofibrosarcomas. TRIO amplification was significantly (P=0.0218) higher in G3 myxofibrosarcomas indicating a late genetic event. These findings support the use of expanded molecular profiling in myxofibrosarcomas to detect drug-able targets to allow patients to participate in basket trials.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/modpathol.2017.94DOI Listing
December 2017

MUG-Mel2, a novel highly pigmented and well characterized NRAS mutated human melanoma cell line.

Sci Rep 2017 05 18;7(1):2098. Epub 2017 May 18.

Division of Dermatology, Graz, Medical University Graz, Graz, Austria.

NRAS mutation in melanoma has been associated with aggressive tumor biology and poor prognosis. Although targeted therapy has been tested for NRAS mutated melanoma, response rates still appear much weaker, than in BRAF mutated melanoma. While plenty of cell lines exist, however, only few melanogenic cell lines retain their in vivo characteristics. In this work we present an intensively pigmented and well-characterized cell line derived from a highly aggressive NRAS mutated cutaneous melanoma, named MUG-Mel2. We present the clinical course, unique morphology, angiogenic properties, growth characteristics using in vivo experiments and 3D cell culture, and results of the exome gene sequencing of an intensively pigmented melanogenic cell line MUG-Mel2, derived from a cutaneous metastasis of an aggressive NRAS p. Q61R mutated melanoma. Amongst several genetic alterations, mutations in GRIN2A, CREBP, PIK3C2G, ATM, and ATR were present. These mutations, known to reinforce DNA repair problems in melanoma, might serve as potential treatment targets. The aggressive and fast growing behavior in animal models and the obtained phenotype in 3D culture reveal a perfect model for research in the field of NRAS mutated melanoma.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-017-02197-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5437015PMC
May 2017

Functionalized, biocompatible, and impermeable nanoscale coatings for PEEK.

Mater Sci Eng C Mater Biol Appl 2017 Jul 18;76:865-870. Epub 2017 Mar 18.

Department of Applied Physics, Ghent University, Sint-Pietersnieuwstraat 41 B4, 9000 Gent, Belgium.

Biologically compatible coatings that provide hermetic seal could resolve a major technological hurdle in the attempt to replace metals with polymers for biochips and active medical implants. The use of amorphous carbon/diamond like carbon (a-C:H) coatings to hermetically seal and biologically enhance polyether-ether-ketone (PEEK) for biomedical device integration in the human body was investigated. The PEEK coating functionality (sp3/sp2 ratio), hardness and thickness (70-200nm) were controlled, by varying H and N concentration during the plasma operation with CH. a-C:H coatings having the highest indentation modulus of 13.5GPa, originate out of a CH (90%) rich composition. Even in a mixture of 70/30 H/CH the hardness is 4.76GPa, corresponding to hard and dense coatings. In all tested conditions of deposition coatings hardens was sufficient for the purpose of PEEK implants modification. The synthesized (a-C:H) nanoscale coatings were not water permeable as measured by the hydrolysis test, resolving the traditional challenge of swelling in wet environment. The hardness of the coatings showed strong correlations with the thickness, surprisingly however, with no correlations with the sp3/sp2 ratio. Selected non water permeable nanoscale coating on PEEK showed strong bioactivity by being viable for human osteoblast (hFOB) and human fibroblast (hGF) cells without toxicity issues. No correlation was observed between the coatings sp3/sp2 ratio and biological performance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.msec.2017.03.153DOI Listing
July 2017

Establishment of a novel cellular model for myxofibrosarcoma heterogeneity.

Sci Rep 2017 03 17;7:44700. Epub 2017 Mar 17.

Institute of Pathology, Medical University of Graz, Graz, Austria.

Human cancers frequently display substantial intra-tumoural heterogeneity in virtually all distinguishable phenotypic features, such as cellular morphology, gene expression, and metastatic potential. In order to investigate tumour heterogeneity in myxofibrosarcoma, we established a novel myxofibrosarcoma cell line with two well defined sub-clones named MUG-Myx2a and MUG-Myx2b. The parental tumour tissue and both MUG-Myx2 cell lines showed the same STR profile. The fact that MUG-Myx2a showed higher proliferation activity, faster migration and enhanced tumourigenicity was of particular interest. NGS mutation analysis revealed corresponding mutations in the FGFR3, KIT, KDR and TP53 genes. In contrast, the MUG-Myx2a cell lines showed an additional PTEN mutation. Analysis of CNV uncovered a highly aberrant karyotype with frequent losses and gains in the tumour sample. The two MUG-Myx2 cell lines share several CNV features of the tumour tissue, while some CNVs are present only in the two cell lines. Furthermore, certain CNV gains and losses that are exclusive to either MUG-Myx2a or MUG-Myx2b, distinguish the two cell lines. As it is currently not possible to purchase two different sarcoma cell lines derived from the same patient, the novel myxofibrosarcoma cell lines MUG-Myx2a and MUG-Myx2b will be useful tools to study pathogenesis, tumour heterogeneity and treatment options.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep44700DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5356330PMC
March 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0168193PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5158315PMC
July 2017

Establishment of clival chordoma cell line MUG-CC1 and lymphoblastoid cells as a model for potential new treatment strategies.

Sci Rep 2016 Apr 13;6:24195. Epub 2016 Apr 13.

Division of Biomedical Research, Medical University of Graz, 8036 Graz, Austria.

Chordomas are rare malignant tumors that develop from embryonic remnants of the notochord and arise only in the midline from the clivus to the sacrum. Surgery followed by radiotherapy is the standard treatment. As chordomas are resistant to standard chemotherapy, further treatment options are urgently needed. We describe the establishment of a clivus chordoma cell line, MUG-CC1. The cell line is characterized according to its morphology, immunohistochemistry, and growth kinetics. During establishment, cell culture supernatants were collected, and the growth factors HGF, SDF-1, FGF2, and PDGF analyzed using xMAP(®) technology. A spontaneous lymphoblastoid EBV-positive cell line was also developed and characterized. MUG-CC1 is strongly positive for brachyury, cytokeratin, and S100. The cell line showed gains of the entire chromosomes 7, 8, 12, 13, 16, 18, and 20, and high level gains on chromosomes 1q21-1q24 and 17q21-17q25. During cultivation, there was significant expression of HGF and SDF-1 compared to continuous chordoma cell lines. A new, well-characterized clival chordoma cell line, as well as a non-tumorigenic lymphoblastoid cell line should serve as an in vitro model for the development of potential new treatment strategies for patients suffering from this disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep24195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4829844PMC
April 2016

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12885-015-1915-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4641423PMC
November 2015

Impact of cyclic mechanical stimulation on the expression of extracellular matrix proteins in human primary rotator cuff fibroblasts.

Knee Surg Sports Traumatol Arthrosc 2016 Dec 21;24(12):3884-3891. Epub 2015 Sep 21.

Department of Orthopaedic Surgery, Medical University of Graz, Auenbruggerplatz 5, 8036, Graz, Austria.

Purpose: Mechanical stimulation plays an important role in the development and remodelling of tendons. The aim of the study was to evaluate the effects of mechanical stimulation on the expression of extracellular matrix proteins in human primary rotator cuff (RC) fibroblasts.

Methods: RC fibroblasts were isolated from patients with degenerative RC tears and characterized using flow cytometry and immunohistochemistry. Cells were stimulated using the Flexcell FX5K™ Tension System. The stimulation regime was a uniaxial sinusoidal waveform with 10 % elongation and a frequency of 0.5 Hz, whereby each cycle consists of 10-s strain and 30-s relaxation. Data were normalized to mechanically unstimulated control groups for every experimental condition. RT-qPCR was performed to determine relative mRNA levels, and collagen production was measured by a colorimetric assay.

Results: The positive expression of CD91 and CD10, and negativity for CD45 and CD4 confirmed the fibroblast phenotype of RC primary cells. RT-qPCR revealed that 10 % continuous cyclic strain for 7 and 14 days induced a significant increase in the mRNA expression both on the matrix metalloproteinases MMP1, MMP3, MMP13, and MMP14 and on the extracellular matrix proteins decorin, tenascin-C, and scleraxis. Furthermore, mechanically stimulated groups produced significantly higher amounts of total collagen.

Conclusion: These results may contribute to a better understanding of strain-induced tendon remodelling and will form the basis for the correct choice of applied force in rehabilitation after orthopaedic surgery. These findings underline the fact that early passive motion of the joint in order to induce remodelling of the tendon should be included within a rehabilitation protocol for rotator cuff repair.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00167-015-3790-6DOI Listing
December 2016

Mutation Analysis of Nine Chordoma Specimens by Targeted Next-Generation Cancer Panel Sequencing.

J Cancer 2015 20;6(10):984-9. Epub 2015 Aug 20.

2. Center for Medical Research, Cell Culture Facility, Medical University of Graz, Stiftingtalstrasse 24, 8010 Graz, Austria.

Background: Chordoma is a rare primary malignant bone tumour. Treatment options are mainly restricted to surgical excision, since chordomas are largely resistant to conventional ionising radiation and chemotherapy. Thus, there is a strong need to gain more thorough insights into the molecular biology and genetics of chordoma to allow for the development of new therapeutic options. We performed an ultra-deep sequencing analysis to find novel mutations in cancer associated genes in chordomas to date unseen with Sanger sequencing.

Material And Methods: Nine chordomas (skull base (n=3), mobile spine (n=4), and sacrum/coccyx (n=2) were screened for mutations in 48 cancer genes using the Hot Spot Cancer Panel (Illumina). All putative mutations were compared against multiple databases (e.g. NCBI, COSMIC, PolyPhen, EGB, SIFT) and published Copy Number Variation (CNV) data for chordoma.

Results: Our results showed mutations with a frequency above 5% in tumorsuppressor- and onco-genes, revealing new possible driver genes for chordomas. We detected three different variants accounting for 11 point mutations in three cancer associated genes (KIT, KDR and TP53). None of the detected mutations was found in all samples investigated. However, all genes affected interact or are connected in pathway analysis. There were no correlations to already reported CNVs in the samples analysed.

Conclusions: We identified mutations in the associated genes KIT, KDR, and TP53. These mutations have been described previously and have been predicted to be tolerated. Further results on a larger series are warranted. The driver mechanisms of chordoma still have to be identified.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7150/jca.11371DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4565847PMC
September 2015

25-O-acetyl-23,24-dihydro-cucurbitacin F induces cell cycle G2/M arrest and apoptosis in human soft tissue sarcoma cells.

J Ethnopharmacol 2015 Apr 19;164:265-72. Epub 2015 Feb 19.

Department of Orthopaedic Surgery, Medical University Graz, Auenbruggerplatz 5, 8036 Graz, Austria.

Ethnopharmacological Relevance: Quisqualis indica is used in traditional Chinese medicine to treat cancer and related syndromes and also known for its anthelminthic effects.

Aim Of The Study: Soft tissue sarcomas represent a rare group of malignant tumors that frequently exhibit chemotherapeutic resistance and increased metastatic potential. In this study, we evaluated the cytotoxic, apoptosis inducing and cell cycle arresting effects of 25-O-acetyl-23,24-dihydro-cucurbitacin F which has been isolated from leaves and twigs of Q. indica.

Material And Methods: The present study investigates the effects of 25-O-acetyl-23,24-dihydro-cucurbitacin F (1) on cell viability, cell cycle distribution, and apoptotic induction of three human sarcoma cell lines of various origins by using the CellTiter 96(®) AQueous One Solution Cell Proliferation Assay, flow cytometrical experiments, real-time RT-PCR, Western blotting, and the Caspase-Glo(®) 3/7 Assay

Results: We could show that 1 reduced cell viability in a dose-dependent manner and arrested the cells at the G2/M interface. The accumulation of cells at the G2/M phase resulted in a significant decrease of the cell cycle checkpoint regulators cyclin B1, cyclin A, CDK1, and CDK2. Interestingly, 1 inhibited survivin expression significantly, which functions as a key regulator of mitosis and programmed cell death, and is overexpressed in many tumor types including sarcomas. Moreover, 1 induced apoptosis in liposarcoma and rhabdomyosarcoma cells caspase-3 dependently.

Conclusion: Our data strongly support 1 as a very interesting target for further investigation and development of novel therapeutics in sarcoma research.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jep.2015.02.023DOI Listing
April 2015